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BioLegend anti cd8
Comparison of <t>CD8</t> + T cells and its subsets in patients with HNSCC in T1 + T2 and T3 + T4 groups. CD8 + T cells gated on the lymphocyte population and then the frequencies of T cell subpopulations were determined within CD8 + T cell gate. Horizontal bar is representative of the Mean ± SEM, * P value
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1) Product Images from "Regulatory and effector T cell subsets in tumor-draining lymph nodes of patients with squamous cell carcinoma of head and neck"

Article Title: Regulatory and effector T cell subsets in tumor-draining lymph nodes of patients with squamous cell carcinoma of head and neck

Journal: BMC Immunology

doi: 10.1186/s12865-022-00530-3

Comparison of CD8 + T cells and its subsets in patients with HNSCC in T1 + T2 and T3 + T4 groups. CD8 + T cells gated on the lymphocyte population and then the frequencies of T cell subpopulations were determined within CD8 + T cell gate. Horizontal bar is representative of the Mean ± SEM, * P value
Figure Legend Snippet: Comparison of CD8 + T cells and its subsets in patients with HNSCC in T1 + T2 and T3 + T4 groups. CD8 + T cells gated on the lymphocyte population and then the frequencies of T cell subpopulations were determined within CD8 + T cell gate. Horizontal bar is representative of the Mean ± SEM, * P value

Techniques Used:

Comparison of CD8 + T cells and its subsets in TDLNs of HNSCC patients in low stages (I + II) and advanced stages (III + IV) of the disease. CD8 + T cells gated on the lymphocyte population and then the frequencies of T cell subpopulations were determined within CD8 + T cell gate. Horizontal bar is representative of the Mean ± SEM, * P value
Figure Legend Snippet: Comparison of CD8 + T cells and its subsets in TDLNs of HNSCC patients in low stages (I + II) and advanced stages (III + IV) of the disease. CD8 + T cells gated on the lymphocyte population and then the frequencies of T cell subpopulations were determined within CD8 + T cell gate. Horizontal bar is representative of the Mean ± SEM, * P value

Techniques Used:

Flow cytometry analysis of CD8 + T cell subsets in the tumor draining lymph nodes of patients with HNSCC. A Lymphocytes were gated, B CD8 + T cells were gated followed by defining these subpopulations in CD8 + T cells gate: C TNF-α + cells, D IFN-γ + cells, E (1) IFN-γ + TNF-α + cells, (2) IFN-γ + TNF-α − cells, F IL-4 + cells, G IL-17 + cells, H IL-10 + cells, I TGF-β + cells subsets according to their cognate cytokine expression
Figure Legend Snippet: Flow cytometry analysis of CD8 + T cell subsets in the tumor draining lymph nodes of patients with HNSCC. A Lymphocytes were gated, B CD8 + T cells were gated followed by defining these subpopulations in CD8 + T cells gate: C TNF-α + cells, D IFN-γ + cells, E (1) IFN-γ + TNF-α + cells, (2) IFN-γ + TNF-α − cells, F IL-4 + cells, G IL-17 + cells, H IL-10 + cells, I TGF-β + cells subsets according to their cognate cytokine expression

Techniques Used: Flow Cytometry, Expressing

2) Product Images from "A multi-enhancer hub at the Ets1 locus controls T cell differentiation and allergic inflammation through 3D genome topology"

Article Title: A multi-enhancer hub at the Ets1 locus controls T cell differentiation and allergic inflammation through 3D genome topology

Journal: bioRxiv

doi: 10.1101/2022.10.28.514213

Ets1 -SE deletion limits Th1-mediated inflammation in vivo a, Schematic representation of the CD45RB High -induced colitis model. 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve CD4 + T cells from wildtype or Ets1- SE −/− were transferred into Rag1 −/− recipients to induce colitis. Weight was monitored once a week for six weeks post injection. b, Weight loss tracking of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells as compared to PBS-injected animals (controls) during 6 weeks post transfer. Weight was measured once per week. Three independent experiments were pooled and repeated four times. Dots represent the mean of individual mouse (PBS - > Rag1 −/− ; n= 5; wildtype - > Rag1 −/− ; n=25 and Ets1-SE −/− - > Rag1 −/− ; n=27). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Two-way ANOVA; Mixed-effect REML model with Fisher’s LSD Test). c, Quantification of colon length (cm) of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. Three independent experiments were pooled and repeated four times. Dots represent the mean of individual mouse (wildtype - > Rag1 −/− ; n=25 and Ets1-SE −/− - > Rag1 −/− ; n=27). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Mann-Whitney U Test). d,e Quantification of histological score of paraffin-embedded colon rolls from Rag1 −/− mice that received 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve CD4 + T cells from either wildtype or Ets1- SE −/− mice at week 6 post transfer and as compared to PBS-injected mice (control). Histological sections were obtained from two independent experiments and were scored in a blinded manner. Each dot represents an individual mouse (PBS - > Rag1 −/− ; n= 2, wildtype - > Rag1 −/− ; n=5 and Ets1-SE −/− - > Rag1 −/− ; n=8). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (One-way ANOVA with multiple comparisons and Bonferroni correction). e , Histological section of colon rolls stained with H E of Rag1 −/− mice that received either 1×10 6 FACS sorted TCR + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells 6 weeks post transfer. Scale = 100μm; Magnification = 100X. f, (left) Quantification of infiltrating colon lamina propria (cLP) colitogenic CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (middle) Quantification of Th1 (T-bet + ), Th2 (GATA-3 + ), Th17 (RORψ-t + ) and Tregs (FoxP3 + ) CD4 + T cells among infiltrating cLP CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (right) Quantification of Th1 (IFNψ + ), Th2 (IL-13 + ), Th17 (IL-17 + ) and Granzyme B (GzmB + ) producing CD4 + T cells among infiltrating cLP CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. Two independent experiments were pooled. Dots represent an individual mouse ( Ets1-SE +/+ - > Rag1 −/− ; n=7 and Ets1-SE −/− - > Rag1 −/− ; n=7). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (CD4 + T cells: Mann-Whitney U Test; Th1/Th2/Th17/Treg and cytokines production: Two-way ANOVA with multiple comparisons and Bonferroni correction). g, (left) Quantification CD4 + T cells in the spleen of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (middle) Quantification in the spleen of Th1 (T-bet + ), Th2 (GATA-3 + ), Th17 (RORψ-t + ) and Tregs (FoxP3 + ) CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (right) Quantification of ex vivo stimulated splenic Th1 (IFNψ + ), Th2 (IL-13 + ), Th17 (IL-17 + ) and Granzyme B (Gzm B + ) producing CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. Data are a representative of one experiment which was repeated three times. Dots represent an individual mouse (wildtype - > Rag1 −/− ; n=7 and Ets1- SE −/− - > Rag1 −/− ; n=7). Error bars = SEM; and p-values: ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (CD4 + T cells: Mann-Whitney U Test; Th1/Th2/Th17/Treg and cytokines production: Two-way ANOVA with multiple comparisons and Bonferroni correction). h, Representative flow cytometry contour plot of Granzyme B- and IFNψ-producing ex vivo stimulated cLP CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. i, Schematic representation of the house dust mite (HDM) extract challenge. Arrows represent days by which intranasal HDM was administered. Mice were euthanized 16 days after the initial sensitization and immune cell infiltration was checked. Two independent experiments were used to measure immune cell infiltration in the lung parenchyma. j, Quantification of lung parenchyma infiltrating CD4 + T cells (TCRβ + , CD4 + ), activated CD4 + T cells (TCRβ + , CD4 + , CD44 + ), CD8 + T cells (TCRβ + , CD8 + ), activated CD4 + T cells (TCRβ + , CD8 + , CD44 + ), CD4 + Th2 cells (TCRβ + , CD4 + , GATA-3 + ) and eosinophils (MHC-II − , Siglec-F + ) from wildtype or Ets1- SE −/− mice 16 days after HDM challenge. All cells were pre-gated on SSC-A/FSC-A, Singlets, Live cell (Viability − ). Two independent experiments were pooled. Each Dot represents an individual mouse (wildtype n=11 and Ets1-SE −/− n=11). Error bars = SEM; and ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Mann-Whitney). k, Quantification of Th2 cells numbers from wildtype or Ets1- SE −/− mice producing IL-5, IL-13 or IL-5/IL-13 four hours after PMA / Ionomycin stimulation. All cells were pre-gated on SSC-A/FSC-A, Singlets, Live cell (Viability − ), CD3 + , CD90.2 + , CD4 + , ST2 + . Data are representative of one independent experiment and repeated twice. Each dot represents an individual mouse (wildtype n=6 and Ets1- SE −/− n=6). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Two-way ANOVA with multiple comparison and Bonferroni correction).
Figure Legend Snippet: Ets1 -SE deletion limits Th1-mediated inflammation in vivo a, Schematic representation of the CD45RB High -induced colitis model. 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve CD4 + T cells from wildtype or Ets1- SE −/− were transferred into Rag1 −/− recipients to induce colitis. Weight was monitored once a week for six weeks post injection. b, Weight loss tracking of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells as compared to PBS-injected animals (controls) during 6 weeks post transfer. Weight was measured once per week. Three independent experiments were pooled and repeated four times. Dots represent the mean of individual mouse (PBS - > Rag1 −/− ; n= 5; wildtype - > Rag1 −/− ; n=25 and Ets1-SE −/− - > Rag1 −/− ; n=27). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Two-way ANOVA; Mixed-effect REML model with Fisher’s LSD Test). c, Quantification of colon length (cm) of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. Three independent experiments were pooled and repeated four times. Dots represent the mean of individual mouse (wildtype - > Rag1 −/− ; n=25 and Ets1-SE −/− - > Rag1 −/− ; n=27). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Mann-Whitney U Test). d,e Quantification of histological score of paraffin-embedded colon rolls from Rag1 −/− mice that received 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve CD4 + T cells from either wildtype or Ets1- SE −/− mice at week 6 post transfer and as compared to PBS-injected mice (control). Histological sections were obtained from two independent experiments and were scored in a blinded manner. Each dot represents an individual mouse (PBS - > Rag1 −/− ; n= 2, wildtype - > Rag1 −/− ; n=5 and Ets1-SE −/− - > Rag1 −/− ; n=8). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (One-way ANOVA with multiple comparisons and Bonferroni correction). e , Histological section of colon rolls stained with H E of Rag1 −/− mice that received either 1×10 6 FACS sorted TCR + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells 6 weeks post transfer. Scale = 100μm; Magnification = 100X. f, (left) Quantification of infiltrating colon lamina propria (cLP) colitogenic CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (middle) Quantification of Th1 (T-bet + ), Th2 (GATA-3 + ), Th17 (RORψ-t + ) and Tregs (FoxP3 + ) CD4 + T cells among infiltrating cLP CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (right) Quantification of Th1 (IFNψ + ), Th2 (IL-13 + ), Th17 (IL-17 + ) and Granzyme B (GzmB + ) producing CD4 + T cells among infiltrating cLP CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. Two independent experiments were pooled. Dots represent an individual mouse ( Ets1-SE +/+ - > Rag1 −/− ; n=7 and Ets1-SE −/− - > Rag1 −/− ; n=7). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (CD4 + T cells: Mann-Whitney U Test; Th1/Th2/Th17/Treg and cytokines production: Two-way ANOVA with multiple comparisons and Bonferroni correction). g, (left) Quantification CD4 + T cells in the spleen of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (middle) Quantification in the spleen of Th1 (T-bet + ), Th2 (GATA-3 + ), Th17 (RORψ-t + ) and Tregs (FoxP3 + ) CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. (right) Quantification of ex vivo stimulated splenic Th1 (IFNψ + ), Th2 (IL-13 + ), Th17 (IL-17 + ) and Granzyme B (Gzm B + ) producing CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. Data are a representative of one experiment which was repeated three times. Dots represent an individual mouse (wildtype - > Rag1 −/− ; n=7 and Ets1- SE −/− - > Rag1 −/− ; n=7). Error bars = SEM; and p-values: ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (CD4 + T cells: Mann-Whitney U Test; Th1/Th2/Th17/Treg and cytokines production: Two-way ANOVA with multiple comparisons and Bonferroni correction). h, Representative flow cytometry contour plot of Granzyme B- and IFNψ-producing ex vivo stimulated cLP CD4 + T cells of Rag1 −/− mice that received either 1×10 6 FACS sorted TCRβ + , CD4 + , CD45RB High naïve wildtype or Ets1- SE −/− CD4 + T cells at week 6 post transfer. i, Schematic representation of the house dust mite (HDM) extract challenge. Arrows represent days by which intranasal HDM was administered. Mice were euthanized 16 days after the initial sensitization and immune cell infiltration was checked. Two independent experiments were used to measure immune cell infiltration in the lung parenchyma. j, Quantification of lung parenchyma infiltrating CD4 + T cells (TCRβ + , CD4 + ), activated CD4 + T cells (TCRβ + , CD4 + , CD44 + ), CD8 + T cells (TCRβ + , CD8 + ), activated CD4 + T cells (TCRβ + , CD8 + , CD44 + ), CD4 + Th2 cells (TCRβ + , CD4 + , GATA-3 + ) and eosinophils (MHC-II − , Siglec-F + ) from wildtype or Ets1- SE −/− mice 16 days after HDM challenge. All cells were pre-gated on SSC-A/FSC-A, Singlets, Live cell (Viability − ). Two independent experiments were pooled. Each Dot represents an individual mouse (wildtype n=11 and Ets1-SE −/− n=11). Error bars = SEM; and ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Mann-Whitney). k, Quantification of Th2 cells numbers from wildtype or Ets1- SE −/− mice producing IL-5, IL-13 or IL-5/IL-13 four hours after PMA / Ionomycin stimulation. All cells were pre-gated on SSC-A/FSC-A, Singlets, Live cell (Viability − ), CD3 + , CD90.2 + , CD4 + , ST2 + . Data are representative of one independent experiment and repeated twice. Each dot represents an individual mouse (wildtype n=6 and Ets1- SE −/− n=6). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Two-way ANOVA with multiple comparison and Bonferroni correction).

