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Thymus cellularity and proportions of thymocyte subsets in both WT and EphA4-deficient mice grouped according to their proportions of DP cells. a Representative flow cytometry dot plots of both WT (EphA4 +/+ ) and EphA4 −/− mutant thymuses according to their proportions of DP thymocytes (DP hi , DP int , DP low ). b Total thymus cellularity. c Proportions of different thymocyte subsets according to the <t>CD4/CD8</t> expression. CD4 + CD8 − SP cells (CD4), DP cells, DN cells, CD4 − CD8 + SP cells (CD8). d Percentage of total TCRβ hi thymocytes. e Percentage of total mature thymocyte subsets defined by the expression of TCRβ hi and CD4/CD8 markers. f Data represent the percentage of total mature positive selected TCRβ hi CD69 + thymocytes and in different thymocyte subsets ( g ). h Percentage of total negative selected Cas3 + CD5 + CD69 + thymocytes according to the expression of Cleaved caspase-3 (Cas3), CD5 and CD69 cell markers. i Frequency of CD4 + T regulatory cells according to FoxP3 expression (TCRβ hi CD4 + FoxP3 + ). The proportions of total CD4 + ( j ) and CD8 + ( k ) CD69 − CD62L + cells. Data are presented as mean ± standard deviation (SD) and analyzed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc test ( b , h , j , k ), Brown-Forsythe and Welch ANOVA test with Tamhane’s T2 post hoc test ( d ) and one-way ( f , i ) or two-way ( c , e , g ) ANOVA test with Tukey’s post hoc test. * p
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1) Product Images from "Altered thymocyte development observed in EphA4-deficient mice courses with changes in both thymic epithelial and extracellular matrix organization"

Article Title: Altered thymocyte development observed in EphA4-deficient mice courses with changes in both thymic epithelial and extracellular matrix organization

Journal: Cellular and Molecular Life Sciences

doi: 10.1007/s00018-022-04610-w

Thymus cellularity and proportions of thymocyte subsets in both WT and EphA4-deficient mice grouped according to their proportions of DP cells. a Representative flow cytometry dot plots of both WT (EphA4 +/+ ) and EphA4 −/− mutant thymuses according to their proportions of DP thymocytes (DP hi , DP int , DP low ). b Total thymus cellularity. c Proportions of different thymocyte subsets according to the CD4/CD8 expression. CD4 + CD8 − SP cells (CD4), DP cells, DN cells, CD4 − CD8 + SP cells (CD8). d Percentage of total TCRβ hi thymocytes. e Percentage of total mature thymocyte subsets defined by the expression of TCRβ hi and CD4/CD8 markers. f Data represent the percentage of total mature positive selected TCRβ hi CD69 + thymocytes and in different thymocyte subsets ( g ). h Percentage of total negative selected Cas3 + CD5 + CD69 + thymocytes according to the expression of Cleaved caspase-3 (Cas3), CD5 and CD69 cell markers. i Frequency of CD4 + T regulatory cells according to FoxP3 expression (TCRβ hi CD4 + FoxP3 + ). The proportions of total CD4 + ( j ) and CD8 + ( k ) CD69 − CD62L + cells. Data are presented as mean ± standard deviation (SD) and analyzed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc test ( b , h , j , k ), Brown-Forsythe and Welch ANOVA test with Tamhane’s T2 post hoc test ( d ) and one-way ( f , i ) or two-way ( c , e , g ) ANOVA test with Tukey’s post hoc test. * p
Figure Legend Snippet: Thymus cellularity and proportions of thymocyte subsets in both WT and EphA4-deficient mice grouped according to their proportions of DP cells. a Representative flow cytometry dot plots of both WT (EphA4 +/+ ) and EphA4 −/− mutant thymuses according to their proportions of DP thymocytes (DP hi , DP int , DP low ). b Total thymus cellularity. c Proportions of different thymocyte subsets according to the CD4/CD8 expression. CD4 + CD8 − SP cells (CD4), DP cells, DN cells, CD4 − CD8 + SP cells (CD8). d Percentage of total TCRβ hi thymocytes. e Percentage of total mature thymocyte subsets defined by the expression of TCRβ hi and CD4/CD8 markers. f Data represent the percentage of total mature positive selected TCRβ hi CD69 + thymocytes and in different thymocyte subsets ( g ). h Percentage of total negative selected Cas3 + CD5 + CD69 + thymocytes according to the expression of Cleaved caspase-3 (Cas3), CD5 and CD69 cell markers. i Frequency of CD4 + T regulatory cells according to FoxP3 expression (TCRβ hi CD4 + FoxP3 + ). The proportions of total CD4 + ( j ) and CD8 + ( k ) CD69 − CD62L + cells. Data are presented as mean ± standard deviation (SD) and analyzed using the Kruskal–Wallis nonparametric test with Dunn’s post hoc test ( b , h , j , k ), Brown-Forsythe and Welch ANOVA test with Tamhane’s T2 post hoc test ( d ) and one-way ( f , i ) or two-way ( c , e , g ) ANOVA test with Tukey’s post hoc test. * p

Techniques Used: Mouse Assay, Flow Cytometry, Mutagenesis, Expressing, Standard Deviation

Proportions of different cell subsets included in the DN cell compartment of both WT and EphA4-deficient mice. a Flow cytometry strategy to analyze DN1-DN4 thymocyte subsets based on CD44 and CD25 expression gated on Lin − ckit lo/+ CD44 −/+ cells. b Percentage of DN1-DN4 thymocyte subsets. Proportions of total TCRγδ + thymocytes ( c ), CD4 − CD8 − CD45 + CD11c + DCs (CD11c + ) ( d ), CD4 − CD8 − CD45 + F4/80 + macrophages (F4/80 + ) ( e ) and CD4 − CD8 − CD45 + CD49b + NK cells (CD49b + ) ( f ). Data are presented as mean ± SD and analyzed using Kruskal–Wallis nonparametric test with Dunn’s post hoc test ( c , f ) and one-way ( d , e ) or two-way ( b ) ANOVA test with Tukey’s post hoc test. * p
Figure Legend Snippet: Proportions of different cell subsets included in the DN cell compartment of both WT and EphA4-deficient mice. a Flow cytometry strategy to analyze DN1-DN4 thymocyte subsets based on CD44 and CD25 expression gated on Lin − ckit lo/+ CD44 −/+ cells. b Percentage of DN1-DN4 thymocyte subsets. Proportions of total TCRγδ + thymocytes ( c ), CD4 − CD8 − CD45 + CD11c + DCs (CD11c + ) ( d ), CD4 − CD8 − CD45 + F4/80 + macrophages (F4/80 + ) ( e ) and CD4 − CD8 − CD45 + CD49b + NK cells (CD49b + ) ( f ). Data are presented as mean ± SD and analyzed using Kruskal–Wallis nonparametric test with Dunn’s post hoc test ( c , f ) and one-way ( d , e ) or two-way ( b ) ANOVA test with Tukey’s post hoc test. * p

Techniques Used: Mouse Assay, Flow Cytometry, Expressing

Apoptotic and cycling cells in both WT and EphA4-deficient mice. Apoptosis in total thymocytes ( a ) or in different cell subsets ( b ) on the basis of CD4/CD8 expression. Percentage of apoptosis in total mature TCRβ hi cells ( c ) or in DP and SP thymocytes ( d ). Apoptotic cells were defined as AnnexinV + SytoxBlue − . Proportions of cycling cells in total thymocytes ( e ), CD4/CD8 cell subsets ( f ), total TCRβ hi cells ( g ) and both DP and SP thymocytes ( h ). Cycling cells were defined as cells in S + G 2 /M cell cycle phase. Data are presented as mean ± SD and analyzed using one-way ( a , c , e , g ) or two-way ( b , d , f , h ) ANOVA test with Tukey’s post hoc test. * p
Figure Legend Snippet: Apoptotic and cycling cells in both WT and EphA4-deficient mice. Apoptosis in total thymocytes ( a ) or in different cell subsets ( b ) on the basis of CD4/CD8 expression. Percentage of apoptosis in total mature TCRβ hi cells ( c ) or in DP and SP thymocytes ( d ). Apoptotic cells were defined as AnnexinV + SytoxBlue − . Proportions of cycling cells in total thymocytes ( e ), CD4/CD8 cell subsets ( f ), total TCRβ hi cells ( g ) and both DP and SP thymocytes ( h ). Cycling cells were defined as cells in S + G 2 /M cell cycle phase. Data are presented as mean ± SD and analyzed using one-way ( a , c , e , g ) or two-way ( b , d , f , h ) ANOVA test with Tukey’s post hoc test. * p

Techniques Used: Mouse Assay, Expressing

2) Product Images from "Nociceptor neurons affect cancer immunosurveillance"

Article Title: Nociceptor neurons affect cancer immunosurveillance

Journal: Nature

doi: 10.1038/s41586-022-05374-w

RAMP1 expression in patient melanoma-infiltrating T cells correlates with worsened survival and poor responsiveness to ICIs. ( a–l ) In silico analysis of Cancer Genome Atlas (TCGA) data linked the survival rate among 459 patients with melanoma with their relative expression levels of various genes of interest (determined by bulk RNA sequencing of tumour biopsy). Kaplan–Meier curves show the patients’ survival after segregation in two groups defined by their low or high expression of a gene of interest. Increased gene expression (labelled as high; red curve) of TUBB3 ( b ), PGP9.5 ( c ), Nav 1.7 ( E ), SLPI ( k ) and RAMP1 ( l ) in biopsy correlate with decreased patient survival (p≤0.05). The mantel–Haenszel hazard ratio and number of patients included in each analysis are shown in the figure ( a–l ). Experimental details were defined in Cancer Genome Atlas (TCGA) 40 . ( m ) In silico analysis of single-cell RNA sequencing of human melanoma-infiltrating T cells revealed that RAMP1 + T cells downregulated Il-2 expression and strongly overexpressed several immune checkpoint receptors ( PD-1 , TIM3 , LAG3 , CTLA4 , CD28 , ICOS , BTLA , CD27 ) in comparison to RAMP1 - T cells. Individual cell data are shown as a log 2 of 1 + (transcript per million / 10). Experimental details and cell clustering were defined in Tirosh et al 42 . N are defined in each panel. ( n–p ) On the basis of the clinical response of patients with melanoma to immune checkpoint blocker, patients were clustered into two groups defined as ICI-responsive or ICI-resistant 41 . In silico analysis of single-cell RNA sequencing of patients’ biopsies revealed that tumour-infiltrating CD8 + T cells from patients who were resistant to ICIs significantly overexpressed RAMP1 (2.0-fold), PD-1 (1.7-fold), LAG3 (1.6-fold), CTLA4 (1.6-fold), and TIM3 (1.7-fold; n–p ). Individual cell data are shown as a log 2 (1+(transcript per million/10). Experimental details and cell clustering were defined in Jerby-Arnon et al 41 . P-values are shown in the figure and determined by two-sided unpaired Student’s t-test. N are defined in each panel ( n–o ). Source Data
Figure Legend Snippet: RAMP1 expression in patient melanoma-infiltrating T cells correlates with worsened survival and poor responsiveness to ICIs. ( a–l ) In silico analysis of Cancer Genome Atlas (TCGA) data linked the survival rate among 459 patients with melanoma with their relative expression levels of various genes of interest (determined by bulk RNA sequencing of tumour biopsy). Kaplan–Meier curves show the patients’ survival after segregation in two groups defined by their low or high expression of a gene of interest. Increased gene expression (labelled as high; red curve) of TUBB3 ( b ), PGP9.5 ( c ), Nav 1.7 ( E ), SLPI ( k ) and RAMP1 ( l ) in biopsy correlate with decreased patient survival (p≤0.05). The mantel–Haenszel hazard ratio and number of patients included in each analysis are shown in the figure ( a–l ). Experimental details were defined in Cancer Genome Atlas (TCGA) 40 . ( m ) In silico analysis of single-cell RNA sequencing of human melanoma-infiltrating T cells revealed that RAMP1 + T cells downregulated Il-2 expression and strongly overexpressed several immune checkpoint receptors ( PD-1 , TIM3 , LAG3 , CTLA4 , CD28 , ICOS , BTLA , CD27 ) in comparison to RAMP1 - T cells. Individual cell data are shown as a log 2 of 1 + (transcript per million / 10). Experimental details and cell clustering were defined in Tirosh et al 42 . N are defined in each panel. ( n–p ) On the basis of the clinical response of patients with melanoma to immune checkpoint blocker, patients were clustered into two groups defined as ICI-responsive or ICI-resistant 41 . In silico analysis of single-cell RNA sequencing of patients’ biopsies revealed that tumour-infiltrating CD8 + T cells from patients who were resistant to ICIs significantly overexpressed RAMP1 (2.0-fold), PD-1 (1.7-fold), LAG3 (1.6-fold), CTLA4 (1.6-fold), and TIM3 (1.7-fold; n–p ). Individual cell data are shown as a log 2 (1+(transcript per million/10). Experimental details and cell clustering were defined in Jerby-Arnon et al 41 . P-values are shown in the figure and determined by two-sided unpaired Student’s t-test. N are defined in each panel ( n–o ). Source Data

Techniques Used: Expressing, In Silico, RNA Sequencing Assay

B16F10-secreted SLPI activates nociceptor neurons. ( a–e ) Naive DRG neurons ( Trpv1 cre ::CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA, and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48h, the cells were collected, FACS purified, and RNA sequenced. DEGs were calculated, and Fgfr1 (fibroblast growth factor receptor 1) was found to be overexpressed in OVA-specific cytotoxic CD8 + T cells when co-cultured with cancer cells and DRG neurons ( a ). Conversely, OVA-specific cytotoxic CD8 + T cells downregulates the expression of the pro-nociceptive factor Hmgb1 (High–mobility group box 1; b ), Braf ( c ) , as well as Fgfr3 ( d ) when co-cultured with B16F10-mCherry-OVA and DRG neurons. Tslp expression level was not affected in any of tested groups ( e ). ( f–i ) Using calcium microscopy, we probed whether SLPI directly activates cultured DRG neurons. We found that SLPI (0.01-10 ng/mL) induces a significant calcium influx in DRG neurons ( f ). SLPI-responsive neurons are mostly small-sized neurons ( g-h ; mean area = 151 μm 2 ) and largely capsaicin-responsive ( i ; ~42%). ( j ) The right hindpaw of naive mice was injected with saline (20 μL) or SLPI (i.d., 1 μg/20 μL), and the mice’s noxious thermal nociceptive threshold was measured (0-6h). The ipsilateral paw injected with SLPI showed thermal hypersensitivity in contrast with the contralateral paw. Saline had no effect on the mice’s thermal sensitivity. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f , h , j ), stacked bar graph on a logarithmic scale ( g ), and Venn Diagram ( i ). N are as follows: a–e : n = 2–4/groups, f : vehicle (n = 28), 10pg/ml (n = 28), 100 pg/ml (n = 132), 1,000 pg/ml (n = 191), 10 ng/ml (n = 260), capsaicin (n = 613), KCl (n = 1,139), g : 0-100 (SLPI=19; KCl=177), 100-200 (SLPI = 45; KCl = 390), 200-300 (SLPI = 16; KCl =216), 300-400 (SLPI=11; KCl = 138), 400-500 (SLPI = 5; KCl = 68), 500-600 (SLPI=2, KCl = 18), 600-700 (SLPI = 0; KCl = 10), 700-800 (SLPI=0; KCl=13), 800+ (SLPI = 0; KCl = 12), h : n = 98, i : KCl + =1139, KCl + Caps + =614, KCl + Caps + SLPI + =261, KCl + Caps - SLPI + =29, j : 0h (n = 9), SLPI at 1h (n = 6), saline at 1h (n = 3), SLPI at 3h (n = 6), saline at 3h (n=3), SLPI at 6h (n = 6), saline at 6h (n = 3). Experiments were independently repeated two ( j ) or three ( f–i ) times with similar results. Sequencing experiment was not repeated ( a–e ). P-values were determined by one-way ANOVA post-hoc Bonferroni ( a–f ); or two-sided unpaired Student’s t-test ( j ). P-values are shown in the figure or indicated by * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001. Source Data
Figure Legend Snippet: B16F10-secreted SLPI activates nociceptor neurons. ( a–e ) Naive DRG neurons ( Trpv1 cre ::CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA, and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48h, the cells were collected, FACS purified, and RNA sequenced. DEGs were calculated, and Fgfr1 (fibroblast growth factor receptor 1) was found to be overexpressed in OVA-specific cytotoxic CD8 + T cells when co-cultured with cancer cells and DRG neurons ( a ). Conversely, OVA-specific cytotoxic CD8 + T cells downregulates the expression of the pro-nociceptive factor Hmgb1 (High–mobility group box 1; b ), Braf ( c ) , as well as Fgfr3 ( d ) when co-cultured with B16F10-mCherry-OVA and DRG neurons. Tslp expression level was not affected in any of tested groups ( e ). ( f–i ) Using calcium microscopy, we probed whether SLPI directly activates cultured DRG neurons. We found that SLPI (0.01-10 ng/mL) induces a significant calcium influx in DRG neurons ( f ). SLPI-responsive neurons are mostly small-sized neurons ( g-h ; mean area = 151 μm 2 ) and largely capsaicin-responsive ( i ; ~42%). ( j ) The right hindpaw of naive mice was injected with saline (20 μL) or SLPI (i.d., 1 μg/20 μL), and the mice’s noxious thermal nociceptive threshold was measured (0-6h). The ipsilateral paw injected with SLPI showed thermal hypersensitivity in contrast with the contralateral paw. Saline had no effect on the mice’s thermal sensitivity. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f , h , j ), stacked bar graph on a logarithmic scale ( g ), and Venn Diagram ( i ). N are as follows: a–e : n = 2–4/groups, f : vehicle (n = 28), 10pg/ml (n = 28), 100 pg/ml (n = 132), 1,000 pg/ml (n = 191), 10 ng/ml (n = 260), capsaicin (n = 613), KCl (n = 1,139), g : 0-100 (SLPI=19; KCl=177), 100-200 (SLPI = 45; KCl = 390), 200-300 (SLPI = 16; KCl =216), 300-400 (SLPI=11; KCl = 138), 400-500 (SLPI = 5; KCl = 68), 500-600 (SLPI=2, KCl = 18), 600-700 (SLPI = 0; KCl = 10), 700-800 (SLPI=0; KCl=13), 800+ (SLPI = 0; KCl = 12), h : n = 98, i : KCl + =1139, KCl + Caps + =614, KCl + Caps + SLPI + =261, KCl + Caps - SLPI + =29, j : 0h (n = 9), SLPI at 1h (n = 6), saline at 1h (n = 3), SLPI at 3h (n = 6), saline at 3h (n=3), SLPI at 6h (n = 6), saline at 6h (n = 3). Experiments were independently repeated two ( j ) or three ( f–i ) times with similar results. Sequencing experiment was not repeated ( a–e ). P-values were determined by one-way ANOVA post-hoc Bonferroni ( a–f ); or two-sided unpaired Student’s t-test ( j ). P-values are shown in the figure or indicated by * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001. Source Data

Techniques Used: Cell Culture, FACS, Purification, Expressing, Microscopy, Mouse Assay, Injection, Whisker Assay, Sequencing

CGRP attenuates the anti-tumour immunity of RAMP1 + CD8 + T cells. a – c , Splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. Eight-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5 × 10 6 cells) with activated Ramp1 −/ − or Ramp1 WT CD8 + T cells or a 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week after transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.). Ten days after B16F10 inoculation, we observed greater tumour growth ( a ) in Ramp1 WT transplanted mice. Intratumoral Ramp1 −/ − (CD45.2 + ) and Ramp1 WT (CD45.1 + ) CD8 + T cells were FACS purified, immunophenotyped ( b ) and RNA sequenced ( c ). Ramp1 −/ − CD8 + T cells showed a lower proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( b ) as well as reduced transcript expression of exhaustion markers ( c ). d , In silico analysis of The Cancer Genome Atlas (TCGA) data 40 was used to correlate the survival rate of 459 patients with melanoma with the relative RAMP1 expression (primary biopsy bulk RNA sequencing). In comparison to patients with low RAMP1 expression, higher RAMP1 levels correlate with decreased patient survival. e , In silico analysis of single-cell RNA sequencing of human melanoma 41 reveals that intratumoral RAMP1 -expressing CD8 + T cells strongly overexpress several immune checkpoint receptors ( PD-1 (also known as PDCD1 ) TIM3 , LAG3 , CTLA4 ) in comparison to Ramp1 -negative CD8 + T cells. Data are shown as mean ± s.e.m. ( a ), slopegraph ( b ), as a heat map showing normalized gene expression (log 10 (10 3 × TPM) ( c ), as a Mantel–Cox regression ( d ) or as a violin plot ( e ). n as follows: a–c : n = 5 per group; d : high ( n = 45), low ( n = 68); e : RAMP1 − CD8 ( n = 1,732), RAMP1 + CD8 ( n = 25). Experiments were independently repeated two ( a , b ) times with similar results. The sequencing experiment was not repeated ( c ). P values were determined by two-way ANOVA with post-hoc Bonferroni ( a ), two-sided unpaired Student’s t -test ( b ) or Mantel–Cox regression ( d ). Source Data
Figure Legend Snippet: CGRP attenuates the anti-tumour immunity of RAMP1 + CD8 + T cells. a – c , Splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. Eight-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5 × 10 6 cells) with activated Ramp1 −/ − or Ramp1 WT CD8 + T cells or a 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week after transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.). Ten days after B16F10 inoculation, we observed greater tumour growth ( a ) in Ramp1 WT transplanted mice. Intratumoral Ramp1 −/ − (CD45.2 + ) and Ramp1 WT (CD45.1 + ) CD8 + T cells were FACS purified, immunophenotyped ( b ) and RNA sequenced ( c ). Ramp1 −/ − CD8 + T cells showed a lower proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( b ) as well as reduced transcript expression of exhaustion markers ( c ). d , In silico analysis of The Cancer Genome Atlas (TCGA) data 40 was used to correlate the survival rate of 459 patients with melanoma with the relative RAMP1 expression (primary biopsy bulk RNA sequencing). In comparison to patients with low RAMP1 expression, higher RAMP1 levels correlate with decreased patient survival. e , In silico analysis of single-cell RNA sequencing of human melanoma 41 reveals that intratumoral RAMP1 -expressing CD8 + T cells strongly overexpress several immune checkpoint receptors ( PD-1 (also known as PDCD1 ) TIM3 , LAG3 , CTLA4 ) in comparison to Ramp1 -negative CD8 + T cells. Data are shown as mean ± s.e.m. ( a ), slopegraph ( b ), as a heat map showing normalized gene expression (log 10 (10 3 × TPM) ( c ), as a Mantel–Cox regression ( d ) or as a violin plot ( e ). n as follows: a–c : n = 5 per group; d : high ( n = 45), low ( n = 68); e : RAMP1 − CD8 ( n = 1,732), RAMP1 + CD8 ( n = 25). Experiments were independently repeated two ( a , b ) times with similar results. The sequencing experiment was not repeated ( c ). P values were determined by two-way ANOVA with post-hoc Bonferroni ( a ), two-sided unpaired Student’s t -test ( b ) or Mantel–Cox regression ( d ). Source Data

Techniques Used: FACS, Purification, Mouse Assay, In Vitro, Transplantation Assay, Expressing, In Silico, RNA Sequencing Assay, Sequencing

The CGRP–RAMP1 axis promotes intratumoral CD8 + T cell exhaustion. ( a–e ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Nav 1.8 WT ::DTA fl/WT ) and ablated mice ( Nav 1.8 cre ::DTA fl/WT ). As measured fifteen days post inoculation, Na V 1.8 + nociceptor-ablated mice had lower proportion of PD-1 + LAG3 + TIM3 + ( a ) CD8 + T cells, but increased levels of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) CD8 + T cells. B16F10-mCherry-OVA (5x10 5 cells; i.d.)-tumour surrounding skin was also collected and capsaicin-induced CGRP release assessed by ELISA. Intratumoral CGRP levels positively correlate with the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ). ( f ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old female sensory neuron intact or ablated mice. In nociceptor-ablated mice, recombinant CGRP injection (100nM, i.d., once daily) rescues intratumoral CD8 + T cells exhaustion (PD-1 + LAG3 + TIM3 + ). ( g ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. We found that blocking the action of CGRP on RAMP1-expressing cells, increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( h–m ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. In another group of mice, BIBN4096 (5 mg/kg, i.p., every other day) injections were started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). The effect of nociceptor neuron-silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. As assessed thirteen days post tumour inoculation, BIBN4096 decreased tumour volume ( h ) and weight ( i ) but increased the relative proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BIBN4096 had no effect on the number of intratumoral CD8 + T cells ( j ). When administered as therapeutic, BIBN4096 reduced tumour volume ( h ) and weight ( i ) but had limited effect on CD8 + T cells’ cytotoxicity ( j–m ). ( n ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron-intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Starting one day post inoculation, BIBN4096 (5 mg/kg) or its vehicle was administered (i.p.) on alternate days; effects on tumour volume were measured. Fourteen days post tumour inoculation, we found that tumour growth was reduced in sensory neuron-ablated mice and in BIBN4096-treated mice. BIBN4096 had no additive effect when given to sensory neuron-ablated mice. ( o–s ) Splenocytes-isolated CD8 + T cells from naïve C57BL6J mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BIBN4096 (1–4 μM) for 24h; effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BIBN4096 did not affect the survival ( o ) of cultured cytotoxic CD8 + T cells, nor their relative expression of PD-1 + LAG3 + TIM3 + ( p ), IFNγ + ( q ), TNF + ( r ), and IL-2 + ( s ). ( t ) B16F10 cells (1x10 5 cells) were cultured for 24h. The cells were then exposed (or not) to BIBN4096 (1-8 μM) for an additional 24h; effects on apoptosis were measured by flow cytometry. BIBN4096 did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( u-w ) Naive splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated ( CD3 and CD28 + IL-2 ) in vitro . 8-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5x10 6 cells) with either Ramp1 −/ − or Ramp1 WT CD8 + T cells or 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week post transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5x10 5 cells; i.d.). Ten days post tumour inoculation, we retrieved a similar number of tumours draining lymph node CD8 + T cells across the three tested groups ( u ). The relative proportion of intra-tumour PD-1 + LAG3 + TIM3 + CD8 + T cells was lower in Ramp1 −/ − transplanted mice ( v ). Within the same tumour, intratumoral CD8 + T cell exhaustion was immunophenotyped by flow cytometry ( representative panel shown in w ) and showed that the relative proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells was ~3-fold lower in Ramp1 −/ − CD8 + T cells than in Ramp1 WT CD8 + T cells ( w ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–d , f , h–m , o–v ), linear regression ( e ), Mantel–Cox regression ( g ), mean ± S.E.M ( n ), or as FACS plot ( w ). N are as follows a–e : Nav 1.8 WT ::DTA fl/WT (n = 18), Nav 1.8 cre ::DTA fl/WT (n = 10), f : Trpv1 WT ::DTA fl/WT (n = 16), Trpv1 cre ::DTA fl/WT +CGRP (n = 11), g : vehicle (n = 89), BIBN4096 (n = 16), h–m : Vehicle (n = 13), BIBN4096 therapeutic (n = 18), BIBN4096 prophylactic (n = 16), n : Trpv1 WT ::DTA fl/WT + vehicle (n = 8), Trpv1 WT ::DTA fl/WT + BIBN4096 (n = 9), Trpv1 cre ::DTA fl/WT + vehicle (n = 7), Trpv1 cre ::DTA fl/WT + BIBN4096 (n = 7), o : vehicle (n = 5), 1µM BIBN4096 (n = 3), 4 µM BIBN4096 (n = 5), p–s : n = 5/groups, t : n = 4/groups, u–w : n = 5/groups. Experiments were independently repeated twice ( a–f , n–w ) or four ( g–m ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( a–d , f , v ), simple linear regression analysis ( e ), Mantel–Cox regression ( g ), by one-way ANOVA posthoc Bonferroni ( h–m; o–u ), or two-way ANOVA post-hoc Bonferroni ( n ). Source Data
Figure Legend Snippet: The CGRP–RAMP1 axis promotes intratumoral CD8 + T cell exhaustion. ( a–e ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Nav 1.8 WT ::DTA fl/WT ) and ablated mice ( Nav 1.8 cre ::DTA fl/WT ). As measured fifteen days post inoculation, Na V 1.8 + nociceptor-ablated mice had lower proportion of PD-1 + LAG3 + TIM3 + ( a ) CD8 + T cells, but increased levels of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) CD8 + T cells. B16F10-mCherry-OVA (5x10 5 cells; i.d.)-tumour surrounding skin was also collected and capsaicin-induced CGRP release assessed by ELISA. Intratumoral CGRP levels positively correlate with the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ). ( f ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old female sensory neuron intact or ablated mice. In nociceptor-ablated mice, recombinant CGRP injection (100nM, i.d., once daily) rescues intratumoral CD8 + T cells exhaustion (PD-1 + LAG3 + TIM3 + ). ( g ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. We found that blocking the action of CGRP on RAMP1-expressing cells, increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( h–m ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. In another group of mice, BIBN4096 (5 mg/kg, i.p., every other day) injections were started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). The effect of nociceptor neuron-silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. As assessed thirteen days post tumour inoculation, BIBN4096 decreased tumour volume ( h ) and weight ( i ) but increased the relative proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BIBN4096 had no effect on the number of intratumoral CD8 + T cells ( j ). When administered as therapeutic, BIBN4096 reduced tumour volume ( h ) and weight ( i ) but had limited effect on CD8 + T cells’ cytotoxicity ( j–m ). ( n ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron-intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Starting one day post inoculation, BIBN4096 (5 mg/kg) or its vehicle was administered (i.p.) on alternate days; effects on tumour volume were measured. Fourteen days post tumour inoculation, we found that tumour growth was reduced in sensory neuron-ablated mice and in BIBN4096-treated mice. BIBN4096 had no additive effect when given to sensory neuron-ablated mice. ( o–s ) Splenocytes-isolated CD8 + T cells from naïve C57BL6J mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BIBN4096 (1–4 μM) for 24h; effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BIBN4096 did not affect the survival ( o ) of cultured cytotoxic CD8 + T cells, nor their relative expression of PD-1 + LAG3 + TIM3 + ( p ), IFNγ + ( q ), TNF + ( r ), and IL-2 + ( s ). ( t ) B16F10 cells (1x10 5 cells) were cultured for 24h. The cells were then exposed (or not) to BIBN4096 (1-8 μM) for an additional 24h; effects on apoptosis were measured by flow cytometry. BIBN4096 did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( u-w ) Naive splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated ( CD3 and CD28 + IL-2 ) in vitro . 8-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5x10 6 cells) with either Ramp1 −/ − or Ramp1 WT CD8 + T cells or 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week post transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5x10 5 cells; i.d.). Ten days post tumour inoculation, we retrieved a similar number of tumours draining lymph node CD8 + T cells across the three tested groups ( u ). The relative proportion of intra-tumour PD-1 + LAG3 + TIM3 + CD8 + T cells was lower in Ramp1 −/ − transplanted mice ( v ). Within the same tumour, intratumoral CD8 + T cell exhaustion was immunophenotyped by flow cytometry ( representative panel shown in w ) and showed that the relative proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells was ~3-fold lower in Ramp1 −/ − CD8 + T cells than in Ramp1 WT CD8 + T cells ( w ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–d , f , h–m , o–v ), linear regression ( e ), Mantel–Cox regression ( g ), mean ± S.E.M ( n ), or as FACS plot ( w ). N are as follows a–e : Nav 1.8 WT ::DTA fl/WT (n = 18), Nav 1.8 cre ::DTA fl/WT (n = 10), f : Trpv1 WT ::DTA fl/WT (n = 16), Trpv1 cre ::DTA fl/WT +CGRP (n = 11), g : vehicle (n = 89), BIBN4096 (n = 16), h–m : Vehicle (n = 13), BIBN4096 therapeutic (n = 18), BIBN4096 prophylactic (n = 16), n : Trpv1 WT ::DTA fl/WT + vehicle (n = 8), Trpv1 WT ::DTA fl/WT + BIBN4096 (n = 9), Trpv1 cre ::DTA fl/WT + vehicle (n = 7), Trpv1 cre ::DTA fl/WT + BIBN4096 (n = 7), o : vehicle (n = 5), 1µM BIBN4096 (n = 3), 4 µM BIBN4096 (n = 5), p–s : n = 5/groups, t : n = 4/groups, u–w : n = 5/groups. Experiments were independently repeated twice ( a–f , n–w ) or four ( g–m ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( a–d , f , v ), simple linear regression analysis ( e ), Mantel–Cox regression ( g ), by one-way ANOVA posthoc Bonferroni ( h–m; o–u ), or two-way ANOVA post-hoc Bonferroni ( n ). Source Data

Techniques Used: Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Expressing, Isolation, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Fluorescence, FACS, Purification, In Vitro, Transplantation Assay, Whisker Assay

CGRP modulates the activation of CD8 + T cells. a , b , Splenocyte CD8 + T cells from wild-type ( a ), Ramp1 −/ − ( a ) or naive OT-I ( b ) mice were cultured under T c1 -stimulating conditions (ex-vivo-activated by CD3 and CD28, IL-12 and anti-IL4) for 48 h to generate cytotoxic CD8 + T cells. In the presence of IL-2 (10 ng ml −1 ), the cells were stimulated with CGRP (100 nM; challenged once every two days) for 96 h. Wild-type cytotoxic CD8 + T cells showed an increased proportion of PD-1 + LAG3 + TIM3 + cells; this effect was absent when treating cytotoxic CD8 + T cells that were collected from Ramp1 −/ − mice ( a ). In co-culture (48 h), CGRP (100 nM; once daily) also reduced the ability of OT-I cytotoxic CD8 + T cells (4 × 10 5 cells) to eliminate B16F10-mCherry-OVA cancer cells ( b ). c , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old female mice with sensory neurons intact or ablated. In nociceptor-ablated mice, peritumoral recombinant CGRP injection (100 nM, i.d., once daily) rescues B16F10 growth (assessed until day 12). d , e , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old male and female mice. Starting one day after inoculation (defined as prophylactic), the RAMP1 antagonist BIBN4096 (5 mg kg −1 ) was administered systemically (i.p.) once every two days. In another group of mice, BIBN4096 (5 mg kg −1 , i.p., every two days) injections were started once the tumour reached a volume of around 200 mm 3 (defined as therapeutic). Prophylactic or therapeutic BIBN4096 treatments decreased tumour growth ( d ) and reduced the proportion of intratumoral PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ; assessed until day 13). Data are shown as box-and-whisker plots (as defined in Fig. 1b, c ), for which individual data points are given ( a , b , e ), or as mean ± s.e.m. ( c , d ). n as follows: a : Ramp1 WT CD8 + vehicle ( n = 9), Ramp1 WT CD8 + CGRP ( n = 10), Ramp1 −/ − CD8 + vehicle ( n = 10), Ramp1 −/ − CD8 + CGRP ( n = 9); b : n = 4 per group; c : intact + vehicle ( n = 15), ablated + CGRP ( n = 11); d : vehicle ( n = 13), BIBN prophylactic ( n = 16), BIBN therapeutic ( n = 18); e : vehicle ( n = 10), BIBN prophylactic ( n = 13), BIBN therapeutic ( n = 16). Experiments were independently repeated three times with similar results. P values were determined by one-way ANOVA with post-hoc Bonferroni ( a , e ), two-sided unpaired Student’s t -test ( b ) or two-way ANOVA with post-hoc Bonferroni ( c , d ). Source Data
Figure Legend Snippet: CGRP modulates the activation of CD8 + T cells. a , b , Splenocyte CD8 + T cells from wild-type ( a ), Ramp1 −/ − ( a ) or naive OT-I ( b ) mice were cultured under T c1 -stimulating conditions (ex-vivo-activated by CD3 and CD28, IL-12 and anti-IL4) for 48 h to generate cytotoxic CD8 + T cells. In the presence of IL-2 (10 ng ml −1 ), the cells were stimulated with CGRP (100 nM; challenged once every two days) for 96 h. Wild-type cytotoxic CD8 + T cells showed an increased proportion of PD-1 + LAG3 + TIM3 + cells; this effect was absent when treating cytotoxic CD8 + T cells that were collected from Ramp1 −/ − mice ( a ). In co-culture (48 h), CGRP (100 nM; once daily) also reduced the ability of OT-I cytotoxic CD8 + T cells (4 × 10 5 cells) to eliminate B16F10-mCherry-OVA cancer cells ( b ). c , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old female mice with sensory neurons intact or ablated. In nociceptor-ablated mice, peritumoral recombinant CGRP injection (100 nM, i.d., once daily) rescues B16F10 growth (assessed until day 12). d , e , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old male and female mice. Starting one day after inoculation (defined as prophylactic), the RAMP1 antagonist BIBN4096 (5 mg kg −1 ) was administered systemically (i.p.) once every two days. In another group of mice, BIBN4096 (5 mg kg −1 , i.p., every two days) injections were started once the tumour reached a volume of around 200 mm 3 (defined as therapeutic). Prophylactic or therapeutic BIBN4096 treatments decreased tumour growth ( d ) and reduced the proportion of intratumoral PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ; assessed until day 13). Data are shown as box-and-whisker plots (as defined in Fig. 1b, c ), for which individual data points are given ( a , b , e ), or as mean ± s.e.m. ( c , d ). n as follows: a : Ramp1 WT CD8 + vehicle ( n = 9), Ramp1 WT CD8 + CGRP ( n = 10), Ramp1 −/ − CD8 + vehicle ( n = 10), Ramp1 −/ − CD8 + CGRP ( n = 9); b : n = 4 per group; c : intact + vehicle ( n = 15), ablated + CGRP ( n = 11); d : vehicle ( n = 13), BIBN prophylactic ( n = 16), BIBN therapeutic ( n = 18); e : vehicle ( n = 10), BIBN prophylactic ( n = 13), BIBN therapeutic ( n = 16). Experiments were independently repeated three times with similar results. P values were determined by one-way ANOVA with post-hoc Bonferroni ( a , e ), two-sided unpaired Student’s t -test ( b ) or two-way ANOVA with post-hoc Bonferroni ( c , d ). Source Data

Techniques Used: Activation Assay, Mouse Assay, Cell Culture, Ex Vivo, Co-Culture Assay, Recombinant, Injection, Whisker Assay

Nociceptor-released CGRP increases cytotoxic CD8 + T cell exhaustion. ( a–b ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating condition ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then cultured or not with wild-type DRG neurons and exposed to capsaicin (1 μM, challenged once every two days) or its vehicle. As measured after 4 days stimulation, capsaicin-stimulated intact neuron increased the proportion of PD-1 + LAG3 + TIM3 + ( a ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( b ). ( c–d ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 μL/mL), naive DRG neurons were cultured in the presence of BoNT/A (50 pg/mL) or its vehicle for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed or not to a RAMP1 blocker (CGRP 8–37 ; 2 μg/mL) and challenge (1:2 dilution) with fresh KCl-induced conditioned medium from naive, or BoNT/A-silenced neurons. As measured after 4 days stimulation, KCl-stimulated neuron-conditioned medium increased the proportion of PD-1 + LAG3 + TIM3 + ( c ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( d ). Such effect was absent when cytotoxic CD8 + T cells were co-exposed to the RAMP1 blocker CGRP 8–37 or challenged with the neuron conditioned medium collected from BoNT/A-silenced neurons ( c–d ). ( e–f ) Splenocytes-isolated CD8 + T cells from wild-type and Ramp1 −/ − mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed to CGRP (0.1 μM) or its vehicle. As measured after 4 days stimulation, representative flow cytometry plots ( f ) show that CGRP decrease Ramp WT cytotoxic CD8 + T cells expression of IFNγ + ( e , f ), TNF + ( f ), and IL-2 + ( f ) when exposed to CGRP. Inversely, CGRP increase the proportion of PD-1 + LAG3 + TIM3 + in Ramp1 W T cytotoxic CD8 + T cells ( f ). Ramp1 −/ − cytotoxic CD8 + T cells were protected from the effect of CGRP ( e–f ). ( g–i ) Splenocytes-isolated CD8 + T cells from naive OT-I mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. B16F10-mCherry-OVA cells (1×10 5 cells) were then cultured with or without OT-I cytotoxic CD8 + T cells (4×10 5 cells). Tc1-stimulated OT-I-CD8 + T cells lead to B16F10-OVA cell apoptosis (AnnexinV + 7AAD + ; g , measured after 48h; h–i , measured after 24h). B16F10-mCherry-OVA cells elimination by cytotoxic CD8 + T cells was reduced when the co-cultures were challenged (1:2 dilution; once daily for two consecutive days) with fresh conditioned medium collected from capsaicin (1 μM)-stimulated naive DRG neurons ( g ; measured after 48h). Similarly, KCl (50mM)-stimulated naive DRG neurons conditioned medium (1:2 dilution) reduced B16F10-mCherry-OVA apoptosis ( h ; measured after 24h). This effect was blunted when the cells were co-exposed to the RAMP1 blocker CGRP 8-37 ( h ; 2 μg/mL; measured after 24h). CGRP (0.1 μM) challenges also reduced OT-I cytotoxic CD8 + T cells elimination of B16F10-OVA cell ( i ; measured after 24h). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g–h ), or representative FACS plot ( f , i ). N are as follows: a : CD8 + vehicle (n = 4), CD8 + capsaicin (n = 9), CD8 + neuron + capsaicin (n = 9), b : n = 5/groups, c : CD8 (n = 6), CD8 + KCl-induced neurons CM (n = 5), CD8 + KCl-induced neurons CM + CGRP 8-37 (n = 6), CD8 + KCl-induced neurons CM + BoNT/A (n = 6), d : n = 5/groups, e : Ramp1 WT CD8 + vehicle (n = 7), Ramp1 WT CD8 + CGRP (n = 8), Ramp1 −/ − CD8 + vehicle (n = 6), Ramp1 −/ − CD8 + CGRP (n = 6), g : B16F10 (n = 3), B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), h : B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM + CGRP 8–37 (n = 5). Experiments were repeated a minimum of three independent times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferroni ( a–e , g–h ). Source Data
Figure Legend Snippet: Nociceptor-released CGRP increases cytotoxic CD8 + T cell exhaustion. ( a–b ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating condition ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then cultured or not with wild-type DRG neurons and exposed to capsaicin (1 μM, challenged once every two days) or its vehicle. As measured after 4 days stimulation, capsaicin-stimulated intact neuron increased the proportion of PD-1 + LAG3 + TIM3 + ( a ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( b ). ( c–d ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 μL/mL), naive DRG neurons were cultured in the presence of BoNT/A (50 pg/mL) or its vehicle for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed or not to a RAMP1 blocker (CGRP 8–37 ; 2 μg/mL) and challenge (1:2 dilution) with fresh KCl-induced conditioned medium from naive, or BoNT/A-silenced neurons. As measured after 4 days stimulation, KCl-stimulated neuron-conditioned medium increased the proportion of PD-1 + LAG3 + TIM3 + ( c ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( d ). Such effect was absent when cytotoxic CD8 + T cells were co-exposed to the RAMP1 blocker CGRP 8–37 or challenged with the neuron conditioned medium collected from BoNT/A-silenced neurons ( c–d ). ( e–f ) Splenocytes-isolated CD8 + T cells from wild-type and Ramp1 −/ − mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed to CGRP (0.1 μM) or its vehicle. As measured after 4 days stimulation, representative flow cytometry plots ( f ) show that CGRP decrease Ramp WT cytotoxic CD8 + T cells expression of IFNγ + ( e , f ), TNF + ( f ), and IL-2 + ( f ) when exposed to CGRP. Inversely, CGRP increase the proportion of PD-1 + LAG3 + TIM3 + in Ramp1 W T cytotoxic CD8 + T cells ( f ). Ramp1 −/ − cytotoxic CD8 + T cells were protected from the effect of CGRP ( e–f ). ( g–i ) Splenocytes-isolated CD8 + T cells from naive OT-I mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. B16F10-mCherry-OVA cells (1×10 5 cells) were then cultured with or without OT-I cytotoxic CD8 + T cells (4×10 5 cells). Tc1-stimulated OT-I-CD8 + T cells lead to B16F10-OVA cell apoptosis (AnnexinV + 7AAD + ; g , measured after 48h; h–i , measured after 24h). B16F10-mCherry-OVA cells elimination by cytotoxic CD8 + T cells was reduced when the co-cultures were challenged (1:2 dilution; once daily for two consecutive days) with fresh conditioned medium collected from capsaicin (1 μM)-stimulated naive DRG neurons ( g ; measured after 48h). Similarly, KCl (50mM)-stimulated naive DRG neurons conditioned medium (1:2 dilution) reduced B16F10-mCherry-OVA apoptosis ( h ; measured after 24h). This effect was blunted when the cells were co-exposed to the RAMP1 blocker CGRP 8-37 ( h ; 2 μg/mL; measured after 24h). CGRP (0.1 μM) challenges also reduced OT-I cytotoxic CD8 + T cells elimination of B16F10-OVA cell ( i ; measured after 24h). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g–h ), or representative FACS plot ( f , i ). N are as follows: a : CD8 + vehicle (n = 4), CD8 + capsaicin (n = 9), CD8 + neuron + capsaicin (n = 9), b : n = 5/groups, c : CD8 (n = 6), CD8 + KCl-induced neurons CM (n = 5), CD8 + KCl-induced neurons CM + CGRP 8-37 (n = 6), CD8 + KCl-induced neurons CM + BoNT/A (n = 6), d : n = 5/groups, e : Ramp1 WT CD8 + vehicle (n = 7), Ramp1 WT CD8 + CGRP (n = 8), Ramp1 −/ − CD8 + vehicle (n = 6), Ramp1 −/ − CD8 + CGRP (n = 6), g : B16F10 (n = 3), B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), h : B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM + CGRP 8–37 (n = 5). Experiments were repeated a minimum of three independent times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferroni ( a–e , g–h ). Source Data