Techniques Used: In Vivo, FACS, Injection, Mouse Assay, MANN-WHITNEY, Staining, Ex Vivo, Flow Cytometry

Ets1 -SE is dispensable for thymic T cell development and periphery colonization a, Gating strategy used to identify thymic T cell population of wildtype or Ets1-SE −/− related to Figure. 2a . Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Colored boxes indicate sequential gating. b, Gating strategy used to identify lung parenchyma immune cell populations of wildtype or Ets1- SE −/− at steady state related to Figure 2b . Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. c, Quantification of mature CD4 + and CD8 + T cells in the spleen of wildtype or Ets1- SE −/− animals at steady state. Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Data are representative of one experiment. Each dot represents an individual mouse (wildtype n= 4 and Ets1- SE −/− n=4). Error bars = SEM; and P : ns = not significant (Mann-Whitney U test). d, Quantification of mature liver CD4 + and CD8 + T cells of wildtype or Ets1- SE −/− animals at steady state. Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Data are representative of one experiment. Each dot represents an individual mouse (wildtype n= 4 and Ets1- SE −/− n=4). Error bars = SEM; and P : ns = not significant (Mann-Whitney U test). e, Quantification of mature bone marrow (BM) CD8 + T cells of wildtype or Ets1- SE −/− animals at steady state. Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Data are representative of one experiment. Each dot represents an individual mouse (wildtype n= 4 and Ets1- SE −/− n=4). Error bars = SEM; and P : ns = not significant (Mann-Whitney U test).
Figure Legend Snippet: Ets1 -SE is dispensable for thymic T cell development and periphery colonization a, Gating strategy used to identify thymic T cell population of wildtype or Ets1-SE −/− related to Figure. 2a . Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Colored boxes indicate sequential gating. b, Gating strategy used to identify lung parenchyma immune cell populations of wildtype or Ets1- SE −/− at steady state related to Figure 2b . Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. c, Quantification of mature CD4 + and CD8 + T cells in the spleen of wildtype or Ets1- SE −/− animals at steady state. Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Data are representative of one experiment. Each dot represents an individual mouse (wildtype n= 4 and Ets1- SE −/− n=4). Error bars = SEM; and P : ns = not significant (Mann-Whitney U test). d, Quantification of mature liver CD4 + and CD8 + T cells of wildtype or Ets1- SE −/− animals at steady state. Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Data are representative of one experiment. Each dot represents an individual mouse (wildtype n= 4 and Ets1- SE −/− n=4). Error bars = SEM; and P : ns = not significant (Mann-Whitney U test). e, Quantification of mature bone marrow (BM) CD8 + T cells of wildtype or Ets1- SE −/− animals at steady state. Cells were pre-gated on SSC-A/FSC-A, Singlets and Live (Viability − ) cells. Data are representative of one experiment. Each dot represents an individual mouse (wildtype n= 4 and Ets1- SE −/− n=4). Error bars = SEM; and P : ns = not significant (Mann-Whitney U test).

Techniques Used: MANN-WHITNEY

Ets1 -SE is dispensable for thymic T cell generation but is required for CD4 + Th1 differentiation a, Plots demonstrate percentage of cells defined by flow cytometry analysis in the thymus. Quantification in the thymus is defined as: Double Negative (DN; TCRβ + , CD4 − , CD8 − ), DN1 (TCRβ + , CD4 − , CD8 − , CD44 + , CD25 − ), DN2 (TCRβ + , CD4 − , CD8 − , CD44 + , CD25 + ), DN3 (TCRβ + , CD4 − , CD8 − , CD44 − , CD25 + ), DN4 (TCRβ + , CD4 − , CD8 − , CD44 − , CD25 − ), TCRβ low (Live, TCRβ low ), TCRβ high (Live, TCRβ high ), Double Positive (DP; TCRβ + , CD4 + , CD8 + ), single-positive CD4 + T cells (CD4SP; TCRβ + , CD4 + , CD8 − ), semi mature CD4 + T cells (TCRβ + , CD4 + , CD24 + ), Mature CD4 + T cells (TCRβ + , CD4 + , CD24 − ), single positive CD8 + T cells (CD8SP; TCRβ + , CD4 − , CD8 + ), semi-mature CD8 + T cells (TCRβ + , CD8 + , CD24 + ), mature CD8 + T cells (TCRβ + , CD8 + , CD24 − ), immature single positive CD8 + T cells (iSP CD8; TCRβ low , CD8 + , CD24 high ) and thymic regulatory CD4 + T cells (TCRβ + , CD4 + , FoxP3 + ) from age matched wildtype and Ets1- SE −/− female mice. All cell populations were pre-gated on SSC-A/FSC-A, Singlets and Viability − (Live) cells and complete gating strategy used to identify each population is displayed in Figure S2a . Data are representative of three independent experiments. Each dot represents an individual mouse (wildtype; n=7 - Ets1- SE −/− ; n=7). Error bars = SEM; and P : ns = not significant, (DN, DP, nTregs, CD4 + /CD8 + SP: Mann-Whitney U test; DN1-DN4, TCRβ low/high , Semi-Mature/Mature CD4 + /CD8 + : Two-way ANOVA with multiple comparisons and Bonferroni correction). b, Plots demonstrate percentages of cells defined by flow cytometry analysis in the lung Frequencies at steady state of lungs parenchyma CD4 + T cells (TCRβ + , CD4 + ), activated CD4 + T cells (TCRβ + , CD4 + , CD44 + ), CD8 + T cells (TCRβ + , CD8 + ), activated CD8 + T cells (TCRβ + , CD8 + , CD44 + ), CD4 + Th2 cells (TCRβ + , CD4 + , GATA-3 + ) and regulatory CD4 + T cells (TCRβ + , CD4 + , Foxp3 + ) from age-matched wildtype and Ets1- SE −/− male mice. All populations were pre-gated on SSC-A/FSC-A, Singlets and Viability − (Live) cells and complete gating strategy used to identify each population is displayed in Figure S2b . Data are representative of two independent experiments. Each dot represents an individual mouse (wildtype; n=4 - Ets1-SE −/− ; n=4). Error bars = SEM; and P : ns = not significant, * = P ≤0.05 (Mann-Whitney U test). c, (left) Representative flow cytometry contour plot of naive CD4 + T cells from wildtype or Ets1- SE −/− mice cultured under Th1, Th2 or Th17 polarizing conditions for 6 days. Unstained wildtype cells are shown for each polarizing condition as a negative control (wildtype FMO Control). (right) Frequencies of Th1 (IFNψ + ), Th2 (IL-13 + ) or Th17 (IL-17 + ) cytokines producing CD4 + T cells cultured under Th1, Th2 or Th17 polarizing conditions for 6 days. All populations were pre-gated on SSC-A/FSC-A, Singlets and Viability − (Live), TCRβ + and CD4 + cells. Two independent experiments were pooled and were repeated five times. Each dot represents an individual mouse (wildtype; n=8 - Ets1-SE −/− ; n=8). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Two-way ANOVA with multiple comparisons and Bonferroni correction).
Figure Legend Snippet: Ets1 -SE is dispensable for thymic T cell generation but is required for CD4 + Th1 differentiation a, Plots demonstrate percentage of cells defined by flow cytometry analysis in the thymus. Quantification in the thymus is defined as: Double Negative (DN; TCRβ + , CD4 − , CD8 − ), DN1 (TCRβ + , CD4 − , CD8 − , CD44 + , CD25 − ), DN2 (TCRβ + , CD4 − , CD8 − , CD44 + , CD25 + ), DN3 (TCRβ + , CD4 − , CD8 − , CD44 − , CD25 + ), DN4 (TCRβ + , CD4 − , CD8 − , CD44 − , CD25 − ), TCRβ low (Live, TCRβ low ), TCRβ high (Live, TCRβ high ), Double Positive (DP; TCRβ + , CD4 + , CD8 + ), single-positive CD4 + T cells (CD4SP; TCRβ + , CD4 + , CD8 − ), semi mature CD4 + T cells (TCRβ + , CD4 + , CD24 + ), Mature CD4 + T cells (TCRβ + , CD4 + , CD24 − ), single positive CD8 + T cells (CD8SP; TCRβ + , CD4 − , CD8 + ), semi-mature CD8 + T cells (TCRβ + , CD8 + , CD24 + ), mature CD8 + T cells (TCRβ + , CD8 + , CD24 − ), immature single positive CD8 + T cells (iSP CD8; TCRβ low , CD8 + , CD24 high ) and thymic regulatory CD4 + T cells (TCRβ + , CD4 + , FoxP3 + ) from age matched wildtype and Ets1- SE −/− female mice. All cell populations were pre-gated on SSC-A/FSC-A, Singlets and Viability − (Live) cells and complete gating strategy used to identify each population is displayed in Figure S2a . Data are representative of three independent experiments. Each dot represents an individual mouse (wildtype; n=7 - Ets1- SE −/− ; n=7). Error bars = SEM; and P : ns = not significant, (DN, DP, nTregs, CD4 + /CD8 + SP: Mann-Whitney U test; DN1-DN4, TCRβ low/high , Semi-Mature/Mature CD4 + /CD8 + : Two-way ANOVA with multiple comparisons and Bonferroni correction). b, Plots demonstrate percentages of cells defined by flow cytometry analysis in the lung Frequencies at steady state of lungs parenchyma CD4 + T cells (TCRβ + , CD4 + ), activated CD4 + T cells (TCRβ + , CD4 + , CD44 + ), CD8 + T cells (TCRβ + , CD8 + ), activated CD8 + T cells (TCRβ + , CD8 + , CD44 + ), CD4 + Th2 cells (TCRβ + , CD4 + , GATA-3 + ) and regulatory CD4 + T cells (TCRβ + , CD4 + , Foxp3 + ) from age-matched wildtype and Ets1- SE −/− male mice. All populations were pre-gated on SSC-A/FSC-A, Singlets and Viability − (Live) cells and complete gating strategy used to identify each population is displayed in Figure S2b . Data are representative of two independent experiments. Each dot represents an individual mouse (wildtype; n=4 - Ets1-SE −/− ; n=4). Error bars = SEM; and P : ns = not significant, * = P ≤0.05 (Mann-Whitney U test). c, (left) Representative flow cytometry contour plot of naive CD4 + T cells from wildtype or Ets1- SE −/− mice cultured under Th1, Th2 or Th17 polarizing conditions for 6 days. Unstained wildtype cells are shown for each polarizing condition as a negative control (wildtype FMO Control). (right) Frequencies of Th1 (IFNψ + ), Th2 (IL-13 + ) or Th17 (IL-17 + ) cytokines producing CD4 + T cells cultured under Th1, Th2 or Th17 polarizing conditions for 6 days. All populations were pre-gated on SSC-A/FSC-A, Singlets and Viability − (Live), TCRβ + and CD4 + cells. Two independent experiments were pooled and were repeated five times. Each dot represents an individual mouse (wildtype; n=8 - Ets1-SE −/− ; n=8). Error bars = SEM; and P : ns = not significant, * = P ≤0.05, ** = P ≤0.01, *** = P ≤0.0005, **** = P ≤0.0001 (Two-way ANOVA with multiple comparisons and Bonferroni correction).