Techniques Used: Isolation, Cell Culture, Ex Vivo, Mouse Assay, Flow Cytometry, Expressing, Whisker Assay, FACS

Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data
Figure Legend Snippet: Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data

Techniques Used: Injection, Mouse Assay, Activation Assay, FACS, Whisker Assay

BoNT/A silencing of B16F10-innervating neurons decreases tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BoNT/A (10–50 pg/μL) for 24h; effects on apoptosis, exhaustion, and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BoNT/A did not affect the survival ( a ) of cultured cytotoxic CD8 + T cells, nor their relative expression of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) and PD-1 + LAG3 + TIM3 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h and subsequently exposed to BoNT/A (1.6-50 pg/μL) or its vehicle for an additional 24h. BoNT/A did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( g–n ) One and three days prior to tumour inoculation ( defined as prophylactic ), the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day after the last injection, orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. In another group of mice, BoNT/A was administered (25 pg/μL; i.d.) one and three days after the tumour reached a volume of ~200mm3 (defined as therapeutic ). The effect of neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. Nineteen days post tumour inoculation, we found that the tumour volume ( g , h ) and weight ( i ) were reduced in mice treated with BoNT/A ( Prophylactic group ). In parallel, we found that silencing tumour-innervating neurons increased the proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BoNT/A had no effect on the total number of intratumoral CD8 T cells ( j ) or the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. ( o ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female sensory neuron-intact or ablated mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic YUMMER1.7 cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. The effects of nociceptor neuron ablation on tumour size and volume were measured. Thirteen days post tumour inoculation, we found that the tumour growth was lower in mice treated with BoNT/A or in sensory neuron-ablated mice. BoNT/A had no additive effects when administered to sensory neuron-ablated mice. ( p ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic B16F10-mCherry-OVA cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. On days 7, 10, 13 and 16 post tumour inoculation, the mice were exposed to αPD-L1 (6 mg/kg, i.p.) or its isotype control. Eighteen days post tumour inoculation, we found that neuron silencing using BoNT/A potentiated αPD-L1-mediated tumour reduction. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f; h–n ) or as mean ± S.E.M ( g , o , p ). N are as follows: a-e : n = 5/groups, f : n = 3/groups, g–i : vehicle (n = 12), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 10), j : vehicle (n = 11), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), k–n : vehicle (n = 10), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), o : intact + vehicle (n = 9), ablated + vehicle (n = 8), intact + BoNT/A (n = 10), ablated + BoNT/A (n = 8), p : vehicle (n = 7), αPD-L1 (n = 8), αPD-L1 + BoNT/A (n = 7). Experiments were independently repeated two ( a–f , o–p ) or four ( g–n ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–f , h–n ) or two-way ANOVA post-hoc Bonferroni ( g , o , p ). Source Data
Figure Legend Snippet: BoNT/A silencing of B16F10-innervating neurons decreases tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BoNT/A (10–50 pg/μL) for 24h; effects on apoptosis, exhaustion, and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BoNT/A did not affect the survival ( a ) of cultured cytotoxic CD8 + T cells, nor their relative expression of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) and PD-1 + LAG3 + TIM3 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h and subsequently exposed to BoNT/A (1.6-50 pg/μL) or its vehicle for an additional 24h. BoNT/A did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( g–n ) One and three days prior to tumour inoculation ( defined as prophylactic ), the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day after the last injection, orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. In another group of mice, BoNT/A was administered (25 pg/μL; i.d.) one and three days after the tumour reached a volume of ~200mm3 (defined as therapeutic ). The effect of neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. Nineteen days post tumour inoculation, we found that the tumour volume ( g , h ) and weight ( i ) were reduced in mice treated with BoNT/A ( Prophylactic group ). In parallel, we found that silencing tumour-innervating neurons increased the proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BoNT/A had no effect on the total number of intratumoral CD8 T cells ( j ) or the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. ( o ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female sensory neuron-intact or ablated mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic YUMMER1.7 cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. The effects of nociceptor neuron ablation on tumour size and volume were measured. Thirteen days post tumour inoculation, we found that the tumour growth was lower in mice treated with BoNT/A or in sensory neuron-ablated mice. BoNT/A had no additive effects when administered to sensory neuron-ablated mice. ( p ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic B16F10-mCherry-OVA cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. On days 7, 10, 13 and 16 post tumour inoculation, the mice were exposed to αPD-L1 (6 mg/kg, i.p.) or its isotype control. Eighteen days post tumour inoculation, we found that neuron silencing using BoNT/A potentiated αPD-L1-mediated tumour reduction. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f; h–n ) or as mean ± S.E.M ( g , o , p ). N are as follows: a-e : n = 5/groups, f : n = 3/groups, g–i : vehicle (n = 12), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 10), j : vehicle (n = 11), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), k–n : vehicle (n = 10), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), o : intact + vehicle (n = 9), ablated + vehicle (n = 8), intact + BoNT/A (n = 10), ablated + BoNT/A (n = 8), p : vehicle (n = 7), αPD-L1 (n = 8), αPD-L1 + BoNT/A (n = 7). Experiments were independently repeated two ( a–f , o–p ) or four ( g–n ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–f , h–n ) or two-way ANOVA post-hoc Bonferroni ( g , o , p ). Source Data

Techniques Used: Isolation, Mouse Assay, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Expressing, Fluorescence, Injection, Whisker Assay

Cancer-secreted SLPI drives the release of CGRP by nociceptor neurons. a – c , Naive DRG neurons ( Trpv1 cre ::-CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA cells and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48 h, the cells were collected, FACS purified and RNA sequenced. a , Hierarchical clustering of sorted neuron molecular profiles depicts distinct groups of transcripts enriched in each group. b , DEGs were calculated, and Slpi was found to be overexpressed in cancer cells when co-cultured with OVA-specific cytotoxic CD8 + T cells, DRG neurons or both populations. c , SLPI is secreted by B16F10-mCherry-OVA cells when co-cultured (24 h or 48 h) with naive DRG neurons and OVA-specific cytotoxic CD8 + T cells, with a maximal effect after 48 h. d – f , Using calcium microscopy, we found that SLPI (10 pg ml −1 –10 ng ml −1 ) activated around 20% of cultured naive DRG neurons ( d , e ). Activation of cultured neurons (3 h) with SLPI also leads to significant release of CGRP ( f ). Data are shown as a heat map showing normalized gene expression (log 2 (1 + TPM) − mean ( a ), as box-and-whisters plots (as defined in Fig. 1b,c ) ( b ) or as mean ± s.e.m. ( c – f ). n as follows: a , b : n = 2–4 per groups; c : n = 3 for all groups except CD8 + T cells ( n = 8); d : n = 17; e : n = 8 per group; f : 0 ng ml −1 ( n = 4), 0.1 ng ml −1 ( n = 5), 1 ng ml −1 ( n = 5), 5 ng ml −1 ( n = 4). Experiments in c – f were independently repeated three times with similar results. The sequencing experiment was not repeated ( a , b ). P values were determined by one-way ANOVA with post-hoc Bonferroni ( b , e , f ) or two-sided unpaired Student’s t -test ( c ). * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source Data
Figure Legend Snippet: Cancer-secreted SLPI drives the release of CGRP by nociceptor neurons. a – c , Naive DRG neurons ( Trpv1 cre ::-CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA cells and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48 h, the cells were collected, FACS purified and RNA sequenced. a , Hierarchical clustering of sorted neuron molecular profiles depicts distinct groups of transcripts enriched in each group. b , DEGs were calculated, and Slpi was found to be overexpressed in cancer cells when co-cultured with OVA-specific cytotoxic CD8 + T cells, DRG neurons or both populations. c , SLPI is secreted by B16F10-mCherry-OVA cells when co-cultured (24 h or 48 h) with naive DRG neurons and OVA-specific cytotoxic CD8 + T cells, with a maximal effect after 48 h. d – f , Using calcium microscopy, we found that SLPI (10 pg ml −1 –10 ng ml −1 ) activated around 20% of cultured naive DRG neurons ( d , e ). Activation of cultured neurons (3 h) with SLPI also leads to significant release of CGRP ( f ). Data are shown as a heat map showing normalized gene expression (log 2 (1 + TPM) − mean ( a ), as box-and-whisters plots (as defined in Fig. 1b,c ) ( b ) or as mean ± s.e.m. ( c – f ). n as follows: a , b : n = 2–4 per groups; c : n = 3 for all groups except CD8 + T cells ( n = 8); d : n = 17; e : n = 8 per group; f : 0 ng ml −1 ( n = 4), 0.1 ng ml −1 ( n = 5), 1 ng ml −1 ( n = 5), 5 ng ml −1 ( n = 4). Experiments in c – f were independently repeated three times with similar results. The sequencing experiment was not repeated ( a , b ). P values were determined by one-way ANOVA with post-hoc Bonferroni ( b , e , f ) or two-sided unpaired Student’s t -test ( c ). * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source Data

Techniques Used: Cell Culture, FACS, Purification, Microscopy, Activation Assay, Expressing, Sequencing

QX-314 silencing of B16F10-innervating neurons reduces tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to QX-314 (50–150 μM) for 24h, effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, QX-314 did not affect the survival of cultured cytotoxic CD8 + T cells ( a ), nor their relative expression of PD-1 + LAG3 + TIM3 + ( b ), IFNγ + ( c ), TNF + ( d ) and IL-2 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h. The cells were then exposed or not to QX-314 (0.1-1%) for an additional 24-72h, and cell count was analysed by bright-field microscopy. QX-314 did not affect B16F10 cells’ survival, as measured by relative cell count changes (at each time point) in comparison to vehicle-exposed cells. ( g–i ) One and three days prior to tumour inoculation, 8-week-old male and female wild-type mice’s right hindpaws or flanks were injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. On the following day, orthotopic B16F10 cells ( g : 5x10 5 cells; i.d.; h–i : 2x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. Starting one day post inoculation, QX-314 (0.3%) or its vehicle was administered (i.d.) once daily in another group of mice. The effects of sensory neuron silencing were tested on neuropeptide release ( g ), as well as mechanical ( h ) and thermal pain hypersensitivity ( i ). First, CGRP levels were increased in B16F10 tumour surrounding skin explant (assessed on day 15) in comparison to control skin; an effect further enhanced by capsaicin (1 μM; 3h) but was absent in skin pre-treated with BoNT/A (25 pg/μL) or QX-314 (0.3%; g ). We also found that B16F10 injection induced mechanical ( h ) and thermal pain hypersensitivities ( i ) fourteen days post tumour inoculation. These effects were stopped by sensory neuron silencing with QX-314 or BoNT/A ( h–i ). ( j ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, QX-314 (0.3%; i.d.; 5 sites) was injected once daily around the tumour. The effect of nociceptor neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. We found that silencing tumour-innervating neurons increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( k–r ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), In other groups of mice, QX-314 daily injection started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). As measured seventeen days post tumour inoculation, silencing tumour innervation also decreased tumour volume ( k , l ) and weight ( m ), as well as the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. QX-314 treatment also increased the total number of intratumoral CD8 + T cells ( o ), as well as relative proportion of IFNγ + ( p ), TNF + ( q ), and IL-2 + ( r ) CD8 + T cells. ( s–t ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into mice treated with QX-314 (0.3%; i.d.) 2-3 days prior to being injected into vehicle-exposed mice. Mice from each group with similar tumour size (~100mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~61%) in nociceptor silenced mice than was observed in isotype vehicle-exposed mice (~49%; s-t ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g , l–r ), as mean ± S.E.M ( f , h , i , k , s , t ), or as Mantel–Cox regression analysis ( j ). N are as follows: a : n = 4/groups, b–e : n = 5/groups, f : n = 3/groups, g : naïve (n = 4), vehicle (n = 7), B16F10+vehicle (n = 5), B16F10+BoNT/A (n = 5), B16F10+QX-314 (n = 5), h–i : n = 6/groups, j : vehicle (n = 89), QX-314 (n = 12), k : vehicle (n = 21), QX-314 prophylactic (n = 21), QX-314 therapeutic (n = 17), l : vehicle (n = 26), QX-314 therapeutic (n = 26), QX-314 prophylactic (n = 28), m : vehicle (n = 25), QX-314 therapeutic (n = 22), QX-314 prophylactic (n = 25), n : vehicle (n = 31), QX-314 therapeutic (n = 29), QX-314 prophylactic (n = 28), o : n = 30/groups, p–r : vehicle (n = 24), QX-314 therapeutic (n = 23), QX-314 prophylactic (n = 25), s : vehicle (n = 9), QX-314 (n = 13), t : vehicle + αPD-L1 (n = 18), QX-314 + αPLD1 (n = 13). Experiments were independently repeated two ( a–i , s–t ) or four ( j–r ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–g , l–r ), two-sided unpaired Student’s t-test ( h–i ), Mantel–Cox regression ( j ), or two-way ANOVA posthoc Bonferroni ( k , s–t ). Source Data
Figure Legend Snippet: QX-314 silencing of B16F10-innervating neurons reduces tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to QX-314 (50–150 μM) for 24h, effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, QX-314 did not affect the survival of cultured cytotoxic CD8 + T cells ( a ), nor their relative expression of PD-1 + LAG3 + TIM3 + ( b ), IFNγ + ( c ), TNF + ( d ) and IL-2 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h. The cells were then exposed or not to QX-314 (0.1-1%) for an additional 24-72h, and cell count was analysed by bright-field microscopy. QX-314 did not affect B16F10 cells’ survival, as measured by relative cell count changes (at each time point) in comparison to vehicle-exposed cells. ( g–i ) One and three days prior to tumour inoculation, 8-week-old male and female wild-type mice’s right hindpaws or flanks were injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. On the following day, orthotopic B16F10 cells ( g : 5x10 5 cells; i.d.; h–i : 2x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. Starting one day post inoculation, QX-314 (0.3%) or its vehicle was administered (i.d.) once daily in another group of mice. The effects of sensory neuron silencing were tested on neuropeptide release ( g ), as well as mechanical ( h ) and thermal pain hypersensitivity ( i ). First, CGRP levels were increased in B16F10 tumour surrounding skin explant (assessed on day 15) in comparison to control skin; an effect further enhanced by capsaicin (1 μM; 3h) but was absent in skin pre-treated with BoNT/A (25 pg/μL) or QX-314 (0.3%; g ). We also found that B16F10 injection induced mechanical ( h ) and thermal pain hypersensitivities ( i ) fourteen days post tumour inoculation. These effects were stopped by sensory neuron silencing with QX-314 or BoNT/A ( h–i ). ( j ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, QX-314 (0.3%; i.d.; 5 sites) was injected once daily around the tumour. The effect of nociceptor neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. We found that silencing tumour-innervating neurons increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( k–r ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), In other groups of mice, QX-314 daily injection started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). As measured seventeen days post tumour inoculation, silencing tumour innervation also decreased tumour volume ( k , l ) and weight ( m ), as well as the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. QX-314 treatment also increased the total number of intratumoral CD8 + T cells ( o ), as well as relative proportion of IFNγ + ( p ), TNF + ( q ), and IL-2 + ( r ) CD8 + T cells. ( s–t ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into mice treated with QX-314 (0.3%; i.d.) 2-3 days prior to being injected into vehicle-exposed mice. Mice from each group with similar tumour size (~100mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~61%) in nociceptor silenced mice than was observed in isotype vehicle-exposed mice (~49%; s-t ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g , l–r ), as mean ± S.E.M ( f , h , i , k , s , t ), or as Mantel–Cox regression analysis ( j ). N are as follows: a : n = 4/groups, b–e : n = 5/groups, f : n = 3/groups, g : naïve (n = 4), vehicle (n = 7), B16F10+vehicle (n = 5), B16F10+BoNT/A (n = 5), B16F10+QX-314 (n = 5), h–i : n = 6/groups, j : vehicle (n = 89), QX-314 (n = 12), k : vehicle (n = 21), QX-314 prophylactic (n = 21), QX-314 therapeutic (n = 17), l : vehicle (n = 26), QX-314 therapeutic (n = 26), QX-314 prophylactic (n = 28), m : vehicle (n = 25), QX-314 therapeutic (n = 22), QX-314 prophylactic (n = 25), n : vehicle (n = 31), QX-314 therapeutic (n = 29), QX-314 prophylactic (n = 28), o : n = 30/groups, p–r : vehicle (n = 24), QX-314 therapeutic (n = 23), QX-314 prophylactic (n = 25), s : vehicle (n = 9), QX-314 (n = 13), t : vehicle + αPD-L1 (n = 18), QX-314 + αPLD1 (n = 13). Experiments were independently repeated two ( a–i , s–t ) or four ( j–r ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–g , l–r ), two-sided unpaired Student’s t-test ( h–i ), Mantel–Cox regression ( j ), or two-way ANOVA posthoc Bonferroni ( k , s–t ). Source Data

Techniques Used: Isolation, Mouse Assay, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Expressing, Cell Counting, Microscopy, Injection, Whisker Assay

Melanoma-innervating nociceptors attenuate cancer immunosurveillance. Melanoma growth sets off anti-tumour immune responses, including the infiltration of effector CD8 T cells and their subsequent release of cytotoxic cytokines (i.e., IFNγ, TNF, Granzyme B). By acting on tissue-resident nociceptor neurons, melanoma-produced SLPI promotes pain hypersensitivity, tweaks the neurons’ transcriptome, and drives neurite outgrowth. These effects culminate in dense melanoma innervation by nociceptors and abundant release of immunomodulatory neuropeptides. CGRP, one such peptide, acts on tumour-infiltrating effector CD8 + T cells that express the CGRP receptor RAMP1, increasing their expression of immune checkpoint receptors (i.e., PD-1, LAG3, TIM3). Therefore, along with the immunosuppressive environment present in the tumour, nociceptor-produced CGRP leads to the functional exhaustion of tumour-infiltrating CD8 + T cells, which opens the door to unchecked proliferation of melanoma cells. Genetically ablating (i.e., TRPV1 lineage) or pharmacologically silencing (i.e., QX-314, BoNT/A) nociceptor neurons as well as blocking the action of CGRP on RAMP1 using a selective antagonist (i.e., BIBN4096) prevents effector CD8 + T cells from undergoing exhaustion. Therefore, targeting melanoma-innervating nociceptor neurons constitutes a novel strategy to safeguard host anti-tumour immunity and stop tumour growth.
Figure Legend Snippet: Melanoma-innervating nociceptors attenuate cancer immunosurveillance. Melanoma growth sets off anti-tumour immune responses, including the infiltration of effector CD8 T cells and their subsequent release of cytotoxic cytokines (i.e., IFNγ, TNF, Granzyme B). By acting on tissue-resident nociceptor neurons, melanoma-produced SLPI promotes pain hypersensitivity, tweaks the neurons’ transcriptome, and drives neurite outgrowth. These effects culminate in dense melanoma innervation by nociceptors and abundant release of immunomodulatory neuropeptides. CGRP, one such peptide, acts on tumour-infiltrating effector CD8 + T cells that express the CGRP receptor RAMP1, increasing their expression of immune checkpoint receptors (i.e., PD-1, LAG3, TIM3). Therefore, along with the immunosuppressive environment present in the tumour, nociceptor-produced CGRP leads to the functional exhaustion of tumour-infiltrating CD8 + T cells, which opens the door to unchecked proliferation of melanoma cells. Genetically ablating (i.e., TRPV1 lineage) or pharmacologically silencing (i.e., QX-314, BoNT/A) nociceptor neurons as well as blocking the action of CGRP on RAMP1 using a selective antagonist (i.e., BIBN4096) prevents effector CD8 + T cells from undergoing exhaustion. Therefore, targeting melanoma-innervating nociceptor neurons constitutes a novel strategy to safeguard host anti-tumour immunity and stop tumour growth.

Techniques Used: Produced, Expressing, Functional Assay, Blocking Assay

Genetic ablation of nociceptors safeguards anti-tumour immunity. a , Orthotopic B16F10-mCherry-OVA cells (2 × 10 5 cells, i.d.) were injected into the left hindpaw of wild-type mice. As measured on day 13 after tumour inoculation, intratumoral CD8 + T cell exhaustion positively correlated with thermal hypersensitivity ( R 2 = 0.55, P ≤ 0.0001). The thermal pain hypersensitivity represents the withdrawal latency ratio of the ipsilateral paw (tumour-inoculated) to the contralateral paw. b , Orthotopic B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) were inoculated into the flank of eight-week-old male and female mice with sensory neurons intact ( Trpv1 WT ::DTA fl/WT ) or ablated ( Trpv1 cre ::DTA fl/WT ). The median length of survival was increased by around 250% in nociceptor-ablated mice (measured until 22 days after inoculation). c – f , Sixteen days after tumour inoculation, sensory-neuron-ablated mice have reduced tumour growth ( c ) and increased tumour infiltration of IFNγ + CD8 + T cells ( d ), and the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells is decreased ( e ). This reduction in B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) tumour volume was absent in nociceptor-ablated mice whose CD8 + T cells were systemically depleted ( f ; assessed until day 14; anti-CD8, 200 μg per mouse, i.p., every 3 days). g , h , To chemically deplete their nociceptor neurons, Rag1 −/ − mice were injected with RTX. Twenty-eight days later, the mice were inoculated with B16F10-mCherry-OVA (5 × 10 5 cells, i.d.). RTX-injected mice that were adoptively transferred with naive OVA-specific CD8 + T cells (i.v., 1 × 10 6 cells, when tumour reached around 500 mm 3 ) showed reduced tumour growth ( g ; assessed until day 19) and exhaustion ( h ) compared to vehicle-exposed Rag1 −/ − mice. Data are shown as a linear regression analysis ± s.e. ( a ), as a Mantel–Cox regression ( b ), as mean ± s.e.m. ( c , f , g ) or as box-and-whisker plots (as defined in Fig. 1b,c ), for which individual data points are given ( d , e , h ). n as follows: a : n = 60; b : intact ( n = 62), ablated ( n = 73); c : intact ( n = 20), ablated ( n = 25); d : intact ( n = 24), ablated ( n = 23); e : intact ( n = 23), ablated ( n = 26); f : intact + anti-CD8 ( n = 10), ablated + anti-CD8 ( n = 8); g : vehicle ( n = 12), RTX ( n = 10); h : vehicle ( n = 11), RTX ( n = 10). Experiments were independently repeated two ( a , f – h ) or six ( b – e ) times with similar results. P values were determined by simple linear regression analysis ( a ), Mantel–Cox regression ( b ), two-way ANOVA with post-hoc Bonferroni ( c , f , g ) or two-sided unpaired Student’s t -test ( d , e , h ). Source Data
Figure Legend Snippet: Genetic ablation of nociceptors safeguards anti-tumour immunity. a , Orthotopic B16F10-mCherry-OVA cells (2 × 10 5 cells, i.d.) were injected into the left hindpaw of wild-type mice. As measured on day 13 after tumour inoculation, intratumoral CD8 + T cell exhaustion positively correlated with thermal hypersensitivity ( R 2 = 0.55, P ≤ 0.0001). The thermal pain hypersensitivity represents the withdrawal latency ratio of the ipsilateral paw (tumour-inoculated) to the contralateral paw. b , Orthotopic B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) were inoculated into the flank of eight-week-old male and female mice with sensory neurons intact ( Trpv1 WT ::DTA fl/WT ) or ablated ( Trpv1 cre ::DTA fl/WT ). The median length of survival was increased by around 250% in nociceptor-ablated mice (measured until 22 days after inoculation). c – f , Sixteen days after tumour inoculation, sensory-neuron-ablated mice have reduced tumour growth ( c ) and increased tumour infiltration of IFNγ + CD8 + T cells ( d ), and the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells is decreased ( e ). This reduction in B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) tumour volume was absent in nociceptor-ablated mice whose CD8 + T cells were systemically depleted ( f ; assessed until day 14; anti-CD8, 200 μg per mouse, i.p., every 3 days). g , h , To chemically deplete their nociceptor neurons, Rag1 −/ − mice were injected with RTX. Twenty-eight days later, the mice were inoculated with B16F10-mCherry-OVA (5 × 10 5 cells, i.d.). RTX-injected mice that were adoptively transferred with naive OVA-specific CD8 + T cells (i.v., 1 × 10 6 cells, when tumour reached around 500 mm 3 ) showed reduced tumour growth ( g ; assessed until day 19) and exhaustion ( h ) compared to vehicle-exposed Rag1 −/ − mice. Data are shown as a linear regression analysis ± s.e. ( a ), as a Mantel–Cox regression ( b ), as mean ± s.e.m. ( c , f , g ) or as box-and-whisker plots (as defined in Fig. 1b,c ), for which individual data points are given ( d , e , h ). n as follows: a : n = 60; b : intact ( n = 62), ablated ( n = 73); c : intact ( n = 20), ablated ( n = 25); d : intact ( n = 24), ablated ( n = 23); e : intact ( n = 23), ablated ( n = 26); f : intact + anti-CD8 ( n = 10), ablated + anti-CD8 ( n = 8); g : vehicle ( n = 12), RTX ( n = 10); h : vehicle ( n = 11), RTX ( n = 10). Experiments were independently repeated two ( a , f – h ) or six ( b – e ) times with similar results. P values were determined by simple linear regression analysis ( a ), Mantel–Cox regression ( b ), two-way ANOVA with post-hoc Bonferroni ( c , f , g ) or two-sided unpaired Student’s t -test ( d , e , h ). Source Data

Techniques Used: Injection, Mouse Assay, Whisker Assay

3) Product Images from "Nociceptor neurons affect cancer immunosurveillance"

Article Title: Nociceptor neurons affect cancer immunosurveillance

Journal: Nature

doi: 10.1038/s41586-022-05374-w

RAMP1 expression in patient melanoma-infiltrating T cells correlates with worsened survival and poor responsiveness to ICIs. ( a–l ) In silico analysis of Cancer Genome Atlas (TCGA) data linked the survival rate among 459 patients with melanoma with their relative expression levels of various genes of interest (determined by bulk RNA sequencing of tumour biopsy). Kaplan–Meier curves show the patients’ survival after segregation in two groups defined by their low or high expression of a gene of interest. Increased gene expression (labelled as high; red curve) of TUBB3 ( b ), PGP9.5 ( c ), Nav 1.7 ( E ), SLPI ( k ) and RAMP1 ( l ) in biopsy correlate with decreased patient survival (p≤0.05). The mantel–Haenszel hazard ratio and number of patients included in each analysis are shown in the figure ( a–l ). Experimental details were defined in Cancer Genome Atlas (TCGA) 40 . ( m ) In silico analysis of single-cell RNA sequencing of human melanoma-infiltrating T cells revealed that RAMP1 + T cells downregulated Il-2 expression and strongly overexpressed several immune checkpoint receptors ( PD-1 , TIM3 , LAG3 , CTLA4 , CD28 , ICOS , BTLA , CD27 ) in comparison to RAMP1 - T cells. Individual cell data are shown as a log 2 of 1 + (transcript per million / 10). Experimental details and cell clustering were defined in Tirosh et al 42 . N are defined in each panel. ( n–p ) On the basis of the clinical response of patients with melanoma to immune checkpoint blocker, patients were clustered into two groups defined as ICI-responsive or ICI-resistant 41 . In silico analysis of single-cell RNA sequencing of patients’ biopsies revealed that tumour-infiltrating CD8 + T cells from patients who were resistant to ICIs significantly overexpressed RAMP1 (2.0-fold), PD-1 (1.7-fold), LAG3 (1.6-fold), CTLA4 (1.6-fold), and TIM3 (1.7-fold; n–p ). Individual cell data are shown as a log 2 (1+(transcript per million/10). Experimental details and cell clustering were defined in Jerby-Arnon et al 41 . P-values are shown in the figure and determined by two-sided unpaired Student’s t-test. N are defined in each panel ( n–o ). Source Data
Figure Legend Snippet: RAMP1 expression in patient melanoma-infiltrating T cells correlates with worsened survival and poor responsiveness to ICIs. ( a–l ) In silico analysis of Cancer Genome Atlas (TCGA) data linked the survival rate among 459 patients with melanoma with their relative expression levels of various genes of interest (determined by bulk RNA sequencing of tumour biopsy). Kaplan–Meier curves show the patients’ survival after segregation in two groups defined by their low or high expression of a gene of interest. Increased gene expression (labelled as high; red curve) of TUBB3 ( b ), PGP9.5 ( c ), Nav 1.7 ( E ), SLPI ( k ) and RAMP1 ( l ) in biopsy correlate with decreased patient survival (p≤0.05). The mantel–Haenszel hazard ratio and number of patients included in each analysis are shown in the figure ( a–l ). Experimental details were defined in Cancer Genome Atlas (TCGA) 40 . ( m ) In silico analysis of single-cell RNA sequencing of human melanoma-infiltrating T cells revealed that RAMP1 + T cells downregulated Il-2 expression and strongly overexpressed several immune checkpoint receptors ( PD-1 , TIM3 , LAG3 , CTLA4 , CD28 , ICOS , BTLA , CD27 ) in comparison to RAMP1 - T cells. Individual cell data are shown as a log 2 of 1 + (transcript per million / 10). Experimental details and cell clustering were defined in Tirosh et al 42 . N are defined in each panel. ( n–p ) On the basis of the clinical response of patients with melanoma to immune checkpoint blocker, patients were clustered into two groups defined as ICI-responsive or ICI-resistant 41 . In silico analysis of single-cell RNA sequencing of patients’ biopsies revealed that tumour-infiltrating CD8 + T cells from patients who were resistant to ICIs significantly overexpressed RAMP1 (2.0-fold), PD-1 (1.7-fold), LAG3 (1.6-fold), CTLA4 (1.6-fold), and TIM3 (1.7-fold; n–p ). Individual cell data are shown as a log 2 (1+(transcript per million/10). Experimental details and cell clustering were defined in Jerby-Arnon et al 41 . P-values are shown in the figure and determined by two-sided unpaired Student’s t-test. N are defined in each panel ( n–o ). Source Data

Techniques Used: Expressing, In Silico, RNA Sequencing Assay

B16F10-secreted SLPI activates nociceptor neurons. ( a–e ) Naive DRG neurons ( Trpv1 cre ::CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA, and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48h, the cells were collected, FACS purified, and RNA sequenced. DEGs were calculated, and Fgfr1 (fibroblast growth factor receptor 1) was found to be overexpressed in OVA-specific cytotoxic CD8 + T cells when co-cultured with cancer cells and DRG neurons ( a ). Conversely, OVA-specific cytotoxic CD8 + T cells downregulates the expression of the pro-nociceptive factor Hmgb1 (High–mobility group box 1; b ), Braf ( c ) , as well as Fgfr3 ( d ) when co-cultured with B16F10-mCherry-OVA and DRG neurons. Tslp expression level was not affected in any of tested groups ( e ). ( f–i ) Using calcium microscopy, we probed whether SLPI directly activates cultured DRG neurons. We found that SLPI (0.01-10 ng/mL) induces a significant calcium influx in DRG neurons ( f ). SLPI-responsive neurons are mostly small-sized neurons ( g-h ; mean area = 151 μm 2 ) and largely capsaicin-responsive ( i ; ~42%). ( j ) The right hindpaw of naive mice was injected with saline (20 μL) or SLPI (i.d., 1 μg/20 μL), and the mice’s noxious thermal nociceptive threshold was measured (0-6h). The ipsilateral paw injected with SLPI showed thermal hypersensitivity in contrast with the contralateral paw. Saline had no effect on the mice’s thermal sensitivity. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f , h , j ), stacked bar graph on a logarithmic scale ( g ), and Venn Diagram ( i ). N are as follows: a–e : n = 2–4/groups, f : vehicle (n = 28), 10pg/ml (n = 28), 100 pg/ml (n = 132), 1,000 pg/ml (n = 191), 10 ng/ml (n = 260), capsaicin (n = 613), KCl (n = 1,139), g : 0-100 (SLPI=19; KCl=177), 100-200 (SLPI = 45; KCl = 390), 200-300 (SLPI = 16; KCl =216), 300-400 (SLPI=11; KCl = 138), 400-500 (SLPI = 5; KCl = 68), 500-600 (SLPI=2, KCl = 18), 600-700 (SLPI = 0; KCl = 10), 700-800 (SLPI=0; KCl=13), 800+ (SLPI = 0; KCl = 12), h : n = 98, i : KCl + =1139, KCl + Caps + =614, KCl + Caps + SLPI + =261, KCl + Caps - SLPI + =29, j : 0h (n = 9), SLPI at 1h (n = 6), saline at 1h (n = 3), SLPI at 3h (n = 6), saline at 3h (n=3), SLPI at 6h (n = 6), saline at 6h (n = 3). Experiments were independently repeated two ( j ) or three ( f–i ) times with similar results. Sequencing experiment was not repeated ( a–e ). P-values were determined by one-way ANOVA post-hoc Bonferroni ( a–f ); or two-sided unpaired Student’s t-test ( j ). P-values are shown in the figure or indicated by * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001. Source Data
Figure Legend Snippet: B16F10-secreted SLPI activates nociceptor neurons. ( a–e ) Naive DRG neurons ( Trpv1 cre ::CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA, and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48h, the cells were collected, FACS purified, and RNA sequenced. DEGs were calculated, and Fgfr1 (fibroblast growth factor receptor 1) was found to be overexpressed in OVA-specific cytotoxic CD8 + T cells when co-cultured with cancer cells and DRG neurons ( a ). Conversely, OVA-specific cytotoxic CD8 + T cells downregulates the expression of the pro-nociceptive factor Hmgb1 (High–mobility group box 1; b ), Braf ( c ) , as well as Fgfr3 ( d ) when co-cultured with B16F10-mCherry-OVA and DRG neurons. Tslp expression level was not affected in any of tested groups ( e ). ( f–i ) Using calcium microscopy, we probed whether SLPI directly activates cultured DRG neurons. We found that SLPI (0.01-10 ng/mL) induces a significant calcium influx in DRG neurons ( f ). SLPI-responsive neurons are mostly small-sized neurons ( g-h ; mean area = 151 μm 2 ) and largely capsaicin-responsive ( i ; ~42%). ( j ) The right hindpaw of naive mice was injected with saline (20 μL) or SLPI (i.d., 1 μg/20 μL), and the mice’s noxious thermal nociceptive threshold was measured (0-6h). The ipsilateral paw injected with SLPI showed thermal hypersensitivity in contrast with the contralateral paw. Saline had no effect on the mice’s thermal sensitivity. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f , h , j ), stacked bar graph on a logarithmic scale ( g ), and Venn Diagram ( i ). N are as follows: a–e : n = 2–4/groups, f : vehicle (n = 28), 10pg/ml (n = 28), 100 pg/ml (n = 132), 1,000 pg/ml (n = 191), 10 ng/ml (n = 260), capsaicin (n = 613), KCl (n = 1,139), g : 0-100 (SLPI=19; KCl=177), 100-200 (SLPI = 45; KCl = 390), 200-300 (SLPI = 16; KCl =216), 300-400 (SLPI=11; KCl = 138), 400-500 (SLPI = 5; KCl = 68), 500-600 (SLPI=2, KCl = 18), 600-700 (SLPI = 0; KCl = 10), 700-800 (SLPI=0; KCl=13), 800+ (SLPI = 0; KCl = 12), h : n = 98, i : KCl + =1139, KCl + Caps + =614, KCl + Caps + SLPI + =261, KCl + Caps - SLPI + =29, j : 0h (n = 9), SLPI at 1h (n = 6), saline at 1h (n = 3), SLPI at 3h (n = 6), saline at 3h (n=3), SLPI at 6h (n = 6), saline at 6h (n = 3). Experiments were independently repeated two ( j ) or three ( f–i ) times with similar results. Sequencing experiment was not repeated ( a–e ). P-values were determined by one-way ANOVA post-hoc Bonferroni ( a–f ); or two-sided unpaired Student’s t-test ( j ). P-values are shown in the figure or indicated by * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001. Source Data

Techniques Used: Cell Culture, FACS, Purification, Expressing, Microscopy, Mouse Assay, Injection, Whisker Assay, Sequencing

CGRP attenuates the anti-tumour immunity of RAMP1 + CD8 + T cells. a – c , Splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. Eight-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5 × 10 6 cells) with activated Ramp1 −/ − or Ramp1 WT CD8 + T cells or a 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week after transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.). Ten days after B16F10 inoculation, we observed greater tumour growth ( a ) in Ramp1 WT transplanted mice. Intratumoral Ramp1 −/ − (CD45.2 + ) and Ramp1 WT (CD45.1 + ) CD8 + T cells were FACS purified, immunophenotyped ( b ) and RNA sequenced ( c ). Ramp1 −/ − CD8 + T cells showed a lower proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( b ) as well as reduced transcript expression of exhaustion markers ( c ). d , In silico analysis of The Cancer Genome Atlas (TCGA) data 40 was used to correlate the survival rate of 459 patients with melanoma with the relative RAMP1 expression (primary biopsy bulk RNA sequencing). In comparison to patients with low RAMP1 expression, higher RAMP1 levels correlate with decreased patient survival. e , In silico analysis of single-cell RNA sequencing of human melanoma 41 reveals that intratumoral RAMP1 -expressing CD8 + T cells strongly overexpress several immune checkpoint receptors ( PD-1 (also known as PDCD1 ) TIM3 , LAG3 , CTLA4 ) in comparison to Ramp1 -negative CD8 + T cells. Data are shown as mean ± s.e.m. ( a ), slopegraph ( b ), as a heat map showing normalized gene expression (log 10 (10 3 × TPM) ( c ), as a Mantel–Cox regression ( d ) or as a violin plot ( e ). n as follows: a–c : n = 5 per group; d : high ( n = 45), low ( n = 68); e : RAMP1 − CD8 ( n = 1,732), RAMP1 + CD8 ( n = 25). Experiments were independently repeated two ( a , b ) times with similar results. The sequencing experiment was not repeated ( c ). P values were determined by two-way ANOVA with post-hoc Bonferroni ( a ), two-sided unpaired Student’s t -test ( b ) or Mantel–Cox regression ( d ). Source Data
Figure Legend Snippet: CGRP attenuates the anti-tumour immunity of RAMP1 + CD8 + T cells. a – c , Splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. Eight-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5 × 10 6 cells) with activated Ramp1 −/ − or Ramp1 WT CD8 + T cells or a 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week after transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.). Ten days after B16F10 inoculation, we observed greater tumour growth ( a ) in Ramp1 WT transplanted mice. Intratumoral Ramp1 −/ − (CD45.2 + ) and Ramp1 WT (CD45.1 + ) CD8 + T cells were FACS purified, immunophenotyped ( b ) and RNA sequenced ( c ). Ramp1 −/ − CD8 + T cells showed a lower proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( b ) as well as reduced transcript expression of exhaustion markers ( c ). d , In silico analysis of The Cancer Genome Atlas (TCGA) data 40 was used to correlate the survival rate of 459 patients with melanoma with the relative RAMP1 expression (primary biopsy bulk RNA sequencing). In comparison to patients with low RAMP1 expression, higher RAMP1 levels correlate with decreased patient survival. e , In silico analysis of single-cell RNA sequencing of human melanoma 41 reveals that intratumoral RAMP1 -expressing CD8 + T cells strongly overexpress several immune checkpoint receptors ( PD-1 (also known as PDCD1 ) TIM3 , LAG3 , CTLA4 ) in comparison to Ramp1 -negative CD8 + T cells. Data are shown as mean ± s.e.m. ( a ), slopegraph ( b ), as a heat map showing normalized gene expression (log 10 (10 3 × TPM) ( c ), as a Mantel–Cox regression ( d ) or as a violin plot ( e ). n as follows: a–c : n = 5 per group; d : high ( n = 45), low ( n = 68); e : RAMP1 − CD8 ( n = 1,732), RAMP1 + CD8 ( n = 25). Experiments were independently repeated two ( a , b ) times with similar results. The sequencing experiment was not repeated ( c ). P values were determined by two-way ANOVA with post-hoc Bonferroni ( a ), two-sided unpaired Student’s t -test ( b ) or Mantel–Cox regression ( d ). Source Data

Techniques Used: FACS, Purification, Mouse Assay, In Vitro, Transplantation Assay, Expressing, In Silico, RNA Sequencing Assay, Sequencing

The CGRP–RAMP1 axis promotes intratumoral CD8 + T cell exhaustion. ( a–e ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Nav 1.8 WT ::DTA fl/WT ) and ablated mice ( Nav 1.8 cre ::DTA fl/WT ). As measured fifteen days post inoculation, Na V 1.8 + nociceptor-ablated mice had lower proportion of PD-1 + LAG3 + TIM3 + ( a ) CD8 + T cells, but increased levels of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) CD8 + T cells. B16F10-mCherry-OVA (5x10 5 cells; i.d.)-tumour surrounding skin was also collected and capsaicin-induced CGRP release assessed by ELISA. Intratumoral CGRP levels positively correlate with the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ). ( f ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old female sensory neuron intact or ablated mice. In nociceptor-ablated mice, recombinant CGRP injection (100nM, i.d., once daily) rescues intratumoral CD8 + T cells exhaustion (PD-1 + LAG3 + TIM3 + ). ( g ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. We found that blocking the action of CGRP on RAMP1-expressing cells, increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( h–m ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. In another group of mice, BIBN4096 (5 mg/kg, i.p., every other day) injections were started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). The effect of nociceptor neuron-silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. As assessed thirteen days post tumour inoculation, BIBN4096 decreased tumour volume ( h ) and weight ( i ) but increased the relative proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BIBN4096 had no effect on the number of intratumoral CD8 + T cells ( j ). When administered as therapeutic, BIBN4096 reduced tumour volume ( h ) and weight ( i ) but had limited effect on CD8 + T cells’ cytotoxicity ( j–m ). ( n ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron-intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Starting one day post inoculation, BIBN4096 (5 mg/kg) or its vehicle was administered (i.p.) on alternate days; effects on tumour volume were measured. Fourteen days post tumour inoculation, we found that tumour growth was reduced in sensory neuron-ablated mice and in BIBN4096-treated mice. BIBN4096 had no additive effect when given to sensory neuron-ablated mice. ( o–s ) Splenocytes-isolated CD8 + T cells from naïve C57BL6J mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BIBN4096 (1–4 μM) for 24h; effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BIBN4096 did not affect the survival ( o ) of cultured cytotoxic CD8 + T cells, nor their relative expression of PD-1 + LAG3 + TIM3 + ( p ), IFNγ + ( q ), TNF + ( r ), and IL-2 + ( s ). ( t ) B16F10 cells (1x10 5 cells) were cultured for 24h. The cells were then exposed (or not) to BIBN4096 (1-8 μM) for an additional 24h; effects on apoptosis were measured by flow cytometry. BIBN4096 did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( u-w ) Naive splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated ( CD3 and CD28 + IL-2 ) in vitro . 8-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5x10 6 cells) with either Ramp1 −/ − or Ramp1 WT CD8 + T cells or 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week post transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5x10 5 cells; i.d.). Ten days post tumour inoculation, we retrieved a similar number of tumours draining lymph node CD8 + T cells across the three tested groups ( u ). The relative proportion of intra-tumour PD-1 + LAG3 + TIM3 + CD8 + T cells was lower in Ramp1 −/ − transplanted mice ( v ). Within the same tumour, intratumoral CD8 + T cell exhaustion was immunophenotyped by flow cytometry ( representative panel shown in w ) and showed that the relative proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells was ~3-fold lower in Ramp1 −/ − CD8 + T cells than in Ramp1 WT CD8 + T cells ( w ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–d , f , h–m , o–v ), linear regression ( e ), Mantel–Cox regression ( g ), mean ± S.E.M ( n ), or as FACS plot ( w ). N are as follows a–e : Nav 1.8 WT ::DTA fl/WT (n = 18), Nav 1.8 cre ::DTA fl/WT (n = 10), f : Trpv1 WT ::DTA fl/WT (n = 16), Trpv1 cre ::DTA fl/WT +CGRP (n = 11), g : vehicle (n = 89), BIBN4096 (n = 16), h–m : Vehicle (n = 13), BIBN4096 therapeutic (n = 18), BIBN4096 prophylactic (n = 16), n : Trpv1 WT ::DTA fl/WT + vehicle (n = 8), Trpv1 WT ::DTA fl/WT + BIBN4096 (n = 9), Trpv1 cre ::DTA fl/WT + vehicle (n = 7), Trpv1 cre ::DTA fl/WT + BIBN4096 (n = 7), o : vehicle (n = 5), 1µM BIBN4096 (n = 3), 4 µM BIBN4096 (n = 5), p–s : n = 5/groups, t : n = 4/groups, u–w : n = 5/groups. Experiments were independently repeated twice ( a–f , n–w ) or four ( g–m ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( a–d , f , v ), simple linear regression analysis ( e ), Mantel–Cox regression ( g ), by one-way ANOVA posthoc Bonferroni ( h–m; o–u ), or two-way ANOVA post-hoc Bonferroni ( n ). Source Data
Figure Legend Snippet: The CGRP–RAMP1 axis promotes intratumoral CD8 + T cell exhaustion. ( a–e ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Nav 1.8 WT ::DTA fl/WT ) and ablated mice ( Nav 1.8 cre ::DTA fl/WT ). As measured fifteen days post inoculation, Na V 1.8 + nociceptor-ablated mice had lower proportion of PD-1 + LAG3 + TIM3 + ( a ) CD8 + T cells, but increased levels of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) CD8 + T cells. B16F10-mCherry-OVA (5x10 5 cells; i.d.)-tumour surrounding skin was also collected and capsaicin-induced CGRP release assessed by ELISA. Intratumoral CGRP levels positively correlate with the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ). ( f ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old female sensory neuron intact or ablated mice. In nociceptor-ablated mice, recombinant CGRP injection (100nM, i.d., once daily) rescues intratumoral CD8 + T cells exhaustion (PD-1 + LAG3 + TIM3 + ). ( g ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. We found that blocking the action of CGRP on RAMP1-expressing cells, increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( h–m ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. In another group of mice, BIBN4096 (5 mg/kg, i.p., every other day) injections were started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). The effect of nociceptor neuron-silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. As assessed thirteen days post tumour inoculation, BIBN4096 decreased tumour volume ( h ) and weight ( i ) but increased the relative proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BIBN4096 had no effect on the number of intratumoral CD8 + T cells ( j ). When administered as therapeutic, BIBN4096 reduced tumour volume ( h ) and weight ( i ) but had limited effect on CD8 + T cells’ cytotoxicity ( j–m ). ( n ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron-intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Starting one day post inoculation, BIBN4096 (5 mg/kg) or its vehicle was administered (i.p.) on alternate days; effects on tumour volume were measured. Fourteen days post tumour inoculation, we found that tumour growth was reduced in sensory neuron-ablated mice and in BIBN4096-treated mice. BIBN4096 had no additive effect when given to sensory neuron-ablated mice. ( o–s ) Splenocytes-isolated CD8 + T cells from naïve C57BL6J mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BIBN4096 (1–4 μM) for 24h; effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BIBN4096 did not affect the survival ( o ) of cultured cytotoxic CD8 + T cells, nor their relative expression of PD-1 + LAG3 + TIM3 + ( p ), IFNγ + ( q ), TNF + ( r ), and IL-2 + ( s ). ( t ) B16F10 cells (1x10 5 cells) were cultured for 24h. The cells were then exposed (or not) to BIBN4096 (1-8 μM) for an additional 24h; effects on apoptosis were measured by flow cytometry. BIBN4096 did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( u-w ) Naive splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated ( CD3 and CD28 + IL-2 ) in vitro . 8-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5x10 6 cells) with either Ramp1 −/ − or Ramp1 WT CD8 + T cells or 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week post transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5x10 5 cells; i.d.). Ten days post tumour inoculation, we retrieved a similar number of tumours draining lymph node CD8 + T cells across the three tested groups ( u ). The relative proportion of intra-tumour PD-1 + LAG3 + TIM3 + CD8 + T cells was lower in Ramp1 −/ − transplanted mice ( v ). Within the same tumour, intratumoral CD8 + T cell exhaustion was immunophenotyped by flow cytometry ( representative panel shown in w ) and showed that the relative proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells was ~3-fold lower in Ramp1 −/ − CD8 + T cells than in Ramp1 WT CD8 + T cells ( w ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–d , f , h–m , o–v ), linear regression ( e ), Mantel–Cox regression ( g ), mean ± S.E.M ( n ), or as FACS plot ( w ). N are as follows a–e : Nav 1.8 WT ::DTA fl/WT (n = 18), Nav 1.8 cre ::DTA fl/WT (n = 10), f : Trpv1 WT ::DTA fl/WT (n = 16), Trpv1 cre ::DTA fl/WT +CGRP (n = 11), g : vehicle (n = 89), BIBN4096 (n = 16), h–m : Vehicle (n = 13), BIBN4096 therapeutic (n = 18), BIBN4096 prophylactic (n = 16), n : Trpv1 WT ::DTA fl/WT + vehicle (n = 8), Trpv1 WT ::DTA fl/WT + BIBN4096 (n = 9), Trpv1 cre ::DTA fl/WT + vehicle (n = 7), Trpv1 cre ::DTA fl/WT + BIBN4096 (n = 7), o : vehicle (n = 5), 1µM BIBN4096 (n = 3), 4 µM BIBN4096 (n = 5), p–s : n = 5/groups, t : n = 4/groups, u–w : n = 5/groups. Experiments were independently repeated twice ( a–f , n–w ) or four ( g–m ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( a–d , f , v ), simple linear regression analysis ( e ), Mantel–Cox regression ( g ), by one-way ANOVA posthoc Bonferroni ( h–m; o–u ), or two-way ANOVA post-hoc Bonferroni ( n ). Source Data

Techniques Used: Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Expressing, Isolation, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Fluorescence, FACS, Purification, In Vitro, Transplantation Assay, Whisker Assay

CGRP modulates the activation of CD8 + T cells. a , b , Splenocyte CD8 + T cells from wild-type ( a ), Ramp1 −/ − ( a ) or naive OT-I ( b ) mice were cultured under T c1 -stimulating conditions (ex-vivo-activated by CD3 and CD28, IL-12 and anti-IL4) for 48 h to generate cytotoxic CD8 + T cells. In the presence of IL-2 (10 ng ml −1 ), the cells were stimulated with CGRP (100 nM; challenged once every two days) for 96 h. Wild-type cytotoxic CD8 + T cells showed an increased proportion of PD-1 + LAG3 + TIM3 + cells; this effect was absent when treating cytotoxic CD8 + T cells that were collected from Ramp1 −/ − mice ( a ). In co-culture (48 h), CGRP (100 nM; once daily) also reduced the ability of OT-I cytotoxic CD8 + T cells (4 × 10 5 cells) to eliminate B16F10-mCherry-OVA cancer cells ( b ). c , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old female mice with sensory neurons intact or ablated. In nociceptor-ablated mice, peritumoral recombinant CGRP injection (100 nM, i.d., once daily) rescues B16F10 growth (assessed until day 12). d , e , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old male and female mice. Starting one day after inoculation (defined as prophylactic), the RAMP1 antagonist BIBN4096 (5 mg kg −1 ) was administered systemically (i.p.) once every two days. In another group of mice, BIBN4096 (5 mg kg −1 , i.p., every two days) injections were started once the tumour reached a volume of around 200 mm 3 (defined as therapeutic). Prophylactic or therapeutic BIBN4096 treatments decreased tumour growth ( d ) and reduced the proportion of intratumoral PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ; assessed until day 13). Data are shown as box-and-whisker plots (as defined in Fig. 1b, c ), for which individual data points are given ( a , b , e ), or as mean ± s.e.m. ( c , d ). n as follows: a : Ramp1 WT CD8 + vehicle ( n = 9), Ramp1 WT CD8 + CGRP ( n = 10), Ramp1 −/ − CD8 + vehicle ( n = 10), Ramp1 −/ − CD8 + CGRP ( n = 9); b : n = 4 per group; c : intact + vehicle ( n = 15), ablated + CGRP ( n = 11); d : vehicle ( n = 13), BIBN prophylactic ( n = 16), BIBN therapeutic ( n = 18); e : vehicle ( n = 10), BIBN prophylactic ( n = 13), BIBN therapeutic ( n = 16). Experiments were independently repeated three times with similar results. P values were determined by one-way ANOVA with post-hoc Bonferroni ( a , e ), two-sided unpaired Student’s t -test ( b ) or two-way ANOVA with post-hoc Bonferroni ( c , d ). Source Data
Figure Legend Snippet: CGRP modulates the activation of CD8 + T cells. a , b , Splenocyte CD8 + T cells from wild-type ( a ), Ramp1 −/ − ( a ) or naive OT-I ( b ) mice were cultured under T c1 -stimulating conditions (ex-vivo-activated by CD3 and CD28, IL-12 and anti-IL4) for 48 h to generate cytotoxic CD8 + T cells. In the presence of IL-2 (10 ng ml −1 ), the cells were stimulated with CGRP (100 nM; challenged once every two days) for 96 h. Wild-type cytotoxic CD8 + T cells showed an increased proportion of PD-1 + LAG3 + TIM3 + cells; this effect was absent when treating cytotoxic CD8 + T cells that were collected from Ramp1 −/ − mice ( a ). In co-culture (48 h), CGRP (100 nM; once daily) also reduced the ability of OT-I cytotoxic CD8 + T cells (4 × 10 5 cells) to eliminate B16F10-mCherry-OVA cancer cells ( b ). c , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old female mice with sensory neurons intact or ablated. In nociceptor-ablated mice, peritumoral recombinant CGRP injection (100 nM, i.d., once daily) rescues B16F10 growth (assessed until day 12). d , e , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old male and female mice. Starting one day after inoculation (defined as prophylactic), the RAMP1 antagonist BIBN4096 (5 mg kg −1 ) was administered systemically (i.p.) once every two days. In another group of mice, BIBN4096 (5 mg kg −1 , i.p., every two days) injections were started once the tumour reached a volume of around 200 mm 3 (defined as therapeutic). Prophylactic or therapeutic BIBN4096 treatments decreased tumour growth ( d ) and reduced the proportion of intratumoral PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ; assessed until day 13). Data are shown as box-and-whisker plots (as defined in Fig. 1b, c ), for which individual data points are given ( a , b , e ), or as mean ± s.e.m. ( c , d ). n as follows: a : Ramp1 WT CD8 + vehicle ( n = 9), Ramp1 WT CD8 + CGRP ( n = 10), Ramp1 −/ − CD8 + vehicle ( n = 10), Ramp1 −/ − CD8 + CGRP ( n = 9); b : n = 4 per group; c : intact + vehicle ( n = 15), ablated + CGRP ( n = 11); d : vehicle ( n = 13), BIBN prophylactic ( n = 16), BIBN therapeutic ( n = 18); e : vehicle ( n = 10), BIBN prophylactic ( n = 13), BIBN therapeutic ( n = 16). Experiments were independently repeated three times with similar results. P values were determined by one-way ANOVA with post-hoc Bonferroni ( a , e ), two-sided unpaired Student’s t -test ( b ) or two-way ANOVA with post-hoc Bonferroni ( c , d ). Source Data

Techniques Used: Activation Assay, Mouse Assay, Cell Culture, Ex Vivo, Co-Culture Assay, Recombinant, Injection, Whisker Assay

Nociceptor-released CGRP increases cytotoxic CD8 + T cell exhaustion. ( a–b ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating condition ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then cultured or not with wild-type DRG neurons and exposed to capsaicin (1 μM, challenged once every two days) or its vehicle. As measured after 4 days stimulation, capsaicin-stimulated intact neuron increased the proportion of PD-1 + LAG3 + TIM3 + ( a ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( b ). ( c–d ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 μL/mL), naive DRG neurons were cultured in the presence of BoNT/A (50 pg/mL) or its vehicle for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed or not to a RAMP1 blocker (CGRP 8–37 ; 2 μg/mL) and challenge (1:2 dilution) with fresh KCl-induced conditioned medium from naive, or BoNT/A-silenced neurons. As measured after 4 days stimulation, KCl-stimulated neuron-conditioned medium increased the proportion of PD-1 + LAG3 + TIM3 + ( c ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( d ). Such effect was absent when cytotoxic CD8 + T cells were co-exposed to the RAMP1 blocker CGRP 8–37 or challenged with the neuron conditioned medium collected from BoNT/A-silenced neurons ( c–d ). ( e–f ) Splenocytes-isolated CD8 + T cells from wild-type and Ramp1 −/ − mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed to CGRP (0.1 μM) or its vehicle. As measured after 4 days stimulation, representative flow cytometry plots ( f ) show that CGRP decrease Ramp WT cytotoxic CD8 + T cells expression of IFNγ + ( e , f ), TNF + ( f ), and IL-2 + ( f ) when exposed to CGRP. Inversely, CGRP increase the proportion of PD-1 + LAG3 + TIM3 + in Ramp1 W T cytotoxic CD8 + T cells ( f ). Ramp1 −/ − cytotoxic CD8 + T cells were protected from the effect of CGRP ( e–f ). ( g–i ) Splenocytes-isolated CD8 + T cells from naive OT-I mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. B16F10-mCherry-OVA cells (1×10 5 cells) were then cultured with or without OT-I cytotoxic CD8 + T cells (4×10 5 cells). Tc1-stimulated OT-I-CD8 + T cells lead to B16F10-OVA cell apoptosis (AnnexinV + 7AAD + ; g , measured after 48h; h–i , measured after 24h). B16F10-mCherry-OVA cells elimination by cytotoxic CD8 + T cells was reduced when the co-cultures were challenged (1:2 dilution; once daily for two consecutive days) with fresh conditioned medium collected from capsaicin (1 μM)-stimulated naive DRG neurons ( g ; measured after 48h). Similarly, KCl (50mM)-stimulated naive DRG neurons conditioned medium (1:2 dilution) reduced B16F10-mCherry-OVA apoptosis ( h ; measured after 24h). This effect was blunted when the cells were co-exposed to the RAMP1 blocker CGRP 8-37 ( h ; 2 μg/mL; measured after 24h). CGRP (0.1 μM) challenges also reduced OT-I cytotoxic CD8 + T cells elimination of B16F10-OVA cell ( i ; measured after 24h). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g–h ), or representative FACS plot ( f , i ). N are as follows: a : CD8 + vehicle (n = 4), CD8 + capsaicin (n = 9), CD8 + neuron + capsaicin (n = 9), b : n = 5/groups, c : CD8 (n = 6), CD8 + KCl-induced neurons CM (n = 5), CD8 + KCl-induced neurons CM + CGRP 8-37 (n = 6), CD8 + KCl-induced neurons CM + BoNT/A (n = 6), d : n = 5/groups, e : Ramp1 WT CD8 + vehicle (n = 7), Ramp1 WT CD8 + CGRP (n = 8), Ramp1 −/ − CD8 + vehicle (n = 6), Ramp1 −/ − CD8 + CGRP (n = 6), g : B16F10 (n = 3), B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), h : B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM + CGRP 8–37 (n = 5). Experiments were repeated a minimum of three independent times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferroni ( a–e , g–h ). Source Data
Figure Legend Snippet: Nociceptor-released CGRP increases cytotoxic CD8 + T cell exhaustion. ( a–b ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating condition ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then cultured or not with wild-type DRG neurons and exposed to capsaicin (1 μM, challenged once every two days) or its vehicle. As measured after 4 days stimulation, capsaicin-stimulated intact neuron increased the proportion of PD-1 + LAG3 + TIM3 + ( a ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( b ). ( c–d ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 μL/mL), naive DRG neurons were cultured in the presence of BoNT/A (50 pg/mL) or its vehicle for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed or not to a RAMP1 blocker (CGRP 8–37 ; 2 μg/mL) and challenge (1:2 dilution) with fresh KCl-induced conditioned medium from naive, or BoNT/A-silenced neurons. As measured after 4 days stimulation, KCl-stimulated neuron-conditioned medium increased the proportion of PD-1 + LAG3 + TIM3 + ( c ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( d ). Such effect was absent when cytotoxic CD8 + T cells were co-exposed to the RAMP1 blocker CGRP 8–37 or challenged with the neuron conditioned medium collected from BoNT/A-silenced neurons ( c–d ). ( e–f ) Splenocytes-isolated CD8 + T cells from wild-type and Ramp1 −/ − mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed to CGRP (0.1 μM) or its vehicle. As measured after 4 days stimulation, representative flow cytometry plots ( f ) show that CGRP decrease Ramp WT cytotoxic CD8 + T cells expression of IFNγ + ( e , f ), TNF + ( f ), and IL-2 + ( f ) when exposed to CGRP. Inversely, CGRP increase the proportion of PD-1 + LAG3 + TIM3 + in Ramp1 W T cytotoxic CD8 + T cells ( f ). Ramp1 −/ − cytotoxic CD8 + T cells were protected from the effect of CGRP ( e–f ). ( g–i ) Splenocytes-isolated CD8 + T cells from naive OT-I mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. B16F10-mCherry-OVA cells (1×10 5 cells) were then cultured with or without OT-I cytotoxic CD8 + T cells (4×10 5 cells). Tc1-stimulated OT-I-CD8 + T cells lead to B16F10-OVA cell apoptosis (AnnexinV + 7AAD + ; g , measured after 48h; h–i , measured after 24h). B16F10-mCherry-OVA cells elimination by cytotoxic CD8 + T cells was reduced when the co-cultures were challenged (1:2 dilution; once daily for two consecutive days) with fresh conditioned medium collected from capsaicin (1 μM)-stimulated naive DRG neurons ( g ; measured after 48h). Similarly, KCl (50mM)-stimulated naive DRG neurons conditioned medium (1:2 dilution) reduced B16F10-mCherry-OVA apoptosis ( h ; measured after 24h). This effect was blunted when the cells were co-exposed to the RAMP1 blocker CGRP 8-37 ( h ; 2 μg/mL; measured after 24h). CGRP (0.1 μM) challenges also reduced OT-I cytotoxic CD8 + T cells elimination of B16F10-OVA cell ( i ; measured after 24h). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g–h ), or representative FACS plot ( f , i ). N are as follows: a : CD8 + vehicle (n = 4), CD8 + capsaicin (n = 9), CD8 + neuron + capsaicin (n = 9), b : n = 5/groups, c : CD8 (n = 6), CD8 + KCl-induced neurons CM (n = 5), CD8 + KCl-induced neurons CM + CGRP 8-37 (n = 6), CD8 + KCl-induced neurons CM + BoNT/A (n = 6), d : n = 5/groups, e : Ramp1 WT CD8 + vehicle (n = 7), Ramp1 WT CD8 + CGRP (n = 8), Ramp1 −/ − CD8 + vehicle (n = 6), Ramp1 −/ − CD8 + CGRP (n = 6), g : B16F10 (n = 3), B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), h : B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM + CGRP 8–37 (n = 5). Experiments were repeated a minimum of three independent times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferroni ( a–e , g–h ). Source Data

Techniques Used: Isolation, Cell Culture, Ex Vivo, Mouse Assay, Flow Cytometry, Expressing, Whisker Assay, FACS

Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data
Figure Legend Snippet: Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data

Techniques Used: Injection, Mouse Assay, Activation Assay, FACS, Whisker Assay

BoNT/A silencing of B16F10-innervating neurons decreases tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BoNT/A (10–50 pg/μL) for 24h; effects on apoptosis, exhaustion, and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BoNT/A did not affect the survival ( a ) of cultured cytotoxic CD8 + T cells, nor their relative expression of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) and PD-1 + LAG3 + TIM3 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h and subsequently exposed to BoNT/A (1.6-50 pg/μL) or its vehicle for an additional 24h. BoNT/A did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( g–n ) One and three days prior to tumour inoculation ( defined as prophylactic ), the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day after the last injection, orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. In another group of mice, BoNT/A was administered (25 pg/μL; i.d.) one and three days after the tumour reached a volume of ~200mm3 (defined as therapeutic ). The effect of neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. Nineteen days post tumour inoculation, we found that the tumour volume ( g , h ) and weight ( i ) were reduced in mice treated with BoNT/A ( Prophylactic group ). In parallel, we found that silencing tumour-innervating neurons increased the proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BoNT/A had no effect on the total number of intratumoral CD8 T cells ( j ) or the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. ( o ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female sensory neuron-intact or ablated mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic YUMMER1.7 cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. The effects of nociceptor neuron ablation on tumour size and volume were measured. Thirteen days post tumour inoculation, we found that the tumour growth was lower in mice treated with BoNT/A or in sensory neuron-ablated mice. BoNT/A had no additive effects when administered to sensory neuron-ablated mice. ( p ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic B16F10-mCherry-OVA cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. On days 7, 10, 13 and 16 post tumour inoculation, the mice were exposed to αPD-L1 (6 mg/kg, i.p.) or its isotype control. Eighteen days post tumour inoculation, we found that neuron silencing using BoNT/A potentiated αPD-L1-mediated tumour reduction. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f; h–n ) or as mean ± S.E.M ( g , o , p ). N are as follows: a-e : n = 5/groups, f : n = 3/groups, g–i : vehicle (n = 12), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 10), j : vehicle (n = 11), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), k–n : vehicle (n = 10), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), o : intact + vehicle (n = 9), ablated + vehicle (n = 8), intact + BoNT/A (n = 10), ablated + BoNT/A (n = 8), p : vehicle (n = 7), αPD-L1 (n = 8), αPD-L1 + BoNT/A (n = 7). Experiments were independently repeated two ( a–f , o–p ) or four ( g–n ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–f , h–n ) or two-way ANOVA post-hoc Bonferroni ( g , o , p ). Source Data
Figure Legend Snippet: BoNT/A silencing of B16F10-innervating neurons decreases tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BoNT/A (10–50 pg/μL) for 24h; effects on apoptosis, exhaustion, and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BoNT/A did not affect the survival ( a ) of cultured cytotoxic CD8 + T cells, nor their relative expression of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) and PD-1 + LAG3 + TIM3 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h and subsequently exposed to BoNT/A (1.6-50 pg/μL) or its vehicle for an additional 24h. BoNT/A did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( g–n ) One and three days prior to tumour inoculation ( defined as prophylactic ), the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day after the last injection, orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. In another group of mice, BoNT/A was administered (25 pg/μL; i.d.) one and three days after the tumour reached a volume of ~200mm3 (defined as therapeutic ). The effect of neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. Nineteen days post tumour inoculation, we found that the tumour volume ( g , h ) and weight ( i ) were reduced in mice treated with BoNT/A ( Prophylactic group ). In parallel, we found that silencing tumour-innervating neurons increased the proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BoNT/A had no effect on the total number of intratumoral CD8 T cells ( j ) or the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. ( o ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female sensory neuron-intact or ablated mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic YUMMER1.7 cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. The effects of nociceptor neuron ablation on tumour size and volume were measured. Thirteen days post tumour inoculation, we found that the tumour growth was lower in mice treated with BoNT/A or in sensory neuron-ablated mice. BoNT/A had no additive effects when administered to sensory neuron-ablated mice. ( p ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic B16F10-mCherry-OVA cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. On days 7, 10, 13 and 16 post tumour inoculation, the mice were exposed to αPD-L1 (6 mg/kg, i.p.) or its isotype control. Eighteen days post tumour inoculation, we found that neuron silencing using BoNT/A potentiated αPD-L1-mediated tumour reduction. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f; h–n ) or as mean ± S.E.M ( g , o , p ). N are as follows: a-e : n = 5/groups, f : n = 3/groups, g–i : vehicle (n = 12), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 10), j : vehicle (n = 11), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), k–n : vehicle (n = 10), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), o : intact + vehicle (n = 9), ablated + vehicle (n = 8), intact + BoNT/A (n = 10), ablated + BoNT/A (n = 8), p : vehicle (n = 7), αPD-L1 (n = 8), αPD-L1 + BoNT/A (n = 7). Experiments were independently repeated two ( a–f , o–p ) or four ( g–n ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–f , h–n ) or two-way ANOVA post-hoc Bonferroni ( g , o , p ). Source Data

Techniques Used: Isolation, Mouse Assay, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Expressing, Fluorescence, Injection, Whisker Assay

Cancer-secreted SLPI drives the release of CGRP by nociceptor neurons. a – c , Naive DRG neurons ( Trpv1 cre ::-CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA cells and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48 h, the cells were collected, FACS purified and RNA sequenced. a , Hierarchical clustering of sorted neuron molecular profiles depicts distinct groups of transcripts enriched in each group. b , DEGs were calculated, and Slpi was found to be overexpressed in cancer cells when co-cultured with OVA-specific cytotoxic CD8 + T cells, DRG neurons or both populations. c , SLPI is secreted by B16F10-mCherry-OVA cells when co-cultured (24 h or 48 h) with naive DRG neurons and OVA-specific cytotoxic CD8 + T cells, with a maximal effect after 48 h. d – f , Using calcium microscopy, we found that SLPI (10 pg ml −1 –10 ng ml −1 ) activated around 20% of cultured naive DRG neurons ( d , e ). Activation of cultured neurons (3 h) with SLPI also leads to significant release of CGRP ( f ). Data are shown as a heat map showing normalized gene expression (log 2 (1 + TPM) − mean ( a ), as box-and-whisters plots (as defined in Fig. 1b,c ) ( b ) or as mean ± s.e.m. ( c – f ). n as follows: a , b : n = 2–4 per groups; c : n = 3 for all groups except CD8 + T cells ( n = 8); d : n = 17; e : n = 8 per group; f : 0 ng ml −1 ( n = 4), 0.1 ng ml −1 ( n = 5), 1 ng ml −1 ( n = 5), 5 ng ml −1 ( n = 4). Experiments in c – f were independently repeated three times with similar results. The sequencing experiment was not repeated ( a , b ). P values were determined by one-way ANOVA with post-hoc Bonferroni ( b , e , f ) or two-sided unpaired Student’s t -test ( c ). * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source Data
Figure Legend Snippet: Cancer-secreted SLPI drives the release of CGRP by nociceptor neurons. a – c , Naive DRG neurons ( Trpv1 cre ::-CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA cells and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48 h, the cells were collected, FACS purified and RNA sequenced. a , Hierarchical clustering of sorted neuron molecular profiles depicts distinct groups of transcripts enriched in each group. b , DEGs were calculated, and Slpi was found to be overexpressed in cancer cells when co-cultured with OVA-specific cytotoxic CD8 + T cells, DRG neurons or both populations. c , SLPI is secreted by B16F10-mCherry-OVA cells when co-cultured (24 h or 48 h) with naive DRG neurons and OVA-specific cytotoxic CD8 + T cells, with a maximal effect after 48 h. d – f , Using calcium microscopy, we found that SLPI (10 pg ml −1 –10 ng ml −1 ) activated around 20% of cultured naive DRG neurons ( d , e ). Activation of cultured neurons (3 h) with SLPI also leads to significant release of CGRP ( f ). Data are shown as a heat map showing normalized gene expression (log 2 (1 + TPM) − mean ( a ), as box-and-whisters plots (as defined in Fig. 1b,c ) ( b ) or as mean ± s.e.m. ( c – f ). n as follows: a , b : n = 2–4 per groups; c : n = 3 for all groups except CD8 + T cells ( n = 8); d : n = 17; e : n = 8 per group; f : 0 ng ml −1 ( n = 4), 0.1 ng ml −1 ( n = 5), 1 ng ml −1 ( n = 5), 5 ng ml −1 ( n = 4). Experiments in c – f were independently repeated three times with similar results. The sequencing experiment was not repeated ( a , b ). P values were determined by one-way ANOVA with post-hoc Bonferroni ( b , e , f ) or two-sided unpaired Student’s t -test ( c ). * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source Data

Techniques Used: Cell Culture, FACS, Purification, Microscopy, Activation Assay, Expressing, Sequencing

QX-314 silencing of B16F10-innervating neurons reduces tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to QX-314 (50–150 μM) for 24h, effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, QX-314 did not affect the survival of cultured cytotoxic CD8 + T cells ( a ), nor their relative expression of PD-1 + LAG3 + TIM3 + ( b ), IFNγ + ( c ), TNF + ( d ) and IL-2 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h. The cells were then exposed or not to QX-314 (0.1-1%) for an additional 24-72h, and cell count was analysed by bright-field microscopy. QX-314 did not affect B16F10 cells’ survival, as measured by relative cell count changes (at each time point) in comparison to vehicle-exposed cells. ( g–i ) One and three days prior to tumour inoculation, 8-week-old male and female wild-type mice’s right hindpaws or flanks were injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. On the following day, orthotopic B16F10 cells ( g : 5x10 5 cells; i.d.; h–i : 2x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. Starting one day post inoculation, QX-314 (0.3%) or its vehicle was administered (i.d.) once daily in another group of mice. The effects of sensory neuron silencing were tested on neuropeptide release ( g ), as well as mechanical ( h ) and thermal pain hypersensitivity ( i ). First, CGRP levels were increased in B16F10 tumour surrounding skin explant (assessed on day 15) in comparison to control skin; an effect further enhanced by capsaicin (1 μM; 3h) but was absent in skin pre-treated with BoNT/A (25 pg/μL) or QX-314 (0.3%; g ). We also found that B16F10 injection induced mechanical ( h ) and thermal pain hypersensitivities ( i ) fourteen days post tumour inoculation. These effects were stopped by sensory neuron silencing with QX-314 or BoNT/A ( h–i ). ( j ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, QX-314 (0.3%; i.d.; 5 sites) was injected once daily around the tumour. The effect of nociceptor neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. We found that silencing tumour-innervating neurons increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( k–r ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), In other groups of mice, QX-314 daily injection started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). As measured seventeen days post tumour inoculation, silencing tumour innervation also decreased tumour volume ( k , l ) and weight ( m ), as well as the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. QX-314 treatment also increased the total number of intratumoral CD8 + T cells ( o ), as well as relative proportion of IFNγ + ( p ), TNF + ( q ), and IL-2 + ( r ) CD8 + T cells. ( s–t ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into mice treated with QX-314 (0.3%; i.d.) 2-3 days prior to being injected into vehicle-exposed mice. Mice from each group with similar tumour size (~100mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~61%) in nociceptor silenced mice than was observed in isotype vehicle-exposed mice (~49%; s-t ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g , l–r ), as mean ± S.E.M ( f , h , i , k , s , t ), or as Mantel–Cox regression analysis ( j ). N are as follows: a : n = 4/groups, b–e : n = 5/groups, f : n = 3/groups, g : naïve (n = 4), vehicle (n = 7), B16F10+vehicle (n = 5), B16F10+BoNT/A (n = 5), B16F10+QX-314 (n = 5), h–i : n = 6/groups, j : vehicle (n = 89), QX-314 (n = 12), k : vehicle (n = 21), QX-314 prophylactic (n = 21), QX-314 therapeutic (n = 17), l : vehicle (n = 26), QX-314 therapeutic (n = 26), QX-314 prophylactic (n = 28), m : vehicle (n = 25), QX-314 therapeutic (n = 22), QX-314 prophylactic (n = 25), n : vehicle (n = 31), QX-314 therapeutic (n = 29), QX-314 prophylactic (n = 28), o : n = 30/groups, p–r : vehicle (n = 24), QX-314 therapeutic (n = 23), QX-314 prophylactic (n = 25), s : vehicle (n = 9), QX-314 (n = 13), t : vehicle + αPD-L1 (n = 18), QX-314 + αPLD1 (n = 13). Experiments were independently repeated two ( a–i , s–t ) or four ( j–r ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–g , l–r ), two-sided unpaired Student’s t-test ( h–i ), Mantel–Cox regression ( j ), or two-way ANOVA posthoc Bonferroni ( k , s–t ). Source Data
Figure Legend Snippet: QX-314 silencing of B16F10-innervating neurons reduces tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to QX-314 (50–150 μM) for 24h, effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, QX-314 did not affect the survival of cultured cytotoxic CD8 + T cells ( a ), nor their relative expression of PD-1 + LAG3 + TIM3 + ( b ), IFNγ + ( c ), TNF + ( d ) and IL-2 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h. The cells were then exposed or not to QX-314 (0.1-1%) for an additional 24-72h, and cell count was analysed by bright-field microscopy. QX-314 did not affect B16F10 cells’ survival, as measured by relative cell count changes (at each time point) in comparison to vehicle-exposed cells. ( g–i ) One and three days prior to tumour inoculation, 8-week-old male and female wild-type mice’s right hindpaws or flanks were injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. On the following day, orthotopic B16F10 cells ( g : 5x10 5 cells; i.d.; h–i : 2x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. Starting one day post inoculation, QX-314 (0.3%) or its vehicle was administered (i.d.) once daily in another group of mice. The effects of sensory neuron silencing were tested on neuropeptide release ( g ), as well as mechanical ( h ) and thermal pain hypersensitivity ( i ). First, CGRP levels were increased in B16F10 tumour surrounding skin explant (assessed on day 15) in comparison to control skin; an effect further enhanced by capsaicin (1 μM; 3h) but was absent in skin pre-treated with BoNT/A (25 pg/μL) or QX-314 (0.3%; g ). We also found that B16F10 injection induced mechanical ( h ) and thermal pain hypersensitivities ( i ) fourteen days post tumour inoculation. These effects were stopped by sensory neuron silencing with QX-314 or BoNT/A ( h–i ). ( j ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, QX-314 (0.3%; i.d.; 5 sites) was injected once daily around the tumour. The effect of nociceptor neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. We found that silencing tumour-innervating neurons increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( k–r ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), In other groups of mice, QX-314 daily injection started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). As measured seventeen days post tumour inoculation, silencing tumour innervation also decreased tumour volume ( k , l ) and weight ( m ), as well as the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. QX-314 treatment also increased the total number of intratumoral CD8 + T cells ( o ), as well as relative proportion of IFNγ + ( p ), TNF + ( q ), and IL-2 + ( r ) CD8 + T cells. ( s–t ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into mice treated with QX-314 (0.3%; i.d.) 2-3 days prior to being injected into vehicle-exposed mice. Mice from each group with similar tumour size (~100mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~61%) in nociceptor silenced mice than was observed in isotype vehicle-exposed mice (~49%; s-t ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g , l–r ), as mean ± S.E.M ( f , h , i , k , s , t ), or as Mantel–Cox regression analysis ( j ). N are as follows: a : n = 4/groups, b–e : n = 5/groups, f : n = 3/groups, g : naïve (n = 4), vehicle (n = 7), B16F10+vehicle (n = 5), B16F10+BoNT/A (n = 5), B16F10+QX-314 (n = 5), h–i : n = 6/groups, j : vehicle (n = 89), QX-314 (n = 12), k : vehicle (n = 21), QX-314 prophylactic (n = 21), QX-314 therapeutic (n = 17), l : vehicle (n = 26), QX-314 therapeutic (n = 26), QX-314 prophylactic (n = 28), m : vehicle (n = 25), QX-314 therapeutic (n = 22), QX-314 prophylactic (n = 25), n : vehicle (n = 31), QX-314 therapeutic (n = 29), QX-314 prophylactic (n = 28), o : n = 30/groups, p–r : vehicle (n = 24), QX-314 therapeutic (n = 23), QX-314 prophylactic (n = 25), s : vehicle (n = 9), QX-314 (n = 13), t : vehicle + αPD-L1 (n = 18), QX-314 + αPLD1 (n = 13). Experiments were independently repeated two ( a–i , s–t ) or four ( j–r ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–g , l–r ), two-sided unpaired Student’s t-test ( h–i ), Mantel–Cox regression ( j ), or two-way ANOVA posthoc Bonferroni ( k , s–t ). Source Data

Techniques Used: Isolation, Mouse Assay, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Expressing, Cell Counting, Microscopy, Injection, Whisker Assay

Melanoma-innervating nociceptors attenuate cancer immunosurveillance. Melanoma growth sets off anti-tumour immune responses, including the infiltration of effector CD8 T cells and their subsequent release of cytotoxic cytokines (i.e., IFNγ, TNF, Granzyme B). By acting on tissue-resident nociceptor neurons, melanoma-produced SLPI promotes pain hypersensitivity, tweaks the neurons’ transcriptome, and drives neurite outgrowth. These effects culminate in dense melanoma innervation by nociceptors and abundant release of immunomodulatory neuropeptides. CGRP, one such peptide, acts on tumour-infiltrating effector CD8 + T cells that express the CGRP receptor RAMP1, increasing their expression of immune checkpoint receptors (i.e., PD-1, LAG3, TIM3). Therefore, along with the immunosuppressive environment present in the tumour, nociceptor-produced CGRP leads to the functional exhaustion of tumour-infiltrating CD8 + T cells, which opens the door to unchecked proliferation of melanoma cells. Genetically ablating (i.e., TRPV1 lineage) or pharmacologically silencing (i.e., QX-314, BoNT/A) nociceptor neurons as well as blocking the action of CGRP on RAMP1 using a selective antagonist (i.e., BIBN4096) prevents effector CD8 + T cells from undergoing exhaustion. Therefore, targeting melanoma-innervating nociceptor neurons constitutes a novel strategy to safeguard host anti-tumour immunity and stop tumour growth.
Figure Legend Snippet: Melanoma-innervating nociceptors attenuate cancer immunosurveillance. Melanoma growth sets off anti-tumour immune responses, including the infiltration of effector CD8 T cells and their subsequent release of cytotoxic cytokines (i.e., IFNγ, TNF, Granzyme B). By acting on tissue-resident nociceptor neurons, melanoma-produced SLPI promotes pain hypersensitivity, tweaks the neurons’ transcriptome, and drives neurite outgrowth. These effects culminate in dense melanoma innervation by nociceptors and abundant release of immunomodulatory neuropeptides. CGRP, one such peptide, acts on tumour-infiltrating effector CD8 + T cells that express the CGRP receptor RAMP1, increasing their expression of immune checkpoint receptors (i.e., PD-1, LAG3, TIM3). Therefore, along with the immunosuppressive environment present in the tumour, nociceptor-produced CGRP leads to the functional exhaustion of tumour-infiltrating CD8 + T cells, which opens the door to unchecked proliferation of melanoma cells. Genetically ablating (i.e., TRPV1 lineage) or pharmacologically silencing (i.e., QX-314, BoNT/A) nociceptor neurons as well as blocking the action of CGRP on RAMP1 using a selective antagonist (i.e., BIBN4096) prevents effector CD8 + T cells from undergoing exhaustion. Therefore, targeting melanoma-innervating nociceptor neurons constitutes a novel strategy to safeguard host anti-tumour immunity and stop tumour growth.