Techniques Used: Flow Cytometry, Mouse Assay, MANN-WHITNEY, Cell Culture, Negative Control

3) Product Images from "APRIL/BLyS deficient rats prevent donor specific antibody (DSA) production and cell proliferation in rodent kidney transplant model"

Article Title: APRIL/BLyS deficient rats prevent donor specific antibody (DSA) production and cell proliferation in rodent kidney transplant model

Journal: PLoS ONE

doi: 10.1371/journal.pone.0275564

Sensitized BLyS -/- demonstrated significantly fewer naïve and MZ B lymphocytes compared to sensitized WT and APRIL -/- rodents. (A) Naïve B lymphocytes defined as CD3 - IgD + CD45R + CD27 - . (B) MZ B lymphocytes defined as HIS57 + CD45RA + . (C) Memory B lymphocytes defined as CD3 - CD27 + CD45R + . (D) Plasma cells defined as IgD - CD45R - CD27 + IgM - CD138 + . Data analyzed using ANOVA.
Figure Legend Snippet: Sensitized BLyS -/- demonstrated significantly fewer naïve and MZ B lymphocytes compared to sensitized WT and APRIL -/- rodents. (A) Naïve B lymphocytes defined as CD3 - IgD + CD45R + CD27 - . (B) MZ B lymphocytes defined as HIS57 + CD45RA + . (C) Memory B lymphocytes defined as CD3 - CD27 + CD45R + . (D) Plasma cells defined as IgD - CD45R - CD27 + IgM - CD138 + . Data analyzed using ANOVA.

Techniques Used:

Depleted antibody secreting cells did not result in decreased donor specific antibody in non-sensitized animals. Mean fluorescence intensity (MFI) was determined for the population of interest. Data analyzed using ANOVA. (A) T (CD3 + ) and (B) B (CD45R + ) cell flow crossmatch was performed in non-sensitized animals. IgG1 + in the T cell flow crossmatch was the only DSA that BLyS -/- significantly decreased compared to APRIL -/- (p
Figure Legend Snippet: Depleted antibody secreting cells did not result in decreased donor specific antibody in non-sensitized animals. Mean fluorescence intensity (MFI) was determined for the population of interest. Data analyzed using ANOVA. (A) T (CD3 + ) and (B) B (CD45R + ) cell flow crossmatch was performed in non-sensitized animals. IgG1 + in the T cell flow crossmatch was the only DSA that BLyS -/- significantly decreased compared to APRIL -/- (p

Techniques Used: Fluorescence

B lymphocytes accumulate at tolerogenic transitional zone stage when BLyS not present. Transitional Zone B lymphocytes defined as CD3 + IgD + CD45R + CD38 + CD24 ++ . Data analyzed using ANOVA.
Figure Legend Snippet: B lymphocytes accumulate at tolerogenic transitional zone stage when BLyS not present. Transitional Zone B lymphocytes defined as CD3 + IgD + CD45R + CD38 + CD24 ++ . Data analyzed using ANOVA.

Techniques Used:

Sensitized BLyS -/- produce significantly less alloantibody against foreign antigens. (A) T (CD3 + ) and (B) B (CD45R + ) cell flow crossmatch was performed in sensitized animals. Mean fluorescence intensity (MFI) was determined for the population of interest. Data analyzed using ANOVA. Sensitized BLyS -/- produced less DSA compared to WT, which was significant for CD3 + IgG2a, CD3 + IgG2c, CD45R + IgG2a, and CD45R + IgG2c.
Figure Legend Snippet: Sensitized BLyS -/- produce significantly less alloantibody against foreign antigens. (A) T (CD3 + ) and (B) B (CD45R + ) cell flow crossmatch was performed in sensitized animals. Mean fluorescence intensity (MFI) was determined for the population of interest. Data analyzed using ANOVA. Sensitized BLyS -/- produced less DSA compared to WT, which was significant for CD3 + IgG2a, CD3 + IgG2c, CD45R + IgG2a, and CD45R + IgG2c.

Techniques Used: Fluorescence, Produced

T lymphocyte populations unchanged by APRIL -/- and BLyS -/- . (A) Regulatory T cells defined as CD4 + CD3 + CD25 + FoxP3 + . (B) CD3+CD4+ T lymphocytes. (C) CD3+CD8+ lymphocytes. Data analyzed using ANOVA.
Figure Legend Snippet: T lymphocyte populations unchanged by APRIL -/- and BLyS -/- . (A) Regulatory T cells defined as CD4 + CD3 + CD25 + FoxP3 + . (B) CD3+CD4+ T lymphocytes. (C) CD3+CD8+ lymphocytes. Data analyzed using ANOVA.

Techniques Used:

4) Product Images from "Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine"

Article Title: Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine

Journal: Open Life Sciences

doi: 10.1515/biol-2022-0478

(a) After the first dose, the activated CD8+ T cells were significantly enhanced in the spleen of the mice. The CD8+ T cells were also significantly activated (lower than the 1 ug and 15 ug groups) in the PBS group after boost. All CD8+ T cells decreased to no significant difference in day 56. (b) The differences in CD4+ T cells of splenocytes were not significant at day 28, 32, and 56.
Figure Legend Snippet: (a) After the first dose, the activated CD8+ T cells were significantly enhanced in the spleen of the mice. The CD8+ T cells were also significantly activated (lower than the 1 ug and 15 ug groups) in the PBS group after boost. All CD8+ T cells decreased to no significant difference in day 56. (b) The differences in CD4+ T cells of splenocytes were not significant at day 28, 32, and 56.

Techniques Used: Mouse Assay

5) Product Images from "Immunogenicity and protectivity of intranasally delivered vector-based heterologous prime-boost COVID-19 vaccine Sputnik V in mice and non-human primates"

Article Title: Immunogenicity and protectivity of intranasally delivered vector-based heterologous prime-boost COVID-19 vaccine Sputnik V in mice and non-human primates

Journal: Emerging Microbes & Infections

doi: 10.1080/22221751.2022.2119169

Antigen-stimulated CD4 + and CD8 + T cell proliferation and cytokine production in splenocytes from С57BL/6 mice that received Sputnik V via the intramuscular (IM) or intranasal (IN) route. Placebo mice were injected twice with PBS. (A) CD4 + and CD8 + T cell proliferation was calculated as the difference (Δ) in % of proliferating (CFSE dim) lymphocytes between stimulated vs non-stimulated cells for each animal. (B) Graphs show the absolute cytokine levels in pg/mL. Dots show individual data points. Each bar represents the mean value per group ± SD (error bars). P -values in cytokine response between stimulated and non-stimulated cells within one group were calculated using the Wilcoxon signed-rank test (* p
Figure Legend Snippet: Antigen-stimulated CD4 + and CD8 + T cell proliferation and cytokine production in splenocytes from С57BL/6 mice that received Sputnik V via the intramuscular (IM) or intranasal (IN) route. Placebo mice were injected twice with PBS. (A) CD4 + and CD8 + T cell proliferation was calculated as the difference (Δ) in % of proliferating (CFSE dim) lymphocytes between stimulated vs non-stimulated cells for each animal. (B) Graphs show the absolute cytokine levels in pg/mL. Dots show individual data points. Each bar represents the mean value per group ± SD (error bars). P -values in cytokine response between stimulated and non-stimulated cells within one group were calculated using the Wilcoxon signed-rank test (* p