Techniques Used: Produced, Expressing, Functional Assay, Blocking Assay

Genetic ablation of nociceptors safeguards anti-tumour immunity. a , Orthotopic B16F10-mCherry-OVA cells (2 × 10 5 cells, i.d.) were injected into the left hindpaw of wild-type mice. As measured on day 13 after tumour inoculation, intratumoral CD8 + T cell exhaustion positively correlated with thermal hypersensitivity ( R 2 = 0.55, P ≤ 0.0001). The thermal pain hypersensitivity represents the withdrawal latency ratio of the ipsilateral paw (tumour-inoculated) to the contralateral paw. b , Orthotopic B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) were inoculated into the flank of eight-week-old male and female mice with sensory neurons intact ( Trpv1 WT ::DTA fl/WT ) or ablated ( Trpv1 cre ::DTA fl/WT ). The median length of survival was increased by around 250% in nociceptor-ablated mice (measured until 22 days after inoculation). c – f , Sixteen days after tumour inoculation, sensory-neuron-ablated mice have reduced tumour growth ( c ) and increased tumour infiltration of IFNγ + CD8 + T cells ( d ), and the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells is decreased ( e ). This reduction in B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) tumour volume was absent in nociceptor-ablated mice whose CD8 + T cells were systemically depleted ( f ; assessed until day 14; anti-CD8, 200 μg per mouse, i.p., every 3 days). g , h , To chemically deplete their nociceptor neurons, Rag1 −/ − mice were injected with RTX. Twenty-eight days later, the mice were inoculated with B16F10-mCherry-OVA (5 × 10 5 cells, i.d.). RTX-injected mice that were adoptively transferred with naive OVA-specific CD8 + T cells (i.v., 1 × 10 6 cells, when tumour reached around 500 mm 3 ) showed reduced tumour growth ( g ; assessed until day 19) and exhaustion ( h ) compared to vehicle-exposed Rag1 −/ − mice. Data are shown as a linear regression analysis ± s.e. ( a ), as a Mantel–Cox regression ( b ), as mean ± s.e.m. ( c , f , g ) or as box-and-whisker plots (as defined in Fig. 1b,c ), for which individual data points are given ( d , e , h ). n as follows: a : n = 60; b : intact ( n = 62), ablated ( n = 73); c : intact ( n = 20), ablated ( n = 25); d : intact ( n = 24), ablated ( n = 23); e : intact ( n = 23), ablated ( n = 26); f : intact + anti-CD8 ( n = 10), ablated + anti-CD8 ( n = 8); g : vehicle ( n = 12), RTX ( n = 10); h : vehicle ( n = 11), RTX ( n = 10). Experiments were independently repeated two ( a , f – h ) or six ( b – e ) times with similar results. P values were determined by simple linear regression analysis ( a ), Mantel–Cox regression ( b ), two-way ANOVA with post-hoc Bonferroni ( c , f , g ) or two-sided unpaired Student’s t -test ( d , e , h ). Source Data
Figure Legend Snippet: Genetic ablation of nociceptors safeguards anti-tumour immunity. a , Orthotopic B16F10-mCherry-OVA cells (2 × 10 5 cells, i.d.) were injected into the left hindpaw of wild-type mice. As measured on day 13 after tumour inoculation, intratumoral CD8 + T cell exhaustion positively correlated with thermal hypersensitivity ( R 2 = 0.55, P ≤ 0.0001). The thermal pain hypersensitivity represents the withdrawal latency ratio of the ipsilateral paw (tumour-inoculated) to the contralateral paw. b , Orthotopic B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) were inoculated into the flank of eight-week-old male and female mice with sensory neurons intact ( Trpv1 WT ::DTA fl/WT ) or ablated ( Trpv1 cre ::DTA fl/WT ). The median length of survival was increased by around 250% in nociceptor-ablated mice (measured until 22 days after inoculation). c – f , Sixteen days after tumour inoculation, sensory-neuron-ablated mice have reduced tumour growth ( c ) and increased tumour infiltration of IFNγ + CD8 + T cells ( d ), and the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells is decreased ( e ). This reduction in B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) tumour volume was absent in nociceptor-ablated mice whose CD8 + T cells were systemically depleted ( f ; assessed until day 14; anti-CD8, 200 μg per mouse, i.p., every 3 days). g , h , To chemically deplete their nociceptor neurons, Rag1 −/ − mice were injected with RTX. Twenty-eight days later, the mice were inoculated with B16F10-mCherry-OVA (5 × 10 5 cells, i.d.). RTX-injected mice that were adoptively transferred with naive OVA-specific CD8 + T cells (i.v., 1 × 10 6 cells, when tumour reached around 500 mm 3 ) showed reduced tumour growth ( g ; assessed until day 19) and exhaustion ( h ) compared to vehicle-exposed Rag1 −/ − mice. Data are shown as a linear regression analysis ± s.e. ( a ), as a Mantel–Cox regression ( b ), as mean ± s.e.m. ( c , f , g ) or as box-and-whisker plots (as defined in Fig. 1b,c ), for which individual data points are given ( d , e , h ). n as follows: a : n = 60; b : intact ( n = 62), ablated ( n = 73); c : intact ( n = 20), ablated ( n = 25); d : intact ( n = 24), ablated ( n = 23); e : intact ( n = 23), ablated ( n = 26); f : intact + anti-CD8 ( n = 10), ablated + anti-CD8 ( n = 8); g : vehicle ( n = 12), RTX ( n = 10); h : vehicle ( n = 11), RTX ( n = 10). Experiments were independently repeated two ( a , f – h ) or six ( b – e ) times with similar results. P values were determined by simple linear regression analysis ( a ), Mantel–Cox regression ( b ), two-way ANOVA with post-hoc Bonferroni ( c , f , g ) or two-sided unpaired Student’s t -test ( d , e , h ). Source Data

Techniques Used: Injection, Mouse Assay, Whisker Assay

4) Product Images from "Nociceptor neurons affect cancer immunosurveillance"

Article Title: Nociceptor neurons affect cancer immunosurveillance

Journal: Nature

doi: 10.1038/s41586-022-05374-w

RAMP1 expression in patient melanoma-infiltrating T cells correlates with worsened survival and poor responsiveness to ICIs. ( a–l ) In silico analysis of Cancer Genome Atlas (TCGA) data linked the survival rate among 459 patients with melanoma with their relative expression levels of various genes of interest (determined by bulk RNA sequencing of tumour biopsy). Kaplan–Meier curves show the patients’ survival after segregation in two groups defined by their low or high expression of a gene of interest. Increased gene expression (labelled as high; red curve) of TUBB3 ( b ), PGP9.5 ( c ), Nav 1.7 ( E ), SLPI ( k ) and RAMP1 ( l ) in biopsy correlate with decreased patient survival (p≤0.05). The mantel–Haenszel hazard ratio and number of patients included in each analysis are shown in the figure ( a–l ). Experimental details were defined in Cancer Genome Atlas (TCGA) 40 . ( m ) In silico analysis of single-cell RNA sequencing of human melanoma-infiltrating T cells revealed that RAMP1 + T cells downregulated Il-2 expression and strongly overexpressed several immune checkpoint receptors ( PD-1 , TIM3 , LAG3 , CTLA4 , CD28 , ICOS , BTLA , CD27 ) in comparison to RAMP1 - T cells. Individual cell data are shown as a log 2 of 1 + (transcript per million / 10). Experimental details and cell clustering were defined in Tirosh et al 42 . N are defined in each panel. ( n–p ) On the basis of the clinical response of patients with melanoma to immune checkpoint blocker, patients were clustered into two groups defined as ICI-responsive or ICI-resistant 41 . In silico analysis of single-cell RNA sequencing of patients’ biopsies revealed that tumour-infiltrating CD8 + T cells from patients who were resistant to ICIs significantly overexpressed RAMP1 (2.0-fold), PD-1 (1.7-fold), LAG3 (1.6-fold), CTLA4 (1.6-fold), and TIM3 (1.7-fold; n–p ). Individual cell data are shown as a log 2 (1+(transcript per million/10). Experimental details and cell clustering were defined in Jerby-Arnon et al 41 . P-values are shown in the figure and determined by two-sided unpaired Student’s t-test. N are defined in each panel ( n–o ). Source Data
Figure Legend Snippet: RAMP1 expression in patient melanoma-infiltrating T cells correlates with worsened survival and poor responsiveness to ICIs. ( a–l ) In silico analysis of Cancer Genome Atlas (TCGA) data linked the survival rate among 459 patients with melanoma with their relative expression levels of various genes of interest (determined by bulk RNA sequencing of tumour biopsy). Kaplan–Meier curves show the patients’ survival after segregation in two groups defined by their low or high expression of a gene of interest. Increased gene expression (labelled as high; red curve) of TUBB3 ( b ), PGP9.5 ( c ), Nav 1.7 ( E ), SLPI ( k ) and RAMP1 ( l ) in biopsy correlate with decreased patient survival (p≤0.05). The mantel–Haenszel hazard ratio and number of patients included in each analysis are shown in the figure ( a–l ). Experimental details were defined in Cancer Genome Atlas (TCGA) 40 . ( m ) In silico analysis of single-cell RNA sequencing of human melanoma-infiltrating T cells revealed that RAMP1 + T cells downregulated Il-2 expression and strongly overexpressed several immune checkpoint receptors ( PD-1 , TIM3 , LAG3 , CTLA4 , CD28 , ICOS , BTLA , CD27 ) in comparison to RAMP1 - T cells. Individual cell data are shown as a log 2 of 1 + (transcript per million / 10). Experimental details and cell clustering were defined in Tirosh et al 42 . N are defined in each panel. ( n–p ) On the basis of the clinical response of patients with melanoma to immune checkpoint blocker, patients were clustered into two groups defined as ICI-responsive or ICI-resistant 41 . In silico analysis of single-cell RNA sequencing of patients’ biopsies revealed that tumour-infiltrating CD8 + T cells from patients who were resistant to ICIs significantly overexpressed RAMP1 (2.0-fold), PD-1 (1.7-fold), LAG3 (1.6-fold), CTLA4 (1.6-fold), and TIM3 (1.7-fold; n–p ). Individual cell data are shown as a log 2 (1+(transcript per million/10). Experimental details and cell clustering were defined in Jerby-Arnon et al 41 . P-values are shown in the figure and determined by two-sided unpaired Student’s t-test. N are defined in each panel ( n–o ). Source Data

Techniques Used: Expressing, In Silico, RNA Sequencing Assay

B16F10-secreted SLPI activates nociceptor neurons. ( a–e ) Naive DRG neurons ( Trpv1 cre ::CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA, and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48h, the cells were collected, FACS purified, and RNA sequenced. DEGs were calculated, and Fgfr1 (fibroblast growth factor receptor 1) was found to be overexpressed in OVA-specific cytotoxic CD8 + T cells when co-cultured with cancer cells and DRG neurons ( a ). Conversely, OVA-specific cytotoxic CD8 + T cells downregulates the expression of the pro-nociceptive factor Hmgb1 (High–mobility group box 1; b ), Braf ( c ) , as well as Fgfr3 ( d ) when co-cultured with B16F10-mCherry-OVA and DRG neurons. Tslp expression level was not affected in any of tested groups ( e ). ( f–i ) Using calcium microscopy, we probed whether SLPI directly activates cultured DRG neurons. We found that SLPI (0.01-10 ng/mL) induces a significant calcium influx in DRG neurons ( f ). SLPI-responsive neurons are mostly small-sized neurons ( g-h ; mean area = 151 μm 2 ) and largely capsaicin-responsive ( i ; ~42%). ( j ) The right hindpaw of naive mice was injected with saline (20 μL) or SLPI (i.d., 1 μg/20 μL), and the mice’s noxious thermal nociceptive threshold was measured (0-6h). The ipsilateral paw injected with SLPI showed thermal hypersensitivity in contrast with the contralateral paw. Saline had no effect on the mice’s thermal sensitivity. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f , h , j ), stacked bar graph on a logarithmic scale ( g ), and Venn Diagram ( i ). N are as follows: a–e : n = 2–4/groups, f : vehicle (n = 28), 10pg/ml (n = 28), 100 pg/ml (n = 132), 1,000 pg/ml (n = 191), 10 ng/ml (n = 260), capsaicin (n = 613), KCl (n = 1,139), g : 0-100 (SLPI=19; KCl=177), 100-200 (SLPI = 45; KCl = 390), 200-300 (SLPI = 16; KCl =216), 300-400 (SLPI=11; KCl = 138), 400-500 (SLPI = 5; KCl = 68), 500-600 (SLPI=2, KCl = 18), 600-700 (SLPI = 0; KCl = 10), 700-800 (SLPI=0; KCl=13), 800+ (SLPI = 0; KCl = 12), h : n = 98, i : KCl + =1139, KCl + Caps + =614, KCl + Caps + SLPI + =261, KCl + Caps - SLPI + =29, j : 0h (n = 9), SLPI at 1h (n = 6), saline at 1h (n = 3), SLPI at 3h (n = 6), saline at 3h (n=3), SLPI at 6h (n = 6), saline at 6h (n = 3). Experiments were independently repeated two ( j ) or three ( f–i ) times with similar results. Sequencing experiment was not repeated ( a–e ). P-values were determined by one-way ANOVA post-hoc Bonferroni ( a–f ); or two-sided unpaired Student’s t-test ( j ). P-values are shown in the figure or indicated by * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001. Source Data
Figure Legend Snippet: B16F10-secreted SLPI activates nociceptor neurons. ( a–e ) Naive DRG neurons ( Trpv1 cre ::CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA, and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48h, the cells were collected, FACS purified, and RNA sequenced. DEGs were calculated, and Fgfr1 (fibroblast growth factor receptor 1) was found to be overexpressed in OVA-specific cytotoxic CD8 + T cells when co-cultured with cancer cells and DRG neurons ( a ). Conversely, OVA-specific cytotoxic CD8 + T cells downregulates the expression of the pro-nociceptive factor Hmgb1 (High–mobility group box 1; b ), Braf ( c ) , as well as Fgfr3 ( d ) when co-cultured with B16F10-mCherry-OVA and DRG neurons. Tslp expression level was not affected in any of tested groups ( e ). ( f–i ) Using calcium microscopy, we probed whether SLPI directly activates cultured DRG neurons. We found that SLPI (0.01-10 ng/mL) induces a significant calcium influx in DRG neurons ( f ). SLPI-responsive neurons are mostly small-sized neurons ( g-h ; mean area = 151 μm 2 ) and largely capsaicin-responsive ( i ; ~42%). ( j ) The right hindpaw of naive mice was injected with saline (20 μL) or SLPI (i.d., 1 μg/20 μL), and the mice’s noxious thermal nociceptive threshold was measured (0-6h). The ipsilateral paw injected with SLPI showed thermal hypersensitivity in contrast with the contralateral paw. Saline had no effect on the mice’s thermal sensitivity. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f , h , j ), stacked bar graph on a logarithmic scale ( g ), and Venn Diagram ( i ). N are as follows: a–e : n = 2–4/groups, f : vehicle (n = 28), 10pg/ml (n = 28), 100 pg/ml (n = 132), 1,000 pg/ml (n = 191), 10 ng/ml (n = 260), capsaicin (n = 613), KCl (n = 1,139), g : 0-100 (SLPI=19; KCl=177), 100-200 (SLPI = 45; KCl = 390), 200-300 (SLPI = 16; KCl =216), 300-400 (SLPI=11; KCl = 138), 400-500 (SLPI = 5; KCl = 68), 500-600 (SLPI=2, KCl = 18), 600-700 (SLPI = 0; KCl = 10), 700-800 (SLPI=0; KCl=13), 800+ (SLPI = 0; KCl = 12), h : n = 98, i : KCl + =1139, KCl + Caps + =614, KCl + Caps + SLPI + =261, KCl + Caps - SLPI + =29, j : 0h (n = 9), SLPI at 1h (n = 6), saline at 1h (n = 3), SLPI at 3h (n = 6), saline at 3h (n=3), SLPI at 6h (n = 6), saline at 6h (n = 3). Experiments were independently repeated two ( j ) or three ( f–i ) times with similar results. Sequencing experiment was not repeated ( a–e ). P-values were determined by one-way ANOVA post-hoc Bonferroni ( a–f ); or two-sided unpaired Student’s t-test ( j ). P-values are shown in the figure or indicated by * for p ≤ 0.05; ** for p ≤ 0.01; *** for p ≤ 0.001. Source Data

Techniques Used: Cell Culture, FACS, Purification, Expressing, Microscopy, Mouse Assay, Injection, Whisker Assay, Sequencing

CGRP attenuates the anti-tumour immunity of RAMP1 + CD8 + T cells. a – c , Splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. Eight-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5 × 10 6 cells) with activated Ramp1 −/ − or Ramp1 WT CD8 + T cells or a 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week after transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.). Ten days after B16F10 inoculation, we observed greater tumour growth ( a ) in Ramp1 WT transplanted mice. Intratumoral Ramp1 −/ − (CD45.2 + ) and Ramp1 WT (CD45.1 + ) CD8 + T cells were FACS purified, immunophenotyped ( b ) and RNA sequenced ( c ). Ramp1 −/ − CD8 + T cells showed a lower proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( b ) as well as reduced transcript expression of exhaustion markers ( c ). d , In silico analysis of The Cancer Genome Atlas (TCGA) data 40 was used to correlate the survival rate of 459 patients with melanoma with the relative RAMP1 expression (primary biopsy bulk RNA sequencing). In comparison to patients with low RAMP1 expression, higher RAMP1 levels correlate with decreased patient survival. e , In silico analysis of single-cell RNA sequencing of human melanoma 41 reveals that intratumoral RAMP1 -expressing CD8 + T cells strongly overexpress several immune checkpoint receptors ( PD-1 (also known as PDCD1 ) TIM3 , LAG3 , CTLA4 ) in comparison to Ramp1 -negative CD8 + T cells. Data are shown as mean ± s.e.m. ( a ), slopegraph ( b ), as a heat map showing normalized gene expression (log 10 (10 3 × TPM) ( c ), as a Mantel–Cox regression ( d ) or as a violin plot ( e ). n as follows: a–c : n = 5 per group; d : high ( n = 45), low ( n = 68); e : RAMP1 − CD8 ( n = 1,732), RAMP1 + CD8 ( n = 25). Experiments were independently repeated two ( a , b ) times with similar results. The sequencing experiment was not repeated ( c ). P values were determined by two-way ANOVA with post-hoc Bonferroni ( a ), two-sided unpaired Student’s t -test ( b ) or Mantel–Cox regression ( d ). Source Data
Figure Legend Snippet: CGRP attenuates the anti-tumour immunity of RAMP1 + CD8 + T cells. a – c , Splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated (CD3 and CD28 + IL-2) in vitro. Eight-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5 × 10 6 cells) with activated Ramp1 −/ − or Ramp1 WT CD8 + T cells or a 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week after transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.). Ten days after B16F10 inoculation, we observed greater tumour growth ( a ) in Ramp1 WT transplanted mice. Intratumoral Ramp1 −/ − (CD45.2 + ) and Ramp1 WT (CD45.1 + ) CD8 + T cells were FACS purified, immunophenotyped ( b ) and RNA sequenced ( c ). Ramp1 −/ − CD8 + T cells showed a lower proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( b ) as well as reduced transcript expression of exhaustion markers ( c ). d , In silico analysis of The Cancer Genome Atlas (TCGA) data 40 was used to correlate the survival rate of 459 patients with melanoma with the relative RAMP1 expression (primary biopsy bulk RNA sequencing). In comparison to patients with low RAMP1 expression, higher RAMP1 levels correlate with decreased patient survival. e , In silico analysis of single-cell RNA sequencing of human melanoma 41 reveals that intratumoral RAMP1 -expressing CD8 + T cells strongly overexpress several immune checkpoint receptors ( PD-1 (also known as PDCD1 ) TIM3 , LAG3 , CTLA4 ) in comparison to Ramp1 -negative CD8 + T cells. Data are shown as mean ± s.e.m. ( a ), slopegraph ( b ), as a heat map showing normalized gene expression (log 10 (10 3 × TPM) ( c ), as a Mantel–Cox regression ( d ) or as a violin plot ( e ). n as follows: a–c : n = 5 per group; d : high ( n = 45), low ( n = 68); e : RAMP1 − CD8 ( n = 1,732), RAMP1 + CD8 ( n = 25). Experiments were independently repeated two ( a , b ) times with similar results. The sequencing experiment was not repeated ( c ). P values were determined by two-way ANOVA with post-hoc Bonferroni ( a ), two-sided unpaired Student’s t -test ( b ) or Mantel–Cox regression ( d ). Source Data

Techniques Used: FACS, Purification, Mouse Assay, In Vitro, Transplantation Assay, Expressing, In Silico, RNA Sequencing Assay, Sequencing

The CGRP–RAMP1 axis promotes intratumoral CD8 + T cell exhaustion. ( a–e ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Nav 1.8 WT ::DTA fl/WT ) and ablated mice ( Nav 1.8 cre ::DTA fl/WT ). As measured fifteen days post inoculation, Na V 1.8 + nociceptor-ablated mice had lower proportion of PD-1 + LAG3 + TIM3 + ( a ) CD8 + T cells, but increased levels of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) CD8 + T cells. B16F10-mCherry-OVA (5x10 5 cells; i.d.)-tumour surrounding skin was also collected and capsaicin-induced CGRP release assessed by ELISA. Intratumoral CGRP levels positively correlate with the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ). ( f ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old female sensory neuron intact or ablated mice. In nociceptor-ablated mice, recombinant CGRP injection (100nM, i.d., once daily) rescues intratumoral CD8 + T cells exhaustion (PD-1 + LAG3 + TIM3 + ). ( g ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. We found that blocking the action of CGRP on RAMP1-expressing cells, increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( h–m ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. In another group of mice, BIBN4096 (5 mg/kg, i.p., every other day) injections were started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). The effect of nociceptor neuron-silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. As assessed thirteen days post tumour inoculation, BIBN4096 decreased tumour volume ( h ) and weight ( i ) but increased the relative proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BIBN4096 had no effect on the number of intratumoral CD8 + T cells ( j ). When administered as therapeutic, BIBN4096 reduced tumour volume ( h ) and weight ( i ) but had limited effect on CD8 + T cells’ cytotoxicity ( j–m ). ( n ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron-intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Starting one day post inoculation, BIBN4096 (5 mg/kg) or its vehicle was administered (i.p.) on alternate days; effects on tumour volume were measured. Fourteen days post tumour inoculation, we found that tumour growth was reduced in sensory neuron-ablated mice and in BIBN4096-treated mice. BIBN4096 had no additive effect when given to sensory neuron-ablated mice. ( o–s ) Splenocytes-isolated CD8 + T cells from naïve C57BL6J mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BIBN4096 (1–4 μM) for 24h; effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BIBN4096 did not affect the survival ( o ) of cultured cytotoxic CD8 + T cells, nor their relative expression of PD-1 + LAG3 + TIM3 + ( p ), IFNγ + ( q ), TNF + ( r ), and IL-2 + ( s ). ( t ) B16F10 cells (1x10 5 cells) were cultured for 24h. The cells were then exposed (or not) to BIBN4096 (1-8 μM) for an additional 24h; effects on apoptosis were measured by flow cytometry. BIBN4096 did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( u-w ) Naive splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated ( CD3 and CD28 + IL-2 ) in vitro . 8-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5x10 6 cells) with either Ramp1 −/ − or Ramp1 WT CD8 + T cells or 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week post transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5x10 5 cells; i.d.). Ten days post tumour inoculation, we retrieved a similar number of tumours draining lymph node CD8 + T cells across the three tested groups ( u ). The relative proportion of intra-tumour PD-1 + LAG3 + TIM3 + CD8 + T cells was lower in Ramp1 −/ − transplanted mice ( v ). Within the same tumour, intratumoral CD8 + T cell exhaustion was immunophenotyped by flow cytometry ( representative panel shown in w ) and showed that the relative proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells was ~3-fold lower in Ramp1 −/ − CD8 + T cells than in Ramp1 WT CD8 + T cells ( w ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–d , f , h–m , o–v ), linear regression ( e ), Mantel–Cox regression ( g ), mean ± S.E.M ( n ), or as FACS plot ( w ). N are as follows a–e : Nav 1.8 WT ::DTA fl/WT (n = 18), Nav 1.8 cre ::DTA fl/WT (n = 10), f : Trpv1 WT ::DTA fl/WT (n = 16), Trpv1 cre ::DTA fl/WT +CGRP (n = 11), g : vehicle (n = 89), BIBN4096 (n = 16), h–m : Vehicle (n = 13), BIBN4096 therapeutic (n = 18), BIBN4096 prophylactic (n = 16), n : Trpv1 WT ::DTA fl/WT + vehicle (n = 8), Trpv1 WT ::DTA fl/WT + BIBN4096 (n = 9), Trpv1 cre ::DTA fl/WT + vehicle (n = 7), Trpv1 cre ::DTA fl/WT + BIBN4096 (n = 7), o : vehicle (n = 5), 1µM BIBN4096 (n = 3), 4 µM BIBN4096 (n = 5), p–s : n = 5/groups, t : n = 4/groups, u–w : n = 5/groups. Experiments were independently repeated twice ( a–f , n–w ) or four ( g–m ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( a–d , f , v ), simple linear regression analysis ( e ), Mantel–Cox regression ( g ), by one-way ANOVA posthoc Bonferroni ( h–m; o–u ), or two-way ANOVA post-hoc Bonferroni ( n ). Source Data
Figure Legend Snippet: The CGRP–RAMP1 axis promotes intratumoral CD8 + T cell exhaustion. ( a–e ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Nav 1.8 WT ::DTA fl/WT ) and ablated mice ( Nav 1.8 cre ::DTA fl/WT ). As measured fifteen days post inoculation, Na V 1.8 + nociceptor-ablated mice had lower proportion of PD-1 + LAG3 + TIM3 + ( a ) CD8 + T cells, but increased levels of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) CD8 + T cells. B16F10-mCherry-OVA (5x10 5 cells; i.d.)-tumour surrounding skin was also collected and capsaicin-induced CGRP release assessed by ELISA. Intratumoral CGRP levels positively correlate with the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ). ( f ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old female sensory neuron intact or ablated mice. In nociceptor-ablated mice, recombinant CGRP injection (100nM, i.d., once daily) rescues intratumoral CD8 + T cells exhaustion (PD-1 + LAG3 + TIM3 + ). ( g ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. We found that blocking the action of CGRP on RAMP1-expressing cells, increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( h–m ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), the RAMP1 antagonist BIBN4096 (5 mg/kg, i.p., every other day) was administered systemically. In another group of mice, BIBN4096 (5 mg/kg, i.p., every other day) injections were started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). The effect of nociceptor neuron-silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. As assessed thirteen days post tumour inoculation, BIBN4096 decreased tumour volume ( h ) and weight ( i ) but increased the relative proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BIBN4096 had no effect on the number of intratumoral CD8 + T cells ( j ). When administered as therapeutic, BIBN4096 reduced tumour volume ( h ) and weight ( i ) but had limited effect on CD8 + T cells’ cytotoxicity ( j–m ). ( n ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron-intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Starting one day post inoculation, BIBN4096 (5 mg/kg) or its vehicle was administered (i.p.) on alternate days; effects on tumour volume were measured. Fourteen days post tumour inoculation, we found that tumour growth was reduced in sensory neuron-ablated mice and in BIBN4096-treated mice. BIBN4096 had no additive effect when given to sensory neuron-ablated mice. ( o–s ) Splenocytes-isolated CD8 + T cells from naïve C57BL6J mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BIBN4096 (1–4 μM) for 24h; effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BIBN4096 did not affect the survival ( o ) of cultured cytotoxic CD8 + T cells, nor their relative expression of PD-1 + LAG3 + TIM3 + ( p ), IFNγ + ( q ), TNF + ( r ), and IL-2 + ( s ). ( t ) B16F10 cells (1x10 5 cells) were cultured for 24h. The cells were then exposed (or not) to BIBN4096 (1-8 μM) for an additional 24h; effects on apoptosis were measured by flow cytometry. BIBN4096 did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( u-w ) Naive splenocyte CD8 + T cells were FACS purified from Ramp1 WT (CD45.1 + ) or Ramp1 −/ − (CD45.2 + ) mice, expanded and stimulated ( CD3 and CD28 + IL-2 ) in vitro . 8-week-old female Rag1 −/ − mice were transplanted (i.v., 2.5x10 6 cells) with either Ramp1 −/ − or Ramp1 WT CD8 + T cells or 1:1 mix of Ramp1 −/ − and Ramp1 WT CD8 + T cells. One week post transplantation, the mice were inoculated with B16F10-mCherry-OVA cells (5x10 5 cells; i.d.). Ten days post tumour inoculation, we retrieved a similar number of tumours draining lymph node CD8 + T cells across the three tested groups ( u ). The relative proportion of intra-tumour PD-1 + LAG3 + TIM3 + CD8 + T cells was lower in Ramp1 −/ − transplanted mice ( v ). Within the same tumour, intratumoral CD8 + T cell exhaustion was immunophenotyped by flow cytometry ( representative panel shown in w ) and showed that the relative proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells was ~3-fold lower in Ramp1 −/ − CD8 + T cells than in Ramp1 WT CD8 + T cells ( w ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–d , f , h–m , o–v ), linear regression ( e ), Mantel–Cox regression ( g ), mean ± S.E.M ( n ), or as FACS plot ( w ). N are as follows a–e : Nav 1.8 WT ::DTA fl/WT (n = 18), Nav 1.8 cre ::DTA fl/WT (n = 10), f : Trpv1 WT ::DTA fl/WT (n = 16), Trpv1 cre ::DTA fl/WT +CGRP (n = 11), g : vehicle (n = 89), BIBN4096 (n = 16), h–m : Vehicle (n = 13), BIBN4096 therapeutic (n = 18), BIBN4096 prophylactic (n = 16), n : Trpv1 WT ::DTA fl/WT + vehicle (n = 8), Trpv1 WT ::DTA fl/WT + BIBN4096 (n = 9), Trpv1 cre ::DTA fl/WT + vehicle (n = 7), Trpv1 cre ::DTA fl/WT + BIBN4096 (n = 7), o : vehicle (n = 5), 1µM BIBN4096 (n = 3), 4 µM BIBN4096 (n = 5), p–s : n = 5/groups, t : n = 4/groups, u–w : n = 5/groups. Experiments were independently repeated twice ( a–f , n–w ) or four ( g–m ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( a–d , f , v ), simple linear regression analysis ( e ), Mantel–Cox regression ( g ), by one-way ANOVA posthoc Bonferroni ( h–m; o–u ), or two-way ANOVA post-hoc Bonferroni ( n ). Source Data

Techniques Used: Injection, Mouse Assay, Enzyme-linked Immunosorbent Assay, Recombinant, Blocking Assay, Expressing, Isolation, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Fluorescence, FACS, Purification, In Vitro, Transplantation Assay, Whisker Assay

CGRP modulates the activation of CD8 + T cells. a , b , Splenocyte CD8 + T cells from wild-type ( a ), Ramp1 −/ − ( a ) or naive OT-I ( b ) mice were cultured under T c1 -stimulating conditions (ex-vivo-activated by CD3 and CD28, IL-12 and anti-IL4) for 48 h to generate cytotoxic CD8 + T cells. In the presence of IL-2 (10 ng ml −1 ), the cells were stimulated with CGRP (100 nM; challenged once every two days) for 96 h. Wild-type cytotoxic CD8 + T cells showed an increased proportion of PD-1 + LAG3 + TIM3 + cells; this effect was absent when treating cytotoxic CD8 + T cells that were collected from Ramp1 −/ − mice ( a ). In co-culture (48 h), CGRP (100 nM; once daily) also reduced the ability of OT-I cytotoxic CD8 + T cells (4 × 10 5 cells) to eliminate B16F10-mCherry-OVA cancer cells ( b ). c , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old female mice with sensory neurons intact or ablated. In nociceptor-ablated mice, peritumoral recombinant CGRP injection (100 nM, i.d., once daily) rescues B16F10 growth (assessed until day 12). d , e , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old male and female mice. Starting one day after inoculation (defined as prophylactic), the RAMP1 antagonist BIBN4096 (5 mg kg −1 ) was administered systemically (i.p.) once every two days. In another group of mice, BIBN4096 (5 mg kg −1 , i.p., every two days) injections were started once the tumour reached a volume of around 200 mm 3 (defined as therapeutic). Prophylactic or therapeutic BIBN4096 treatments decreased tumour growth ( d ) and reduced the proportion of intratumoral PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ; assessed until day 13). Data are shown as box-and-whisker plots (as defined in Fig. 1b, c ), for which individual data points are given ( a , b , e ), or as mean ± s.e.m. ( c , d ). n as follows: a : Ramp1 WT CD8 + vehicle ( n = 9), Ramp1 WT CD8 + CGRP ( n = 10), Ramp1 −/ − CD8 + vehicle ( n = 10), Ramp1 −/ − CD8 + CGRP ( n = 9); b : n = 4 per group; c : intact + vehicle ( n = 15), ablated + CGRP ( n = 11); d : vehicle ( n = 13), BIBN prophylactic ( n = 16), BIBN therapeutic ( n = 18); e : vehicle ( n = 10), BIBN prophylactic ( n = 13), BIBN therapeutic ( n = 16). Experiments were independently repeated three times with similar results. P values were determined by one-way ANOVA with post-hoc Bonferroni ( a , e ), two-sided unpaired Student’s t -test ( b ) or two-way ANOVA with post-hoc Bonferroni ( c , d ). Source Data
Figure Legend Snippet: CGRP modulates the activation of CD8 + T cells. a , b , Splenocyte CD8 + T cells from wild-type ( a ), Ramp1 −/ − ( a ) or naive OT-I ( b ) mice were cultured under T c1 -stimulating conditions (ex-vivo-activated by CD3 and CD28, IL-12 and anti-IL4) for 48 h to generate cytotoxic CD8 + T cells. In the presence of IL-2 (10 ng ml −1 ), the cells were stimulated with CGRP (100 nM; challenged once every two days) for 96 h. Wild-type cytotoxic CD8 + T cells showed an increased proportion of PD-1 + LAG3 + TIM3 + cells; this effect was absent when treating cytotoxic CD8 + T cells that were collected from Ramp1 −/ − mice ( a ). In co-culture (48 h), CGRP (100 nM; once daily) also reduced the ability of OT-I cytotoxic CD8 + T cells (4 × 10 5 cells) to eliminate B16F10-mCherry-OVA cancer cells ( b ). c , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old female mice with sensory neurons intact or ablated. In nociceptor-ablated mice, peritumoral recombinant CGRP injection (100 nM, i.d., once daily) rescues B16F10 growth (assessed until day 12). d , e , Orthotopic B16F10-mCherry-OVA cells (5 × 10 5 cells, i.d.) were inoculated into eight-week-old male and female mice. Starting one day after inoculation (defined as prophylactic), the RAMP1 antagonist BIBN4096 (5 mg kg −1 ) was administered systemically (i.p.) once every two days. In another group of mice, BIBN4096 (5 mg kg −1 , i.p., every two days) injections were started once the tumour reached a volume of around 200 mm 3 (defined as therapeutic). Prophylactic or therapeutic BIBN4096 treatments decreased tumour growth ( d ) and reduced the proportion of intratumoral PD-1 + LAG3 + TIM3 + CD8 + T cells ( e ; assessed until day 13). Data are shown as box-and-whisker plots (as defined in Fig. 1b, c ), for which individual data points are given ( a , b , e ), or as mean ± s.e.m. ( c , d ). n as follows: a : Ramp1 WT CD8 + vehicle ( n = 9), Ramp1 WT CD8 + CGRP ( n = 10), Ramp1 −/ − CD8 + vehicle ( n = 10), Ramp1 −/ − CD8 + CGRP ( n = 9); b : n = 4 per group; c : intact + vehicle ( n = 15), ablated + CGRP ( n = 11); d : vehicle ( n = 13), BIBN prophylactic ( n = 16), BIBN therapeutic ( n = 18); e : vehicle ( n = 10), BIBN prophylactic ( n = 13), BIBN therapeutic ( n = 16). Experiments were independently repeated three times with similar results. P values were determined by one-way ANOVA with post-hoc Bonferroni ( a , e ), two-sided unpaired Student’s t -test ( b ) or two-way ANOVA with post-hoc Bonferroni ( c , d ). Source Data

Techniques Used: Activation Assay, Mouse Assay, Cell Culture, Ex Vivo, Co-Culture Assay, Recombinant, Injection, Whisker Assay

Nociceptor-released CGRP increases cytotoxic CD8 + T cell exhaustion. ( a–b ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating condition ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then cultured or not with wild-type DRG neurons and exposed to capsaicin (1 μM, challenged once every two days) or its vehicle. As measured after 4 days stimulation, capsaicin-stimulated intact neuron increased the proportion of PD-1 + LAG3 + TIM3 + ( a ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( b ). ( c–d ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 μL/mL), naive DRG neurons were cultured in the presence of BoNT/A (50 pg/mL) or its vehicle for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed or not to a RAMP1 blocker (CGRP 8–37 ; 2 μg/mL) and challenge (1:2 dilution) with fresh KCl-induced conditioned medium from naive, or BoNT/A-silenced neurons. As measured after 4 days stimulation, KCl-stimulated neuron-conditioned medium increased the proportion of PD-1 + LAG3 + TIM3 + ( c ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( d ). Such effect was absent when cytotoxic CD8 + T cells were co-exposed to the RAMP1 blocker CGRP 8–37 or challenged with the neuron conditioned medium collected from BoNT/A-silenced neurons ( c–d ). ( e–f ) Splenocytes-isolated CD8 + T cells from wild-type and Ramp1 −/ − mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed to CGRP (0.1 μM) or its vehicle. As measured after 4 days stimulation, representative flow cytometry plots ( f ) show that CGRP decrease Ramp WT cytotoxic CD8 + T cells expression of IFNγ + ( e , f ), TNF + ( f ), and IL-2 + ( f ) when exposed to CGRP. Inversely, CGRP increase the proportion of PD-1 + LAG3 + TIM3 + in Ramp1 W T cytotoxic CD8 + T cells ( f ). Ramp1 −/ − cytotoxic CD8 + T cells were protected from the effect of CGRP ( e–f ). ( g–i ) Splenocytes-isolated CD8 + T cells from naive OT-I mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. B16F10-mCherry-OVA cells (1×10 5 cells) were then cultured with or without OT-I cytotoxic CD8 + T cells (4×10 5 cells). Tc1-stimulated OT-I-CD8 + T cells lead to B16F10-OVA cell apoptosis (AnnexinV + 7AAD + ; g , measured after 48h; h–i , measured after 24h). B16F10-mCherry-OVA cells elimination by cytotoxic CD8 + T cells was reduced when the co-cultures were challenged (1:2 dilution; once daily for two consecutive days) with fresh conditioned medium collected from capsaicin (1 μM)-stimulated naive DRG neurons ( g ; measured after 48h). Similarly, KCl (50mM)-stimulated naive DRG neurons conditioned medium (1:2 dilution) reduced B16F10-mCherry-OVA apoptosis ( h ; measured after 24h). This effect was blunted when the cells were co-exposed to the RAMP1 blocker CGRP 8-37 ( h ; 2 μg/mL; measured after 24h). CGRP (0.1 μM) challenges also reduced OT-I cytotoxic CD8 + T cells elimination of B16F10-OVA cell ( i ; measured after 24h). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g–h ), or representative FACS plot ( f , i ). N are as follows: a : CD8 + vehicle (n = 4), CD8 + capsaicin (n = 9), CD8 + neuron + capsaicin (n = 9), b : n = 5/groups, c : CD8 (n = 6), CD8 + KCl-induced neurons CM (n = 5), CD8 + KCl-induced neurons CM + CGRP 8-37 (n = 6), CD8 + KCl-induced neurons CM + BoNT/A (n = 6), d : n = 5/groups, e : Ramp1 WT CD8 + vehicle (n = 7), Ramp1 WT CD8 + CGRP (n = 8), Ramp1 −/ − CD8 + vehicle (n = 6), Ramp1 −/ − CD8 + CGRP (n = 6), g : B16F10 (n = 3), B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), h : B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM + CGRP 8–37 (n = 5). Experiments were repeated a minimum of three independent times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferroni ( a–e , g–h ). Source Data
Figure Legend Snippet: Nociceptor-released CGRP increases cytotoxic CD8 + T cell exhaustion. ( a–b ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating condition ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then cultured or not with wild-type DRG neurons and exposed to capsaicin (1 μM, challenged once every two days) or its vehicle. As measured after 4 days stimulation, capsaicin-stimulated intact neuron increased the proportion of PD-1 + LAG3 + TIM3 + ( a ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( b ). ( c–d ) Splenocytes-isolated CD8 + T cells were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. In the presence of peptidase inhibitors (1 μL/mL), naive DRG neurons were cultured in the presence of BoNT/A (50 pg/mL) or its vehicle for 24h. The cells were then washed, stimulated (30 min) with KCl (50mM), and the conditioned medium collected. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed or not to a RAMP1 blocker (CGRP 8–37 ; 2 μg/mL) and challenge (1:2 dilution) with fresh KCl-induced conditioned medium from naive, or BoNT/A-silenced neurons. As measured after 4 days stimulation, KCl-stimulated neuron-conditioned medium increased the proportion of PD-1 + LAG3 + TIM3 + ( c ) cytotoxic CD8 + T cells, while it decreased the one of IFNγ + ( d ). Such effect was absent when cytotoxic CD8 + T cells were co-exposed to the RAMP1 blocker CGRP 8–37 or challenged with the neuron conditioned medium collected from BoNT/A-silenced neurons ( c–d ). ( e–f ) Splenocytes-isolated CD8 + T cells from wild-type and Ramp1 −/ − mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. On alternate days for 4 days, the cytotoxic CD8 + T cells were exposed to CGRP (0.1 μM) or its vehicle. As measured after 4 days stimulation, representative flow cytometry plots ( f ) show that CGRP decrease Ramp WT cytotoxic CD8 + T cells expression of IFNγ + ( e , f ), TNF + ( f ), and IL-2 + ( f ) when exposed to CGRP. Inversely, CGRP increase the proportion of PD-1 + LAG3 + TIM3 + in Ramp1 W T cytotoxic CD8 + T cells ( f ). Ramp1 −/ − cytotoxic CD8 + T cells were protected from the effect of CGRP ( e–f ). ( g–i ) Splenocytes-isolated CD8 + T cells from naive OT-I mice were cultured under Tc1-stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. B16F10-mCherry-OVA cells (1×10 5 cells) were then cultured with or without OT-I cytotoxic CD8 + T cells (4×10 5 cells). Tc1-stimulated OT-I-CD8 + T cells lead to B16F10-OVA cell apoptosis (AnnexinV + 7AAD + ; g , measured after 48h; h–i , measured after 24h). B16F10-mCherry-OVA cells elimination by cytotoxic CD8 + T cells was reduced when the co-cultures were challenged (1:2 dilution; once daily for two consecutive days) with fresh conditioned medium collected from capsaicin (1 μM)-stimulated naive DRG neurons ( g ; measured after 48h). Similarly, KCl (50mM)-stimulated naive DRG neurons conditioned medium (1:2 dilution) reduced B16F10-mCherry-OVA apoptosis ( h ; measured after 24h). This effect was blunted when the cells were co-exposed to the RAMP1 blocker CGRP 8-37 ( h ; 2 μg/mL; measured after 24h). CGRP (0.1 μM) challenges also reduced OT-I cytotoxic CD8 + T cells elimination of B16F10-OVA cell ( i ; measured after 24h). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g–h ), or representative FACS plot ( f , i ). N are as follows: a : CD8 + vehicle (n = 4), CD8 + capsaicin (n = 9), CD8 + neuron + capsaicin (n = 9), b : n = 5/groups, c : CD8 (n = 6), CD8 + KCl-induced neurons CM (n = 5), CD8 + KCl-induced neurons CM + CGRP 8-37 (n = 6), CD8 + KCl-induced neurons CM + BoNT/A (n = 6), d : n = 5/groups, e : Ramp1 WT CD8 + vehicle (n = 7), Ramp1 WT CD8 + CGRP (n = 8), Ramp1 −/ − CD8 + vehicle (n = 6), Ramp1 −/ − CD8 + CGRP (n = 6), g : B16F10 (n = 3), B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), h : B16F10 + OT-I CD8 (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM (n = 4), B16F10 + OT-I CD8 + KCl-induced neuron CM + CGRP 8–37 (n = 5). Experiments were repeated a minimum of three independent times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferroni ( a–e , g–h ). Source Data

Techniques Used: Isolation, Cell Culture, Ex Vivo, Mouse Assay, Flow Cytometry, Expressing, Whisker Assay, FACS

Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data
Figure Legend Snippet: Nociceptor ablation reduces the exhaustion of intratumoral CD8 + T cells. ( a-b ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated ( Trpv1 cre ::DTA fl/WT ) mice. Sixteen days post-B16F10-mCherry-OVA cells inoculation (5x10 5 cells; i.d.), tumour-infiltrating CD8 + T cells were immunophenotyped ( a ) and were found to be more numerous in sensory neuron depleted tumours ( b ). ( c-g ) Orthotopic B16F10-mCherry-OVA (2x10 5 cells; i.d.) cells were injected into the left hindpaw paw of nociceptor intact (n = 96; Trpv1 WT ::DTA fl/WT ) or ablated (n = 18; Trpv1 cre ::DTA fl/WT ) mice. When compared to their baseline threshold, littermate control mice showed significant thermal hypersensitivity on day 7, an effect that peaks on day 21 ( c ). In these mice, intratumoral frequency of PD-1 + LAG3 + TIM3 + ( d ) and IFNγ + ( e ) CD8 + T cells increased 12 days post tumour inoculation, an effect that peaked on day 19. Finally, B16F10 tumour volume peaked on day 22 ( f ). When compared with littermate control mice, sensory neuron ablated mice inoculated with B16F10 cells showed no thermal pain hypersensitivity ( c ), reduced intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells ( d ) and tumour volume ( f ). In littermate control mice, thermal pain hypersensitivity (day 7) precedes the increase in intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells (day 12), and significant tumour growth (day 12; g ). ( h ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female sensory neuron intact or ablated mice. The mice were treated with αPD-L1 (6 mg/kg, i.p.; days 7, 10, 13, 16 post tumour inoculation) or its isotype control. On day 19, αPD-L1 potentiated the nociceptor ablation mediated reduction in B16F10-OVA tumour volume. ( i–k ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into a cohort of nociceptor neuron-ablated mice 3 days prior to the injection given to nociceptor intact mice. Mice from each group with similar tumour size (~85mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~47%) in nociceptor-ablated mice than was observed in nociceptor-intact mice (~32%; i–j ). In addition, nociceptor ablation increased the proportion of intratumoral tumour-specific ( k ; defined as H-2Kb + ) CD8 + T cells. These differences were further enhanced by αPD-L1 treatment ( i–k ). ( l–m ) Sensory neurons ablation ( Trpv1 cre ::DTA fl/WT ) decreased growth of YUMMER1.7 cells (5×10 5 cells; i.d.) an immunogenic version of a Braf V600E Cdkn2a −/− Pten −/− melanoma cell line ( l ; assessed until day 12). The non-immunogenic YUMM1.7 cell line (5×10 5 cells; i.d.; assessed until day 14) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). Nociceptor ablation had no effect on YUMM1.7 growth ( m ). ( n ) Orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) cells were injected to nociceptor intact ( Trpv1 WT ::DTA fl/WT ) and ablated mice ( Trpv1 cre ::DTA fl/WT ). The reduction in B16F10-mCherry-OVA (5×10 5 cells; i.d.) tumour growth observed in nociceptors ablated mice was absent following systemic CD3 depletion (assessed until day 15; αCD3, 200 μg/mouse; i.p.; every 3 days). ( o ) To deplete their nociceptor neurons, C57BL6J mice were injected with RTX (s.c., 30, 70, 100 μg/kg) and were subsequently (28 days later) inoculated with B16F10-mCherry-OVA (2×10 5 cells). RTX-injected mice showed reduced tumour growth when compared to vehicle-exposed mice (assessed until day 13). ( p–q ) Orthotopic B16F10-mCherry-OVA (5×10 5 cells; i.d.) cells were injected to light-sensitive mice ( Nav 1.8 cre ::ChR2 fl/WT ). As opposed to unstimulated mice, the optogenetic activation (3.5 ms, 10Hz, 478nm, 60 mW, giving approx. 2-6 mW/mm 2 with a 0.39-NA fibre placed 5–10 mm from the skin, 20 min) of tumour-innervating nociceptor neurons, when started once B16F10 tumours were visible (~20 mm 3 ) or well established (~200 mm 3 ), resulted in enhanced tumour growth ( p , as measured until day 14) and intratumoral CGRP release ( q ). Data are shown as FACS plot ( a ; depict the gating strategy used in fig. 3d,e ), as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median) for which individual data points are given ( b , k , q ), scatter dot plot ( c–f ), percentage change from maximal thermal hypersensitivity, intratumoral frequency of PD-1 + LAG3 + TIM3 + CD8 + T cells and tumour volume ( g ), or mean ± S.E.M ( h–j , l–p ). N are as follows: a–b : intact (n = 29), ablated (n = 33), c : intact (n = 96), ablated (n = 19), d : intact (n = 92), ablated (n = 15), e : intact (n = 96), ablated (n = 15), f : intact (n = 96), ablated (n = 16), g : n=96, h : intact (n = 9), ablated (n = 10), intact+αPD-L1 (n = 9), ablated+αPD-L1 (n = 8), i : intact (n = 14), ablated (n = 4), j : intact+αPD-L1 (n = 12), ablated+αPD-L1 (n = 12), k : intact (n = 5), ablated (n = 6), intact+αPD-L1 (n = 5), ablated+αPD-L1 (n = 5), l : intact (n = 8), ablated (n = 11), m : intact (n = 6), ablated (n = 13), n : intact (n = 5), ablated (n = 5), intact+αCD3 (n = 6), ablated+αCD3 (n = 5), o : vehicle (n = 11), RTX (n = 10), p : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 8), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 8), q : Nav 1.8 cre ::ChR2 fl/WT (n = 12), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~200 mm 3 ) (n = 7), Nav 1.8 cre ::ChR2 fl/WT + Light (vol. ~20 mm 3 ) (n = 9). Experiments were independently repeated two ( c–g ), three ( h–q ) or six ( a , b ) times with similar results. P-values are shown in the figure and determined by two-sided unpaired Student’s t-test ( b–f , k , q ), or two-way ANOVA post-hoc Bonferroni ( h–j , l–p ). Source Data

Techniques Used: Injection, Mouse Assay, Activation Assay, FACS, Whisker Assay

BoNT/A silencing of B16F10-innervating neurons decreases tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BoNT/A (10–50 pg/μL) for 24h; effects on apoptosis, exhaustion, and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BoNT/A did not affect the survival ( a ) of cultured cytotoxic CD8 + T cells, nor their relative expression of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) and PD-1 + LAG3 + TIM3 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h and subsequently exposed to BoNT/A (1.6-50 pg/μL) or its vehicle for an additional 24h. BoNT/A did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( g–n ) One and three days prior to tumour inoculation ( defined as prophylactic ), the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day after the last injection, orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. In another group of mice, BoNT/A was administered (25 pg/μL; i.d.) one and three days after the tumour reached a volume of ~200mm3 (defined as therapeutic ). The effect of neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. Nineteen days post tumour inoculation, we found that the tumour volume ( g , h ) and weight ( i ) were reduced in mice treated with BoNT/A ( Prophylactic group ). In parallel, we found that silencing tumour-innervating neurons increased the proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BoNT/A had no effect on the total number of intratumoral CD8 T cells ( j ) or the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. ( o ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female sensory neuron-intact or ablated mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic YUMMER1.7 cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. The effects of nociceptor neuron ablation on tumour size and volume were measured. Thirteen days post tumour inoculation, we found that the tumour growth was lower in mice treated with BoNT/A or in sensory neuron-ablated mice. BoNT/A had no additive effects when administered to sensory neuron-ablated mice. ( p ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic B16F10-mCherry-OVA cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. On days 7, 10, 13 and 16 post tumour inoculation, the mice were exposed to αPD-L1 (6 mg/kg, i.p.) or its isotype control. Eighteen days post tumour inoculation, we found that neuron silencing using BoNT/A potentiated αPD-L1-mediated tumour reduction. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f; h–n ) or as mean ± S.E.M ( g , o , p ). N are as follows: a-e : n = 5/groups, f : n = 3/groups, g–i : vehicle (n = 12), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 10), j : vehicle (n = 11), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), k–n : vehicle (n = 10), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), o : intact + vehicle (n = 9), ablated + vehicle (n = 8), intact + BoNT/A (n = 10), ablated + BoNT/A (n = 8), p : vehicle (n = 7), αPD-L1 (n = 8), αPD-L1 + BoNT/A (n = 7). Experiments were independently repeated two ( a–f , o–p ) or four ( g–n ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–f , h–n ) or two-way ANOVA post-hoc Bonferroni ( g , o , p ). Source Data
Figure Legend Snippet: BoNT/A silencing of B16F10-innervating neurons decreases tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to BoNT/A (10–50 pg/μL) for 24h; effects on apoptosis, exhaustion, and activation were measured by flow cytometry. When compared to vehicle-exposed cells, BoNT/A did not affect the survival ( a ) of cultured cytotoxic CD8 + T cells, nor their relative expression of IFNγ + ( b ), TNF + ( c ), IL-2 + ( d ) and PD-1 + LAG3 + TIM3 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h and subsequently exposed to BoNT/A (1.6-50 pg/μL) or its vehicle for an additional 24h. BoNT/A did not trigger B16F10 cells apoptosis, as measured by the mean fluorescence intensity of Annexin V. ( g–n ) One and three days prior to tumour inoculation ( defined as prophylactic ), the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day after the last injection, orthotopic B16F10-mCherry-OVA (5x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. In another group of mice, BoNT/A was administered (25 pg/μL; i.d.) one and three days after the tumour reached a volume of ~200mm3 (defined as therapeutic ). The effect of neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. Nineteen days post tumour inoculation, we found that the tumour volume ( g , h ) and weight ( i ) were reduced in mice treated with BoNT/A ( Prophylactic group ). In parallel, we found that silencing tumour-innervating neurons increased the proportion of IFNγ + ( k ), TNF + ( l ), and IL-2 + ( m ) CD8 + T cells. BoNT/A had no effect on the total number of intratumoral CD8 T cells ( j ) or the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. ( o ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female sensory neuron-intact or ablated mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic YUMMER1.7 cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. The effects of nociceptor neuron ablation on tumour size and volume were measured. Thirteen days post tumour inoculation, we found that the tumour growth was lower in mice treated with BoNT/A or in sensory neuron-ablated mice. BoNT/A had no additive effects when administered to sensory neuron-ablated mice. ( p ) One and three days prior to tumour inoculation, the skin of 8-week-old male and female mice was injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. One day following the last injection, orthotopic B16F10-mCherry-OVA cells (5×10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. On days 7, 10, 13 and 16 post tumour inoculation, the mice were exposed to αPD-L1 (6 mg/kg, i.p.) or its isotype control. Eighteen days post tumour inoculation, we found that neuron silencing using BoNT/A potentiated αPD-L1-mediated tumour reduction. Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–f; h–n ) or as mean ± S.E.M ( g , o , p ). N are as follows: a-e : n = 5/groups, f : n = 3/groups, g–i : vehicle (n = 12), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 10), j : vehicle (n = 11), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), k–n : vehicle (n = 10), BoNT/A therapeutic (n = 12), BoNT/A prophylactic (n = 8), o : intact + vehicle (n = 9), ablated + vehicle (n = 8), intact + BoNT/A (n = 10), ablated + BoNT/A (n = 8), p : vehicle (n = 7), αPD-L1 (n = 8), αPD-L1 + BoNT/A (n = 7). Experiments were independently repeated two ( a–f , o–p ) or four ( g–n ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–f , h–n ) or two-way ANOVA post-hoc Bonferroni ( g , o , p ). Source Data

Techniques Used: Isolation, Mouse Assay, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Expressing, Fluorescence, Injection, Whisker Assay

Cancer-secreted SLPI drives the release of CGRP by nociceptor neurons. a – c , Naive DRG neurons ( Trpv1 cre ::-CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA cells and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48 h, the cells were collected, FACS purified and RNA sequenced. a , Hierarchical clustering of sorted neuron molecular profiles depicts distinct groups of transcripts enriched in each group. b , DEGs were calculated, and Slpi was found to be overexpressed in cancer cells when co-cultured with OVA-specific cytotoxic CD8 + T cells, DRG neurons or both populations. c , SLPI is secreted by B16F10-mCherry-OVA cells when co-cultured (24 h or 48 h) with naive DRG neurons and OVA-specific cytotoxic CD8 + T cells, with a maximal effect after 48 h. d – f , Using calcium microscopy, we found that SLPI (10 pg ml −1 –10 ng ml −1 ) activated around 20% of cultured naive DRG neurons ( d , e ). Activation of cultured neurons (3 h) with SLPI also leads to significant release of CGRP ( f ). Data are shown as a heat map showing normalized gene expression (log 2 (1 + TPM) − mean ( a ), as box-and-whisters plots (as defined in Fig. 1b,c ) ( b ) or as mean ± s.e.m. ( c – f ). n as follows: a , b : n = 2–4 per groups; c : n = 3 for all groups except CD8 + T cells ( n = 8); d : n = 17; e : n = 8 per group; f : 0 ng ml −1 ( n = 4), 0.1 ng ml −1 ( n = 5), 1 ng ml −1 ( n = 5), 5 ng ml −1 ( n = 4). Experiments in c – f were independently repeated three times with similar results. The sequencing experiment was not repeated ( a , b ). P values were determined by one-way ANOVA with post-hoc Bonferroni ( b , e , f ) or two-sided unpaired Student’s t -test ( c ). * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source Data
Figure Legend Snippet: Cancer-secreted SLPI drives the release of CGRP by nociceptor neurons. a – c , Naive DRG neurons ( Trpv1 cre ::-CheRiff-eGFP fl/WT ), B16F10-mCherry-OVA cells and OVA-specific cytotoxic CD8 + T cells were cultured alone or in combination. After 48 h, the cells were collected, FACS purified and RNA sequenced. a , Hierarchical clustering of sorted neuron molecular profiles depicts distinct groups of transcripts enriched in each group. b , DEGs were calculated, and Slpi was found to be overexpressed in cancer cells when co-cultured with OVA-specific cytotoxic CD8 + T cells, DRG neurons or both populations. c , SLPI is secreted by B16F10-mCherry-OVA cells when co-cultured (24 h or 48 h) with naive DRG neurons and OVA-specific cytotoxic CD8 + T cells, with a maximal effect after 48 h. d – f , Using calcium microscopy, we found that SLPI (10 pg ml −1 –10 ng ml −1 ) activated around 20% of cultured naive DRG neurons ( d , e ). Activation of cultured neurons (3 h) with SLPI also leads to significant release of CGRP ( f ). Data are shown as a heat map showing normalized gene expression (log 2 (1 + TPM) − mean ( a ), as box-and-whisters plots (as defined in Fig. 1b,c ) ( b ) or as mean ± s.e.m. ( c – f ). n as follows: a , b : n = 2–4 per groups; c : n = 3 for all groups except CD8 + T cells ( n = 8); d : n = 17; e : n = 8 per group; f : 0 ng ml −1 ( n = 4), 0.1 ng ml −1 ( n = 5), 1 ng ml −1 ( n = 5), 5 ng ml −1 ( n = 4). Experiments in c – f were independently repeated three times with similar results. The sequencing experiment was not repeated ( a , b ). P values were determined by one-way ANOVA with post-hoc Bonferroni ( b , e , f ) or two-sided unpaired Student’s t -test ( c ). * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001. Source Data

Techniques Used: Cell Culture, FACS, Purification, Microscopy, Activation Assay, Expressing, Sequencing

QX-314 silencing of B16F10-innervating neurons reduces tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to QX-314 (50–150 μM) for 24h, effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, QX-314 did not affect the survival of cultured cytotoxic CD8 + T cells ( a ), nor their relative expression of PD-1 + LAG3 + TIM3 + ( b ), IFNγ + ( c ), TNF + ( d ) and IL-2 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h. The cells were then exposed or not to QX-314 (0.1-1%) for an additional 24-72h, and cell count was analysed by bright-field microscopy. QX-314 did not affect B16F10 cells’ survival, as measured by relative cell count changes (at each time point) in comparison to vehicle-exposed cells. ( g–i ) One and three days prior to tumour inoculation, 8-week-old male and female wild-type mice’s right hindpaws or flanks were injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. On the following day, orthotopic B16F10 cells ( g : 5x10 5 cells; i.d.; h–i : 2x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. Starting one day post inoculation, QX-314 (0.3%) or its vehicle was administered (i.d.) once daily in another group of mice. The effects of sensory neuron silencing were tested on neuropeptide release ( g ), as well as mechanical ( h ) and thermal pain hypersensitivity ( i ). First, CGRP levels were increased in B16F10 tumour surrounding skin explant (assessed on day 15) in comparison to control skin; an effect further enhanced by capsaicin (1 μM; 3h) but was absent in skin pre-treated with BoNT/A (25 pg/μL) or QX-314 (0.3%; g ). We also found that B16F10 injection induced mechanical ( h ) and thermal pain hypersensitivities ( i ) fourteen days post tumour inoculation. These effects were stopped by sensory neuron silencing with QX-314 or BoNT/A ( h–i ). ( j ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, QX-314 (0.3%; i.d.; 5 sites) was injected once daily around the tumour. The effect of nociceptor neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. We found that silencing tumour-innervating neurons increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( k–r ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), In other groups of mice, QX-314 daily injection started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). As measured seventeen days post tumour inoculation, silencing tumour innervation also decreased tumour volume ( k , l ) and weight ( m ), as well as the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. QX-314 treatment also increased the total number of intratumoral CD8 + T cells ( o ), as well as relative proportion of IFNγ + ( p ), TNF + ( q ), and IL-2 + ( r ) CD8 + T cells. ( s–t ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into mice treated with QX-314 (0.3%; i.d.) 2-3 days prior to being injected into vehicle-exposed mice. Mice from each group with similar tumour size (~100mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~61%) in nociceptor silenced mice than was observed in isotype vehicle-exposed mice (~49%; s-t ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g , l–r ), as mean ± S.E.M ( f , h , i , k , s , t ), or as Mantel–Cox regression analysis ( j ). N are as follows: a : n = 4/groups, b–e : n = 5/groups, f : n = 3/groups, g : naïve (n = 4), vehicle (n = 7), B16F10+vehicle (n = 5), B16F10+BoNT/A (n = 5), B16F10+QX-314 (n = 5), h–i : n = 6/groups, j : vehicle (n = 89), QX-314 (n = 12), k : vehicle (n = 21), QX-314 prophylactic (n = 21), QX-314 therapeutic (n = 17), l : vehicle (n = 26), QX-314 therapeutic (n = 26), QX-314 prophylactic (n = 28), m : vehicle (n = 25), QX-314 therapeutic (n = 22), QX-314 prophylactic (n = 25), n : vehicle (n = 31), QX-314 therapeutic (n = 29), QX-314 prophylactic (n = 28), o : n = 30/groups, p–r : vehicle (n = 24), QX-314 therapeutic (n = 23), QX-314 prophylactic (n = 25), s : vehicle (n = 9), QX-314 (n = 13), t : vehicle + αPD-L1 (n = 18), QX-314 + αPLD1 (n = 13). Experiments were independently repeated two ( a–i , s–t ) or four ( j–r ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–g , l–r ), two-sided unpaired Student’s t-test ( h–i ), Mantel–Cox regression ( j ), or two-way ANOVA posthoc Bonferroni ( k , s–t ). Source Data
Figure Legend Snippet: QX-314 silencing of B16F10-innervating neurons reduces tumour growth. ( a–e ) Splenocytes-isolated CD8 + T cells from naive C57BL6J mice were cultured under T c1 -stimulating conditions ( ex vivo activated by CD3 and CD28, IL-12, and anti-IL4) for 48h. The cells were then exposed to QX-314 (50–150 μM) for 24h, effects on apoptosis, exhaustion and activation were measured by flow cytometry. When compared to vehicle-exposed cells, QX-314 did not affect the survival of cultured cytotoxic CD8 + T cells ( a ), nor their relative expression of PD-1 + LAG3 + TIM3 + ( b ), IFNγ + ( c ), TNF + ( d ) and IL-2 + ( e ). ( f ) B16F10 (1x10 5 cells) were cultured for 24h. The cells were then exposed or not to QX-314 (0.1-1%) for an additional 24-72h, and cell count was analysed by bright-field microscopy. QX-314 did not affect B16F10 cells’ survival, as measured by relative cell count changes (at each time point) in comparison to vehicle-exposed cells. ( g–i ) One and three days prior to tumour inoculation, 8-week-old male and female wild-type mice’s right hindpaws or flanks were injected with BoNT/A (25 pg/μL; i.d.) or its vehicle. On the following day, orthotopic B16F10 cells ( g : 5x10 5 cells; i.d.; h–i : 2x10 5 cells; i.d.) were inoculated into the area pre-exposed to BoNT/A. Starting one day post inoculation, QX-314 (0.3%) or its vehicle was administered (i.d.) once daily in another group of mice. The effects of sensory neuron silencing were tested on neuropeptide release ( g ), as well as mechanical ( h ) and thermal pain hypersensitivity ( i ). First, CGRP levels were increased in B16F10 tumour surrounding skin explant (assessed on day 15) in comparison to control skin; an effect further enhanced by capsaicin (1 μM; 3h) but was absent in skin pre-treated with BoNT/A (25 pg/μL) or QX-314 (0.3%; g ). We also found that B16F10 injection induced mechanical ( h ) and thermal pain hypersensitivities ( i ) fourteen days post tumour inoculation. These effects were stopped by sensory neuron silencing with QX-314 or BoNT/A ( h–i ). ( j ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation, QX-314 (0.3%; i.d.; 5 sites) was injected once daily around the tumour. The effect of nociceptor neuron silencing on tumour size and tumour-infiltrating CD8 + T cell exhaustion was measured. We found that silencing tumour-innervating neurons increased the mice’s median length of survival (~270% Mantel–Haenszel hazard ratio; measured on day 19). ( k–r ) Orthotopic B16F10-mCherry-OVA cells (5x10 5 cells; i.d.) were inoculated into 8-week-old male and female mice. Starting one day post inoculation ( defined as prophylactic ), In other groups of mice, QX-314 daily injection started once the tumour reached a volume of ~200mm 3 ( defined as therapeutic ). As measured seventeen days post tumour inoculation, silencing tumour innervation also decreased tumour volume ( k , l ) and weight ( m ), as well as the relative proportion of PD-1 + LAG3 + TIM3 + ( n ) CD8 + T cells. QX-314 treatment also increased the total number of intratumoral CD8 + T cells ( o ), as well as relative proportion of IFNγ + ( p ), TNF + ( q ), and IL-2 + ( r ) CD8 + T cells. ( s–t ) Orthotropic B16F10-mCherry-OVA cells (5x10 5 cells, i.d.) were injected into mice treated with QX-314 (0.3%; i.d.) 2-3 days prior to being injected into vehicle-exposed mice. Mice from each group with similar tumour size (~100mm 3 ) were selected and exposed to αPD-L1 (6 mg/kg, i.p.) once every 3 days for a total of 9 days. Eighteen days post tumour inoculation, we found that αPD-L1-reduced tumour growth was higher (~61%) in nociceptor silenced mice than was observed in isotype vehicle-exposed mice (~49%; s-t ). Data are shown as box-and-whisker plots (runs from minimal to maximal values; the box extends from 25 th to 75 th percentile and the middle line indicates the median), for which individual data points are given ( a–e , g , l–r ), as mean ± S.E.M ( f , h , i , k , s , t ), or as Mantel–Cox regression analysis ( j ). N are as follows: a : n = 4/groups, b–e : n = 5/groups, f : n = 3/groups, g : naïve (n = 4), vehicle (n = 7), B16F10+vehicle (n = 5), B16F10+BoNT/A (n = 5), B16F10+QX-314 (n = 5), h–i : n = 6/groups, j : vehicle (n = 89), QX-314 (n = 12), k : vehicle (n = 21), QX-314 prophylactic (n = 21), QX-314 therapeutic (n = 17), l : vehicle (n = 26), QX-314 therapeutic (n = 26), QX-314 prophylactic (n = 28), m : vehicle (n = 25), QX-314 therapeutic (n = 22), QX-314 prophylactic (n = 25), n : vehicle (n = 31), QX-314 therapeutic (n = 29), QX-314 prophylactic (n = 28), o : n = 30/groups, p–r : vehicle (n = 24), QX-314 therapeutic (n = 23), QX-314 prophylactic (n = 25), s : vehicle (n = 9), QX-314 (n = 13), t : vehicle + αPD-L1 (n = 18), QX-314 + αPLD1 (n = 13). Experiments were independently repeated two ( a–i , s–t ) or four ( j–r ) times with similar results. P-values are shown in the figure and determined by one-way ANOVA posthoc Bonferonni ( a–g , l–r ), two-sided unpaired Student’s t-test ( h–i ), Mantel–Cox regression ( j ), or two-way ANOVA posthoc Bonferroni ( k , s–t ). Source Data

Techniques Used: Isolation, Mouse Assay, Cell Culture, Ex Vivo, Activation Assay, Flow Cytometry, Expressing, Cell Counting, Microscopy, Injection, Whisker Assay

Melanoma-innervating nociceptors attenuate cancer immunosurveillance. Melanoma growth sets off anti-tumour immune responses, including the infiltration of effector CD8 T cells and their subsequent release of cytotoxic cytokines (i.e., IFNγ, TNF, Granzyme B). By acting on tissue-resident nociceptor neurons, melanoma-produced SLPI promotes pain hypersensitivity, tweaks the neurons’ transcriptome, and drives neurite outgrowth. These effects culminate in dense melanoma innervation by nociceptors and abundant release of immunomodulatory neuropeptides. CGRP, one such peptide, acts on tumour-infiltrating effector CD8 + T cells that express the CGRP receptor RAMP1, increasing their expression of immune checkpoint receptors (i.e., PD-1, LAG3, TIM3). Therefore, along with the immunosuppressive environment present in the tumour, nociceptor-produced CGRP leads to the functional exhaustion of tumour-infiltrating CD8 + T cells, which opens the door to unchecked proliferation of melanoma cells. Genetically ablating (i.e., TRPV1 lineage) or pharmacologically silencing (i.e., QX-314, BoNT/A) nociceptor neurons as well as blocking the action of CGRP on RAMP1 using a selective antagonist (i.e., BIBN4096) prevents effector CD8 + T cells from undergoing exhaustion. Therefore, targeting melanoma-innervating nociceptor neurons constitutes a novel strategy to safeguard host anti-tumour immunity and stop tumour growth.
Figure Legend Snippet: Melanoma-innervating nociceptors attenuate cancer immunosurveillance. Melanoma growth sets off anti-tumour immune responses, including the infiltration of effector CD8 T cells and their subsequent release of cytotoxic cytokines (i.e., IFNγ, TNF, Granzyme B). By acting on tissue-resident nociceptor neurons, melanoma-produced SLPI promotes pain hypersensitivity, tweaks the neurons’ transcriptome, and drives neurite outgrowth. These effects culminate in dense melanoma innervation by nociceptors and abundant release of immunomodulatory neuropeptides. CGRP, one such peptide, acts on tumour-infiltrating effector CD8 + T cells that express the CGRP receptor RAMP1, increasing their expression of immune checkpoint receptors (i.e., PD-1, LAG3, TIM3). Therefore, along with the immunosuppressive environment present in the tumour, nociceptor-produced CGRP leads to the functional exhaustion of tumour-infiltrating CD8 + T cells, which opens the door to unchecked proliferation of melanoma cells. Genetically ablating (i.e., TRPV1 lineage) or pharmacologically silencing (i.e., QX-314, BoNT/A) nociceptor neurons as well as blocking the action of CGRP on RAMP1 using a selective antagonist (i.e., BIBN4096) prevents effector CD8 + T cells from undergoing exhaustion. Therefore, targeting melanoma-innervating nociceptor neurons constitutes a novel strategy to safeguard host anti-tumour immunity and stop tumour growth.

Techniques Used: Produced, Expressing, Functional Assay, Blocking Assay

Genetic ablation of nociceptors safeguards anti-tumour immunity. a , Orthotopic B16F10-mCherry-OVA cells (2 × 10 5 cells, i.d.) were injected into the left hindpaw of wild-type mice. As measured on day 13 after tumour inoculation, intratumoral CD8 + T cell exhaustion positively correlated with thermal hypersensitivity ( R 2 = 0.55, P ≤ 0.0001). The thermal pain hypersensitivity represents the withdrawal latency ratio of the ipsilateral paw (tumour-inoculated) to the contralateral paw. b , Orthotopic B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) were inoculated into the flank of eight-week-old male and female mice with sensory neurons intact ( Trpv1 WT ::DTA fl/WT ) or ablated ( Trpv1 cre ::DTA fl/WT ). The median length of survival was increased by around 250% in nociceptor-ablated mice (measured until 22 days after inoculation). c – f , Sixteen days after tumour inoculation, sensory-neuron-ablated mice have reduced tumour growth ( c ) and increased tumour infiltration of IFNγ + CD8 + T cells ( d ), and the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells is decreased ( e ). This reduction in B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) tumour volume was absent in nociceptor-ablated mice whose CD8 + T cells were systemically depleted ( f ; assessed until day 14; anti-CD8, 200 μg per mouse, i.p., every 3 days). g , h , To chemically deplete their nociceptor neurons, Rag1 −/ − mice were injected with RTX. Twenty-eight days later, the mice were inoculated with B16F10-mCherry-OVA (5 × 10 5 cells, i.d.). RTX-injected mice that were adoptively transferred with naive OVA-specific CD8 + T cells (i.v., 1 × 10 6 cells, when tumour reached around 500 mm 3 ) showed reduced tumour growth ( g ; assessed until day 19) and exhaustion ( h ) compared to vehicle-exposed Rag1 −/ − mice. Data are shown as a linear regression analysis ± s.e. ( a ), as a Mantel–Cox regression ( b ), as mean ± s.e.m. ( c , f , g ) or as box-and-whisker plots (as defined in Fig. 1b,c ), for which individual data points are given ( d , e , h ). n as follows: a : n = 60; b : intact ( n = 62), ablated ( n = 73); c : intact ( n = 20), ablated ( n = 25); d : intact ( n = 24), ablated ( n = 23); e : intact ( n = 23), ablated ( n = 26); f : intact + anti-CD8 ( n = 10), ablated + anti-CD8 ( n = 8); g : vehicle ( n = 12), RTX ( n = 10); h : vehicle ( n = 11), RTX ( n = 10). Experiments were independently repeated two ( a , f – h ) or six ( b – e ) times with similar results. P values were determined by simple linear regression analysis ( a ), Mantel–Cox regression ( b ), two-way ANOVA with post-hoc Bonferroni ( c , f , g ) or two-sided unpaired Student’s t -test ( d , e , h ). Source Data
Figure Legend Snippet: Genetic ablation of nociceptors safeguards anti-tumour immunity. a , Orthotopic B16F10-mCherry-OVA cells (2 × 10 5 cells, i.d.) were injected into the left hindpaw of wild-type mice. As measured on day 13 after tumour inoculation, intratumoral CD8 + T cell exhaustion positively correlated with thermal hypersensitivity ( R 2 = 0.55, P ≤ 0.0001). The thermal pain hypersensitivity represents the withdrawal latency ratio of the ipsilateral paw (tumour-inoculated) to the contralateral paw. b , Orthotopic B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) were inoculated into the flank of eight-week-old male and female mice with sensory neurons intact ( Trpv1 WT ::DTA fl/WT ) or ablated ( Trpv1 cre ::DTA fl/WT ). The median length of survival was increased by around 250% in nociceptor-ablated mice (measured until 22 days after inoculation). c – f , Sixteen days after tumour inoculation, sensory-neuron-ablated mice have reduced tumour growth ( c ) and increased tumour infiltration of IFNγ + CD8 + T cells ( d ), and the proportion of PD-1 + LAG3 + TIM3 + CD8 + T cells is decreased ( e ). This reduction in B16F10-mCherry-OVA (5 × 10 5 cells, i.d.) tumour volume was absent in nociceptor-ablated mice whose CD8 + T cells were systemically depleted ( f ; assessed until day 14; anti-CD8, 200 μg per mouse, i.p., every 3 days). g , h , To chemically deplete their nociceptor neurons, Rag1 −/ − mice were injected with RTX. Twenty-eight days later, the mice were inoculated with B16F10-mCherry-OVA (5 × 10 5 cells, i.d.). RTX-injected mice that were adoptively transferred with naive OVA-specific CD8 + T cells (i.v., 1 × 10 6 cells, when tumour reached around 500 mm 3 ) showed reduced tumour growth ( g ; assessed until day 19) and exhaustion ( h ) compared to vehicle-exposed Rag1 −/ − mice. Data are shown as a linear regression analysis ± s.e. ( a ), as a Mantel–Cox regression ( b ), as mean ± s.e.m. ( c , f , g ) or as box-and-whisker plots (as defined in Fig. 1b,c ), for which individual data points are given ( d , e , h ). n as follows: a : n = 60; b : intact ( n = 62), ablated ( n = 73); c : intact ( n = 20), ablated ( n = 25); d : intact ( n = 24), ablated ( n = 23); e : intact ( n = 23), ablated ( n = 26); f : intact + anti-CD8 ( n = 10), ablated + anti-CD8 ( n = 8); g : vehicle ( n = 12), RTX ( n = 10); h : vehicle ( n = 11), RTX ( n = 10). Experiments were independently repeated two ( a , f – h ) or six ( b – e ) times with similar results. P values were determined by simple linear regression analysis ( a ), Mantel–Cox regression ( b ), two-way ANOVA with post-hoc Bonferroni ( c , f , g ) or two-sided unpaired Student’s t -test ( d , e , h ). Source Data

Techniques Used: Injection, Mouse Assay, Whisker Assay

5) Product Images from "Ageing-resembling phenotype of long-term allogeneic hematopoietic cells recipients compared to their donors"

Article Title: Ageing-resembling phenotype of long-term allogeneic hematopoietic cells recipients compared to their donors

Journal: Immunity & Ageing : I & A

doi: 10.1186/s12979-022-00308-6

Box plots of mean telomeric length (median kb) in recipients (R) of allo-HCT and their donors (D) in main lymphocyte subpopulations CD4 + , CD8 + , CD19 + and CD56 + . The box and whiskers plots are corresponding to median, 25th and 75th quartile and outlayers. Means are marked as X
Figure Legend Snippet: Box plots of mean telomeric length (median kb) in recipients (R) of allo-HCT and their donors (D) in main lymphocyte subpopulations CD4 + , CD8 + , CD19 + and CD56 + . The box and whiskers plots are corresponding to median, 25th and 75th quartile and outlayers. Means are marked as X

Techniques Used:

6) Product Images from "Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations"

Article Title: Natural heteroclitic-like peptides are generated by SARS-CoV-2 mutations

Journal: bioRxiv

doi: 10.1101/2022.10.28.513849

Comparative ex-vivo and in vitro analysis of the variant peptide effect on T cells. A) Dots represent the frequency of spike-specific IFN-g-producing CD8 and CD4 T cells reactive to wild-type and variant spike peptide pools determined by flow-cytometry after 18 hours stimulation; background-subtracted data were analyzed statistically by the Mann-Whitney test. B) Bars represent decreased or increased ( red- and green- shadowed areas, respectively) responses to variant versus wild-type peptide pools measured by IFN-g production (FluoroSPOT and ICS assays) and CD107 degranulation in 11 vaccinated subjects. C) Enhancing and inhibitory effects of mutated versus wild-type peptides measured by ICS on CD8 T cells ex-vivo ( grey bars ) or on in vitro expanded C8 T cell lines ( red bars ); T-cell lines were generated by wild-type and variant peptide pool stimulation for 8 days of PBMCs from six vaccinated donors. D) Representative dot-plots derived from cytofluorimetric analysis of in vitro expanded T cells, confirming that the stimulatory and inhibitory effects of variant peptides on IFN-γ production are totally sustained by CD8 T cells.
Figure Legend Snippet: Comparative ex-vivo and in vitro analysis of the variant peptide effect on T cells. A) Dots represent the frequency of spike-specific IFN-g-producing CD8 and CD4 T cells reactive to wild-type and variant spike peptide pools determined by flow-cytometry after 18 hours stimulation; background-subtracted data were analyzed statistically by the Mann-Whitney test. B) Bars represent decreased or increased ( red- and green- shadowed areas, respectively) responses to variant versus wild-type peptide pools measured by IFN-g production (FluoroSPOT and ICS assays) and CD107 degranulation in 11 vaccinated subjects. C) Enhancing and inhibitory effects of mutated versus wild-type peptides measured by ICS on CD8 T cells ex-vivo ( grey bars ) or on in vitro expanded C8 T cell lines ( red bars ); T-cell lines were generated by wild-type and variant peptide pool stimulation for 8 days of PBMCs from six vaccinated donors. D) Representative dot-plots derived from cytofluorimetric analysis of in vitro expanded T cells, confirming that the stimulatory and inhibitory effects of variant peptides on IFN-γ production are totally sustained by CD8 T cells.

Techniques Used: Ex Vivo, In Vitro, Variant Assay, Flow Cytometry, MANN-WHITNEY, Generated, Derivative Assay

CD8 T cell variant epitopes and their location in the trimeric structure of the prototype spike protein ectodomain. A) List of mutated CD8 epitopes, that negatively (‘ Inhibitory’ ) or positively (‘ Enhancing’ ) modulate the CD8 T cell response compared to the corresponding prototype (‘Wuhan’) epitopes, identified by ex-vivo FluoroSPOT assay in vaccinated subjects (n=8); numbers in the # column allow to identify each peptide in the structure shown below. B) Trimeric structure of the prototype (Wuhan lineage) spike ectodomain (PDB 6ZXN) shown with one protomer (RBD-up) in yellow cartoon and the two other protomers (RBD-down) represented as light blue and ice surfaces. Inhibitory CD8 peptides ( left-side panel) are shown on the yellow protomer as red ribbons, with the mutated residues displayed as blue sticks. The enhancing CD8 epitopes ( right-side panel) are mapped on the yellow protomer as blue ribbons, with the mutated residues displayed as red sticks. Numbers (#) indicate the specific mutated peptides listed in panel (A); the substituted Ser13 residue of the enhancing peptides #6-9 is not displayed because the corresponding region in the spike protein structure is not resolved; NTD, N-terminal domain; RBD, receptor binding domain; S2, subunit 2.
Figure Legend Snippet: CD8 T cell variant epitopes and their location in the trimeric structure of the prototype spike protein ectodomain. A) List of mutated CD8 epitopes, that negatively (‘ Inhibitory’ ) or positively (‘ Enhancing’ ) modulate the CD8 T cell response compared to the corresponding prototype (‘Wuhan’) epitopes, identified by ex-vivo FluoroSPOT assay in vaccinated subjects (n=8); numbers in the # column allow to identify each peptide in the structure shown below. B) Trimeric structure of the prototype (Wuhan lineage) spike ectodomain (PDB 6ZXN) shown with one protomer (RBD-up) in yellow cartoon and the two other protomers (RBD-down) represented as light blue and ice surfaces. Inhibitory CD8 peptides ( left-side panel) are shown on the yellow protomer as red ribbons, with the mutated residues displayed as blue sticks. The enhancing CD8 epitopes ( right-side panel) are mapped on the yellow protomer as blue ribbons, with the mutated residues displayed as red sticks. Numbers (#) indicate the specific mutated peptides listed in panel (A); the substituted Ser13 residue of the enhancing peptides #6-9 is not displayed because the corresponding region in the spike protein structure is not resolved; NTD, N-terminal domain; RBD, receptor binding domain; S2, subunit 2.

Techniques Used: Variant Assay, Ex Vivo, Flurospot, Binding Assay

Effect of VOC and VOI mutations on spike-specific T cell responses in Covid-19 convalescent individuals. A) PBMCs from convalescent patients were stimulated for 18 hours by overlapping wild-type and variant peptides. Cytokine-secreting T cells were measured by FluoroSPOT assays. As in Fig. 1 , only subjects with significant ex-vivo SARS-CoV-2 spike-specific CD8 T cell responses for at least two of the three analyzed cytokines (IFN-γ, TNF-α and IL2; 6 out of 10 tested patients) are illustrated (see Fig. 1 legend for further details). B) Individual stimulatory peptides identified by ex-vivo FluoroSPOT assays in three convalescent patients; grey and black bars represent the responses induced by wild-type and variant peptides, respectively. C) Bars represent the percentage increase of ex-vivo spike-specific IFN-γ production detected by FluoroSPOT ( grey bars ), ICS ( blue bars ) and CD8 T cell degranulation ( red bars ) upon PBMC stimulation with mutated vs wild-type spike peptide pools (n=4). D) Bars represent the percentage increase of IFN-γ detected by ICS either ex-vivo on PBMC ( grey bars ) or in vitro on expanded spike-specific CD8 T cell lines stimulated with variant vs . wild-type peptide pools ( red bars ) (n=4).
Figure Legend Snippet: Effect of VOC and VOI mutations on spike-specific T cell responses in Covid-19 convalescent individuals. A) PBMCs from convalescent patients were stimulated for 18 hours by overlapping wild-type and variant peptides. Cytokine-secreting T cells were measured by FluoroSPOT assays. As in Fig. 1 , only subjects with significant ex-vivo SARS-CoV-2 spike-specific CD8 T cell responses for at least two of the three analyzed cytokines (IFN-γ, TNF-α and IL2; 6 out of 10 tested patients) are illustrated (see Fig. 1 legend for further details). B) Individual stimulatory peptides identified by ex-vivo FluoroSPOT assays in three convalescent patients; grey and black bars represent the responses induced by wild-type and variant peptides, respectively. C) Bars represent the percentage increase of ex-vivo spike-specific IFN-γ production detected by FluoroSPOT ( grey bars ), ICS ( blue bars ) and CD8 T cell degranulation ( red bars ) upon PBMC stimulation with mutated vs wild-type spike peptide pools (n=4). D) Bars represent the percentage increase of IFN-γ detected by ICS either ex-vivo on PBMC ( grey bars ) or in vitro on expanded spike-specific CD8 T cell lines stimulated with variant vs . wild-type peptide pools ( red bars ) (n=4).

Techniques Used: Variant Assay, Ex Vivo, In Vitro

Breadth of the global spike-specific T cell response in vaccinated subjects and in SARS-CoV-2 infection convalescent patients. PBMCs from vaccinated (n=26) and convalescent (n=10) individuals were stimulated for 18 hours with seven pools of overlapping 15-mer peptides spanning the entire spike sequence ( right ) and nine pools of 10 peptides representing CD8 T cell epitopes previously described in the literature varying in length from 9 to 13 AA ( left ). Color intensity in the heatmaps indicates IFN-γ, TNF-α or IL-2 production levels measured as SFCs generated upon stimulation with each spike peptide pool (lower than 25 th , 25 th -50 th , 50 th -75 th , higher than 75 th percentile as specified in the inset) of individual vaccinated ( panel A ) and convalescent ( panel B ) subjects.
Figure Legend Snippet: Breadth of the global spike-specific T cell response in vaccinated subjects and in SARS-CoV-2 infection convalescent patients. PBMCs from vaccinated (n=26) and convalescent (n=10) individuals were stimulated for 18 hours with seven pools of overlapping 15-mer peptides spanning the entire spike sequence ( right ) and nine pools of 10 peptides representing CD8 T cell epitopes previously described in the literature varying in length from 9 to 13 AA ( left ). Color intensity in the heatmaps indicates IFN-γ, TNF-α or IL-2 production levels measured as SFCs generated upon stimulation with each spike peptide pool (lower than 25 th , 25 th -50 th , 50 th -75 th , higher than 75 th percentile as specified in the inset) of individual vaccinated ( panel A ) and convalescent ( panel B ) subjects.

Techniques Used: Infection, Sequencing, Generated

7) Product Images from "Mechanisms of unconventional CD8 Tc2 lymphocyte induction in allergic contact dermatitis: Role of H3/H4 histamine receptors"

Article Title: Mechanisms of unconventional CD8 Tc2 lymphocyte induction in allergic contact dermatitis: Role of H3/H4 histamine receptors

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.999852

Analysis of the induction of a Th1/Tc1 profile. The bars represent mean ± SEM IFN-γ production by CD4 + ( A , panel a) and CD8 + ( B , panel a) cells in the gate of CD4 and CD8 lymphocytes, respectively. The intracytoplasmic determination was made from leukocytes from draining lymph nodes of treated mice after being cultured for 24 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml), with the addition of 10 µg/ml brefeldin A (Golgi plug). Permeabilized cells were stained with PE-labeled IFN-γ. * p
Figure Legend Snippet: Analysis of the induction of a Th1/Tc1 profile. The bars represent mean ± SEM IFN-γ production by CD4 + ( A , panel a) and CD8 + ( B , panel a) cells in the gate of CD4 and CD8 lymphocytes, respectively. The intracytoplasmic determination was made from leukocytes from draining lymph nodes of treated mice after being cultured for 24 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml), with the addition of 10 µg/ml brefeldin A (Golgi plug). Permeabilized cells were stained with PE-labeled IFN-γ. * p

Techniques Used: Mouse Assay, Cell Culture, Staining, Labeling

Induction of a regulatory phenotype in the presence of HA antagonists. (A) Intracytoplasmic staining of mononuclear cells stimulated in vitro for 24 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml). Cells were treated with brefeldin for 6 hours, permeabilized, and then IL-10 was labeled and the cells were counted in a cytometer. Evaluation of proportion of myeloid cells (B) and regulatory T cells in permeabilized cells (C) from draining lymph nodes. ( D, E , panel a) The determination of proliferation by dilution of CFSE is shown. Lymph node cells were labeled with the fluorescent dye CFSE and incubated for 96 hours in the presence of anti-CD3 (0.1 μg/ml) that was applied to the plate. A representative dot plot shows CD4 ( D , panel b) and CD8 lymphocytes ( E , panel b). Bars represent the mean ± SEM. ns (not significant); * p
Figure Legend Snippet: Induction of a regulatory phenotype in the presence of HA antagonists. (A) Intracytoplasmic staining of mononuclear cells stimulated in vitro for 24 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml). Cells were treated with brefeldin for 6 hours, permeabilized, and then IL-10 was labeled and the cells were counted in a cytometer. Evaluation of proportion of myeloid cells (B) and regulatory T cells in permeabilized cells (C) from draining lymph nodes. ( D, E , panel a) The determination of proliferation by dilution of CFSE is shown. Lymph node cells were labeled with the fluorescent dye CFSE and incubated for 96 hours in the presence of anti-CD3 (0.1 μg/ml) that was applied to the plate. A representative dot plot shows CD4 ( D , panel b) and CD8 lymphocytes ( E , panel b). Bars represent the mean ± SEM. ns (not significant); * p

Techniques Used: Staining, In Vitro, Labeling, Cytometry, Incubation

Blockade of signaling by blocking the H 4 R on DCs results in a tolerogenic profile. (A, B) The intracytoplasmic content of IL-13 is shown. Lymphocytes from draining lymph nodes were cultured for 24 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml), with the addition of brefeldin A (10 µg/ml). Finally, cells were permeabilized and stained (C) FOXp3 + CD4 + regulatory T cells stained from the draining lymph nodes of treated mice. Cells were permeabilized, then stained with the corresponding antibodies and analyzed by cytometry ( n = 6). (D, E) IL-10 intracytoplasmic determination in lymphocytes cultured for 18 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml), with the addition of brefeldin (10 µg/ml). Finally, cells were permeabilized, labeled, and analyzed by cytometry ( n = 6). ( F, G , panel a) Proliferation of leukocytes from the lymph node is shown. Cells were labeled with the fluorescent dye CFSE and incubated in plates pretreated with anti-CD3 (0.1 µg/ml). Ninety-six hours later, cells were analyzed by cytometry ( n = 3). A representative experimental histogram is shown for CD4 and CD8 cells ( F, G , panel b). (H) Induration was measured after 9 days of DC treatment. (I) Quantification of allergen-specific antibodies measured in serum of treated mice ( n = 7). Bars represent the mean ± SEM. * p
Figure Legend Snippet: Blockade of signaling by blocking the H 4 R on DCs results in a tolerogenic profile. (A, B) The intracytoplasmic content of IL-13 is shown. Lymphocytes from draining lymph nodes were cultured for 24 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml), with the addition of brefeldin A (10 µg/ml). Finally, cells were permeabilized and stained (C) FOXp3 + CD4 + regulatory T cells stained from the draining lymph nodes of treated mice. Cells were permeabilized, then stained with the corresponding antibodies and analyzed by cytometry ( n = 6). (D, E) IL-10 intracytoplasmic determination in lymphocytes cultured for 18 hours with PMA (10 ng/ml) + ionomycin (1 ng/ml), with the addition of brefeldin (10 µg/ml). Finally, cells were permeabilized, labeled, and analyzed by cytometry ( n = 6). ( F, G , panel a) Proliferation of leukocytes from the lymph node is shown. Cells were labeled with the fluorescent dye CFSE and incubated in plates pretreated with anti-CD3 (0.1 µg/ml). Ninety-six hours later, cells were analyzed by cytometry ( n = 3). A representative experimental histogram is shown for CD4 and CD8 cells ( F, G , panel b). (H) Induration was measured after 9 days of DC treatment. (I) Quantification of allergen-specific antibodies measured in serum of treated mice ( n = 7). Bars represent the mean ± SEM. * p

Techniques Used: Blocking Assay, Cell Culture, Staining, Mouse Assay, Cytometry, Labeling, Incubation

8) Product Images from "Long-term quantitative assessment of anti-SARS-CoV-2 spike protein immunogenicity (QUASI) after COVID-19 vaccination in older people living with HIV (PWH)"

Article Title: Long-term quantitative assessment of anti-SARS-CoV-2 spike protein immunogenicity (QUASI) after COVID-19 vaccination in older people living with HIV (PWH)

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-022-07737-0

Immunologic T cell subset testing. a SARS-CoV-2-specific T cell response after intracellular cytokine staining assay (ICS, 6 h). Cytokine production was defined as IFγ+TNFα–, IFγ+TNFα+ and IFγ-TNFα+ combined. Cytokine production was measured within live CD3+CD4+CD8- cells for CD4 response and live CD3+CD4-CD8+ cells for CD8 response. b SARS-CoV-2-specific T cells after activation induced marker assay (AIM, 20 h). SARS-CoV-2-specific CD4 T cells and CD8 T cells were defined as live CD3+CD4+CD8-OX40+CD137+ cells and CD3+CD4+CD8-CD69+CD137+ cells, respectively
Figure Legend Snippet: Immunologic T cell subset testing. a SARS-CoV-2-specific T cell response after intracellular cytokine staining assay (ICS, 6 h). Cytokine production was defined as IFγ+TNFα–, IFγ+TNFα+ and IFγ-TNFα+ combined. Cytokine production was measured within live CD3+CD4+CD8- cells for CD4 response and live CD3+CD4-CD8+ cells for CD8 response. b SARS-CoV-2-specific T cells after activation induced marker assay (AIM, 20 h). SARS-CoV-2-specific CD4 T cells and CD8 T cells were defined as live CD3+CD4+CD8-OX40+CD137+ cells and CD3+CD4+CD8-CD69+CD137+ cells, respectively

Techniques Used: Staining, Activation Assay, Marker

9) Product Images from "The acid ceramidase/ceramide axis controls parasitemia in Plasmodium yoelii-infected mice by regulating erythropoiesis"

Article Title: The acid ceramidase/ceramide axis controls parasitemia in Plasmodium yoelii-infected mice by regulating erythropoiesis

Journal: eLife

doi: 10.7554/eLife.77975

T cell response of P. yoelii -infected acid ceramidase (Ac) knockout (KO) and Ac wildtype (WT) mice 14 days post infection (p.i.). Percentages of Ki67-, CD49dCD11a/CD11a-, and PD1-expressing CD4+ and CD8+ T cells in spleen of P. yoelii -infected Ac WT mice and Ac KO littermates were determined by flow cytometry 14 days p.i. (n = 9). Data from three independent experiments are presented as mean ± SEM. T cell response of P. yoelii -infected acid ceramidase (Ac) knockout (KO) and Ac wildtype (WT) mice 14 days post infection (p.i.).
Figure Legend Snippet: T cell response of P. yoelii -infected acid ceramidase (Ac) knockout (KO) and Ac wildtype (WT) mice 14 days post infection (p.i.). Percentages of Ki67-, CD49dCD11a/CD11a-, and PD1-expressing CD4+ and CD8+ T cells in spleen of P. yoelii -infected Ac WT mice and Ac KO littermates were determined by flow cytometry 14 days p.i. (n = 9). Data from three independent experiments are presented as mean ± SEM. T cell response of P. yoelii -infected acid ceramidase (Ac) knockout (KO) and Ac wildtype (WT) mice 14 days post infection (p.i.).