Techniques Used: Mouse Assay, Injection

Antigen-stimulated CD4 + and CD8 + T cell proliferation and cytokine production in PBMCs of common marmosets that received Sputnik V via the intramuscular (IM) or intranasal (IN) route. (A) CD4 + and CD8 + T cell proliferation was calculated as the difference (Δ) in % of proliferating (CFSE dim) lymphocytes between stimulated vs non-stimulated cells for each animal. (B) The cytokine data were presented as the difference (delta) in cytokine concentrations between the samples with and without protein stimulation. Dots show individual data points. Each bar represents the mean value per group ± SD (error bars). Significant differences between vaccinated and non-vaccinated animals were calculated using the Mann-Whitney U test (* p
Figure Legend Snippet: Antigen-stimulated CD4 + and CD8 + T cell proliferation and cytokine production in PBMCs of common marmosets that received Sputnik V via the intramuscular (IM) or intranasal (IN) route. (A) CD4 + and CD8 + T cell proliferation was calculated as the difference (Δ) in % of proliferating (CFSE dim) lymphocytes between stimulated vs non-stimulated cells for each animal. (B) The cytokine data were presented as the difference (delta) in cytokine concentrations between the samples with and without protein stimulation. Dots show individual data points. Each bar represents the mean value per group ± SD (error bars). Significant differences between vaccinated and non-vaccinated animals were calculated using the Mann-Whitney U test (* p

Techniques Used: MANN-WHITNEY

Flow cytometry analysis of tissue-resident lung lymphocytes from С57BL/6 mice that received intramuscular (IM) or intranasal (IN) Sputnik V vaccine. (A) Study design. Mice (the number of animals for each group is indicated in the legend) received prime-boost IM or IN vaccination with 3-week interval. Non-vaccinated mice were injected with PBS. On day 35 mice were sacrificed for evaluating the local immune response in lung parenchyma. (B) Gating strategy for assessing IL17 + and IFNɣ+ CD4 and CD8 tissue-resident lymphocytes. Lymphocytes were gated using the forward and side scatter characteristics. Live CD45 + cells were then obtained from the single cells gate. Intravenously administered anti-CD45-FITC antibodies were used for distinguishing tissue-resident (CD45.2-) and intravascular (CD45.2+) live lymphocytes. IL17 and IFNɣ expression was detected in the CD44 + fraction of CD4 + and CD8 + lymphocytes. (C) The individual percentages of IL-17+, IFNy+ CD4 + CD44 + and IFNy+ CD8 lymphocytes are shown by dots. Each bar represents the mean value per group ± 95%CI (error bars). Significant differences between vaccinated and unvaccinated animals were calculated using the Mann-Whitney test and indicated with asterisks (* p
Figure Legend Snippet: Flow cytometry analysis of tissue-resident lung lymphocytes from С57BL/6 mice that received intramuscular (IM) or intranasal (IN) Sputnik V vaccine. (A) Study design. Mice (the number of animals for each group is indicated in the legend) received prime-boost IM or IN vaccination with 3-week interval. Non-vaccinated mice were injected with PBS. On day 35 mice were sacrificed for evaluating the local immune response in lung parenchyma. (B) Gating strategy for assessing IL17 + and IFNɣ+ CD4 and CD8 tissue-resident lymphocytes. Lymphocytes were gated using the forward and side scatter characteristics. Live CD45 + cells were then obtained from the single cells gate. Intravenously administered anti-CD45-FITC antibodies were used for distinguishing tissue-resident (CD45.2-) and intravascular (CD45.2+) live lymphocytes. IL17 and IFNɣ expression was detected in the CD44 + fraction of CD4 + and CD8 + lymphocytes. (C) The individual percentages of IL-17+, IFNy+ CD4 + CD44 + and IFNy+ CD8 lymphocytes are shown by dots. Each bar represents the mean value per group ± 95%CI (error bars). Significant differences between vaccinated and unvaccinated animals were calculated using the Mann-Whitney test and indicated with asterisks (* p

Techniques Used: Flow Cytometry, Mouse Assay, Injection, Expressing, MANN-WHITNEY

6) Product Images from "Ethanol Ablation Therapy Drives Immune-Mediated Antitumor Effects in Murine Breast Cancer Models"

Article Title: Ethanol Ablation Therapy Drives Immune-Mediated Antitumor Effects in Murine Breast Cancer Models

Journal: Cancers

doi: 10.3390/cancers14194669

ECE reduces number and suppressive capabilities of gMDSCs in 4T1 tumor-bearing mice. Myeloid immune cell subsets within ( A ) 4T1 tumors ( n = 5) and ( B ) spleens ( n = 6) 7 days following i.t. saline or ECE therapy, as determined by flow cytometry. Spleens from tumor naïve mice used as controls. Proliferation of anti-CD3/CD28 stimulated ( C ) CD8+ and ( D ) CD4+ T cells in the presence of Ly6G+ cells from i.t. saline- or ECE-treated 4T1-bearing mice. i.t. = intratumoral. * p
Figure Legend Snippet: ECE reduces number and suppressive capabilities of gMDSCs in 4T1 tumor-bearing mice. Myeloid immune cell subsets within ( A ) 4T1 tumors ( n = 5) and ( B ) spleens ( n = 6) 7 days following i.t. saline or ECE therapy, as determined by flow cytometry. Spleens from tumor naïve mice used as controls. Proliferation of anti-CD3/CD28 stimulated ( C ) CD8+ and ( D ) CD4+ T cells in the presence of Ly6G+ cells from i.t. saline- or ECE-treated 4T1-bearing mice. i.t. = intratumoral. * p

Techniques Used: Mouse Assay, Flow Cytometry

ECE promotes enhanced T cell infiltration and CD8+ T cell-dependent antitumor effects in poorly invasive breast cancer models. ( A ) Tumor growth for rats with DMBA-induced tumors receiving either ECE or saline intratumoral injections, n = 8. ( B ) Representative images of CD3 staining in DMBA-induced tumors collected at day 30. ( C ) Average CD3+ cells per field of view (FOV), n = 8. ( D ) Tumor growth for mice with 67NR tumors, n = 10. ( E ) Average CD3+ cells per FOV. ( F ) Kaplan–Meier survival curves of mice receiving either ECE or saline intratumoral injections with CD8 depletion or isotype controls, n = 10. * p
Figure Legend Snippet: ECE promotes enhanced T cell infiltration and CD8+ T cell-dependent antitumor effects in poorly invasive breast cancer models. ( A ) Tumor growth for rats with DMBA-induced tumors receiving either ECE or saline intratumoral injections, n = 8. ( B ) Representative images of CD3 staining in DMBA-induced tumors collected at day 30. ( C ) Average CD3+ cells per field of view (FOV), n = 8. ( D ) Tumor growth for mice with 67NR tumors, n = 10. ( E ) Average CD3+ cells per FOV. ( F ) Kaplan–Meier survival curves of mice receiving either ECE or saline intratumoral injections with CD8 depletion or isotype controls, n = 10. * p

Techniques Used: Staining, Mouse Assay

7) Product Images from "Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy"

Article Title: Transient cell-in-cell formation underlies tumor relapse and resistance to immunotherapy

Journal: eLife

doi: 10.7554/eLife.80315

Cell-in-cell tumor formations are recognized but not killed by reactive T cells, related to Figure 5 . ( A ) Mean percentage of apoptotic B16F10, pre-incubated with secreted granules from activated immune cells, and following overnight incubation with gp100-reactive T cells (n=3). ( B ) Mean percentage of apoptotic B16F10 following overnight incubation with gp100-reactive T cells (n=4). ( C ) Representative images of B16 single cells or cell-in-cell culture gp100-reactive T cells. Dashed lines indicate immunological synapses. ( D ) 3D rendering of B16F10 following incubation with tumor-reactive T cells. (Relates to Figure 5E ). ( E ) Confocal plane image of B16F10 incubated with tumor-reactive CD8 + T cells. ( F ) 3D rendering of B16F10 following incubation with tumor-reactive T cells. (Relates to Figure S6E). ( G ). Granzyme B molecules prevalence inside B16F10 cells following incubation with tumor reactive T cells. ( H ) Relative confluence over time of B16 single cells or cell-in-cell (CiC) culture following incubation with doxorubicin (doxo) or cycloheximide (CH). Experiments were repeated at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (*denotes p
Figure Legend Snippet: Cell-in-cell tumor formations are recognized but not killed by reactive T cells, related to Figure 5 . ( A ) Mean percentage of apoptotic B16F10, pre-incubated with secreted granules from activated immune cells, and following overnight incubation with gp100-reactive T cells (n=3). ( B ) Mean percentage of apoptotic B16F10 following overnight incubation with gp100-reactive T cells (n=4). ( C ) Representative images of B16 single cells or cell-in-cell culture gp100-reactive T cells. Dashed lines indicate immunological synapses. ( D ) 3D rendering of B16F10 following incubation with tumor-reactive T cells. (Relates to Figure 5E ). ( E ) Confocal plane image of B16F10 incubated with tumor-reactive CD8 + T cells. ( F ) 3D rendering of B16F10 following incubation with tumor-reactive T cells. (Relates to Figure S6E). ( G ). Granzyme B molecules prevalence inside B16F10 cells following incubation with tumor reactive T cells. ( H ) Relative confluence over time of B16 single cells or cell-in-cell (CiC) culture following incubation with doxorubicin (doxo) or cycloheximide (CH). Experiments were repeated at least three times. Statistical significance was calculated using ANOVA with Tukey’s correction for multiple comparisons (*denotes p

Techniques Used: Incubation, Cell Culture

Murine tumor cell lines undergo cell-in-cell formation incubation with reactive T cells, Related to Figure 2 and Figure 3 . ( A ) Representative images of B16F10 cells following incubation with different immune cell subsets from naïve mice. ( B ) Mean percentage over time of cell-in-cell and simple cells from B16F10 tumor incubated with gp100-reactive CD8 + or TRP1-reactive CD4 + T cells (n=4). ( C ) Representative images of breast epithelial cancer cells (4T1) incubated with allogeneic T cells (D). Representative images of breast epithelial non-transformed cells (EPH4) incubated with allogeneic T cells. ( E ) Representative confocal images of B16F10 and 4T1 incubated overnight with gp100-reactive or allogenic CD8 + T cells, respectively. ( E-H ). Representative images of murine cancer cell lines incubated overnight with activated allogeneic splenic CD8 + T cells. All experiments were repeated at least three times. Scale bars = 20 μm.
Figure Legend Snippet: Murine tumor cell lines undergo cell-in-cell formation incubation with reactive T cells, Related to Figure 2 and Figure 3 . ( A ) Representative images of B16F10 cells following incubation with different immune cell subsets from naïve mice. ( B ) Mean percentage over time of cell-in-cell and simple cells from B16F10 tumor incubated with gp100-reactive CD8 + or TRP1-reactive CD4 + T cells (n=4). ( C ) Representative images of breast epithelial cancer cells (4T1) incubated with allogeneic T cells (D). Representative images of breast epithelial non-transformed cells (EPH4) incubated with allogeneic T cells. ( E ) Representative confocal images of B16F10 and 4T1 incubated overnight with gp100-reactive or allogenic CD8 + T cells, respectively. ( E-H ). Representative images of murine cancer cell lines incubated overnight with activated allogeneic splenic CD8 + T cells. All experiments were repeated at least three times. Scale bars = 20 μm.