Techniques Used: Infection, Knock-Out, Mouse Assay, Expressing, Flow Cytometry

T cell-specific and myeloid-specific acid ceramidase (Ac) deletion has no impact on the course of P . yoelii infection. ( A ) The knockout of Ac in T cells was confirmed by analyzing Asah1 mRNA expression of sorted splenic CD4 + and CD8 + T cells from naïve Asah1/Cd4 cre/+ (Ac CD4cre knockout [KO]) mice and Asah1/Cd4 +/+ littermates (Ac CD4cre wildtype [WT]) as controls via RT-qPCR (n = 2–4). ( B ) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac CD4cre KO mice and Ac CD4cre WT littermates was determined at indicated time points (n = 7–10). ( C ) The knockout of Ac in myeloid cells was confirmed by analyzing Asah1 mRNA expression of macrophages, dendritic cells, and neutrophils isolated from spleen, peritoneal lavage (pLavage), and blood of naïve Asah1/Lyz2 cre/+ (Ac Lyz2cre KO) mice and Asah1/Lyz2 +/+ littermates (Ac Lyz2cre WT) as controls via RT-qPCR (n = 2–6). ( D ) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac Lyz2cre KO and Ac Lyz2cre WT mice was determined at indicated time points (n = 9). Data from two independent experiments each are presented as mean ± SEM. T cell-specific and myeloid-specific acid ceramidase (Ac) deletion has no impact on the course of P. yoelii infection.
Figure Legend Snippet: T cell-specific and myeloid-specific acid ceramidase (Ac) deletion has no impact on the course of P . yoelii infection. ( A ) The knockout of Ac in T cells was confirmed by analyzing Asah1 mRNA expression of sorted splenic CD4 + and CD8 + T cells from naïve Asah1/Cd4 cre/+ (Ac CD4cre knockout [KO]) mice and Asah1/Cd4 +/+ littermates (Ac CD4cre wildtype [WT]) as controls via RT-qPCR (n = 2–4). ( B ) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac CD4cre KO mice and Ac CD4cre WT littermates was determined at indicated time points (n = 7–10). ( C ) The knockout of Ac in myeloid cells was confirmed by analyzing Asah1 mRNA expression of macrophages, dendritic cells, and neutrophils isolated from spleen, peritoneal lavage (pLavage), and blood of naïve Asah1/Lyz2 cre/+ (Ac Lyz2cre KO) mice and Asah1/Lyz2 +/+ littermates (Ac Lyz2cre WT) as controls via RT-qPCR (n = 2–6). ( D ) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac Lyz2cre KO and Ac Lyz2cre WT mice was determined at indicated time points (n = 9). Data from two independent experiments each are presented as mean ± SEM. T cell-specific and myeloid-specific acid ceramidase (Ac) deletion has no impact on the course of P. yoelii infection.

Techniques Used: Infection, Knock-Out, Expressing, Mouse Assay, Quantitative RT-PCR, Isolation

10) Product Images from "Inducing Ectopic T Cell Clusters Using Stromal Vascular Fraction Spheroid‐Based Immunotherapy to Enhance Anti‐Tumor Immunity, Inducing Ectopic T Cell Clusters Using Stromal Vascular Fraction Spheroid‐Based Immunotherapy to Enhance Anti‐Tumor Immunity"

Article Title: Inducing Ectopic T Cell Clusters Using Stromal Vascular Fraction Spheroid‐Based Immunotherapy to Enhance Anti‐Tumor Immunity, Inducing Ectopic T Cell Clusters Using Stromal Vascular Fraction Spheroid‐Based Immunotherapy to Enhance Anti‐Tumor Immunity

Journal: Advanced Science

doi: 10.1002/advs.202203842

SVF SPH and antigen‐loaded amDCs enhance antigen‐specific T cell response in both systemic and local immunity. a) Experimental design of immunization with SVF SPHs and antigen‐loaded amDCs via sub‐renal capsule transplantation. b) Representative dot plots of OVA‐specific CD4 + T cells stained with peptide‐loaded MHC tetramers in the spleen (top) and the kidney (bottom). Untreated group (UT); SVF SPH (SPH)‐, amDC‐, and SVF SPH + amDC‐transplanted groups; and OT‐II mouse splenocytes as the positive control (Pos Ctrl (OT‐II)). c) Frequency of OVA‐specific CD4 + and CD8 + T cells in the indicated groups, in the spleen to represent systemic immunity. d) Frequency of OVA‐specific CD4 + and CD8 + T cells in the kidney, to represent local immunity, in the indicated groups ( n = 4, mean ± s.e.m.). *: p
Figure Legend Snippet: SVF SPH and antigen‐loaded amDCs enhance antigen‐specific T cell response in both systemic and local immunity. a) Experimental design of immunization with SVF SPHs and antigen‐loaded amDCs via sub‐renal capsule transplantation. b) Representative dot plots of OVA‐specific CD4 + T cells stained with peptide‐loaded MHC tetramers in the spleen (top) and the kidney (bottom). Untreated group (UT); SVF SPH (SPH)‐, amDC‐, and SVF SPH + amDC‐transplanted groups; and OT‐II mouse splenocytes as the positive control (Pos Ctrl (OT‐II)). c) Frequency of OVA‐specific CD4 + and CD8 + T cells in the indicated groups, in the spleen to represent systemic immunity. d) Frequency of OVA‐specific CD4 + and CD8 + T cells in the kidney, to represent local immunity, in the indicated groups ( n = 4, mean ± s.e.m.). *: p

Techniques Used: Transplantation Assay, Staining, Positive Control

SVF SPHs and amDCs exhibit enhanced anti‐tumor effects. a) Experimental scheme for immunization with SVF SPHs and amDCs in mice bearing a tumor (OVA‐expressing B16 melanoma) via sub‐renal capsule transplantation. b) Tumor progression ( n = 8 per group, mean ± s.e.m.). c) Survival curves ( n = 5 per group). d) Experimental scheme for intratumoral injection (i.t) of SVF SPHs and amDCs in mice bearing a tumor (OVA‐expressing B16 melanoma). e) Tumor growth curves ( n = 9 to 10/group, mean ± s.e.m.) f–h) Tumors on day 15–17 were used for flow cytometric analysis and IF, separately. f) Representative images of dissected tumors from the mice group on day 15. g) Number of tumor‐infiltrating OVA‐specific CD4 + effector (CD44 + CD62L – ) and memory (CD44 + CD62L + ) T cells (top row) and CD8 + effector T cells (CD44 + CD62L – ) and memory (CD44 + CD62L + ) T cells (bottom row) per gram of tumor weight ( n = 5, mean ± s.e.m.). h) Confocal images of infiltrating CD3 + T cells in the tumor. The white dotted line represents the boundary of the tumor. Scale bar: 100 µm. *: p
Figure Legend Snippet: SVF SPHs and amDCs exhibit enhanced anti‐tumor effects. a) Experimental scheme for immunization with SVF SPHs and amDCs in mice bearing a tumor (OVA‐expressing B16 melanoma) via sub‐renal capsule transplantation. b) Tumor progression ( n = 8 per group, mean ± s.e.m.). c) Survival curves ( n = 5 per group). d) Experimental scheme for intratumoral injection (i.t) of SVF SPHs and amDCs in mice bearing a tumor (OVA‐expressing B16 melanoma). e) Tumor growth curves ( n = 9 to 10/group, mean ± s.e.m.) f–h) Tumors on day 15–17 were used for flow cytometric analysis and IF, separately. f) Representative images of dissected tumors from the mice group on day 15. g) Number of tumor‐infiltrating OVA‐specific CD4 + effector (CD44 + CD62L – ) and memory (CD44 + CD62L + ) T cells (top row) and CD8 + effector T cells (CD44 + CD62L – ) and memory (CD44 + CD62L + ) T cells (bottom row) per gram of tumor weight ( n = 5, mean ± s.e.m.). h) Confocal images of infiltrating CD3 + T cells in the tumor. The white dotted line represents the boundary of the tumor. Scale bar: 100 µm. *: p

Techniques Used: Mouse Assay, Expressing, Transplantation Assay, Injection

11) Product Images from "Cell-intrinsic ceramides determine T cell function during melanoma progression"

Article Title: Cell-intrinsic ceramides determine T cell function during melanoma progression

Journal: bioRxiv

doi: 10.1101/2022.08.31.505996

Ceramide co-localizes with CD3 and TCR. CD8 + T cells were isolated and stimulated with CD3/CD28 MACSiBead Particles for 2h and stained for ceramide (red) and (A) CD3 or (B) TCR beta (green). Cells were visualized using a Biorevo BZ-9000 fluorescence microscope.
Figure Legend Snippet: Ceramide co-localizes with CD3 and TCR. CD8 + T cells were isolated and stimulated with CD3/CD28 MACSiBead Particles for 2h and stained for ceramide (red) and (A) CD3 or (B) TCR beta (green). Cells were visualized using a Biorevo BZ-9000 fluorescence microscope.

Techniques Used: Isolation, Staining, Fluorescence, Microscopy

Ac-deficient CD8 + T cells have elevated ceramide levels and show increased activation in vitro . (A) Isolated CD8 + T cells from Ac flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. mRNA expression of Ac ( Asah1 ) following activation was analyzed by RT-qPCR (n= 3-4). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) For TCR signaling analysis, splenocytes from Ac flox/flox /CD4cre and WT mice were left unstimulated (0’) or stimulated with anti-CD3 and anti-CD28 for 5 (5’) min. Afterwards, samples we re analyzed for phospho-ZAP70 of gated ZAP70 + CD8 + T cells and phosphor-PLCγ of gated CD8 + T cells by flow cytometry (n= 4). Representative dot plots and FMOs for ZAP70 are shown in the left panel. (D) Western blot analysis of phospho-ZAP70 expression of CD8 + T cells from Ac flox/flox /CD4cre and WT mice after 5 min of stimulation with anti-CD3 and anti-CD28 (n= 3). (E) CD8 + T cells were left untreated as control or stimulated for 24 or 48 h and analyzed for granzyme B expression by flow cytometry (n= 5-8). Representative contour plots are shown in the left panel. (F) Specific killing of antigen-specific CTLs from Ac flox/flox /CD4cre/OTI mice and WT controls was assessed (n= 8-9). Representative histograms are shown in the left panel. Data are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p
Figure Legend Snippet: Ac-deficient CD8 + T cells have elevated ceramide levels and show increased activation in vitro . (A) Isolated CD8 + T cells from Ac flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. mRNA expression of Ac ( Asah1 ) following activation was analyzed by RT-qPCR (n= 3-4). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) For TCR signaling analysis, splenocytes from Ac flox/flox /CD4cre and WT mice were left unstimulated (0’) or stimulated with anti-CD3 and anti-CD28 for 5 (5’) min. Afterwards, samples we re analyzed for phospho-ZAP70 of gated ZAP70 + CD8 + T cells and phosphor-PLCγ of gated CD8 + T cells by flow cytometry (n= 4). Representative dot plots and FMOs for ZAP70 are shown in the left panel. (D) Western blot analysis of phospho-ZAP70 expression of CD8 + T cells from Ac flox/flox /CD4cre and WT mice after 5 min of stimulation with anti-CD3 and anti-CD28 (n= 3). (E) CD8 + T cells were left untreated as control or stimulated for 24 or 48 h and analyzed for granzyme B expression by flow cytometry (n= 5-8). Representative contour plots are shown in the left panel. (F) Specific killing of antigen-specific CTLs from Ac flox/flox /CD4cre/OTI mice and WT controls was assessed (n= 8-9). Representative histograms are shown in the left panel. Data are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p

Techniques Used: Activation Assay, In Vitro, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Mass Spectrometry, Flow Cytometry, Western Blot, MANN-WHITNEY

Impaired CD8 + T cell function in Asm-deficient tumor-bearing mice upon CD4 + T cell depletion. CD4 + T cells were depleted from Asm-WT and Asm-KO mice by repeated i.p. injection of anti-CD4 depleting antibody. Control groups received PBS. B16-F1 tumor cells were transplanted one day later s.c. (A) Tumor volume was determined after tumor establishment based on caliper measurements (n= 3-7). (B) Frequencies of CD4 + and CD8 + T cells in dLN and tumor and (C) expression of IFN-γ, CD44, and granzyme B of CD8 + tumor-infiltrating lymphocytes (TILs) were analyzed using flow cytometry. Representative dots plots are shown in the left panel. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p
Figure Legend Snippet: Impaired CD8 + T cell function in Asm-deficient tumor-bearing mice upon CD4 + T cell depletion. CD4 + T cells were depleted from Asm-WT and Asm-KO mice by repeated i.p. injection of anti-CD4 depleting antibody. Control groups received PBS. B16-F1 tumor cells were transplanted one day later s.c. (A) Tumor volume was determined after tumor establishment based on caliper measurements (n= 3-7). (B) Frequencies of CD4 + and CD8 + T cells in dLN and tumor and (C) expression of IFN-γ, CD44, and granzyme B of CD8 + tumor-infiltrating lymphocytes (TILs) were analyzed using flow cytometry. Representative dots plots are shown in the left panel. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p

Techniques Used: Cell Function Assay, Mouse Assay, Injection, Expressing, Flow Cytometry

Elevated anti-tumor immune response in T cell-specific Ac-deficient mice. (A) B16-F1 melanoma cells were transplanted into Ac flox/flox /CD4cre mice and WT littermates. Tumor volume was monitored once tumors have been established (n= 8-10). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells were determined by flow cytometry and absolute cell numbers were calculated. (C) Frequencies of IFN-γ + and granzyme B + TILs were determined by flow cytometry. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p
Figure Legend Snippet: Elevated anti-tumor immune response in T cell-specific Ac-deficient mice. (A) B16-F1 melanoma cells were transplanted into Ac flox/flox /CD4cre mice and WT littermates. Tumor volume was monitored once tumors have been established (n= 8-10). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells were determined by flow cytometry and absolute cell numbers were calculated. (C) Frequencies of IFN-γ + and granzyme B + TILs were determined by flow cytometry. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p

Techniques Used: Mouse Assay, Flow Cytometry

Ablation of Asm results in decreased T cell activation and enhanced tumor growth. (A) B16-F1 melanoma cells were transplanted into Asm-KO mice and WT littermates. Tumor volume was monitored daily once tumors were palpable (n= 12-18). (B) Frequencies of CD4 + T cells, CD4 + Foxp3 + Tregs, and CD8 + T cells within draining lymph nodes (dLN) and tumor of Asm-KO and Asm-WT mice were determined by flow cytometry. Representative contour plots (dLN) are shown in the left panel. (C) IFN-γ, and CD44 expression of CD4 + and CD8 + tumor-infiltrating lymphocytes (TILs) in tumor-bearing mice. (D) Percentages of Foxp3 + Tregs of CD4 + T cells within spleen of naïve Asm-KO and Asm-WT mice were determined by flow cytometry (n= 16-17). (E) Sorted CD4 + CD25 - T cells from Asm-KO and Asm-WT mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 and TGF-β1 (iTreg). Respective controls (Th0) were only stimulated with anti-CD3 and anti-CD28 antibodies. After 3 days, Treg differentiation was analyzed by Foxp3 expression (n= 3-4). Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p
Figure Legend Snippet: Ablation of Asm results in decreased T cell activation and enhanced tumor growth. (A) B16-F1 melanoma cells were transplanted into Asm-KO mice and WT littermates. Tumor volume was monitored daily once tumors were palpable (n= 12-18). (B) Frequencies of CD4 + T cells, CD4 + Foxp3 + Tregs, and CD8 + T cells within draining lymph nodes (dLN) and tumor of Asm-KO and Asm-WT mice were determined by flow cytometry. Representative contour plots (dLN) are shown in the left panel. (C) IFN-γ, and CD44 expression of CD4 + and CD8 + tumor-infiltrating lymphocytes (TILs) in tumor-bearing mice. (D) Percentages of Foxp3 + Tregs of CD4 + T cells within spleen of naïve Asm-KO and Asm-WT mice were determined by flow cytometry (n= 16-17). (E) Sorted CD4 + CD25 - T cells from Asm-KO and Asm-WT mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 and TGF-β1 (iTreg). Respective controls (Th0) were only stimulated with anti-CD3 and anti-CD28 antibodies. After 3 days, Treg differentiation was analyzed by Foxp3 expression (n= 3-4). Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Expressing

Cell-intrinsic Asm activity determines CD8 + T cell activation in vivo . (A) B16-F1 melanoma cells were transplanted into Asm flox/flox /CD4cre mice and WT littermates and tumor growth was monitored when tumors reached a detectable size (n= 12-16). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells within dLN and tumor were determined by flow cytometry and absolute cell numbers were calculated. Representative dot plots are shown in the upper panel. (C) Expression of IFN-γ, TNF-α, and granzyme B of TILs were determined by flow cytometry. (Results from 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p
Figure Legend Snippet: Cell-intrinsic Asm activity determines CD8 + T cell activation in vivo . (A) B16-F1 melanoma cells were transplanted into Asm flox/flox /CD4cre mice and WT littermates and tumor growth was monitored when tumors reached a detectable size (n= 12-16). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells within dLN and tumor were determined by flow cytometry and absolute cell numbers were calculated. Representative dot plots are shown in the upper panel. (C) Expression of IFN-γ, TNF-α, and granzyme B of TILs were determined by flow cytometry. (Results from 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p

Techniques Used: Activity Assay, Activation Assay, In Vivo, Mouse Assay, Flow Cytometry, Expressing, MANN-WHITNEY

T cell specific Asm deficiency leads to reduced CD8 + T cell activation in vitro . Isolated CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. (A) mRNA expression of Asm ( Smpd1 ) following activation was analyzed by RT-qPCR (n=4-8). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) Expression of CD25, CD69, and CD44 was analyzed by flow cytometry (n= 6-7). Representative histograms for are shown in the upper panel. (D) Ova-specific CTLs were generated and incubated with Ova-peptide 257-264 loaded CFSE high -labeled target and unloaded CFSE low -labeled control cells. Specific killing was evaluated by frequencies of target and control populations determined by flow cytometry (n= 6-7). Representative histograms are shown in the left panel. (E) Frequencies of granzyme B-expressing CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates without and (F) in the presence of C16 ceramide were analyzed by flow cytometry (n= 4-8). Representative contour plots are shown in the left panel. Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p
Figure Legend Snippet: T cell specific Asm deficiency leads to reduced CD8 + T cell activation in vitro . Isolated CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. (A) mRNA expression of Asm ( Smpd1 ) following activation was analyzed by RT-qPCR (n=4-8). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) Expression of CD25, CD69, and CD44 was analyzed by flow cytometry (n= 6-7). Representative histograms for are shown in the upper panel. (D) Ova-specific CTLs were generated and incubated with Ova-peptide 257-264 loaded CFSE high -labeled target and unloaded CFSE low -labeled control cells. Specific killing was evaluated by frequencies of target and control populations determined by flow cytometry (n= 6-7). Representative histograms are shown in the left panel. (E) Frequencies of granzyme B-expressing CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates without and (F) in the presence of C16 ceramide were analyzed by flow cytometry (n= 4-8). Representative contour plots are shown in the left panel. Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p

Techniques Used: Activation Assay, In Vitro, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Mass Spectrometry, Flow Cytometry, Generated, Incubation, Labeling, MANN-WHITNEY

12) Product Images from "Cell-intrinsic ceramides determine T cell function during melanoma progression"

Article Title: Cell-intrinsic ceramides determine T cell function during melanoma progression

Journal: bioRxiv

doi: 10.1101/2022.08.31.505996

Ceramide co-localizes with CD3 and TCR. CD8 + T cells were isolated and stimulated with CD3/CD28 MACSiBead Particles for 2h and stained for ceramide (red) and (A) CD3 or (B) TCR beta (green). Cells were visualized using a Biorevo BZ-9000 fluorescence microscope.
Figure Legend Snippet: Ceramide co-localizes with CD3 and TCR. CD8 + T cells were isolated and stimulated with CD3/CD28 MACSiBead Particles for 2h and stained for ceramide (red) and (A) CD3 or (B) TCR beta (green). Cells were visualized using a Biorevo BZ-9000 fluorescence microscope.

Techniques Used: Isolation, Staining, Fluorescence, Microscopy

Ac-deficient CD8 + T cells have elevated ceramide levels and show increased activation in vitro . (A) Isolated CD8 + T cells from Ac flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. mRNA expression of Ac ( Asah1 ) following activation was analyzed by RT-qPCR (n= 3-4). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) For TCR signaling analysis, splenocytes from Ac flox/flox /CD4cre and WT mice were left unstimulated (0’) or stimulated with anti-CD3 and anti-CD28 for 5 (5’) min. Afterwards, samples we re analyzed for phospho-ZAP70 of gated ZAP70 + CD8 + T cells and phosphor-PLCγ of gated CD8 + T cells by flow cytometry (n= 4). Representative dot plots and FMOs for ZAP70 are shown in the left panel. (D) Western blot analysis of phospho-ZAP70 expression of CD8 + T cells from Ac flox/flox /CD4cre and WT mice after 5 min of stimulation with anti-CD3 and anti-CD28 (n= 3). (E) CD8 + T cells were left untreated as control or stimulated for 24 or 48 h and analyzed for granzyme B expression by flow cytometry (n= 5-8). Representative contour plots are shown in the left panel. (F) Specific killing of antigen-specific CTLs from Ac flox/flox /CD4cre/OTI mice and WT controls was assessed (n= 8-9). Representative histograms are shown in the left panel. Data are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p
Figure Legend Snippet: Ac-deficient CD8 + T cells have elevated ceramide levels and show increased activation in vitro . (A) Isolated CD8 + T cells from Ac flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. mRNA expression of Ac ( Asah1 ) following activation was analyzed by RT-qPCR (n= 3-4). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) For TCR signaling analysis, splenocytes from Ac flox/flox /CD4cre and WT mice were left unstimulated (0’) or stimulated with anti-CD3 and anti-CD28 for 5 (5’) min. Afterwards, samples we re analyzed for phospho-ZAP70 of gated ZAP70 + CD8 + T cells and phosphor-PLCγ of gated CD8 + T cells by flow cytometry (n= 4). Representative dot plots and FMOs for ZAP70 are shown in the left panel. (D) Western blot analysis of phospho-ZAP70 expression of CD8 + T cells from Ac flox/flox /CD4cre and WT mice after 5 min of stimulation with anti-CD3 and anti-CD28 (n= 3). (E) CD8 + T cells were left untreated as control or stimulated for 24 or 48 h and analyzed for granzyme B expression by flow cytometry (n= 5-8). Representative contour plots are shown in the left panel. (F) Specific killing of antigen-specific CTLs from Ac flox/flox /CD4cre/OTI mice and WT controls was assessed (n= 8-9). Representative histograms are shown in the left panel. Data are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p

Techniques Used: Activation Assay, In Vitro, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Mass Spectrometry, Flow Cytometry, Western Blot, MANN-WHITNEY

Impaired CD8 + T cell function in Asm-deficient tumor-bearing mice upon CD4 + T cell depletion. CD4 + T cells were depleted from Asm-WT and Asm-KO mice by repeated i.p. injection of anti-CD4 depleting antibody. Control groups received PBS. B16-F1 tumor cells were transplanted one day later s.c. (A) Tumor volume was determined after tumor establishment based on caliper measurements (n= 3-7). (B) Frequencies of CD4 + and CD8 + T cells in dLN and tumor and (C) expression of IFN-γ, CD44, and granzyme B of CD8 + tumor-infiltrating lymphocytes (TILs) were analyzed using flow cytometry. Representative dots plots are shown in the left panel. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p
Figure Legend Snippet: Impaired CD8 + T cell function in Asm-deficient tumor-bearing mice upon CD4 + T cell depletion. CD4 + T cells were depleted from Asm-WT and Asm-KO mice by repeated i.p. injection of anti-CD4 depleting antibody. Control groups received PBS. B16-F1 tumor cells were transplanted one day later s.c. (A) Tumor volume was determined after tumor establishment based on caliper measurements (n= 3-7). (B) Frequencies of CD4 + and CD8 + T cells in dLN and tumor and (C) expression of IFN-γ, CD44, and granzyme B of CD8 + tumor-infiltrating lymphocytes (TILs) were analyzed using flow cytometry. Representative dots plots are shown in the left panel. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p

Techniques Used: Cell Function Assay, Mouse Assay, Injection, Expressing, Flow Cytometry

Elevated anti-tumor immune response in T cell-specific Ac-deficient mice. (A) B16-F1 melanoma cells were transplanted into Ac flox/flox /CD4cre mice and WT littermates. Tumor volume was monitored once tumors have been established (n= 8-10). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells were determined by flow cytometry and absolute cell numbers were calculated. (C) Frequencies of IFN-γ + and granzyme B + TILs were determined by flow cytometry. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p
Figure Legend Snippet: Elevated anti-tumor immune response in T cell-specific Ac-deficient mice. (A) B16-F1 melanoma cells were transplanted into Ac flox/flox /CD4cre mice and WT littermates. Tumor volume was monitored once tumors have been established (n= 8-10). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells were determined by flow cytometry and absolute cell numbers were calculated. (C) Frequencies of IFN-γ + and granzyme B + TILs were determined by flow cytometry. Results from 2 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p

Techniques Used: Mouse Assay, Flow Cytometry

Ablation of Asm results in decreased T cell activation and enhanced tumor growth. (A) B16-F1 melanoma cells were transplanted into Asm-KO mice and WT littermates. Tumor volume was monitored daily once tumors were palpable (n= 12-18). (B) Frequencies of CD4 + T cells, CD4 + Foxp3 + Tregs, and CD8 + T cells within draining lymph nodes (dLN) and tumor of Asm-KO and Asm-WT mice were determined by flow cytometry. Representative contour plots (dLN) are shown in the left panel. (C) IFN-γ, and CD44 expression of CD4 + and CD8 + tumor-infiltrating lymphocytes (TILs) in tumor-bearing mice. (D) Percentages of Foxp3 + Tregs of CD4 + T cells within spleen of naïve Asm-KO and Asm-WT mice were determined by flow cytometry (n= 16-17). (E) Sorted CD4 + CD25 - T cells from Asm-KO and Asm-WT mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 and TGF-β1 (iTreg). Respective controls (Th0) were only stimulated with anti-CD3 and anti-CD28 antibodies. After 3 days, Treg differentiation was analyzed by Foxp3 expression (n= 3-4). Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p
Figure Legend Snippet: Ablation of Asm results in decreased T cell activation and enhanced tumor growth. (A) B16-F1 melanoma cells were transplanted into Asm-KO mice and WT littermates. Tumor volume was monitored daily once tumors were palpable (n= 12-18). (B) Frequencies of CD4 + T cells, CD4 + Foxp3 + Tregs, and CD8 + T cells within draining lymph nodes (dLN) and tumor of Asm-KO and Asm-WT mice were determined by flow cytometry. Representative contour plots (dLN) are shown in the left panel. (C) IFN-γ, and CD44 expression of CD4 + and CD8 + tumor-infiltrating lymphocytes (TILs) in tumor-bearing mice. (D) Percentages of Foxp3 + Tregs of CD4 + T cells within spleen of naïve Asm-KO and Asm-WT mice were determined by flow cytometry (n= 16-17). (E) Sorted CD4 + CD25 - T cells from Asm-KO and Asm-WT mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-2 and TGF-β1 (iTreg). Respective controls (Th0) were only stimulated with anti-CD3 and anti-CD28 antibodies. After 3 days, Treg differentiation was analyzed by Foxp3 expression (n= 3-4). Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons or Student’s t-test. (*p

Techniques Used: Activation Assay, Mouse Assay, Flow Cytometry, Expressing

Cell-intrinsic Asm activity determines CD8 + T cell activation in vivo . (A) B16-F1 melanoma cells were transplanted into Asm flox/flox /CD4cre mice and WT littermates and tumor growth was monitored when tumors reached a detectable size (n= 12-16). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells within dLN and tumor were determined by flow cytometry and absolute cell numbers were calculated. Representative dot plots are shown in the upper panel. (C) Expression of IFN-γ, TNF-α, and granzyme B of TILs were determined by flow cytometry. (Results from 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p
Figure Legend Snippet: Cell-intrinsic Asm activity determines CD8 + T cell activation in vivo . (A) B16-F1 melanoma cells were transplanted into Asm flox/flox /CD4cre mice and WT littermates and tumor growth was monitored when tumors reached a detectable size (n= 12-16). (B) Percentages of CD4 + T cells, Foxp3 + Tregs, and CD8 + T cells within dLN and tumor were determined by flow cytometry and absolute cell numbers were calculated. Representative dot plots are shown in the upper panel. (C) Expression of IFN-γ, TNF-α, and granzyme B of TILs were determined by flow cytometry. (Results from 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p

Techniques Used: Activity Assay, Activation Assay, In Vivo, Mouse Assay, Flow Cytometry, Expressing, MANN-WHITNEY

T cell specific Asm deficiency leads to reduced CD8 + T cell activation in vitro . Isolated CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. (A) mRNA expression of Asm ( Smpd1 ) following activation was analyzed by RT-qPCR (n=4-8). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) Expression of CD25, CD69, and CD44 was analyzed by flow cytometry (n= 6-7). Representative histograms for are shown in the upper panel. (D) Ova-specific CTLs were generated and incubated with Ova-peptide 257-264 loaded CFSE high -labeled target and unloaded CFSE low -labeled control cells. Specific killing was evaluated by frequencies of target and control populations determined by flow cytometry (n= 6-7). Representative histograms are shown in the left panel. (E) Frequencies of granzyme B-expressing CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates without and (F) in the presence of C16 ceramide were analyzed by flow cytometry (n= 4-8). Representative contour plots are shown in the left panel. Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p
Figure Legend Snippet: T cell specific Asm deficiency leads to reduced CD8 + T cell activation in vitro . Isolated CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates where either left unstimulated or stimulated with anti-CD3 and anti-CD28 for indicated time points. (A) mRNA expression of Asm ( Smpd1 ) following activation was analyzed by RT-qPCR (n=4-8). (B) Ceramide levels of CD8 + T cells were determined by mass spectrometry (n= 4). (C) Expression of CD25, CD69, and CD44 was analyzed by flow cytometry (n= 6-7). Representative histograms for are shown in the upper panel. (D) Ova-specific CTLs were generated and incubated with Ova-peptide 257-264 loaded CFSE high -labeled target and unloaded CFSE low -labeled control cells. Specific killing was evaluated by frequencies of target and control populations determined by flow cytometry (n= 6-7). Representative histograms are shown in the left panel. (E) Frequencies of granzyme B-expressing CD8 + T cells from Asm flox/flox /CD4cre mice and WT littermates without and (F) in the presence of C16 ceramide were analyzed by flow cytometry (n= 4-8). Representative contour plots are shown in the left panel. Results from 2 to 4 independent experiments are depicted as mean ± SEM. Statistical analysis was performed by 2way ANOVA with Sidak’s multiple comparisons, Mann-Whitney U-test or Student’s t-test. (*p

Techniques Used: Activation Assay, In Vitro, Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Mass Spectrometry, Flow Cytometry, Generated, Incubation, Labeling, MANN-WHITNEY

13) Product Images from "Heat-killed Limosilactobacillus reuteri PSC102 Ameliorates Impaired Immunity in Cyclophosphamide-induced Immunosuppressed Mice"

Article Title: Heat-killed Limosilactobacillus reuteri PSC102 Ameliorates Impaired Immunity in Cyclophosphamide-induced Immunosuppressed Mice

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2022.820838

Effects of hLR on thymic and splenic T-lymphocyte subpopulations. (A) Thymic T-lymphocyte subpopulations (CD4 + , region Q1; CD8 + , region Q4). (B) Splenic T-lymphocyte subpopulations (CD4 + , region Q1; CD8 + , region Q4). Data are the mean ± SEM ( n = 4). Different letters above the bars indicate a significant difference between the groups ( p
Figure Legend Snippet: Effects of hLR on thymic and splenic T-lymphocyte subpopulations. (A) Thymic T-lymphocyte subpopulations (CD4 + , region Q1; CD8 + , region Q4). (B) Splenic T-lymphocyte subpopulations (CD4 + , region Q1; CD8 + , region Q4). Data are the mean ± SEM ( n = 4). Different letters above the bars indicate a significant difference between the groups ( p

Techniques Used:

14) Product Images from "The Pioneer Transcription Factor Foxa2 Modulates T Helper Differentiation to Reduce Mouse Allergic Airway Disease"

Article Title: The Pioneer Transcription Factor Foxa2 Modulates T Helper Differentiation to Reduce Mouse Allergic Airway Disease

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.890781

Absence of Foxa2 in T-cells enhances Th2-mediated disease in AAD. Control and Foxa2cKO mice were immunized intranasally with 25μg papain (or PBS as control) at day 0 and day 7 and analysed at day 10. (A, B) Foxa2 expression (QRT-PCR) representative of three independent experiments of sorted (A) CD4+CD3+ T-cells and (B) CD45-CD326+ epithelial cells from lungs of control (n=3) and Foxa2cKO (n=3) groups after PBS and papain-treatment. (C) Flow cytometry: T-cell populations in control and Foxa2cKO BAL, lung and mLN after papain-treatment. (D, E) Barcharts show percentage and cell number of (D) CD8 and (E) CD4 T-cells in BAL, lung and mLN from papain-treated control and Foxa2cKO mice (n=4-7). (F, G) Flow cytometry (lung) from control (n=3) and Foxa2cKO (n=3) after papain-treatment. (F) Dot plots: CD4 expression versus SSC. Contour plots: CD25 and icFoxp3 expression gated on lung CD4+ cells. (G) Barcharts: percentage (left) of CD25+icFoxp3+ cells in CD4+ population; number (right) of CD4+CD25+icFoxp3+ cells. (H) Histogram: icTbet expression in CD4 T-cells in control and Foxa2cKO lungs after papain-treatment. Barchart: mean fluorescence intensity (MFI) of icTbet in CD4 T-cells from control and Foxa2cKO lungs after papain-treatment. (I) Histogram: icGata3 expression from control and Foxa2cKO lungs after papain-treatment. Barchart: percentage of icGata3+ cells in lung CD4 T-cells from control and Foxa2cKO after papain-treatment. For (H, I) , grey histograms show isotype control antibody staining; control (n=4) and Foxa2cKO (n=6). (J) Flow cytometry: icIL-4 (upper) and icIL-13 (lower) in CD4 T-cells from control and Foxa2cKO mLN after papain-treatment. Barchart: percentage of cytokine-positive cells from control (n=5) and Foxa2cKO mice (n=7) after papain-treatment. (K) Flow cytometry: Ccr3 expression on CD4 T-cells from control and Foxa2cKO mLN after papain-treatment. Barchart: percentage of Ccr3+ cells in CD4 T-cell population in mLN from control (n=5) and Foxa2cKO (n=7) after papain-treatment. Au: arbitrary units; Barcharts: mean ± SEM; *p
Figure Legend Snippet: Absence of Foxa2 in T-cells enhances Th2-mediated disease in AAD. Control and Foxa2cKO mice were immunized intranasally with 25μg papain (or PBS as control) at day 0 and day 7 and analysed at day 10. (A, B) Foxa2 expression (QRT-PCR) representative of three independent experiments of sorted (A) CD4+CD3+ T-cells and (B) CD45-CD326+ epithelial cells from lungs of control (n=3) and Foxa2cKO (n=3) groups after PBS and papain-treatment. (C) Flow cytometry: T-cell populations in control and Foxa2cKO BAL, lung and mLN after papain-treatment. (D, E) Barcharts show percentage and cell number of (D) CD8 and (E) CD4 T-cells in BAL, lung and mLN from papain-treated control and Foxa2cKO mice (n=4-7). (F, G) Flow cytometry (lung) from control (n=3) and Foxa2cKO (n=3) after papain-treatment. (F) Dot plots: CD4 expression versus SSC. Contour plots: CD25 and icFoxp3 expression gated on lung CD4+ cells. (G) Barcharts: percentage (left) of CD25+icFoxp3+ cells in CD4+ population; number (right) of CD4+CD25+icFoxp3+ cells. (H) Histogram: icTbet expression in CD4 T-cells in control and Foxa2cKO lungs after papain-treatment. Barchart: mean fluorescence intensity (MFI) of icTbet in CD4 T-cells from control and Foxa2cKO lungs after papain-treatment. (I) Histogram: icGata3 expression from control and Foxa2cKO lungs after papain-treatment. Barchart: percentage of icGata3+ cells in lung CD4 T-cells from control and Foxa2cKO after papain-treatment. For (H, I) , grey histograms show isotype control antibody staining; control (n=4) and Foxa2cKO (n=6). (J) Flow cytometry: icIL-4 (upper) and icIL-13 (lower) in CD4 T-cells from control and Foxa2cKO mLN after papain-treatment. Barchart: percentage of cytokine-positive cells from control (n=5) and Foxa2cKO mice (n=7) after papain-treatment. (K) Flow cytometry: Ccr3 expression on CD4 T-cells from control and Foxa2cKO mLN after papain-treatment. Barchart: percentage of Ccr3+ cells in CD4 T-cell population in mLN from control (n=5) and Foxa2cKO (n=7) after papain-treatment. Au: arbitrary units; Barcharts: mean ± SEM; *p

Techniques Used: Mouse Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Fluorescence, Staining

15) Product Images from "Upregulation of NKG2D ligands impairs hematopoietic stem cell function in Fanconi anemia"

Article Title: Upregulation of NKG2D ligands impairs hematopoietic stem cell function in Fanconi anemia

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI142842

Comparative analysis of BM NK and T cells from HDs and patients with FA. ( A ) Percentage of NK cells (CD56 + CD3 – ; FA n = 22, HD n = 8) and of NK cells with strong cytotoxic capacity (CD56 +/lo CD16 + CD3 – ; FA n = 15, HD n = 6) and activated NK cells (CD56 + CD69 + CD3 – ; FA n = 14, HD n = 6) in mononuclear BM cells from HDs and patients with FA. Lower panels show the corresponding percentages of BM cytotoxic T cells (CD8 + CD3 + ; FA n = 11, HD n = 8), activated cytotoxic T cells (CD8 + CD69 + CD3 + ; FA n = 11, HD n = 6), and Th cells (CD4 + CD3 + ; FA n = 6, HD n = 9). To compare data between HD and FA samples, an unpaired t test was used for comparisons between CD8 + CD3 + cells, with Welch’s correction for CD56 +lo CD16 + CD3 – and CD4 + CD3 + cells, and the Mann-Whitney U test was used for comparisons between CD56 + CD3 – , CD56 + CD69 + CD3 – , and CD8 + CD69 + CD3 + cells. ( B ) Left panels show representative histograms of NKG2D receptor levels in NK and CD8 + T cells from HDs and patients with FA. Right panels show comparative analyses of NKG2D receptor expression in BM samples from HDs and patients with FA ( n = 3). An unpaired t test was used for comparisons of NKG2D receptor expression between HD and FA samples in CD56 + and CD8 + cell populations. Mean values ± SEM are shown in all panels.
Figure Legend Snippet: Comparative analysis of BM NK and T cells from HDs and patients with FA. ( A ) Percentage of NK cells (CD56 + CD3 – ; FA n = 22, HD n = 8) and of NK cells with strong cytotoxic capacity (CD56 +/lo CD16 + CD3 – ; FA n = 15, HD n = 6) and activated NK cells (CD56 + CD69 + CD3 – ; FA n = 14, HD n = 6) in mononuclear BM cells from HDs and patients with FA. Lower panels show the corresponding percentages of BM cytotoxic T cells (CD8 + CD3 + ; FA n = 11, HD n = 8), activated cytotoxic T cells (CD8 + CD69 + CD3 + ; FA n = 11, HD n = 6), and Th cells (CD4 + CD3 + ; FA n = 6, HD n = 9). To compare data between HD and FA samples, an unpaired t test was used for comparisons between CD8 + CD3 + cells, with Welch’s correction for CD56 +lo CD16 + CD3 – and CD4 + CD3 + cells, and the Mann-Whitney U test was used for comparisons between CD56 + CD3 – , CD56 + CD69 + CD3 – , and CD8 + CD69 + CD3 + cells. ( B ) Left panels show representative histograms of NKG2D receptor levels in NK and CD8 + T cells from HDs and patients with FA. Right panels show comparative analyses of NKG2D receptor expression in BM samples from HDs and patients with FA ( n = 3). An unpaired t test was used for comparisons of NKG2D receptor expression between HD and FA samples in CD56 + and CD8 + cell populations. Mean values ± SEM are shown in all panels.