Techniques Used: Incubation, Mouse Assay, Transformation Assay

IFNg-activated T cells induce cell-in-cell tumor formation, Related to Figure 4 . ( A ) SEM analysis of B16F10 incubated with macrophages (left square) or NK cells (right square). ( B ) Transmission electron microscopy of T cells secreted granules, collected 48 hours after activation with immobilized aCD3/aCD28 antibodies. ( C ) Representative image of a single-layer transmitting electron microscopy of T cells incubated overnight with B16F10 cells. ( D ) Representative image of B16F10 incubated overnight with T cell-derived secreted granules containing media. ( E ) Representative images of B16F10 incubated overnight with activated T cells. ( F ) Heatmap of gene expression patterns of naïve and activated CD8 + T cells. ( G ) Confocal microscopy images of human melanoma incubated overnight with allogeneic T cells from healthy donors. Scale bars = 5 μm (A top), 500 nm (A bottom, B), 20 μm (D, E, G).
Figure Legend Snippet: IFNg-activated T cells induce cell-in-cell tumor formation, Related to Figure 4 . ( A ) SEM analysis of B16F10 incubated with macrophages (left square) or NK cells (right square). ( B ) Transmission electron microscopy of T cells secreted granules, collected 48 hours after activation with immobilized aCD3/aCD28 antibodies. ( C ) Representative image of a single-layer transmitting electron microscopy of T cells incubated overnight with B16F10 cells. ( D ) Representative image of B16F10 incubated overnight with T cell-derived secreted granules containing media. ( E ) Representative images of B16F10 incubated overnight with activated T cells. ( F ) Heatmap of gene expression patterns of naïve and activated CD8 + T cells. ( G ) Confocal microscopy images of human melanoma incubated overnight with allogeneic T cells from healthy donors. Scale bars = 5 μm (A top), 500 nm (A bottom, B), 20 μm (D, E, G).

Techniques Used: Incubation, Transmission Assay, Electron Microscopy, Activation Assay, Derivative Assay, Expressing, Confocal Microscopy

8) Product Images from "Induction of T cell exhaustion by JAK1/3 inhibition in the treatment of alopecia areata"

Article Title: Induction of T cell exhaustion by JAK1/3 inhibition in the treatment of alopecia areata

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.955038

γc cytokines regulated effector T cell exhaustion. (A) to (J) T cells from C3H/HeJ mice without AA were stimulated with 500 ng/ml anti-CD3 in the presence of indicated regents in vitro for 4 (d) (A) and (B) The expression of PD-1 on CD8 + T cells was measured by FACS after treated with increasing dose of Ifidancitinib. *P
Figure Legend Snippet: γc cytokines regulated effector T cell exhaustion. (A) to (J) T cells from C3H/HeJ mice without AA were stimulated with 500 ng/ml anti-CD3 in the presence of indicated regents in vitro for 4 (d) (A) and (B) The expression of PD-1 on CD8 + T cells was measured by FACS after treated with increasing dose of Ifidancitinib. *P

Techniques Used: Mouse Assay, In Vitro, Expressing, FACS

Ifidancitinib induced multiple co-inhibitory receptors in CD44 + CD62L - CD8 + T cells. Mice were treated as in Figure 3 . (A) and (B) The frequency of PD-1 + , Tim-3 + and Eomes + T cells within CD44 + CD62L - CD8 + T cells populations within the SDLNs of mice treated with Ifidancitinib or vehicle. **P
Figure Legend Snippet: Ifidancitinib induced multiple co-inhibitory receptors in CD44 + CD62L - CD8 + T cells. Mice were treated as in Figure 3 . (A) and (B) The frequency of PD-1 + , Tim-3 + and Eomes + T cells within CD44 + CD62L - CD8 + T cells populations within the SDLNs of mice treated with Ifidancitinib or vehicle. **P

Techniques Used: Mouse Assay

Single cell RNAseq identifies expansion of exhausted CD8 T cells in skin of mice treated with tofacitinib. (A) UMAP plots of immune cell clusters identified in the skin of Tofa-treated (Treated) and control mice (Untreated). Note the marked decrease in the proportion of the effector CD8+T cells (Eff_CD8T) and TRM cells, and the expansion of exhausted CD8+ T cells (ex_CD8T) in the skin of Tofa-treated mice. (B) Stacked bar plot showing distribution of each cluster relative to the total number of cells per condition. (C) Violin plots showing mRNA transcript expression levels of canonical exhaustion markers including Tox , Pdcd1 , and Tcf7 encoding for TOX, PD1, and TCF1, respectively, across different clusters in Tofa-treated and control mice. RNA velocity analysis reveals that effector CD8+ T cells give rise to TRM and ex_CD8 T cells in AA-affected skin in C3H/HeJ mice. (D) RNA velocity plots of immune cell clusters identified in the skin of Tofa-treated (Treated) and control mice (Untreated). (E) RNA velocity plots and pseudotime analysis of CD8 T cell clusters identified in the skin of Tofa-treated and control mice.
Figure Legend Snippet: Single cell RNAseq identifies expansion of exhausted CD8 T cells in skin of mice treated with tofacitinib. (A) UMAP plots of immune cell clusters identified in the skin of Tofa-treated (Treated) and control mice (Untreated). Note the marked decrease in the proportion of the effector CD8+T cells (Eff_CD8T) and TRM cells, and the expansion of exhausted CD8+ T cells (ex_CD8T) in the skin of Tofa-treated mice. (B) Stacked bar plot showing distribution of each cluster relative to the total number of cells per condition. (C) Violin plots showing mRNA transcript expression levels of canonical exhaustion markers including Tox , Pdcd1 , and Tcf7 encoding for TOX, PD1, and TCF1, respectively, across different clusters in Tofa-treated and control mice. RNA velocity analysis reveals that effector CD8+ T cells give rise to TRM and ex_CD8 T cells in AA-affected skin in C3H/HeJ mice. (D) RNA velocity plots of immune cell clusters identified in the skin of Tofa-treated (Treated) and control mice (Untreated). (E) RNA velocity plots and pseudotime analysis of CD8 T cell clusters identified in the skin of Tofa-treated and control mice.

Techniques Used: Mouse Assay, Expressing

Ifidancitinib inhibited γc cytokine signaling. (A) and (B) In vitro activated T cells were pretreated with various dose of Ifidancitinib or vehicle (DMSO) for 1 h and stimulated with rhIL-2 (20 ng/ml) or rmIFN-γ (40 ng/ml) for 15 min, and then cell lysates were subjected to immunoblotting with the indicated Abs. (C) to (F) CD8 T cells from normal haired C3H/HeJ mice were stimulated with rmIL-15 (50 ng/ml) in the presence of increasing dose of Ifidancitinib or DMSO for 4 (d) On day 5, NKG2D expression was analyzed by flow cytometry (C) and (D) . In (E) and (F) , cells were restimulated with cell stimulation cocktail in the presence of plus protein transport inhibitors and analyzed for the intracellular expression of IFN-γ and IL-17. (G) and (H) CellTrace Violet labeled CD4 + or CD8 + T cells were stimulated with anti-CD3 and IL-2 in the presence of DMSO or increasing dose of Ifidancitinib for 4 (d) Proliferation of the T cells were measured by dilution of CellTrace Violet. (I) and (J) CellTrace Violet dye stained CD45.1 T cell blasts from B6 Cd45.1 were adoptively transferred to B6 CD45.2 recipients. CD45.2 recipients were then treated with Ifidancitinib and 20µg rhIL-2. Proliferation of the CD45.1 T cells were measured by dilution of CellTrace Violet. **P
Figure Legend Snippet: Ifidancitinib inhibited γc cytokine signaling. (A) and (B) In vitro activated T cells were pretreated with various dose of Ifidancitinib or vehicle (DMSO) for 1 h and stimulated with rhIL-2 (20 ng/ml) or rmIFN-γ (40 ng/ml) for 15 min, and then cell lysates were subjected to immunoblotting with the indicated Abs. (C) to (F) CD8 T cells from normal haired C3H/HeJ mice were stimulated with rmIL-15 (50 ng/ml) in the presence of increasing dose of Ifidancitinib or DMSO for 4 (d) On day 5, NKG2D expression was analyzed by flow cytometry (C) and (D) . In (E) and (F) , cells were restimulated with cell stimulation cocktail in the presence of plus protein transport inhibitors and analyzed for the intracellular expression of IFN-γ and IL-17. (G) and (H) CellTrace Violet labeled CD4 + or CD8 + T cells were stimulated with anti-CD3 and IL-2 in the presence of DMSO or increasing dose of Ifidancitinib for 4 (d) Proliferation of the T cells were measured by dilution of CellTrace Violet. (I) and (J) CellTrace Violet dye stained CD45.1 T cell blasts from B6 Cd45.1 were adoptively transferred to B6 CD45.2 recipients. CD45.2 recipients were then treated with Ifidancitinib and 20µg rhIL-2. Proliferation of the CD45.1 T cells were measured by dilution of CellTrace Violet. **P

Techniques Used: In Vitro, Mouse Assay, Expressing, Flow Cytometry, Cell Stimulation, Labeling, Staining

9) Product Images from "Oxaliplatin Induces Immunogenic Cell Death in Human and Murine Laryngeal Cancer"

Article Title: Oxaliplatin Induces Immunogenic Cell Death in Human and Murine Laryngeal Cancer

Journal: Journal of Oncology

doi: 10.1155/2022/3760766

Oxaliplatin-treated primary laryngeal cancer cells pulsed with DCs induced the populations of tumor-specific CD8 + T cells and reduced the population of Treg cells. Cisplatin or oxaliplatin-treated primary laryngeal cancer cells were pulsed with monocyte-derived DCs. After that, the cells were used to stim ulate autologous T cells for 2 weeks. (a-b) The numbers of IFN- γ -producing CD8 + T cells were analyzed by intracellular IFN- γ staining after stimulation. The frequencies of CD4 + CD25 + FoxP3 + Treg cells were analyzed by a flow cytometer. The results are from five independent studies. Data were represented as the means ± SD. ∗∗ p
Figure Legend Snippet: Oxaliplatin-treated primary laryngeal cancer cells pulsed with DCs induced the populations of tumor-specific CD8 + T cells and reduced the population of Treg cells. Cisplatin or oxaliplatin-treated primary laryngeal cancer cells were pulsed with monocyte-derived DCs. After that, the cells were used to stim ulate autologous T cells for 2 weeks. (a-b) The numbers of IFN- γ -producing CD8 + T cells were analyzed by intracellular IFN- γ staining after stimulation. The frequencies of CD4 + CD25 + FoxP3 + Treg cells were analyzed by a flow cytometer. The results are from five independent studies. Data were represented as the means ± SD. ∗∗ p

Techniques Used: Derivative Assay, Staining, Flow Cytometry

10) Product Images from "TSLP promoting B cell proliferation and polarizing follicular helper T cell as a therapeutic target in IgG4-related disease"

Article Title: TSLP promoting B cell proliferation and polarizing follicular helper T cell as a therapeutic target in IgG4-related disease

Journal: Journal of Translational Medicine

doi: 10.1186/s12967-022-03606-1

The expression of TSLP and its receptors in IgG4-RD. A Plasma levels of TSLP in HC, treatment naïve IgG4-RD patients and IgG4-RD patients with disease remission measured by Elisa. B Representative immunohistochemical staining of TSLP in LG from patients with SS (left) and SMG from patients with IgG4-RD (right). C The expression levels of TSLP in SMG estimated by semiquantitative analysis. D Trible staining for CD11c (green), CD4 (green), CD19 (green), CRLF2 (TSLPR, red) and DAPI (blue) in SMG from representative patients with IgG4-RD. E Percentages of TSLPR and IL-7Ra in CD4+, CD8+, CD19+, and Lin-CD11c+HLA-DR+ cells respectively. F Correlations between serum levels of IgG, IgE, IgG1, IgG4, RI and plasma level of TSLP. HC healthy controls, IgG4-RD IgG4-related disease, LG labial gland, SMG submandibular gland, SS primary Sjogren’s syndrome, RI responder index. *p
Figure Legend Snippet: The expression of TSLP and its receptors in IgG4-RD. A Plasma levels of TSLP in HC, treatment naïve IgG4-RD patients and IgG4-RD patients with disease remission measured by Elisa. B Representative immunohistochemical staining of TSLP in LG from patients with SS (left) and SMG from patients with IgG4-RD (right). C The expression levels of TSLP in SMG estimated by semiquantitative analysis. D Trible staining for CD11c (green), CD4 (green), CD19 (green), CRLF2 (TSLPR, red) and DAPI (blue) in SMG from representative patients with IgG4-RD. E Percentages of TSLPR and IL-7Ra in CD4+, CD8+, CD19+, and Lin-CD11c+HLA-DR+ cells respectively. F Correlations between serum levels of IgG, IgE, IgG1, IgG4, RI and plasma level of TSLP. HC healthy controls, IgG4-RD IgG4-related disease, LG labial gland, SMG submandibular gland, SS primary Sjogren’s syndrome, RI responder index. *p

Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

11) Product Images from "Vascular Disruptive Hydrogel Platform for Enhanced Chemotherapy and Anti-Angiogenesis through Alleviation of Immune Surveillance"

Article Title: Vascular Disruptive Hydrogel Platform for Enhanced Chemotherapy and Anti-Angiogenesis through Alleviation of Immune Surveillance

Journal: Pharmaceutics

doi: 10.3390/pharmaceutics14091809

Schematic illustration of V+E@Gel on promoting chemotherapy and anti-angiogenesis therapy through relieving immune surveillance. Local administration of V+E@Gel could promote the infiltration of CD8+ lymphocytes and decrease the number of MDSCs and Tregs, relieve the immune surveillance in tumor tissues and thus induce robust and long-lasting immunogenic cell death.
Figure Legend Snippet: Schematic illustration of V+E@Gel on promoting chemotherapy and anti-angiogenesis therapy through relieving immune surveillance. Local administration of V+E@Gel could promote the infiltration of CD8+ lymphocytes and decrease the number of MDSCs and Tregs, relieve the immune surveillance in tumor tissues and thus induce robust and long-lasting immunogenic cell death.

Techniques Used:

V+E@Gel induced robust immune reaction in tumor tissues. ( A – C ) Flow cytometric analysis and ( D – F ) quantitative analysis of CD8 + T cells after gating on CD3 + CD8 + cells, Treg cells after gating on CD4 + FOXP3 + cells, and MDSCs after gating on Gr-1 + CD11b + cells. All data are shown as mean ± S.D. ( n = 3). * p
Figure Legend Snippet: V+E@Gel induced robust immune reaction in tumor tissues. ( A – C ) Flow cytometric analysis and ( D – F ) quantitative analysis of CD8 + T cells after gating on CD3 + CD8 + cells, Treg cells after gating on CD4 + FOXP3 + cells, and MDSCs after gating on Gr-1 + CD11b + cells. All data are shown as mean ± S.D. ( n = 3). * p

Techniques Used:

12) Product Images from "IL-4 prevents adenosine-mediated immunoregulation by inhibiting CD39 expression"

Article Title: IL-4 prevents adenosine-mediated immunoregulation by inhibiting CD39 expression

Journal: JCI Insight

doi: 10.1172/jci.insight.157509

STAT6 inhibition in a T cell response upregulates CD39 expression. ( A ) Purified total T cells were activated for 5 days by anti-CD3/CD28 Dynabeads. Each day, cells were examined for the expression of pSTAT6, GATA3, and CD39 in CD4 or CD8 T cells by flow cytometry. In addition, the level of pSTAT6 was also compared in T cells with or without IL-4 receptor blocking Abs. ( B ) Purified total T cells were activated for 3 days by anti-CD3/CD28 Dynabeads in the presence or absence of IL-4 (20, 50, or 100 ng/mL), IL-4 receptor blocking Abs (1 or 2 μg/mL), or STAT6 inhibitor AS1517499 (100 nM) as indicated. GATA3 expression was assessed by Western blotting. ( C ) Purified total T cells were activated in the presence or absence of the STAT6 inhibitor AS1517499 (100 nM). After 4 days, CD39 expression was evaluated by flow cytometry. ( D ) Total T cells were activated for 2 days before nucleofection with STAT6-specific siRNA smartpool. After 2 additional days, cells were collected for ENTPD1 and STAT6 mRNA quantification by real-time qPCR. ( E ) CD45.1 + naive SMARTA cells were isolated and injected into CD45.2 B6 WT mice via tail vein, followed by LCMV Armstrong infection and daily i.p. injection of the STAT6 inhibitor AS1517499 or DMSO solvent control. After 6 days, SMARTA cells were isolated from spleen and examined for CD39 expression using flow cytometry. Representative contour plots (upper panel) and summary data (lower panel). Data in D are shown as mean ± SEM and compared by 2-tailed paired or unpaired Student’s t test in C and E .
Figure Legend Snippet: STAT6 inhibition in a T cell response upregulates CD39 expression. ( A ) Purified total T cells were activated for 5 days by anti-CD3/CD28 Dynabeads. Each day, cells were examined for the expression of pSTAT6, GATA3, and CD39 in CD4 or CD8 T cells by flow cytometry. In addition, the level of pSTAT6 was also compared in T cells with or without IL-4 receptor blocking Abs. ( B ) Purified total T cells were activated for 3 days by anti-CD3/CD28 Dynabeads in the presence or absence of IL-4 (20, 50, or 100 ng/mL), IL-4 receptor blocking Abs (1 or 2 μg/mL), or STAT6 inhibitor AS1517499 (100 nM) as indicated. GATA3 expression was assessed by Western blotting. ( C ) Purified total T cells were activated in the presence or absence of the STAT6 inhibitor AS1517499 (100 nM). After 4 days, CD39 expression was evaluated by flow cytometry. ( D ) Total T cells were activated for 2 days before nucleofection with STAT6-specific siRNA smartpool. After 2 additional days, cells were collected for ENTPD1 and STAT6 mRNA quantification by real-time qPCR. ( E ) CD45.1 + naive SMARTA cells were isolated and injected into CD45.2 B6 WT mice via tail vein, followed by LCMV Armstrong infection and daily i.p. injection of the STAT6 inhibitor AS1517499 or DMSO solvent control. After 6 days, SMARTA cells were isolated from spleen and examined for CD39 expression using flow cytometry. Representative contour plots (upper panel) and summary data (lower panel). Data in D are shown as mean ± SEM and compared by 2-tailed paired or unpaired Student’s t test in C and E .

Techniques Used: Inhibition, Expressing, Purification, Flow Cytometry, Blocking Assay, Western Blot, Real-time Polymerase Chain Reaction, Isolation, Injection, Mouse Assay, Infection

IL-4–induced STAT6 signaling inhibits CD39 expression. ( A ) Purified total T cells from human peripheral blood were activated for 4 days by anti-CD3/CD28 Dynabeads in the presence or absence of IL-4 (20 ng/mL). CD39 expression was compared by flow cytometric analysis. Left are representative contour plots and right are summary results. ( B ) Total T cells were treated as described in A and collected for ENTPD1 transcript quantification. ( C ) PBMCs were activated for 4 days with soluble anti-CD3 Ab (2 μg/mL) in the presence or absence of IL-4. Results on CD39 expression on gated CD19 + B cells are shown as representative histograms (left) and summary data (right). ( D ) Mice were implanted with B16 melanoma cells. Splenocytes and TILs were harvested on day 26 and expression of IL-4R on indicated CD4 and CD8 T cell subsets was determined. Contour plots of CD39 and TIM3 expression (left) and histograms of IL-4R expression (right) are representative of 2 experiments. Data in B are shown as mean ± SEM and compared by 2-tailed paired Student’s t test in A – C .
Figure Legend Snippet: IL-4–induced STAT6 signaling inhibits CD39 expression. ( A ) Purified total T cells from human peripheral blood were activated for 4 days by anti-CD3/CD28 Dynabeads in the presence or absence of IL-4 (20 ng/mL). CD39 expression was compared by flow cytometric analysis. Left are representative contour plots and right are summary results. ( B ) Total T cells were treated as described in A and collected for ENTPD1 transcript quantification. ( C ) PBMCs were activated for 4 days with soluble anti-CD3 Ab (2 μg/mL) in the presence or absence of IL-4. Results on CD39 expression on gated CD19 + B cells are shown as representative histograms (left) and summary data (right). ( D ) Mice were implanted with B16 melanoma cells. Splenocytes and TILs were harvested on day 26 and expression of IL-4R on indicated CD4 and CD8 T cell subsets was determined. Contour plots of CD39 and TIM3 expression (left) and histograms of IL-4R expression (right) are representative of 2 experiments. Data in B are shown as mean ± SEM and compared by 2-tailed paired Student’s t test in A – C .