Techniques Used: MANN-WHITNEY, Expressing

Implications of NKG2D–NKG2D-L interactions in a Fanca –/– mouse BMF model. ( A ) Experimental protocol used for evaluating the effect of NKG2D–NKG2D-L interactions in the BMF induced by MMC in Fanca –/– mice. Mice were periodically treated with an isotype control or an anti-NKG2D mAb, prior to and after initiating MMC treatment (2 doses of 0.3 mg/kg). PB samples from Fanca –/– mice were obtained prior to and after treatment. ( B ) Representative flow cytometric analysis showing expression of NKG2D-Ls in Lin – c-Kit + cells from Fanca –/– mice treated with MMC compared with expression in untreated mice. ( C ) Proportion of Lin – c-Kit + cells and LSK cells positive for NKG2D-Ls at the end of the experimental protocol (day 14) in untreated or MMC-treated Fanca –/– mice receiving the isotype control or the anti-NKG2D antibody. ( D ) Proportion of NK cells and CD8 + and CD4 + T cells, and their respective activated subpopulations in mononuclear BM cells from Fanca –/– mice treated as in C . ( E ) Comparative analysis of PB cell parameters in MMC-treated Fanca –/– mice that received the isotype control (blue dots) or the anti-NKG2D mAB (red dots). Each dot shows changes in PB parameters on days 7 and 14 after MMC treatment with respect to the values determined on day –4 (prior to starting mAb and MMC treatments), which were considered as 100%. The mean PB values corresponding to the isotype control– and the anti-NKG2D–treated groups on day –4 were, respectively, hemoglobin: 14.53 and 13.38 g/dL; RBCs: 10.37 and 9.32 × 10 6 cells/μL; WBCs: 4.64 and 5.08 × 10 3 cells/μL; platelets: 1002 and 914 × 10 3 cells/μL. The Mann-Whitney U test was used to compare day 7 mean values and an unpaired t test to compare day 14 mean values according to the respective data distributions. Whiskers represent the minimum and maximum values, the lower and upper box edges correspond to the 25th and 75th percentiles, respectively, and the lines within the boxes correspond to the median.
Figure Legend Snippet: Implications of NKG2D–NKG2D-L interactions in a Fanca –/– mouse BMF model. ( A ) Experimental protocol used for evaluating the effect of NKG2D–NKG2D-L interactions in the BMF induced by MMC in Fanca –/– mice. Mice were periodically treated with an isotype control or an anti-NKG2D mAb, prior to and after initiating MMC treatment (2 doses of 0.3 mg/kg). PB samples from Fanca –/– mice were obtained prior to and after treatment. ( B ) Representative flow cytometric analysis showing expression of NKG2D-Ls in Lin – c-Kit + cells from Fanca –/– mice treated with MMC compared with expression in untreated mice. ( C ) Proportion of Lin – c-Kit + cells and LSK cells positive for NKG2D-Ls at the end of the experimental protocol (day 14) in untreated or MMC-treated Fanca –/– mice receiving the isotype control or the anti-NKG2D antibody. ( D ) Proportion of NK cells and CD8 + and CD4 + T cells, and their respective activated subpopulations in mononuclear BM cells from Fanca –/– mice treated as in C . ( E ) Comparative analysis of PB cell parameters in MMC-treated Fanca –/– mice that received the isotype control (blue dots) or the anti-NKG2D mAB (red dots). Each dot shows changes in PB parameters on days 7 and 14 after MMC treatment with respect to the values determined on day –4 (prior to starting mAb and MMC treatments), which were considered as 100%. The mean PB values corresponding to the isotype control– and the anti-NKG2D–treated groups on day –4 were, respectively, hemoglobin: 14.53 and 13.38 g/dL; RBCs: 10.37 and 9.32 × 10 6 cells/μL; WBCs: 4.64 and 5.08 × 10 3 cells/μL; platelets: 1002 and 914 × 10 3 cells/μL. The Mann-Whitney U test was used to compare day 7 mean values and an unpaired t test to compare day 14 mean values according to the respective data distributions. Whiskers represent the minimum and maximum values, the lower and upper box edges correspond to the 25th and 75th percentiles, respectively, and the lines within the boxes correspond to the median.

Techniques Used: Mouse Assay, Expressing, MANN-WHITNEY

16) Product Images from "Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19"

Article Title: Impaired ketogenesis ties metabolism to T cell dysfunction in COVID-19

Journal: Nature

doi: 10.1038/s41586-022-05128-8

BHB supports mitochondrial fitness and OXPHOS in CD8 + T cells. a , b , Splenic mouse CD8 + T cells were activated in culture for 3 days in T H 1 polarizing conditions with or without 5 mM BHB. Energy metabolism was monitored by extracellular flux analysis ( a ) and by SCENITH ( b ) ( n = 4 ). c , Human CD8 + T cells were isolated from the blood of healthy donors ( n = 9 ) and activated in culture for 1 week in T H 1 polarizing conditions with or without 5 mM BHB. Energy metabolism was monitored by SCENITH. ( c ) Each dot represents a donor. ( a , b ) Data representative of two independent experiments with n = 4 mice in each experimental group. All graphs display mean ± s.e.m. ( a - c ) Statistics were assessed by two-tailed Student’s t -test and, not significant (not indicated) p > 0.05; *p
Figure Legend Snippet: BHB supports mitochondrial fitness and OXPHOS in CD8 + T cells. a , b , Splenic mouse CD8 + T cells were activated in culture for 3 days in T H 1 polarizing conditions with or without 5 mM BHB. Energy metabolism was monitored by extracellular flux analysis ( a ) and by SCENITH ( b ) ( n = 4 ). c , Human CD8 + T cells were isolated from the blood of healthy donors ( n = 9 ) and activated in culture for 1 week in T H 1 polarizing conditions with or without 5 mM BHB. Energy metabolism was monitored by SCENITH. ( c ) Each dot represents a donor. ( a , b ) Data representative of two independent experiments with n = 4 mice in each experimental group. All graphs display mean ± s.e.m. ( a - c ) Statistics were assessed by two-tailed Student’s t -test and, not significant (not indicated) p > 0.05; *p

Techniques Used: Isolation, Mouse Assay, Two Tailed Test

A ketogenic diet promotes the resolution of inflammation. a , b Analysis of CD4 + and CD8 + T cells in the blood of healthy donors ( a , n = 6 ; b , n = 11 , black ) and the blood ( a , n = 11 ; b , n = 8 open red ) or BALF ( a , n = 11 ; b , n = 5 , filled red ) of patients with severe COVID-19. ( a ) Gating strategy (left panel), representative histograms of PD-1 expression (right panel) and percentage of CD4 + and CD8 + PD-1 + cells and gMFI of PD-1 ( n = 9 open red and filled red ) analysed by flow cytometry. ( b ) Metabolic characterization of CD8 + T cells by SCENITH. c - h , C57BL/6 mice were fed a control or ketogenic diet for 7 days followed by infection with IAV (d0). Mice were euthanized and analysed on d10. ( c ) Quantification of ketone bodies in the plasma and lung of mice (BHB lung: n = 5 naïve, n = 11 CD, n = 7 KD; BHB Plasma: n = 7 naïve, n = 11 CD, n = 12 KD). ( d ) Gating strategy (left panel), representative histograms of PD-1 expression (right panel) and percentage of CD4 + PD-1 + cells and gMFI of PD-1 ( n = 4 naïve, n = 8 CD and KD) ( e ) Relative expression of viral PB1 RNA (viral load) on day 7 ( n = 12 CD and n = 11 KD). ( f ) Relative weight loss of infected mice ( n = 4 CD and KD). ( g ) Quantification of total protein (BSA) and matrix metalloproteinases (MMPs) on day 14 ( n = 6 naïve, n = 8 CD and KD. ( h ) Representative images stained with picrosirius red of lungs and quantified for tissue density (shown as pixel count per image) and collagen deposition (total green area per image) ( n = 4 ). ( i ) Representative image of lungs stained by haematoxylin and eosin (H E) and lung injury score analysis ( n = 6 ). Each dot represents a mouse. ( a - b ) Each dot represents a donor. ( c – g ) Pooled data from three independent experiments with n = 4 mice per experimental group. ( h , i ) Representative of three independent experiments with n = 6 in each experimental group. All graphs display mean ± s.e.m. Statistics were assessed by ( a , b ) non-parametric one-way ANOVA (Kruskal–Wallis test), ( c , d , g–i ) ordinary one-away ANOVA (Tukey´s correction) and ( e , f ) two-tailed Student’s t -test, not significant (not indicated) p > 0.05; *p
Figure Legend Snippet: A ketogenic diet promotes the resolution of inflammation. a , b Analysis of CD4 + and CD8 + T cells in the blood of healthy donors ( a , n = 6 ; b , n = 11 , black ) and the blood ( a , n = 11 ; b , n = 8 open red ) or BALF ( a , n = 11 ; b , n = 5 , filled red ) of patients with severe COVID-19. ( a ) Gating strategy (left panel), representative histograms of PD-1 expression (right panel) and percentage of CD4 + and CD8 + PD-1 + cells and gMFI of PD-1 ( n = 9 open red and filled red ) analysed by flow cytometry. ( b ) Metabolic characterization of CD8 + T cells by SCENITH. c - h , C57BL/6 mice were fed a control or ketogenic diet for 7 days followed by infection with IAV (d0). Mice were euthanized and analysed on d10. ( c ) Quantification of ketone bodies in the plasma and lung of mice (BHB lung: n = 5 naïve, n = 11 CD, n = 7 KD; BHB Plasma: n = 7 naïve, n = 11 CD, n = 12 KD). ( d ) Gating strategy (left panel), representative histograms of PD-1 expression (right panel) and percentage of CD4 + PD-1 + cells and gMFI of PD-1 ( n = 4 naïve, n = 8 CD and KD) ( e ) Relative expression of viral PB1 RNA (viral load) on day 7 ( n = 12 CD and n = 11 KD). ( f ) Relative weight loss of infected mice ( n = 4 CD and KD). ( g ) Quantification of total protein (BSA) and matrix metalloproteinases (MMPs) on day 14 ( n = 6 naïve, n = 8 CD and KD. ( h ) Representative images stained with picrosirius red of lungs and quantified for tissue density (shown as pixel count per image) and collagen deposition (total green area per image) ( n = 4 ). ( i ) Representative image of lungs stained by haematoxylin and eosin (H E) and lung injury score analysis ( n = 6 ). Each dot represents a mouse. ( a - b ) Each dot represents a donor. ( c – g ) Pooled data from three independent experiments with n = 4 mice per experimental group. ( h , i ) Representative of three independent experiments with n = 6 in each experimental group. All graphs display mean ± s.e.m. Statistics were assessed by ( a , b ) non-parametric one-way ANOVA (Kruskal–Wallis test), ( c , d , g–i ) ordinary one-away ANOVA (Tukey´s correction) and ( e , f ) two-tailed Student’s t -test, not significant (not indicated) p > 0.05; *p

Techniques Used: Expressing, Flow Cytometry, Mouse Assay, Infection, Staining, Two Tailed Test

BHB promotes T cell function in a BDH1-dependent manner. a , c – e , k , m , Human CD4 + T and CD8 + T cells were isolated from the blood of healthy donors and culture for 1 week in T H 1 polarizing conditions in the presence or absence of 5 mM BHB. b , f – h , l , n , o , Splenic mouse CD4 + and CD8 + T cells were activated in culture for 1 week in T H 1 polarizing conditions with or without 5 mM BHB. Representative flow plots and percentage of human ( n = 13 ) ( a ) and mouse ( n = 6 ) TNF + CD4 + T cells and TNF gMFI ( b ). Total numbers of live human CD8 + T cells ( c ), percentage of IFNγ + , IFNγ gMFI ( d ) and TNF + CD8 + T cells, TNF gMFI ( e ) ( n = 9 ) analysed by flow cytometry. Total number of live mouse CD8 + T cells ( f ) percentage of IFNγ + CD8 + T cells, IFNγ gMFI ( g ) and percentage of TNF + CD8 + T cells, TNF gMFI ( h ) analysed by flow cytometry ( n = 6 ). ( i , j ) Representative histograms and quantification of BDH1 protein by flow cytometry (MFI) and gene expression analysis (fold change) of BDH1 RNA in ( i ) human CD4 + T cells ( n = 6 ) or ( j ) mouse naïve splenic CD4 + T cells ( n = 6 ) mock treated or nucleofected with Bdh1 -targeting (sgBdh1) sgRNA/Cas9 RNPs cultured for 2 days in T H 1 polarizing conditions. Representative flow plots, percentages and gMFI of Ki-67 expression in human CD4 + T cells ( n = 13 ) ( k ), in mouse CD4 + T cells ( n = 6 ) ( l ), in human CD8 + T cells ( n = 9 ) ( m ), in mouse CD8 + T cells ( n = 4 ) ( n ) and the % of mouse Annexin V + CD4 + T cells ( n = 6 ) ( o ) measured by flow cytometry. ( a , c–e , i - k , m ) Each dot represents a donor. ( b , f – h , l , n , o ) Data representative of three independent experiments with ( b , f–h , j , l , o ) n = 6 and ( n ) n = 4 mice in each experimental group. All graphs display mean ± s.e.m. Statistics were assessed by ( a – o ) by two-tailed Student’s t -test, not significant (not indicated) p > 0.05; *p
Figure Legend Snippet: BHB promotes T cell function in a BDH1-dependent manner. a , c – e , k , m , Human CD4 + T and CD8 + T cells were isolated from the blood of healthy donors and culture for 1 week in T H 1 polarizing conditions in the presence or absence of 5 mM BHB. b , f – h , l , n , o , Splenic mouse CD4 + and CD8 + T cells were activated in culture for 1 week in T H 1 polarizing conditions with or without 5 mM BHB. Representative flow plots and percentage of human ( n = 13 ) ( a ) and mouse ( n = 6 ) TNF + CD4 + T cells and TNF gMFI ( b ). Total numbers of live human CD8 + T cells ( c ), percentage of IFNγ + , IFNγ gMFI ( d ) and TNF + CD8 + T cells, TNF gMFI ( e ) ( n = 9 ) analysed by flow cytometry. Total number of live mouse CD8 + T cells ( f ) percentage of IFNγ + CD8 + T cells, IFNγ gMFI ( g ) and percentage of TNF + CD8 + T cells, TNF gMFI ( h ) analysed by flow cytometry ( n = 6 ). ( i , j ) Representative histograms and quantification of BDH1 protein by flow cytometry (MFI) and gene expression analysis (fold change) of BDH1 RNA in ( i ) human CD4 + T cells ( n = 6 ) or ( j ) mouse naïve splenic CD4 + T cells ( n = 6 ) mock treated or nucleofected with Bdh1 -targeting (sgBdh1) sgRNA/Cas9 RNPs cultured for 2 days in T H 1 polarizing conditions. Representative flow plots, percentages and gMFI of Ki-67 expression in human CD4 + T cells ( n = 13 ) ( k ), in mouse CD4 + T cells ( n = 6 ) ( l ), in human CD8 + T cells ( n = 9 ) ( m ), in mouse CD8 + T cells ( n = 4 ) ( n ) and the % of mouse Annexin V + CD4 + T cells ( n = 6 ) ( o ) measured by flow cytometry. ( a , c–e , i - k , m ) Each dot represents a donor. ( b , f – h , l , n , o ) Data representative of three independent experiments with ( b , f–h , j , l , o ) n = 6 and ( n ) n = 4 mice in each experimental group. All graphs display mean ± s.e.m. Statistics were assessed by ( a – o ) by two-tailed Student’s t -test, not significant (not indicated) p > 0.05; *p

Techniques Used: Cell Function Assay, Isolation, Flow Cytometry, Expressing, Cell Culture, Mouse Assay, Two Tailed Test

17) Product Images from "Activated Allogeneic Donor-derived Marrow-infiltrating Lymphocytes Display Measurable In Vitro Antitumor Activity"

Article Title: Activated Allogeneic Donor-derived Marrow-infiltrating Lymphocytes Display Measurable In Vitro Antitumor Activity

Journal: Journal of immunotherapy (Hagerstown, Md. : 1997)

doi: 10.1097/CJI.0000000000000256

ddMILs expansion. A, Fold expansion of CD3 + , CD4 + , and CD8 + ddMILs after activation. Median and interquartile range are shown for each subset. B–D, Comparison of CD3 + , CD4 + , and CD8 + cell counts in paired samples before and after polyclonal expansion with anti-CD3/CD28 beads and the addition of IL2 for 14 days. P -values are indicated in the figure. ddMIL indicates donor-derived marrow-infiltrating lymphocyte.
Figure Legend Snippet: ddMILs expansion. A, Fold expansion of CD3 + , CD4 + , and CD8 + ddMILs after activation. Median and interquartile range are shown for each subset. B–D, Comparison of CD3 + , CD4 + , and CD8 + cell counts in paired samples before and after polyclonal expansion with anti-CD3/CD28 beads and the addition of IL2 for 14 days. P -values are indicated in the figure. ddMIL indicates donor-derived marrow-infiltrating lymphocyte.

Techniques Used: Activation Assay, Derivative Assay

Activated ddMILs show a HLA-dependent reactivity only toward allogeneic antigens. Reactive activated ddMILs were defined as IFNγ + and CFSE low (identifying proliferation). Results are shown for both CD4 + (A) and CD8 + (B) subpopulations after stimulation with AIM-V medium alone (ctrl), autologous and donor-derived antigens (auto), and allogeneic antigens (allo). Median and interquartile range are shown for each parameter. P-values provided by 1-way ANOVA and post hoc analyses are indicated on the figure. CFSE indicates carboxyfluorescein diacetate succinimidyl ester; ddMIL, donor-derived marrow-infiltrating lymphocyte.
Figure Legend Snippet: Activated ddMILs show a HLA-dependent reactivity only toward allogeneic antigens. Reactive activated ddMILs were defined as IFNγ + and CFSE low (identifying proliferation). Results are shown for both CD4 + (A) and CD8 + (B) subpopulations after stimulation with AIM-V medium alone (ctrl), autologous and donor-derived antigens (auto), and allogeneic antigens (allo). Median and interquartile range are shown for each parameter. P-values provided by 1-way ANOVA and post hoc analyses are indicated on the figure. CFSE indicates carboxyfluorescein diacetate succinimidyl ester; ddMIL, donor-derived marrow-infiltrating lymphocyte.

Techniques Used: Derivative Assay

Characterization of T-cell subsets before and after expansion. A, Relative median proportion of CD4 + and CD8 + T-cell subsets before and after expansion. B, Comparison of the percentages of both CD4 + and CD8 + T-cell subsets before and after expansion. P -values indicated refer to 1-way ANOVA. T CM phenotype was significantly more frequent than other subsets in both CD4 + and CD8 + compartments after expansion (post hoc analyses, P
Figure Legend Snippet: Characterization of T-cell subsets before and after expansion. A, Relative median proportion of CD4 + and CD8 + T-cell subsets before and after expansion. B, Comparison of the percentages of both CD4 + and CD8 + T-cell subsets before and after expansion. P -values indicated refer to 1-way ANOVA. T CM phenotype was significantly more frequent than other subsets in both CD4 + and CD8 + compartments after expansion (post hoc analyses, P

Techniques Used:

Polyclonal stimulation leads to reduction of T REG cells and expression of activation markers. A, Percent of CD4 + /CD25 + /CD127 low (T REG ) cells in paired preexpansion and postexpansion samples. B, Percent of CD4 + cells expressing CD69 and IFNγ, respectively. C, Percent of CD8 + cells expressing CD69 and IFNγ, respectively. Each line represents paired samples from the same patient. Median and interquartile range are shown for each parameter. P -values are indicated on the figure.
Figure Legend Snippet: Polyclonal stimulation leads to reduction of T REG cells and expression of activation markers. A, Percent of CD4 + /CD25 + /CD127 low (T REG ) cells in paired preexpansion and postexpansion samples. B, Percent of CD4 + cells expressing CD69 and IFNγ, respectively. C, Percent of CD8 + cells expressing CD69 and IFNγ, respectively. Each line represents paired samples from the same patient. Median and interquartile range are shown for each parameter. P -values are indicated on the figure.

Techniques Used: Expressing, Activation Assay

18) Product Images from "PD-1 Inhibitor Combined With Radiotherapy and GM-CSF (PRaG) in Patients With Metastatic Solid Tumors: An Open-Label Phase II Study"

Article Title: PD-1 Inhibitor Combined With Radiotherapy and GM-CSF (PRaG) in Patients With Metastatic Solid Tumors: An Open-Label Phase II Study

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.952066

Lymphocyte subset percentage changes after treatment from baseline between the three groups (CR+PR, SD, PD). The red boxplot represents percentage changes after one cycle of treatment from baseline. The green boxplot represents percentage changes after two treatment cycles from baseline. The blue boxplot represents percentage changes after three cycles of treatment from baseline. The differences in the proportion of changes after the first cycle of treatment, after the second cycle of treatment, and after the third treatment cycle was compared separately between the three groups (CR+PR, SD, PD). The one-way ANOVA was used for the homogeneity of consistent variance, and the rank-sum test was used for the homogeneity of inconsistent variance. None of the other lymphocyte subset percentage changes showed statistical differences ( p > 0.05). (A) CD3 + T cells percentage changes from baseline. (B) CD3 + CD4 + T cells percentage changes from baseline. (C) CD3 + CD8 + T cells percentage changes from baseline. (D) CD16 + CD56 + T cells percentage changes from baseline.
Figure Legend Snippet: Lymphocyte subset percentage changes after treatment from baseline between the three groups (CR+PR, SD, PD). The red boxplot represents percentage changes after one cycle of treatment from baseline. The green boxplot represents percentage changes after two treatment cycles from baseline. The blue boxplot represents percentage changes after three cycles of treatment from baseline. The differences in the proportion of changes after the first cycle of treatment, after the second cycle of treatment, and after the third treatment cycle was compared separately between the three groups (CR+PR, SD, PD). The one-way ANOVA was used for the homogeneity of consistent variance, and the rank-sum test was used for the homogeneity of inconsistent variance. None of the other lymphocyte subset percentage changes showed statistical differences ( p > 0.05). (A) CD3 + T cells percentage changes from baseline. (B) CD3 + CD4 + T cells percentage changes from baseline. (C) CD3 + CD8 + T cells percentage changes from baseline. (D) CD16 + CD56 + T cells percentage changes from baseline.

Techniques Used:

19) Product Images from "Null Function of Npr1 Disturbs Immune Response in Colonic Inflammation During Early Postnatal Stage"

Article Title: Null Function of Npr1 Disturbs Immune Response in Colonic Inflammation During Early Postnatal Stage

Journal: Inflammation

doi: 10.1007/s10753-022-01702-4

Restructuring of T-cell subpopulation in the colon tissue from Npr1 −/− mice. Immunofluorescent staining for CD3 + , CD4 + , and CD8 + in a WT and Npr1 −/− mice (4 weeks, n = 4–5) and b Npr1 −/− mice treated with 8-Br-cGMP or saline as a control (12 weeks, n = 4). Values are mean ± S.D. * p
Figure Legend Snippet: Restructuring of T-cell subpopulation in the colon tissue from Npr1 −/− mice. Immunofluorescent staining for CD3 + , CD4 + , and CD8 + in a WT and Npr1 −/− mice (4 weeks, n = 4–5) and b Npr1 −/− mice treated with 8-Br-cGMP or saline as a control (12 weeks, n = 4). Values are mean ± S.D. * p

Techniques Used: Mouse Assay, Staining

Restructuring of T-cell subpopulation in the colon tissue from DSS-treated mice. Immunofluorescent staining for CD3 + , CD4 + , and CD8 + in control, DSS-treated mice, and DSS-treated mice with 8-Br-cGMP administration (9 weeks, n = 7). Values are mean ± S.D. * p
Figure Legend Snippet: Restructuring of T-cell subpopulation in the colon tissue from DSS-treated mice. Immunofluorescent staining for CD3 + , CD4 + , and CD8 + in control, DSS-treated mice, and DSS-treated mice with 8-Br-cGMP administration (9 weeks, n = 7). Values are mean ± S.D. * p

Techniques Used: Mouse Assay, Staining

Changes of immune cell composition in the spleen from Npr1 −/− mice at the age of 4 weeks. a The spleen weight and index from WT and Npr1 −/− mice ( n = 7). Spleen index was generated as spleen weight (mg)/body weight (g). b Expression of Il6 mRNA in the spleen from WT and Npr1 −/− mice ( n = 3). c Leukocytes defined as CD45 + in the splenic immune cells from WT and Npr1 −/− mice ( n = 4–6). Population of neutrophils featured by d CD11B + LY-6G + , e NK cells, and f T cells featured by CD3 + from leukocytes in the spleen from WT and Npr1 −/− mice ( n = 4–6). g Population of CD3 + T cells in the splenic cells from WT and Npr1 −/− mice ( n = 4–5). h Population of CD4 + T and CD8 + T differentiated from CD3 + T cells from WT and Npr 1 −/− mice. i Population of Treg cells from CD4 + T cells in WT and Npr1 −/− mice. Values are mean ± S.D. * p
Figure Legend Snippet: Changes of immune cell composition in the spleen from Npr1 −/− mice at the age of 4 weeks. a The spleen weight and index from WT and Npr1 −/− mice ( n = 7). Spleen index was generated as spleen weight (mg)/body weight (g). b Expression of Il6 mRNA in the spleen from WT and Npr1 −/− mice ( n = 3). c Leukocytes defined as CD45 + in the splenic immune cells from WT and Npr1 −/− mice ( n = 4–6). Population of neutrophils featured by d CD11B + LY-6G + , e NK cells, and f T cells featured by CD3 + from leukocytes in the spleen from WT and Npr1 −/− mice ( n = 4–6). g Population of CD3 + T cells in the splenic cells from WT and Npr1 −/− mice ( n = 4–5). h Population of CD4 + T and CD8 + T differentiated from CD3 + T cells from WT and Npr 1 −/− mice. i Population of Treg cells from CD4 + T cells in WT and Npr1 −/− mice. Values are mean ± S.D. * p

Techniques Used: Mouse Assay, Generated, Expressing

20) Product Images from "Transgenic Expression of IL15 Retains CD123-Redirected T Cells in a Less Differentiated State Resulting in Improved Anti-AML Activity in Autologous AML PDX Models"

Article Title: Transgenic Expression of IL15 Retains CD123-Redirected T Cells in a Less Differentiated State Resulting in Improved Anti-AML Activity in Autologous AML PDX Models

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.880108

Transgenic Expression of IL15 improves effector function of CD123-ENG T-cells. (A) Scheme of repeat stimulation assay. AML cells and T cells in co-cultures were enumerated using counting beads by flow cytometry prior to each re-challenge. (B) CD123 + MOLM-13 AML cell count from the repeat stimulation assay prior to the 2nd (D5), 3rd (D10), 4th (D15), and 5th (D20) stimulation. (C, D) Apoptosis measured by Annexin-V + DAPI - flow cytometry (C) of AML and (D) T-cells on day 20. Representative flow cytometry plots and summary data are shown. (E) CD3 + , CD8 + , and CD4 + , T-cell counts (log10) in coculture assay overtime. (F) PD-1 and TIM3 expression in T-cells on day 5. Left panel: representative flow cytometry plots gated on CD3 + T-cells. Right: summary data. (G) Expression PD-1, TIM3, and LAG-3 in T-cells on day 20. Left panel: representative flow cytometry plots gated on CD3 + T-cells. Right: summary data. (H, I) Phenotypic analysis on CD3 + CD20 + of CD123.IL15 and CD19.IL15 T-cells before and on day 30 of co-culture. (H) Representative flow cytometry plots. (I) Summary data, n = 3 for all data shown in panels; data are shown as mean ± SE, ***:p
Figure Legend Snippet: Transgenic Expression of IL15 improves effector function of CD123-ENG T-cells. (A) Scheme of repeat stimulation assay. AML cells and T cells in co-cultures were enumerated using counting beads by flow cytometry prior to each re-challenge. (B) CD123 + MOLM-13 AML cell count from the repeat stimulation assay prior to the 2nd (D5), 3rd (D10), 4th (D15), and 5th (D20) stimulation. (C, D) Apoptosis measured by Annexin-V + DAPI - flow cytometry (C) of AML and (D) T-cells on day 20. Representative flow cytometry plots and summary data are shown. (E) CD3 + , CD8 + , and CD4 + , T-cell counts (log10) in coculture assay overtime. (F) PD-1 and TIM3 expression in T-cells on day 5. Left panel: representative flow cytometry plots gated on CD3 + T-cells. Right: summary data. (G) Expression PD-1, TIM3, and LAG-3 in T-cells on day 20. Left panel: representative flow cytometry plots gated on CD3 + T-cells. Right: summary data. (H, I) Phenotypic analysis on CD3 + CD20 + of CD123.IL15 and CD19.IL15 T-cells before and on day 30 of co-culture. (H) Representative flow cytometry plots. (I) Summary data, n = 3 for all data shown in panels; data are shown as mean ± SE, ***:p

Techniques Used: Transgenic Assay, Expressing, Flow Cytometry, Cell Counting, Co-culture Assay, Co-Culture Assay

21) Product Images from "IL-6 Responsiveness of CD4+ and CD8+ T Cells after Allogeneic Stem Cell Transplantation Differs between Patients and Is Associated with Previous Acute Graft versus Host Disease and Pretransplant Antithymocyte Globulin Therapy"

Article Title: IL-6 Responsiveness of CD4+ and CD8+ T Cells after Allogeneic Stem Cell Transplantation Differs between Patients and Is Associated with Previous Acute Graft versus Host Disease and Pretransplant Antithymocyte Globulin Therapy

Journal: Journal of Clinical Medicine

doi: 10.3390/jcm11092530

The effect of IL-6 stimulation on intracellular signaling in TCR-activated T cells derived from 31 allotransplant recipients on day +90 post-transplant. The T cells were stimulated either with hyper IL-6 (trans-signaling), IL-6+sIL-6R (classical and trans-signaling) or IL-6+sIL-6R+sgp130FC (classical signaling) in the presence of the T cell activation signal anti-CD3 + anti-CD28. The results are presented as the mean fluorescence intensity (MFI), and unstimulated refers to no IL-6 stimulation but only anti-CD3 + anti-CD28 activation. The figure presents the results for ( left ) effects on STAT3 (Ser727) phosphorylation of CD3 + CD4 + T cells derived from patients with GVHD stimulated with hyper-IL-6; ( center ) effects on mTOR (Ser2448) phosphorylation in CD3 + CD8 + T cells derived from patients with GVHD and stimulated with IL-6+sIL-6R or IL-6+sIL-6R+sgp130FC; and ( right ) effects on mTOR (Ser2448) phosphorylation in CD3 + CD8 + T cells derived from patients without GVHD and stimulated with IL-6+sIL-6R or IL-6+sIL-6R+sgp130FC. The Wilcoxon signed-rank test was used for the analyses. * indicates a p -value 0.05 to 0.01, ** indicates a p -value
Figure Legend Snippet: The effect of IL-6 stimulation on intracellular signaling in TCR-activated T cells derived from 31 allotransplant recipients on day +90 post-transplant. The T cells were stimulated either with hyper IL-6 (trans-signaling), IL-6+sIL-6R (classical and trans-signaling) or IL-6+sIL-6R+sgp130FC (classical signaling) in the presence of the T cell activation signal anti-CD3 + anti-CD28. The results are presented as the mean fluorescence intensity (MFI), and unstimulated refers to no IL-6 stimulation but only anti-CD3 + anti-CD28 activation. The figure presents the results for ( left ) effects on STAT3 (Ser727) phosphorylation of CD3 + CD4 + T cells derived from patients with GVHD stimulated with hyper-IL-6; ( center ) effects on mTOR (Ser2448) phosphorylation in CD3 + CD8 + T cells derived from patients with GVHD and stimulated with IL-6+sIL-6R or IL-6+sIL-6R+sgp130FC; and ( right ) effects on mTOR (Ser2448) phosphorylation in CD3 + CD8 + T cells derived from patients without GVHD and stimulated with IL-6+sIL-6R or IL-6+sIL-6R+sgp130FC. The Wilcoxon signed-rank test was used for the analyses. * indicates a p -value 0.05 to 0.01, ** indicates a p -value

Techniques Used: Derivative Assay, Activation Assay, Fluorescence

Overview of the gating strategy for comparison of stimulated and unstimulated cells. ( a ) After exclusion of duplicates and dead cells from the lymphocyte gate, CD3 + and CD3 − gates were selected. ( b ) The CD3 + cells were thereafter further divided into CD4 + and CD8 + cells. ( c ) Stimulated samples were finally compared with the unstimulated control cells; this comparison was carried out for the CD3 − , CD3 + CD4 + and CD3 + CD8 + cells.
Figure Legend Snippet: Overview of the gating strategy for comparison of stimulated and unstimulated cells. ( a ) After exclusion of duplicates and dead cells from the lymphocyte gate, CD3 + and CD3 − gates were selected. ( b ) The CD3 + cells were thereafter further divided into CD4 + and CD8 + cells. ( c ) Stimulated samples were finally compared with the unstimulated control cells; this comparison was carried out for the CD3 − , CD3 + CD4 + and CD3 + CD8 + cells.

Techniques Used:

The IL-6 responsiveness of CD3 + CD4 + and CD3 + CD8 + T cells derived from allotransplant recipients at day +90 post-transplant. We investigated the effects of four different forms of IL-6 stimulation on the T cell subsets: IL-6 alone (classical IL-6 signaling), hyper-IL-6 (trans-signaling), IL-6 plus sIL-6R (classical and trans-signaling) and IL-6+sIL-6R+sgp130FC (classical Il-6 signaling; trans-signaling blocked by sgp130FC). We investigated the effects of these signals on AKT (Thr308), mTOR (Ser2448), STAT3 (Ser727) and STAT3 (Tyr705) phosphorylation in resting cells and in TCR-activated cells (anti-CD3 + anti-CD28). We analyzed the overall results for the 31 patients. All statistical analyses were carried out using the Wilcoxon signed-rank test. The figure summarizes our overall results (see Table 3 and Table 4 ), but due to the overall number of comparisons, we only present differences with a p -value
Figure Legend Snippet: The IL-6 responsiveness of CD3 + CD4 + and CD3 + CD8 + T cells derived from allotransplant recipients at day +90 post-transplant. We investigated the effects of four different forms of IL-6 stimulation on the T cell subsets: IL-6 alone (classical IL-6 signaling), hyper-IL-6 (trans-signaling), IL-6 plus sIL-6R (classical and trans-signaling) and IL-6+sIL-6R+sgp130FC (classical Il-6 signaling; trans-signaling blocked by sgp130FC). We investigated the effects of these signals on AKT (Thr308), mTOR (Ser2448), STAT3 (Ser727) and STAT3 (Tyr705) phosphorylation in resting cells and in TCR-activated cells (anti-CD3 + anti-CD28). We analyzed the overall results for the 31 patients. All statistical analyses were carried out using the Wilcoxon signed-rank test. The figure summarizes our overall results (see Table 3 and Table 4 ), but due to the overall number of comparisons, we only present differences with a p -value

Techniques Used: Derivative Assay

The effects of PMA on STAT3 (Tyr705) phosphorylation of post-transplant CD3 + CD8 + T cells derived from 31 allotransplant recipients on day +90 post-transplant. The T cells were stimulated with PMA either in the presence ( left ) or absence ( right ) of the T cell activation signal anti-CD3 + anti-CD28. The results are presented as the mean fluorescence intensity (MFI). The Wilcoxon signed-rank test was used for the analyses. * indicates p -value 0.01.
Figure Legend Snippet: The effects of PMA on STAT3 (Tyr705) phosphorylation of post-transplant CD3 + CD8 + T cells derived from 31 allotransplant recipients on day +90 post-transplant. The T cells were stimulated with PMA either in the presence ( left ) or absence ( right ) of the T cell activation signal anti-CD3 + anti-CD28. The results are presented as the mean fluorescence intensity (MFI). The Wilcoxon signed-rank test was used for the analyses. * indicates p -value 0.01.

Techniques Used: Derivative Assay, Activation Assay, Fluorescence

(page 8). The effect of classical and trans IL-6 signaling on STAT3 (Tyr705) phosphorylation for post-transplant CD3 + CD4 + T cells derived from 31 allotransplant recipients on day +90 post-transplant; a comparison of patients with and without previous acute GVHD. The T cells were stimulated either with hyper IL-6 ( left , trans-signaling), IL-6 ( middle left , classical signaling), IL-6+IL-6R ( middle right , classical and trans-signaling) and IL-6+sIL-6R+sgp130FC, classical signaling). IL-6 stimulation was tested in the absence ( upper part ) and in the presence ( lower part ) of the T cell activation signal anti-CD3 + anti-CD28. The results are presented as the mean fluorescence intensity (MFI), and for each of the four IL-6 stimulations, we compared patients without (dark symbols) and with previous acute GVHD (open symbols). The Wilcoxon signed-rank test was used for the statistical analyses. * indicates a p -value 0.05 to 0.01, ** indicates a p -value 0.001 to 0.001, *** indicates p -value
Figure Legend Snippet: (page 8). The effect of classical and trans IL-6 signaling on STAT3 (Tyr705) phosphorylation for post-transplant CD3 + CD4 + T cells derived from 31 allotransplant recipients on day +90 post-transplant; a comparison of patients with and without previous acute GVHD. The T cells were stimulated either with hyper IL-6 ( left , trans-signaling), IL-6 ( middle left , classical signaling), IL-6+IL-6R ( middle right , classical and trans-signaling) and IL-6+sIL-6R+sgp130FC, classical signaling). IL-6 stimulation was tested in the absence ( upper part ) and in the presence ( lower part ) of the T cell activation signal anti-CD3 + anti-CD28. The results are presented as the mean fluorescence intensity (MFI), and for each of the four IL-6 stimulations, we compared patients without (dark symbols) and with previous acute GVHD (open symbols). The Wilcoxon signed-rank test was used for the statistical analyses. * indicates a p -value 0.05 to 0.01, ** indicates a p -value 0.001 to 0.001, *** indicates p -value

Techniques Used: Polyacrylamide Gel Electrophoresis, Derivative Assay, Activation Assay, Fluorescence

22) Product Images from "NFAT Factors Are Dispensable for the Development but Are Critical for the Maintenance of Foxp3+ Regulatory T Cells"

Article Title: NFAT Factors Are Dispensable for the Development but Are Critical for the Maintenance of Foxp3+ Regulatory T Cells

Journal: Cells

doi: 10.3390/cells11091397

NFAT signaling is dispensable for the generation of Foxp3 + T reg cells. ( a ) Flow cytometry showing the distribution of thymocyte populations based on CD4 and CD8 stainings from WT, Il7r -/- and Jak3 -/- mice. Numbers atop each plot represent total cellularity and within each plot represent the percent distribution of respective populations. ( b ) Quanification of total thymic cellularity in WT, Il7r -/- and Jak3 -/- mice ( n = 7 each, *** p
Figure Legend Snippet: NFAT signaling is dispensable for the generation of Foxp3 + T reg cells. ( a ) Flow cytometry showing the distribution of thymocyte populations based on CD4 and CD8 stainings from WT, Il7r -/- and Jak3 -/- mice. Numbers atop each plot represent total cellularity and within each plot represent the percent distribution of respective populations. ( b ) Quanification of total thymic cellularity in WT, Il7r -/- and Jak3 -/- mice ( n = 7 each, *** p

Techniques Used: Flow Cytometry, Mouse Assay

23) Product Images from "NFAT Factors Are Dispensable for the Development but Are Critical for the Maintenance of Foxp3+ Regulatory T Cells"

Article Title: NFAT Factors Are Dispensable for the Development but Are Critical for the Maintenance of Foxp3+ Regulatory T Cells

Journal: Cells

doi: 10.3390/cells11091397

NFAT signaling is dispensable for the generation of Foxp3 + T reg cells. ( a ) Flow cytometry showing the distribution of thymocyte populations based on CD4 and CD8 stainings from WT, Il7r -/- and Jak3 -/- mice. Numbers atop each plot represent total cellularity and within each plot represent the percent distribution of respective populations. ( b ) Quanification of total thymic cellularity in WT, Il7r -/- and Jak3 -/- mice ( n = 7 each, *** p
Figure Legend Snippet: NFAT signaling is dispensable for the generation of Foxp3 + T reg cells. ( a ) Flow cytometry showing the distribution of thymocyte populations based on CD4 and CD8 stainings from WT, Il7r -/- and Jak3 -/- mice. Numbers atop each plot represent total cellularity and within each plot represent the percent distribution of respective populations. ( b ) Quanification of total thymic cellularity in WT, Il7r -/- and Jak3 -/- mice ( n = 7 each, *** p

Techniques Used: Flow Cytometry, Mouse Assay

24) Product Images from "Hepatitis B Virus-Specific Cellular Immunity Contributes to the Outcome of Occult Hepatitis B Virus Infection"

Article Title: Hepatitis B Virus-Specific Cellular Immunity Contributes to the Outcome of Occult Hepatitis B Virus Infection

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2022.850665

Intracellular IL-10 or TGF-β secretion CD4 + /CD8 + suppressor T-cell responses to HBV core peptides. (A) Representative flow-cytometric analysis of reactivity of CD4 + /CD8 + T cells from an OBI donor stimulated with negative control and HBV core peptides. (B,C) IL-10 secreting CD4 + or CD8 + T cells. (D,E) TGF-β secreting CD4 + or CD8 + T cells. A median of frequency in each group is indicated compared with the Mann–Whitney test. Statistically significant differences are shown with asterisks (* P
Figure Legend Snippet: Intracellular IL-10 or TGF-β secretion CD4 + /CD8 + suppressor T-cell responses to HBV core peptides. (A) Representative flow-cytometric analysis of reactivity of CD4 + /CD8 + T cells from an OBI donor stimulated with negative control and HBV core peptides. (B,C) IL-10 secreting CD4 + or CD8 + T cells. (D,E) TGF-β secreting CD4 + or CD8 + T cells. A median of frequency in each group is indicated compared with the Mann–Whitney test. Statistically significant differences are shown with asterisks (* P

Techniques Used: Negative Control, MANN-WHITNEY

Frequency of intracellular cytokine expressing CD8 + T cells after stimulation of HBV core peptides. PBMCs were freshly isolated from individual blood donors and tested by ICS for intracellular IFN-γ, TNF-α, IL-2, IL-17A, or IL-21 secreting CD8 + T-cell response, respectively, after simulation with core peptides for 6 h. (A) Representative flow-cytometric analysis of reactivity of CD8 + T cells from an OBI donor stimulated with negative control and HBV core peptides. (B–F) The frequency of intracellular cytokine secreting CD8 + T-cell response was expressed as a mean of triplicates for each individual donor. A median of frequency in each group is indicated and the differences between groups are compared with the Mann–Whitney test. Statistically significant differences are shown with asterisks (* P
Figure Legend Snippet: Frequency of intracellular cytokine expressing CD8 + T cells after stimulation of HBV core peptides. PBMCs were freshly isolated from individual blood donors and tested by ICS for intracellular IFN-γ, TNF-α, IL-2, IL-17A, or IL-21 secreting CD8 + T-cell response, respectively, after simulation with core peptides for 6 h. (A) Representative flow-cytometric analysis of reactivity of CD8 + T cells from an OBI donor stimulated with negative control and HBV core peptides. (B–F) The frequency of intracellular cytokine secreting CD8 + T-cell response was expressed as a mean of triplicates for each individual donor. A median of frequency in each group is indicated and the differences between groups are compared with the Mann–Whitney test. Statistically significant differences are shown with asterisks (* P

Techniques Used: Expressing, Isolation, Negative Control, MANN-WHITNEY

Proliferation rates of CD4 + and CD8 + T cells from blood donors with different outcomes of HBV infection stimulated with HBV core peptides. (A,B) HBV-core specific CD4 + or CD8 + T-cell proliferation rate. Each group median is indicated, and each dot represents a rate for T cell proliferation from four groups of individual blood donors. (C) Comparison of the median frequencies between CD4 + and CD8 + T cell proliferation rates in each study group. P -values are analyzed by one-way ANOVA and two-tailed t -test. Statistically significant differences are shown with asterisks (* P
Figure Legend Snippet: Proliferation rates of CD4 + and CD8 + T cells from blood donors with different outcomes of HBV infection stimulated with HBV core peptides. (A,B) HBV-core specific CD4 + or CD8 + T-cell proliferation rate. Each group median is indicated, and each dot represents a rate for T cell proliferation from four groups of individual blood donors. (C) Comparison of the median frequencies between CD4 + and CD8 + T cell proliferation rates in each study group. P -values are analyzed by one-way ANOVA and two-tailed t -test. Statistically significant differences are shown with asterisks (* P

Techniques Used: Infection, Two Tailed Test

25) Product Images from "Immunomodulatory activity of ethanol extract of Annona reticulata L. leaf in cultured immune cells and in Swiss albino mice"

Article Title: Immunomodulatory activity of ethanol extract of Annona reticulata L. leaf in cultured immune cells and in Swiss albino mice

Journal: Journal of Ayurveda and Integrative Medicine

doi: 10.1016/j.jaim.2022.100554

a. FACS analysis (dot plots) showing Ki67 expression in control, PHA challenged, and extract treated PBMC, gated on lymphocytes. Upper, middle, and lower panels represent CD4 + , CD8 + , and CD19 + lymphocytes respectively. Values in the upper right quadrant (UR) in each dot plot represent the percentage of cells expressing Ki67. The mean of that percentage value in different groups from 5 different experiments was plotted graphically to evaluate the difference between groups. (One way ANOVA; n = 5); ∗( P
Figure Legend Snippet: a. FACS analysis (dot plots) showing Ki67 expression in control, PHA challenged, and extract treated PBMC, gated on lymphocytes. Upper, middle, and lower panels represent CD4 + , CD8 + , and CD19 + lymphocytes respectively. Values in the upper right quadrant (UR) in each dot plot represent the percentage of cells expressing Ki67. The mean of that percentage value in different groups from 5 different experiments was plotted graphically to evaluate the difference between groups. (One way ANOVA; n = 5); ∗( P