Techniques Used: Expressing, Purification, Mouse Assay

13) Product Images from "Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer"

Article Title: Chronic chromosome instability induced by Plk1 results in immune suppression in breast cancer

Journal: bioRxiv

doi: 10.1101/2022.06.16.496429

Role of NK cells in immune response of high CIN tumors. A ) Survival analysis of Her2 (left) and Her2-Plk1 (right) tumors of mice treated with NK1.1 blocking antibody. Survival probability over time (upper graphs) and number and percentage of mice alive over time (lower table). The curves in light blue and light yellow depict untreated mice, whereas the curves in dark blue and dark yellow show mice treated with NK block, respectively. B) Normalized gene expression of CD27 versus Itgam ( CD11b ) in NK cells at the single cell level (individual dots) (Her2: blue, Her2-Plk1: yellow). The Spearman rank correlation coefficient across samples as well as the fold change and p-values adjusted for multiple testing for differential expression between Her2 and Her2-Plk1 samples are provided. C) Violin graphs showing the log-normalized gene expression values of NK cells in Her2 (blue) and Her2-Plk1 (yellow) tumors exemplarily shown for genes involved in cytotoxic response and surface receptors of NK cells. D) Heatmap showing normalized gene expression levels of differentially expressed effector genes, chemokines and their receptors, and activating receptor genes in NK cells. Each line represents a single cell. E) Gene set enrichment analysis for identification of pathways with enrichment of differentially expressed genes for two macrophage subsets and NK cells. Y-axis: hallmark signatures and X-axis: normalized enrichment scores with adjusted p-values, color-coded by the direction of the effect (yellow: upregulation in Her2-Plk1, blue: upregulation in Her2).
Figure Legend Snippet: Role of NK cells in immune response of high CIN tumors. A ) Survival analysis of Her2 (left) and Her2-Plk1 (right) tumors of mice treated with NK1.1 blocking antibody. Survival probability over time (upper graphs) and number and percentage of mice alive over time (lower table). The curves in light blue and light yellow depict untreated mice, whereas the curves in dark blue and dark yellow show mice treated with NK block, respectively. B) Normalized gene expression of CD27 versus Itgam ( CD11b ) in NK cells at the single cell level (individual dots) (Her2: blue, Her2-Plk1: yellow). The Spearman rank correlation coefficient across samples as well as the fold change and p-values adjusted for multiple testing for differential expression between Her2 and Her2-Plk1 samples are provided. C) Violin graphs showing the log-normalized gene expression values of NK cells in Her2 (blue) and Her2-Plk1 (yellow) tumors exemplarily shown for genes involved in cytotoxic response and surface receptors of NK cells. D) Heatmap showing normalized gene expression levels of differentially expressed effector genes, chemokines and their receptors, and activating receptor genes in NK cells. Each line represents a single cell. E) Gene set enrichment analysis for identification of pathways with enrichment of differentially expressed genes for two macrophage subsets and NK cells. Y-axis: hallmark signatures and X-axis: normalized enrichment scores with adjusted p-values, color-coded by the direction of the effect (yellow: upregulation in Her2-Plk1, blue: upregulation in Her2).

Techniques Used: Mouse Assay, Blocking Assay, Expressing

14) Product Images from "Biomimetic doxorubicin/ginsenoside co-loading nanosystem for chemoimmunotherapy of acute myeloid leukemia"

Article Title: Biomimetic doxorubicin/ginsenoside co-loading nanosystem for chemoimmunotherapy of acute myeloid leukemia

Journal: Journal of Nanobiotechnology

doi: 10.1186/s12951-022-01491-w

Immuno-activation in vivo. A DC maturation (CD80 + CD86 + of CD11c + BMDCs) in the lymph nodes detected by flow cytometry. B Statistical analysis of ( A ) ( n = 3). C Proportion of peripheral CD3 + T cells on day 8. D Statistical analysis of ( C ) ( n = 3). E Proportion of CD8 + T cells and CD4 + T cells (gated on CD3 + T cells) on day 8. F Statistical analysis of proportion of peripheral CD8 + T cells ( n = 3). G Proportion of CD3 + T cells in bone marrow. H CD8 + T cells and CD4 + T cells (gated on CD3 + T cells) on day 22. I Statistical analysis of ( G ) ( n = 3). J Statistical analysis of proportion of CD8 + T cells in bone marrow ( n = 3). K TNF- α and ( L ) IFN- γ in serum on day 8 detected by ELISA ( n = 3). Data are shown as mean ± SD. P values were calculated by Student’s t-test, * p
Figure Legend Snippet: Immuno-activation in vivo. A DC maturation (CD80 + CD86 + of CD11c + BMDCs) in the lymph nodes detected by flow cytometry. B Statistical analysis of ( A ) ( n = 3). C Proportion of peripheral CD3 + T cells on day 8. D Statistical analysis of ( C ) ( n = 3). E Proportion of CD8 + T cells and CD4 + T cells (gated on CD3 + T cells) on day 8. F Statistical analysis of proportion of peripheral CD8 + T cells ( n = 3). G Proportion of CD3 + T cells in bone marrow. H CD8 + T cells and CD4 + T cells (gated on CD3 + T cells) on day 22. I Statistical analysis of ( G ) ( n = 3). J Statistical analysis of proportion of CD8 + T cells in bone marrow ( n = 3). K TNF- α and ( L ) IFN- γ in serum on day 8 detected by ELISA ( n = 3). Data are shown as mean ± SD. P values were calculated by Student’s t-test, * p

Techniques Used: Activation Assay, In Vivo, Flow Cytometry, Enzyme-linked Immunosorbent Assay

15) Product Images from "DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D"

Article Title: DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Journal: Molecular Therapy Oncolytics

doi: 10.1016/j.omto.2022.06.003

Killing activity of DAP10-T cells against various cancer cells (A) Schematic diagram of the structures of CAR gene-expression cassettes. (B) Schematic diagram for the expression of DAP10BBz with native NKG2D on T cell membrane. Arrows represent the interaction of NKG2D and DAP10. (C) Expression of NKG2D on CD4+ and CD8+ T cells after DAP10BBz gene transduction detected by flow cytometry. The histogram plot (left) shows one representative experiment from 6 different donors, and the data of NKG2D mean fluorescence intensity (MFI) in CAR-T cells from 6 donors were plotted (right). (D) Results of killing assays performed with DAP10-T cells against multiple solid cancer cell lines. The results shown are from one representative experiment out of two independent experiments with 2 different donors. (E) Detection of granzyme B, IFN-γ and IL-2 secretion by DAP10-T cells and CAR19-T cells stimulated by multiple solid cancer cell lines. Error bars denote the SEM, and the results were compared with Student’s t test. ∗p
Figure Legend Snippet: Killing activity of DAP10-T cells against various cancer cells (A) Schematic diagram of the structures of CAR gene-expression cassettes. (B) Schematic diagram for the expression of DAP10BBz with native NKG2D on T cell membrane. Arrows represent the interaction of NKG2D and DAP10. (C) Expression of NKG2D on CD4+ and CD8+ T cells after DAP10BBz gene transduction detected by flow cytometry. The histogram plot (left) shows one representative experiment from 6 different donors, and the data of NKG2D mean fluorescence intensity (MFI) in CAR-T cells from 6 donors were plotted (right). (D) Results of killing assays performed with DAP10-T cells against multiple solid cancer cell lines. The results shown are from one representative experiment out of two independent experiments with 2 different donors. (E) Detection of granzyme B, IFN-γ and IL-2 secretion by DAP10-T cells and CAR19-T cells stimulated by multiple solid cancer cell lines. Error bars denote the SEM, and the results were compared with Student’s t test. ∗p

Techniques Used: Activity Assay, Expressing, Transduction, Flow Cytometry, Fluorescence

16) Product Images from "Long-term hepatitis B virus infection of rhesus macaques requires suppression of host immunity"

Article Title: Long-term hepatitis B virus infection of rhesus macaques requires suppression of host immunity

Journal: Nature Communications

doi: 10.1038/s41467-022-30593-0

HBV-specific adaptive immune responses in RM. A Anti-HBs concentration in serum. B HBV-specific CD4 + and CD8 + T cell responses in blood, mesenteric lymph nodes, and spleens of HBV-infected RM. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.
Figure Legend Snippet: HBV-specific adaptive immune responses in RM. A Anti-HBs concentration in serum. B HBV-specific CD4 + and CD8 + T cell responses in blood, mesenteric lymph nodes, and spleens of HBV-infected RM. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.

Techniques Used: Concentration Assay, Infection

Sustained HBV replication in RM on immunosuppression. A Timeline for HBV infection and immunosuppression of RM. B HBV DNA levels in serum during and postremoval of immunosuppression. C HBsAg levels in serum. D HBeAg levels in serum. E ALT levels in serum. F Anti-HBc IgG quantification in serum. G Anti-HBs IgG quantification in serum. H–J Longitudinal measurements of anti-HBsAg T cells by IFNγ ELISpot. K Frequency of Ki67-expressing CD4 + and CD8 + T cells in the PBMC. SFC = spot forming cell. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.
Figure Legend Snippet: Sustained HBV replication in RM on immunosuppression. A Timeline for HBV infection and immunosuppression of RM. B HBV DNA levels in serum during and postremoval of immunosuppression. C HBsAg levels in serum. D HBeAg levels in serum. E ALT levels in serum. F Anti-HBc IgG quantification in serum. G Anti-HBs IgG quantification in serum. H–J Longitudinal measurements of anti-HBsAg T cells by IFNγ ELISpot. K Frequency of Ki67-expressing CD4 + and CD8 + T cells in the PBMC. SFC = spot forming cell. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.

Techniques Used: Infection, Enzyme-linked Immunospot, Expressing

17) Product Images from "Long-term hepatitis B virus infection of rhesus macaques requires suppression of host immunity"

Article Title: Long-term hepatitis B virus infection of rhesus macaques requires suppression of host immunity

Journal: Nature Communications

doi: 10.1038/s41467-022-30593-0

HBV-specific adaptive immune responses in RM. A Anti-HBs concentration in serum. B HBV-specific CD4 + and CD8 + T cell responses in blood, mesenteric lymph nodes, and spleens of HBV-infected RM. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.
Figure Legend Snippet: HBV-specific adaptive immune responses in RM. A Anti-HBs concentration in serum. B HBV-specific CD4 + and CD8 + T cell responses in blood, mesenteric lymph nodes, and spleens of HBV-infected RM. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.