Techniques Used: FACS, Expressing

26) Product Images from "Targeting the NANOG/HDAC1 axis reverses resistance to PD-1 blockade by reinvigorating the antitumor immunity cycle"

Article Title: Targeting the NANOG/HDAC1 axis reverses resistance to PD-1 blockade by reinvigorating the antitumor immunity cycle

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI147908

HDAC1 inhibition renders tumors susceptible to an anti–PD-1–mediated antitumor immune response. ( A ) CT26 P0 and CT26 P3 cells were transfected with siGFP or siNANOG. After 16 hours, cells were treated with the indicated concentrations of FK228, MS-275, SAHA, or cisplatin for 48 hours. Cell viability was measured by counting live cells using trypan blue. ( B ) Western blot analysis of NANOG, HDAC1, CXCL10, MCL1, AcH3-K14, and AcH3-K27 expression in CT26 P3 cells treated with DMSO or FK228. β-Actin was used as an internal loading control. ( C – I ) CT26 P3 tumor–bearing mice were administered vehicle or FK228, with or without PD-1 antibody treatment. ( C ) Tumor volume curves and ( D ) changes in tumor growth compared with baseline, 17 days after challenge. ( E ) Survival of mice inoculated with CT26 P3 and treated with the indicated reagents. ( F ) Flow cytometric profiles of tumor-infiltrating CD3 + CD8 + T cells. ( G ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. ( H ) Frequency of apoptotic cells in the tumors. ( I ) Quantification of antigen-specific CTLs in spleens from tumor-bearing mice. Ten mice from each group were used for in vivo experiments. Results in the graphs represent 3 independent experiments performed in triplicate. Data represent the mean ± SD. * P
Figure Legend Snippet: HDAC1 inhibition renders tumors susceptible to an anti–PD-1–mediated antitumor immune response. ( A ) CT26 P0 and CT26 P3 cells were transfected with siGFP or siNANOG. After 16 hours, cells were treated with the indicated concentrations of FK228, MS-275, SAHA, or cisplatin for 48 hours. Cell viability was measured by counting live cells using trypan blue. ( B ) Western blot analysis of NANOG, HDAC1, CXCL10, MCL1, AcH3-K14, and AcH3-K27 expression in CT26 P3 cells treated with DMSO or FK228. β-Actin was used as an internal loading control. ( C – I ) CT26 P3 tumor–bearing mice were administered vehicle or FK228, with or without PD-1 antibody treatment. ( C ) Tumor volume curves and ( D ) changes in tumor growth compared with baseline, 17 days after challenge. ( E ) Survival of mice inoculated with CT26 P3 and treated with the indicated reagents. ( F ) Flow cytometric profiles of tumor-infiltrating CD3 + CD8 + T cells. ( G ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. ( H ) Frequency of apoptotic cells in the tumors. ( I ) Quantification of antigen-specific CTLs in spleens from tumor-bearing mice. Ten mice from each group were used for in vivo experiments. Results in the graphs represent 3 independent experiments performed in triplicate. Data represent the mean ± SD. * P

Techniques Used: Inhibition, Transfection, Western Blot, Expressing, Mouse Assay, In Vivo

CT26 P3 cells display the immune-refractory feature of the TME. ( A – I ) CT26 P0, P3, or N3 tumor–bearing mice were treated with IgG or anti–PD-1 (α–PD-1) antibody. ( A ) Tumor growth curves and ( B ) changes in tumor volume 17 days after challenge compared with baseline. CR, complete response. ( C ) Formalin-fixed, paraffin-embedded (FFPE) sections of CT26 P0 or P3 tumors treated with IgG or anti–PD-1 antibody were stained with the indicated markers by pseudo-coloring. The indicated markers are shown on the right. Scale bars: 100 μm and 20 μm (enlarged insets). ( D ) Frequency of tumor-infiltrating CD8 + T cells. ( E ) Frequency of apoptotic cells in the tumors. ( F ) Flow cytometric profiles of the tumor-infiltrating CD3 + CD8 + T cells. ( G ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. ( H ) Frequency of apoptotic cells in the tumors. ( I ) Quantification of antigen-specific CTLs in spleens from the tumor-bearing mice. Ten mice from each group were used for in vivo experiments. Results shown in the graphs represent 3 independent experiments performed in triplicate. ( D – I ) Data represent the mean ± SD. *** P
Figure Legend Snippet: CT26 P3 cells display the immune-refractory feature of the TME. ( A – I ) CT26 P0, P3, or N3 tumor–bearing mice were treated with IgG or anti–PD-1 (α–PD-1) antibody. ( A ) Tumor growth curves and ( B ) changes in tumor volume 17 days after challenge compared with baseline. CR, complete response. ( C ) Formalin-fixed, paraffin-embedded (FFPE) sections of CT26 P0 or P3 tumors treated with IgG or anti–PD-1 antibody were stained with the indicated markers by pseudo-coloring. The indicated markers are shown on the right. Scale bars: 100 μm and 20 μm (enlarged insets). ( D ) Frequency of tumor-infiltrating CD8 + T cells. ( E ) Frequency of apoptotic cells in the tumors. ( F ) Flow cytometric profiles of the tumor-infiltrating CD3 + CD8 + T cells. ( G ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. ( H ) Frequency of apoptotic cells in the tumors. ( I ) Quantification of antigen-specific CTLs in spleens from the tumor-bearing mice. Ten mice from each group were used for in vivo experiments. Results shown in the graphs represent 3 independent experiments performed in triplicate. ( D – I ) Data represent the mean ± SD. *** P

Techniques Used: Mouse Assay, Formalin-fixed Paraffin-Embedded, Staining, In Vivo

NANOG repression enhances the response to anti–PD-1 therapy by inducing the immune-stimulatory feature in the TME. ( A ) Top: Quantification of NANOG expression in tumor cells at different stages of immune selection (P0–P3). Parallel stages without selection are labeled as N1–N3. Bottom: Representative Western blots. ( B ) Flow cytometric analysis of NANOG + tumor cells and quantification of the frequency of NANOG + tumor cells. ( C – H ) CT26 P3 tumor–bearing mice were administered si GFP or si Nanog with or without anti–PD-1 antibody treatment. ( C ) Tumor growth curves and ( D ) changes in tumor volume 17 days after challenge compared with baseline. ( E ) Flow cytometric profiles of tumor-infiltrating CD3 + CD8 + T cells. ( F ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. ( G ) Frequency of apoptotic cells in the tumors. ( H ) Quantification of antigen-specific CTLs in spleens from tumor-bearing mice. Ten mice from each group were used for in vivo experiments. Results in the graphs represent 3 independent experiments performed in triplicate. Data represent the mean ± SD. ** P
Figure Legend Snippet: NANOG repression enhances the response to anti–PD-1 therapy by inducing the immune-stimulatory feature in the TME. ( A ) Top: Quantification of NANOG expression in tumor cells at different stages of immune selection (P0–P3). Parallel stages without selection are labeled as N1–N3. Bottom: Representative Western blots. ( B ) Flow cytometric analysis of NANOG + tumor cells and quantification of the frequency of NANOG + tumor cells. ( C – H ) CT26 P3 tumor–bearing mice were administered si GFP or si Nanog with or without anti–PD-1 antibody treatment. ( C ) Tumor growth curves and ( D ) changes in tumor volume 17 days after challenge compared with baseline. ( E ) Flow cytometric profiles of tumor-infiltrating CD3 + CD8 + T cells. ( F ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. ( G ) Frequency of apoptotic cells in the tumors. ( H ) Quantification of antigen-specific CTLs in spleens from tumor-bearing mice. Ten mice from each group were used for in vivo experiments. Results in the graphs represent 3 independent experiments performed in triplicate. Data represent the mean ± SD. ** P

Techniques Used: Expressing, Selection, Labeling, Western Blot, Mouse Assay, In Vivo

NANOG blocks CD8 + T cell infiltration through HDAC1-mediated epigenetic repression of CXCL10. ( A ) Correlation plot of NANOG and T cell infiltration signatures in pan-tumor types. Correlation and 2-tailed P values were assessed using the Pearson’s correlation coefficient and unpaired Student’s t test. ( B ) Pearson’s correlation of NANOG signature expression with indicated transcripts of T cell–recruiting chemokines. RSEM, relative SEM. ( C ) qPCR analysis of Cxcl9 and Cxcl10 mRNA expression. ( D ) Western blot analysis of the expression of CXCL10. ( E – G ) CT26-no insert or CT26- Nanog cells were transfected with si GFP or si Hdac1 . WCL, whole-cell lysate; SUP, supernatant. ( E ) Western blot analysis of CXCL10 expression. ( F ) qPCR analysis of Cxcl10 mRNA expression. ( G ) Relative occupancy of AcH3K14, AcH3K27, and HDAC1 in the Cxcl10 promoters was assessed by qChIP analysis. The ChIP data values represent ratios relative to the input. ( H ) Transwell T cell migration assay using CT26- Nanog -si GFP or CT26- Nanog -si Hdac1 cells were treated with IgG or anti-CXCL10. ( I ) Western blot analysis of the expression of GFP (CXCL10) in CT26-no insert or CT26- Nanog WT cells transduced with Cxcl10 WT or Cxcl10 MT. ( J – L ) CT26-no insert, CT26- Nanog - Cxcl10 WT, or CT26- Nanog - Cxcl10 MT tumor–bearing mice were treated with anti–PD-1 antibody. ( J ) Tumor growth curves. ( K ) Flow cytometric profiles of tumor-infiltrating CD3 + CD8 + T cells. ( L ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. Five mice from each group were used for in vivo experiments. ( D , E , and I ) β-Actin was used as an internal loading control. Results in the graphs represent 3 independent experiments performed in triplicate. Data represent the mean ± SD. * P
Figure Legend Snippet: NANOG blocks CD8 + T cell infiltration through HDAC1-mediated epigenetic repression of CXCL10. ( A ) Correlation plot of NANOG and T cell infiltration signatures in pan-tumor types. Correlation and 2-tailed P values were assessed using the Pearson’s correlation coefficient and unpaired Student’s t test. ( B ) Pearson’s correlation of NANOG signature expression with indicated transcripts of T cell–recruiting chemokines. RSEM, relative SEM. ( C ) qPCR analysis of Cxcl9 and Cxcl10 mRNA expression. ( D ) Western blot analysis of the expression of CXCL10. ( E – G ) CT26-no insert or CT26- Nanog cells were transfected with si GFP or si Hdac1 . WCL, whole-cell lysate; SUP, supernatant. ( E ) Western blot analysis of CXCL10 expression. ( F ) qPCR analysis of Cxcl10 mRNA expression. ( G ) Relative occupancy of AcH3K14, AcH3K27, and HDAC1 in the Cxcl10 promoters was assessed by qChIP analysis. The ChIP data values represent ratios relative to the input. ( H ) Transwell T cell migration assay using CT26- Nanog -si GFP or CT26- Nanog -si Hdac1 cells were treated with IgG or anti-CXCL10. ( I ) Western blot analysis of the expression of GFP (CXCL10) in CT26-no insert or CT26- Nanog WT cells transduced with Cxcl10 WT or Cxcl10 MT. ( J – L ) CT26-no insert, CT26- Nanog - Cxcl10 WT, or CT26- Nanog - Cxcl10 MT tumor–bearing mice were treated with anti–PD-1 antibody. ( J ) Tumor growth curves. ( K ) Flow cytometric profiles of tumor-infiltrating CD3 + CD8 + T cells. ( L ) Ratio of granzyme B + to tumor-infiltrating CD3 + CD8 + T cells. Five mice from each group were used for in vivo experiments. ( D , E , and I ) β-Actin was used as an internal loading control. Results in the graphs represent 3 independent experiments performed in triplicate. Data represent the mean ± SD. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Transfection, Chromatin Immunoprecipitation, Cell Migration Assay, Transduction, Mouse Assay, In Vivo

27) Product Images from "The acid ceramidase/ceramide axis controls parasitemia in Plasmodium yoelii-infected mice by regulating erythropoiesis"

Article Title: The acid ceramidase/ceramide axis controls parasitemia in Plasmodium yoelii-infected mice by regulating erythropoiesis

Journal: bioRxiv

doi: 10.1101/2022.03.08.483432

P. yoelii -infected Ac KO mice show decreased parasitemia with less T cell activation in the early phase of infection. (A) Parasitemia of P. yoelii -infected Ac WT and Ac KO mice was determined at indicated time points by microscopy of giemsa-stained blood films (n=9-18). (B) Spleen weight on day 7 and 14 post infection (p.i.) (n=7-9). (C) Frequencies of viable CD4 + , CD8 + , and regulatory T cells (Foxp3 + of CD4 + ) were analysed by flow cytometry 7 days p.i. (n=7-9). (D) Percentages of Ki67-, CD49dCD11a/CD11a-, and PD1-expressing CD4 + and CD8 + T cells were determined by flow cytometry 7 days p.i. (n=7-9). Data from three to five independent experiments are presented as mean ± SEM. Statistical analyses were performed using unpaired Student’s t-test or Mann-Whitney U-test (** p
Figure Legend Snippet: P. yoelii -infected Ac KO mice show decreased parasitemia with less T cell activation in the early phase of infection. (A) Parasitemia of P. yoelii -infected Ac WT and Ac KO mice was determined at indicated time points by microscopy of giemsa-stained blood films (n=9-18). (B) Spleen weight on day 7 and 14 post infection (p.i.) (n=7-9). (C) Frequencies of viable CD4 + , CD8 + , and regulatory T cells (Foxp3 + of CD4 + ) were analysed by flow cytometry 7 days p.i. (n=7-9). (D) Percentages of Ki67-, CD49dCD11a/CD11a-, and PD1-expressing CD4 + and CD8 + T cells were determined by flow cytometry 7 days p.i. (n=7-9). Data from three to five independent experiments are presented as mean ± SEM. Statistical analyses were performed using unpaired Student’s t-test or Mann-Whitney U-test (** p

Techniques Used: Infection, Mouse Assay, Activation Assay, Microscopy, Staining, Flow Cytometry, Expressing, MANN-WHITNEY

T cell-specific and myeloid-specific Ac deletion has no impact on the course of P. yoelii infection. (A) The knockout of Ac in T cells was confirmed by analysing Asah1 mRNA expression of sorted splenic CD4 + and CD8 + T cells from naïve Ac flox/flox /CD4cre tg (Ac CD4cre KO) mice and Ac flox/flox /CD4cre wt littermates (Ac CD4cre WT) as controls via RT-qPCR (n=2-4). (B) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac CD4cre KO mice and Ac CD4cre WT littermates was determined at indicated time points (n=7-10). (C) The knockout of Ac in myeloid cells was confirmed by analysing Asah1 mRNA expression of macrophages, dendritic cells, and neutrophils isolated from spleen, peritoneal lavage (pLavage), and blood of naïve Ac flox/flox /LysMcre tg (Ac LysMcre KO) mice and Ac flox/flox /LysMcre wt littermates (Ac LysMcre WT) as controls via RT-qPCR (n=2-6). (D) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac LysMcre KO and Ac LysMcre WT mice was determined at indicated time points (n=9). Data from two independent experiments each are presented as mean ± SEM.
Figure Legend Snippet: T cell-specific and myeloid-specific Ac deletion has no impact on the course of P. yoelii infection. (A) The knockout of Ac in T cells was confirmed by analysing Asah1 mRNA expression of sorted splenic CD4 + and CD8 + T cells from naïve Ac flox/flox /CD4cre tg (Ac CD4cre KO) mice and Ac flox/flox /CD4cre wt littermates (Ac CD4cre WT) as controls via RT-qPCR (n=2-4). (B) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac CD4cre KO mice and Ac CD4cre WT littermates was determined at indicated time points (n=7-10). (C) The knockout of Ac in myeloid cells was confirmed by analysing Asah1 mRNA expression of macrophages, dendritic cells, and neutrophils isolated from spleen, peritoneal lavage (pLavage), and blood of naïve Ac flox/flox /LysMcre tg (Ac LysMcre KO) mice and Ac flox/flox /LysMcre wt littermates (Ac LysMcre WT) as controls via RT-qPCR (n=2-6). (D) Parasitemia (left panel) and spleen weight (right panel) of P. yoelii -infected Ac LysMcre KO and Ac LysMcre WT mice was determined at indicated time points (n=9). Data from two independent experiments each are presented as mean ± SEM.

Techniques Used: Infection, Knock-Out, Expressing, Mouse Assay, Quantitative RT-PCR, Isolation

28) Product Images from "HRSV prefusion-F protein with Adju-Phos adjuvant induces long-lasting Th2-biased immunity in mice"

Article Title: HRSV prefusion-F protein with Adju-Phos adjuvant induces long-lasting Th2-biased immunity in mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0262231

Intracellular IL-4 and IFN-γ production by CD8+ T cells. (A) The spots in the rectangular box represent IFN-γ-secreting or IL-4-secreting CD8+ T cells. (B) The percentages of CD8+ T cells producing IFN-γ or IL-4. (C) Ratio of IL-4-secreting CD8+ T cells and IFN-γ-secreting CD8+ T cells. Statistically significant differences were measured by one-way ANOVA with Newman–Keuls posttest. *** p
Figure Legend Snippet: Intracellular IL-4 and IFN-γ production by CD8+ T cells. (A) The spots in the rectangular box represent IFN-γ-secreting or IL-4-secreting CD8+ T cells. (B) The percentages of CD8+ T cells producing IFN-γ or IL-4. (C) Ratio of IL-4-secreting CD8+ T cells and IFN-γ-secreting CD8+ T cells. Statistically significant differences were measured by one-way ANOVA with Newman–Keuls posttest. *** p

Techniques Used:

29) Product Images from "Using a Prime-Boost Vaccination Strategy That Proved Effective for High Resolution Epitope Mapping to Characterize the Elusive Immunogenicity of Survivin"

Article Title: Using a Prime-Boost Vaccination Strategy That Proved Effective for High Resolution Epitope Mapping to Characterize the Elusive Immunogenicity of Survivin

Journal: Cancers

doi: 10.3390/cancers13246270

Blocking CTLA4 was unable to rescue the lack of immunogenicity of murine survivin. C57BL/6 mice were given 200 μg of a CTLA4-blocking antibody intraperitoneally 24 h prior to vaccination, as well as 100 μg one hour before vaccination. The mice were then vaccinated with Ad48-T34A-mSurvivin at 1 × 10 8 ifu and boosted 14 days later with MG1-T34A-mSurvivin at 1 × 10 9 pfu. Intracellular cytokine staining following peptide re-stimulation at the expected peak of the secondary T cell response failed to reveal detectable survivin-specific CD8 + ( top ) or CD4 + ( bottom ) T cell responses.
Figure Legend Snippet: Blocking CTLA4 was unable to rescue the lack of immunogenicity of murine survivin. C57BL/6 mice were given 200 μg of a CTLA4-blocking antibody intraperitoneally 24 h prior to vaccination, as well as 100 μg one hour before vaccination. The mice were then vaccinated with Ad48-T34A-mSurvivin at 1 × 10 8 ifu and boosted 14 days later with MG1-T34A-mSurvivin at 1 × 10 9 pfu. Intracellular cytokine staining following peptide re-stimulation at the expected peak of the secondary T cell response failed to reveal detectable survivin-specific CD8 + ( top ) or CD4 + ( bottom ) T cell responses.

Techniques Used: Blocking Assay, Mouse Assay, Staining

Co-vaccination against survivin and dopachrome tautomerase did not reduce the magnitude of a DCT-specific CD8 + T cell response when compared to DCT vaccination alone. Female C57BL/6 mice were vaccinated with Ad-hDCT, Ad-T34A-mSurvivin, or Ad-hDCT + Ad-T34A-mSurvivin at a total dose of 1 × 10 8 ifu intramuscularly. T cell responses were quantified ten days post-vaccination by flow cytometric assessment of intracellular cytokine staining after ex vivo re-stimulation with peptides. Blood-derived DCT 180–188 -specific CD8 + T cell response frequency ( left ) and total number ( right ) were not significantly different in mice vaccinated against DCT alone versus co-vaccination against DCT and murine survivin. Blood-derived mSurvivin 53–67 specific CD8 + T cell responses were not detectable (data not shown). Significance was determined by one-way analysis of variance with Tukey’s multiple comparison test ( n = 4/treatment).
Figure Legend Snippet: Co-vaccination against survivin and dopachrome tautomerase did not reduce the magnitude of a DCT-specific CD8 + T cell response when compared to DCT vaccination alone. Female C57BL/6 mice were vaccinated with Ad-hDCT, Ad-T34A-mSurvivin, or Ad-hDCT + Ad-T34A-mSurvivin at a total dose of 1 × 10 8 ifu intramuscularly. T cell responses were quantified ten days post-vaccination by flow cytometric assessment of intracellular cytokine staining after ex vivo re-stimulation with peptides. Blood-derived DCT 180–188 -specific CD8 + T cell response frequency ( left ) and total number ( right ) were not significantly different in mice vaccinated against DCT alone versus co-vaccination against DCT and murine survivin. Blood-derived mSurvivin 53–67 specific CD8 + T cell responses were not detectable (data not shown). Significance was determined by one-way analysis of variance with Tukey’s multiple comparison test ( n = 4/treatment).

Techniques Used: Mouse Assay, Staining, Ex Vivo, Derivative Assay

Prime-boosting against eGFP with Ad-eGFP and VSV-eGFP induced CD4 + and CD8 + T cell responses of high magnitude. ( a ) C57BL/6 and ( b ) BALB/c mice were vaccinated against eGFP by priming intramuscularly with Ad-eGFP at 1 × 10 8 ifu, and primary responses were assessed 10 days later. Mice were boosted intravenously with VSV-eGFP (1 × 10 9 pfu) 14 days post-Ad, and the seconda ry responses were assessed five days post-VSV. Graphs depict CD8 + (left) and CD4 + (right) T cell responses to the immunodominant and subdominant epitopes of eGFP via intracellular cytokine staining after peptide re-stimulation. Standard errors of the means are shown. Data represent n = 3 and n = 4 C57BL/6 and BALB/c mice per group, respectively.
Figure Legend Snippet: Prime-boosting against eGFP with Ad-eGFP and VSV-eGFP induced CD4 + and CD8 + T cell responses of high magnitude. ( a ) C57BL/6 and ( b ) BALB/c mice were vaccinated against eGFP by priming intramuscularly with Ad-eGFP at 1 × 10 8 ifu, and primary responses were assessed 10 days later. Mice were boosted intravenously with VSV-eGFP (1 × 10 9 pfu) 14 days post-Ad, and the seconda ry responses were assessed five days post-VSV. Graphs depict CD8 + (left) and CD4 + (right) T cell responses to the immunodominant and subdominant epitopes of eGFP via intracellular cytokine staining after peptide re-stimulation. Standard errors of the means are shown. Data represent n = 3 and n = 4 C57BL/6 and BALB/c mice per group, respectively.

Techniques Used: Mouse Assay, Staining

Enhanced green fluorescent protein (eGFP) epitope maps showing dominant and subdominant peptides for CD8 + and CD4 + T cell responses in C57BL/6 and BALB/c mice. C57BL/6 and BALB/c mice were vaccinated with Ad-eGFP, followed by a VSV-eGFP boost, and eGFP-specific responses were analyzed at the peak of the response (five days post-boost) using peptide re-stimulation and intracellular flow cytometry staining. Shown is the complete eGFP amino acid sequence with boxes that show the peptides that contained the immunodominant and subdominant epitopes for ( a ) CD8 + and ( b ) CD4 + T cells in C57BL/6 mice. Similarly, peptides containing eGFP-derived ( c ) CD8 + and ( d ) CD4 + T cell epitopes in BALB/c mice are shown. Sequences containing the complete epitope are boxed with a bolded line. Conversely, sequences containing partial epitopes are boxed in a thin dotted line. Data were derived from a minimum of three mice per group.
Figure Legend Snippet: Enhanced green fluorescent protein (eGFP) epitope maps showing dominant and subdominant peptides for CD8 + and CD4 + T cell responses in C57BL/6 and BALB/c mice. C57BL/6 and BALB/c mice were vaccinated with Ad-eGFP, followed by a VSV-eGFP boost, and eGFP-specific responses were analyzed at the peak of the response (five days post-boost) using peptide re-stimulation and intracellular flow cytometry staining. Shown is the complete eGFP amino acid sequence with boxes that show the peptides that contained the immunodominant and subdominant epitopes for ( a ) CD8 + and ( b ) CD4 + T cells in C57BL/6 mice. Similarly, peptides containing eGFP-derived ( c ) CD8 + and ( d ) CD4 + T cell epitopes in BALB/c mice are shown. Sequences containing the complete epitope are boxed with a bolded line. Conversely, sequences containing partial epitopes are boxed in a thin dotted line. Data were derived from a minimum of three mice per group.

Techniques Used: Mouse Assay, Flow Cytometry, Staining, Sequencing, Derivative Assay

30) Product Images from "Sensitive identification of neoantigens and cognate TCRs in human solid tumors"

Article Title: Sensitive identification of neoantigens and cognate TCRs in human solid tumors

Journal: Nature Biotechnology

doi: 10.1038/s41587-021-01072-6

Frequency, reactivity and efficacy of tumor-reactive TCRs. a , Heatmaps reporting the frequencies of antigen-specific TCRβ clonotypes from patients 2, 5 and 6 within the different bulk TIL populations (top). TCRs are detailed in Supplementary Table 5 . Antitumor-reactivity of TCR-transfected primary CD8 T cells, measured by 4-1BB upregulation following co-culture with autologous tumor cells (bottom). The background levels of 4-1BB expressed by cognate negative controls (TCR-T cells alone) were subtracted (Supplementary Fig. 4 ). b , Representative example of flow cytometry data showing in vitro tumor recognition (4-1BB upregulation) of antigen-specific TCRs (MAGEC1 TCRs A and B and SCM1A L674S TCR C) from patient 1. (MOCK: control of transfection, neg pair: irrelevant TCRα/β pair). c , In vivo efficacy of adoptively-transferred tyrosinase 508-514 TCR-transduced T cells against autologous patient-derived tumor xenografts. The graph shows tumor size of individual hIL-2 NOG mice adoptively-transferred with TCR-transduced (in orange; n = 7) and untransduced (in blue; n = 5) cells. ACT was performed on Day 14.
Figure Legend Snippet: Frequency, reactivity and efficacy of tumor-reactive TCRs. a , Heatmaps reporting the frequencies of antigen-specific TCRβ clonotypes from patients 2, 5 and 6 within the different bulk TIL populations (top). TCRs are detailed in Supplementary Table 5 . Antitumor-reactivity of TCR-transfected primary CD8 T cells, measured by 4-1BB upregulation following co-culture with autologous tumor cells (bottom). The background levels of 4-1BB expressed by cognate negative controls (TCR-T cells alone) were subtracted (Supplementary Fig. 4 ). b , Representative example of flow cytometry data showing in vitro tumor recognition (4-1BB upregulation) of antigen-specific TCRs (MAGEC1 TCRs A and B and SCM1A L674S TCR C) from patient 1. (MOCK: control of transfection, neg pair: irrelevant TCRα/β pair). c , In vivo efficacy of adoptively-transferred tyrosinase 508-514 TCR-transduced T cells against autologous patient-derived tumor xenografts. The graph shows tumor size of individual hIL-2 NOG mice adoptively-transferred with TCR-transduced (in orange; n = 7) and untransduced (in blue; n = 5) cells. ACT was performed on Day 14.

Techniques Used: Transfection, Co-Culture Assay, Flow Cytometry, In Vitro, In Vivo, Derivative Assay, Mouse Assay

Increased detection of tumor antigen-specific CD8 T cells with NeoScreen . a , Frequency of neoepitope-specific CD8 T cells from patients 6 and 7 measured with pMHC multimers (CTRL: control, NA: not available, LP: long peptide, TMG: tandem minigene). b , Cumulative analysis of the frequency of tumor antigen-specific T cells ( n = 4 epitopes, Supplementary Table 4 ) in conventional (x-axis) and NeoScreen (y-axis) cultures of patients 6 and 7. c , Representative example of the frequency of neoepitope- and TAA-specific CD8 T cells from patient 1 measured with pMHC multimers. d , Cumulative analysis of the frequency of tumor antigen-specific T cells ( n = 9 enriched epitopes from seven patients dedicated to antigen discovery, Supplementary Table 2 ) in conventional (x-axis) and NeoScreen (y-axis) cultures, by pMHC multimers. e-g , Magnitude of tumor antigen-specific CD8 T cells (determined by IFNγ Spot Forming Unit per 10 5 cells ( e , n = 22 epitopes), pMHC-multimers staining ( f , n = 13) or upregulation of 4-1BB ( g , n = 20)) obtained with NeoScreen or conventional cultures. Box plots represent median (line), 25% and 75% confidence limit (box limits) and min to max (whiskers). h , Cumulative frequencies of tumor antigen-specific T cells, for enriched epitopes only ( n = 13 epitopes from all nine patients) in conventional (x-axis) and NeoScreen (y-axis) cultures, by pMHC multimers or 4-1BB up-regulation. i , Cumulative frequencies of tumor antigen-specific T cells ( n = 20 epitopes from all nine patients) in x1 NeoScreen (x-axis) and x2 NeoScreen (y-axis) cultures, by pMHC multimers or 4-1BB up-regulation. In d , e , g-i , the background levels of IFNγ Spot Forming Unit ( e ) or 4-1BB expression ( d , g-i ) by cognate negative controls (TILs alone) were subtracted. In b , d , h and i , the highest values between 1x NeoScreen and 2x NeoScreen are considered and data are displayed in logarithmic scale. In b , d and e-i , P-values were determined with one-tailed paired t-tests.
Figure Legend Snippet: Increased detection of tumor antigen-specific CD8 T cells with NeoScreen . a , Frequency of neoepitope-specific CD8 T cells from patients 6 and 7 measured with pMHC multimers (CTRL: control, NA: not available, LP: long peptide, TMG: tandem minigene). b , Cumulative analysis of the frequency of tumor antigen-specific T cells ( n = 4 epitopes, Supplementary Table 4 ) in conventional (x-axis) and NeoScreen (y-axis) cultures of patients 6 and 7. c , Representative example of the frequency of neoepitope- and TAA-specific CD8 T cells from patient 1 measured with pMHC multimers. d , Cumulative analysis of the frequency of tumor antigen-specific T cells ( n = 9 enriched epitopes from seven patients dedicated to antigen discovery, Supplementary Table 2 ) in conventional (x-axis) and NeoScreen (y-axis) cultures, by pMHC multimers. e-g , Magnitude of tumor antigen-specific CD8 T cells (determined by IFNγ Spot Forming Unit per 10 5 cells ( e , n = 22 epitopes), pMHC-multimers staining ( f , n = 13) or upregulation of 4-1BB ( g , n = 20)) obtained with NeoScreen or conventional cultures. Box plots represent median (line), 25% and 75% confidence limit (box limits) and min to max (whiskers). h , Cumulative frequencies of tumor antigen-specific T cells, for enriched epitopes only ( n = 13 epitopes from all nine patients) in conventional (x-axis) and NeoScreen (y-axis) cultures, by pMHC multimers or 4-1BB up-regulation. i , Cumulative frequencies of tumor antigen-specific T cells ( n = 20 epitopes from all nine patients) in x1 NeoScreen (x-axis) and x2 NeoScreen (y-axis) cultures, by pMHC multimers or 4-1BB up-regulation. In d , e , g-i , the background levels of IFNγ Spot Forming Unit ( e ) or 4-1BB expression ( d , g-i ) by cognate negative controls (TILs alone) were subtracted. In b , d , h and i , the highest values between 1x NeoScreen and 2x NeoScreen are considered and data are displayed in logarithmic scale. In b , d and e-i , P-values were determined with one-tailed paired t-tests.

Techniques Used: Staining, Expressing, One-tailed Test

Improved identification of neoantigen-specific CD8 T cells with NeoScreen . a-b , Neoantigen discovery with NeoScreen ( n = 6 patients, Supplementary Table 2 ). a , Cumulative analysis of the frequency of neoepitope-specific T cells ( n = 11 neoepitopes, Supplementary Table 4 ) in conventional (x-axis) and NeoScreen (y-axis) cultures of patients 1, 2, 4, 5, 8 and 9, by pMHC multimers staining or 4-1BB up-regulation. b , Proportion of neoepitopes among enriched versus newly-detected T cell reactivities. c , Cumulative frequencies of neoepitope-specific CD8 T cells ( n = 15 neoepitopes from 8 patients) in in vitro -expanded TIL cultures (x2: re-stimulated). Box plots represent median (line), 25% and 75% confidence limit (box limits) and min to max (whiskers). d , Frequencies of neoepitope-specific CD8 T cells ( n = 15) in conventional (x-axis) and NeoScreen (y-axis) cultures. e , Cumulative frequencies of neoepitope-specific T cells ( n = 13 neoepitopes) in x1 NeoScreen (x-axis) and x2 NeoScreen (y-axis) cultures. In a , c-e , the background levels of 4-1BB expression by cognate negative controls were subtracted. In a and d , the highest values between 1x NeoScreen and 2x NeoScreen are considered. In a and c-e , P-values were determined with one-tailed paired t-tests and data are displayed in logarithmic scale.
Figure Legend Snippet: Improved identification of neoantigen-specific CD8 T cells with NeoScreen . a-b , Neoantigen discovery with NeoScreen ( n = 6 patients, Supplementary Table 2 ). a , Cumulative analysis of the frequency of neoepitope-specific T cells ( n = 11 neoepitopes, Supplementary Table 4 ) in conventional (x-axis) and NeoScreen (y-axis) cultures of patients 1, 2, 4, 5, 8 and 9, by pMHC multimers staining or 4-1BB up-regulation. b , Proportion of neoepitopes among enriched versus newly-detected T cell reactivities. c , Cumulative frequencies of neoepitope-specific CD8 T cells ( n = 15 neoepitopes from 8 patients) in in vitro -expanded TIL cultures (x2: re-stimulated). Box plots represent median (line), 25% and 75% confidence limit (box limits) and min to max (whiskers). d , Frequencies of neoepitope-specific CD8 T cells ( n = 15) in conventional (x-axis) and NeoScreen (y-axis) cultures. e , Cumulative frequencies of neoepitope-specific T cells ( n = 13 neoepitopes) in x1 NeoScreen (x-axis) and x2 NeoScreen (y-axis) cultures. In a , c-e , the background levels of 4-1BB expression by cognate negative controls were subtracted. In a and d , the highest values between 1x NeoScreen and 2x NeoScreen are considered. In a and c-e , P-values were determined with one-tailed paired t-tests and data are displayed in logarithmic scale.

Techniques Used: Staining, In Vitro, Expressing, One-tailed Test

Sensitive tumor antigen discovery. a , NeoScreen pipeline. b – e , Antigen discovery with NeoScreen ( n = 7 patients). b , c , Representative examples of flow cytometry data ( b ) and cumulative frequencies ( c ) of tumor antigen-specific CD8 T cells ( n = 19 epitopes) in conventional ( x axis) and NeoScreen ( y axis) TIL cultures, by pMHC-multimers or 4-1BB upregulation. d , Proportions of neoepitope- versus TAA-specific among enriched versus newly detected T cell reactivities. e , Number of tumor epitopes per patient identified with conventional and NeoScreen strategies (histograms report median values). f , Frequencies of tumor antigen-specific CD8 T cells ( n = 23 epitopes from nine patients) in conventional ( x axis) and NeoScreen ( y axis) cultures. g , Frequencies of antigen-specific CD8 T cells ( n = 23) in in vitro expanded TIL cultures (2×: re-stimulated). Box plots represent the median (line), 25% and 75% confidence limit (box limits) and min to max (whiskers). In c , f and g , the background levels of 4-1BB expressed by cognate negative controls were subtracted. In c and f , the highest values between 1×NeoScreen and 2×NeoScreen are considered, and data are displayed in logarithmic scale. In c and e – g , P values were determined with one-tailed paired t -tests.
Figure Legend Snippet: Sensitive tumor antigen discovery. a , NeoScreen pipeline. b – e , Antigen discovery with NeoScreen ( n = 7 patients). b , c , Representative examples of flow cytometry data ( b ) and cumulative frequencies ( c ) of tumor antigen-specific CD8 T cells ( n = 19 epitopes) in conventional ( x axis) and NeoScreen ( y axis) TIL cultures, by pMHC-multimers or 4-1BB upregulation. d , Proportions of neoepitope- versus TAA-specific among enriched versus newly detected T cell reactivities. e , Number of tumor epitopes per patient identified with conventional and NeoScreen strategies (histograms report median values). f , Frequencies of tumor antigen-specific CD8 T cells ( n = 23 epitopes from nine patients) in conventional ( x axis) and NeoScreen ( y axis) cultures. g , Frequencies of antigen-specific CD8 T cells ( n = 23) in in vitro expanded TIL cultures (2×: re-stimulated). Box plots represent the median (line), 25% and 75% confidence limit (box limits) and min to max (whiskers). In c , f and g , the background levels of 4-1BB expressed by cognate negative controls were subtracted. In c and f , the highest values between 1×NeoScreen and 2×NeoScreen are considered, and data are displayed in logarithmic scale. In c and e – g , P values were determined with one-tailed paired t -tests.

Techniques Used: Flow Cytometry, In Vitro, One-tailed Test

Limited cross-reactivity of neoepitope-specific CD8 T cell responses. a-j , Representative examples of T cell responses of NeoScreen TILs against mutated (MUT) vs wild type (WT) peptides (each at 1 µg/mL) by IFNγ ELISpot assay (mean±SD of duplicate). Sequences are detailed in Supplementary Table 4 . Dose titration curves of T cell responses against neoepitopes and cognate WT peptide ( d , f and h ). (PMA-iono: phorbol 12-myristate 13-acetate ionomycin).
Figure Legend Snippet: Limited cross-reactivity of neoepitope-specific CD8 T cell responses. a-j , Representative examples of T cell responses of NeoScreen TILs against mutated (MUT) vs wild type (WT) peptides (each at 1 µg/mL) by IFNγ ELISpot assay (mean±SD of duplicate). Sequences are detailed in Supplementary Table 4 . Dose titration curves of T cell responses against neoepitopes and cognate WT peptide ( d , f and h ). (PMA-iono: phorbol 12-myristate 13-acetate ionomycin).

Techniques Used: Enzyme-linked Immunospot, Titration

Tumor-reactive TCR identification and validation. a , Representative example of neoepitope-specific CD8 T cell sorting by pMHC-multimer. Manhattan plot depicts TCRβ chain VJ recombination of PHLPP2 N1186Y -specific clonotypes A, B and C. b , Validation of antigen specificity after TCR cloning. c , Superimposition of the modeled TCR-pMHC complexes for TCR-A, TCR-B and TCR-C. The location of CDR3α and CDR3β loops is shown by arrows. d , Violin plots display frequencies of TCRβ-A, TCRβ-B and TCRβ-C in bulk TCR repertoires of the different TIL cultures and of the original tumor. e , Heat maps depict the frequencies of tumor antigen-specific TCRβ clonotypes ( n = 50) within the different bulk TIL populations (top). Overview (bottom) of tumor reactivity of TCR-transfected primary CD8 T cells ( n = 31 and Extended Data Fig. 10a ). The background levels of 4-1BB expressed by cognate negative controls (TCR T cells alone) were subtracted (Supplementary Fig. 4 ). In d and e , NeoScreen TILs from patient 7 were generated with long peptides. f , Cumulative analysis of the frequency of tumor antigen-specific TCRβ detected in conventional ( x axis) and NeoScreen ( y axis) cultures. Highest values between 1×NeoScreen and 2×NeoScreen are considered, and data are displayed in logarithmic scale. P value was determined with a one-tailed paired t -test. g , Proportions of neoepitope- versus TAA-specific TCRβ among enriched versus newly detected clonotypes. h , ACT of TCR-transduced T cells in autologous patient-derived xenograft tumor model. i , In vivo efficacy of adoptively transferred tyrosinase 508–514 TCR-transduced T cells against autologous patient-derived tumor xenografts. The graph shows tumor size (mean ± s.e.m. of replicates) over time. P value was determined with a one-tailed unpaired t -test.
Figure Legend Snippet: Tumor-reactive TCR identification and validation. a , Representative example of neoepitope-specific CD8 T cell sorting by pMHC-multimer. Manhattan plot depicts TCRβ chain VJ recombination of PHLPP2 N1186Y -specific clonotypes A, B and C. b , Validation of antigen specificity after TCR cloning. c , Superimposition of the modeled TCR-pMHC complexes for TCR-A, TCR-B and TCR-C. The location of CDR3α and CDR3β loops is shown by arrows. d , Violin plots display frequencies of TCRβ-A, TCRβ-B and TCRβ-C in bulk TCR repertoires of the different TIL cultures and of the original tumor. e , Heat maps depict the frequencies of tumor antigen-specific TCRβ clonotypes ( n = 50) within the different bulk TIL populations (top). Overview (bottom) of tumor reactivity of TCR-transfected primary CD8 T cells ( n = 31 and Extended Data Fig. 10a ). The background levels of 4-1BB expressed by cognate negative controls (TCR T cells alone) were subtracted (Supplementary Fig. 4 ). In d and e , NeoScreen TILs from patient 7 were generated with long peptides. f , Cumulative analysis of the frequency of tumor antigen-specific TCRβ detected in conventional ( x axis) and NeoScreen ( y axis) cultures. Highest values between 1×NeoScreen and 2×NeoScreen are considered, and data are displayed in logarithmic scale. P value was determined with a one-tailed paired t -test. g , Proportions of neoepitope- versus TAA-specific TCRβ among enriched versus newly detected clonotypes. h , ACT of TCR-transduced T cells in autologous patient-derived xenograft tumor model. i , In vivo efficacy of adoptively transferred tyrosinase 508–514 TCR-transduced T cells against autologous patient-derived tumor xenografts. The graph shows tumor size (mean ± s.e.m. of replicates) over time. P value was determined with a one-tailed unpaired t -test.

Techniques Used: FACS, Clone Assay, Transfection, Generated, One-tailed Test, Derivative Assay, In Vivo

31) Product Images from "Short- and Long-Term Immunological Responses in Chronic HCV/HIV Co-Infected Compared to HCV Mono-Infected Patients after DAA Therapy"

Article Title: Short- and Long-Term Immunological Responses in Chronic HCV/HIV Co-Infected Compared to HCV Mono-Infected Patients after DAA Therapy

Journal: Pathogens

doi: 10.3390/pathogens10111488

Longitudinal analysis of CD4+ and CD8+ T cells and of CD4+/CD8+ T-cell subsets before and after DAA therapy. Whole blood T-cell subsets of HCV mono-infected and HCV/HIV co-infected were compared from T0 up to T4. Levels of CD4 ( A ), CD8 ( B ), and different CD4 and CD8 activation subsets on the basis of CD69 ( C ), CD25 ( D ), HLA-DR ( E ), and CD38 ( F ) expression are shown. Statistically significant differences over time are marked by an arrow and asterisk, and differences between the two groups of patients by an asterisk. One asterisk corresponds to p
Figure Legend Snippet: Longitudinal analysis of CD4+ and CD8+ T cells and of CD4+/CD8+ T-cell subsets before and after DAA therapy. Whole blood T-cell subsets of HCV mono-infected and HCV/HIV co-infected were compared from T0 up to T4. Levels of CD4 ( A ), CD8 ( B ), and different CD4 and CD8 activation subsets on the basis of CD69 ( C ), CD25 ( D ), HLA-DR ( E ), and CD38 ( F ) expression are shown. Statistically significant differences over time are marked by an arrow and asterisk, and differences between the two groups of patients by an asterisk. One asterisk corresponds to p

Techniques Used: Infection, Activation Assay, Expressing

Longitudinal analysis of CD4+/CD8+ T-cell subsets before and after DAA therapy. Whole blood T-cell subsets of HCV mono-infected and HCV/HIV co-infected were compared from T0 up to T4. Levels of expression of CD28 ( A,B ), CD45RO ( C , D ), and exhaustion PD-1 ( E , F ) markers are shown. Statistically significant differences over time are marked by an arrow and asterisk, and differences between the two groups of patients by an asterisk. One asterisk corresponds to p
Figure Legend Snippet: Longitudinal analysis of CD4+/CD8+ T-cell subsets before and after DAA therapy. Whole blood T-cell subsets of HCV mono-infected and HCV/HIV co-infected were compared from T0 up to T4. Levels of expression of CD28 ( A,B ), CD45RO ( C , D ), and exhaustion PD-1 ( E , F ) markers are shown. Statistically significant differences over time are marked by an arrow and asterisk, and differences between the two groups of patients by an asterisk. One asterisk corresponds to p

Techniques Used: Infection, Expressing

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