Techniques Used: Concentration Assay, Infection

Sustained HBV replication in RM on immunosuppression. A Timeline for HBV infection and immunosuppression of RM. B HBV DNA levels in serum during and postremoval of immunosuppression. C HBsAg levels in serum. D HBeAg levels in serum. E ALT levels in serum. F Anti-HBc IgG quantification in serum. G Anti-HBs IgG quantification in serum. H–J Longitudinal measurements of anti-HBsAg T cells by IFNγ ELISpot. K Frequency of Ki67-expressing CD4 + and CD8 + T cells in the PBMC. SFC = spot forming cell. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.
Figure Legend Snippet: Sustained HBV replication in RM on immunosuppression. A Timeline for HBV infection and immunosuppression of RM. B HBV DNA levels in serum during and postremoval of immunosuppression. C HBsAg levels in serum. D HBeAg levels in serum. E ALT levels in serum. F Anti-HBc IgG quantification in serum. G Anti-HBs IgG quantification in serum. H–J Longitudinal measurements of anti-HBsAg T cells by IFNγ ELISpot. K Frequency of Ki67-expressing CD4 + and CD8 + T cells in the PBMC. SFC = spot forming cell. Data are presented as mean values + /− SD. Source data are provided as a Source Data file.

Techniques Used: Infection, Enzyme-linked Immunospot, Expressing

18) Product Images from "Activation of cGAS‐STING by Lethal Malaria N67C Dictates Immunity and Mortality through Induction of CD11b+Ly6Chi Proinflammatory Monocytes, Activation of cGAS‐STING by Lethal Malaria N67C Dictates Immunity and Mortality through Induction of CD11b+Ly6Chi Proinflammatory Monocytes"

Article Title: Activation of cGAS‐STING by Lethal Malaria N67C Dictates Immunity and Mortality through Induction of CD11b+Ly6Chi Proinflammatory Monocytes, Activation of cGAS‐STING by Lethal Malaria N67C Dictates Immunity and Mortality through Induction of CD11b+Ly6Chi Proinflammatory Monocytes

Journal: Advanced Science

doi: 10.1002/advs.202103701

Late IL‐6 signaling inhibits host immunity against N67C infection by suppressing T cell function. A,B) WT, Tmem173 gt and Mb21d1 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of CD3 + cells (A), CD4 + cells and CD8 + cells in splenocytes (B). C,D) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of CD4 + cells and CD8 + cells in splenocytes. E,F) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i. and stimulated with N67C crude antigen in vitro, then subjected to FACS analysis of IFN‐γ + cells in CD4 + cells (E) and in CD8 + cells (F). G) WT, Tmem173 gt , and Mb21d1 –/– mice ( n = 3) were infected with N67C. Sera were collected at day 5 p.i. and subjected to ELISA analysis of IFN‐γ. H) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Sera were collected at day 5 p.i. and subjected to ELISA analysis of IFN‐γ. I) Mavs –/– mice ( n = 3) were infected with N67C, and then treated with or without anti‐IL6R antibody at day 2 p.i. Sera were collected at indicated time points and subjected to ELISA analysis of IFN‐γ. J) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i. RNAs from splenocytes were isolated and used for expression analysis using qPCR. K, L) WT, Tmem173 gt , and Mb21d1 –/– mice ( n = 3) were infected with N67C. Splenocytes (K) and lymph nodes (L) were collected at day 5 p.i. RNAs from splenocytes and lymph nodes were isolated and used for expression analysis using qPCR. M,N) WT mice ( n = 4) were infected with N67C, followed with administration of recombinant IFN‐α/β or control BSA at 18 h p.i., then treated with anti‐PD‐1 antibody. Parasitemia (M) and mortality rates (N) were monitored daily. Data are representative of three independent experiments and are plotted as the mean ± SD. * p
Figure Legend Snippet: Late IL‐6 signaling inhibits host immunity against N67C infection by suppressing T cell function. A,B) WT, Tmem173 gt and Mb21d1 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of CD3 + cells (A), CD4 + cells and CD8 + cells in splenocytes (B). C,D) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of CD4 + cells and CD8 + cells in splenocytes. E,F) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i. and stimulated with N67C crude antigen in vitro, then subjected to FACS analysis of IFN‐γ + cells in CD4 + cells (E) and in CD8 + cells (F). G) WT, Tmem173 gt , and Mb21d1 –/– mice ( n = 3) were infected with N67C. Sera were collected at day 5 p.i. and subjected to ELISA analysis of IFN‐γ. H) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Sera were collected at day 5 p.i. and subjected to ELISA analysis of IFN‐γ. I) Mavs –/– mice ( n = 3) were infected with N67C, and then treated with or without anti‐IL6R antibody at day 2 p.i. Sera were collected at indicated time points and subjected to ELISA analysis of IFN‐γ. J) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i. RNAs from splenocytes were isolated and used for expression analysis using qPCR. K, L) WT, Tmem173 gt , and Mb21d1 –/– mice ( n = 3) were infected with N67C. Splenocytes (K) and lymph nodes (L) were collected at day 5 p.i. RNAs from splenocytes and lymph nodes were isolated and used for expression analysis using qPCR. M,N) WT mice ( n = 4) were infected with N67C, followed with administration of recombinant IFN‐α/β or control BSA at 18 h p.i., then treated with anti‐PD‐1 antibody. Parasitemia (M) and mortality rates (N) were monitored daily. Data are representative of three independent experiments and are plotted as the mean ± SD. * p

Techniques Used: Infection, Cell Function Assay, Mouse Assay, FACS, In Vitro, Enzyme-linked Immunosorbent Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Recombinant

IL‐6 induces CD11b + Ly6C hi proinflammatory monocytes expansion and inhibits T cell function. A) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Gr‐1 + cells in CD11b + cells. B) WT and Tmem173 gt mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Gr‐1 + cells in CD11b + cells. C) WT, Tmem173 gt , and Mb21d1 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Ly6C hi Ly6G – cells and Ly6C lo Ly6G + cells in CD11b + cells. D) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Ly6C hi Ly6G – cells and Ly6C lo Ly6G + cells in CD11b + cells. E) Proliferation of CD4 + and CD8 + cells stimulated with CD3/CD28 antibody in the presence of Ly6C hi cells isolated from spleens of N67C infected WT mice ( n = 3). F,G) WT mice ( n = 5) were infected with N67C, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with or without anti‐Ly6C antibody (rat IgG serves as control) at day 3 p.i. Splenocytes were collected at day 6 p.i., and subjected to FACS analysis of malaria specific T cells %, PD‐1 + cells %, IFN‐γ + cells in indicated cell populations (F). Sera were collected at day 6 p.i. and subjected to ELISA analysis of IFN‐γ (G). H) WT mice ( n = 5) were infected with N67C, followed by administrated with or without recombinant IFN‐α/β at 18 h p.i., and then treated with or without anti‐Ly6C antibody at day 3 p.i., parasitemia and mortality rates were monitored daily. Data are representative of three independent experiments and are plotted as the mean ± SD. * p
Figure Legend Snippet: IL‐6 induces CD11b + Ly6C hi proinflammatory monocytes expansion and inhibits T cell function. A) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Gr‐1 + cells in CD11b + cells. B) WT and Tmem173 gt mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Gr‐1 + cells in CD11b + cells. C) WT, Tmem173 gt , and Mb21d1 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Ly6C hi Ly6G – cells and Ly6C lo Ly6G + cells in CD11b + cells. D) Mavs –/‐ and Mavs –/– Il6 –/– mice ( n = 3) were infected with N67C. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Ly6C hi Ly6G – cells and Ly6C lo Ly6G + cells in CD11b + cells. E) Proliferation of CD4 + and CD8 + cells stimulated with CD3/CD28 antibody in the presence of Ly6C hi cells isolated from spleens of N67C infected WT mice ( n = 3). F,G) WT mice ( n = 5) were infected with N67C, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with or without anti‐Ly6C antibody (rat IgG serves as control) at day 3 p.i. Splenocytes were collected at day 6 p.i., and subjected to FACS analysis of malaria specific T cells %, PD‐1 + cells %, IFN‐γ + cells in indicated cell populations (F). Sera were collected at day 6 p.i. and subjected to ELISA analysis of IFN‐γ (G). H) WT mice ( n = 5) were infected with N67C, followed by administrated with or without recombinant IFN‐α/β at 18 h p.i., and then treated with or without anti‐Ly6C antibody at day 3 p.i., parasitemia and mortality rates were monitored daily. Data are representative of three independent experiments and are plotted as the mean ± SD. * p

Techniques Used: Cell Function Assay, Mouse Assay, Infection, FACS, Isolation, Recombinant, Enzyme-linked Immunosorbent Assay

Administration of IL‐6 is detrimental for host generating immune responses against YM infection. A,B) Mavs –/– mice ( n = 4) were infected with YM, and then treated with or without recombinant IL‐6 at day 3 p.i., parasitemia (A) and survival (B) were monitored daily. C) WT mice ( n = 5) were infected with YM, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i. The parasitemia and survival were monitored daily. D–F) WT mice ( n = 5) were infected with YM, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i. Sera were collected at day 5 p.i. and subjected to ELISA analysis of IFN‐γ (D). Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of IFN‐γ + cells in CD4 + cells (E) and in CD8 + cells (F). G) WT mice ( n = 5) were infected with YM, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Ly6C hi Ly6G – cells and Ly6C lo Ly6G + cells in CD11b + cells. H) WT mice ( n = 5) were infected with YM, followed by administration with or without recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i., followed by treatment with or without anti‐Ly6C antibody every two days starting from day 3 p.i. Parasitemia and mortality rates were monitored daily. I) A schematic model to show that activation of cGAS‐STING by Plasmodium N67C recruits MyD88 to induce p38 MAPK mediated IL‐6 production in macrophages at late stage of infection, which further induces and expands CD11b + Ly6C hi proinflammatory monocytes to inhibit T cell proliferation, function and host anti‐malaria immune responses against N67C infection. Data are representative of three independent experiments and are plotted as the mean ± SD. * p
Figure Legend Snippet: Administration of IL‐6 is detrimental for host generating immune responses against YM infection. A,B) Mavs –/– mice ( n = 4) were infected with YM, and then treated with or without recombinant IL‐6 at day 3 p.i., parasitemia (A) and survival (B) were monitored daily. C) WT mice ( n = 5) were infected with YM, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i. The parasitemia and survival were monitored daily. D–F) WT mice ( n = 5) were infected with YM, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i. Sera were collected at day 5 p.i. and subjected to ELISA analysis of IFN‐γ (D). Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of IFN‐γ + cells in CD4 + cells (E) and in CD8 + cells (F). G) WT mice ( n = 5) were infected with YM, followed by administrated with recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i. Splenocytes were collected at day 5 p.i., and subjected to FACS analysis of Ly6C hi Ly6G – cells and Ly6C lo Ly6G + cells in CD11b + cells. H) WT mice ( n = 5) were infected with YM, followed by administration with or without recombinant IFN‐α/β at 18 h p.i., and then treated with recombinant IL‐6 at day 3 p.i., followed by treatment with or without anti‐Ly6C antibody every two days starting from day 3 p.i. Parasitemia and mortality rates were monitored daily. I) A schematic model to show that activation of cGAS‐STING by Plasmodium N67C recruits MyD88 to induce p38 MAPK mediated IL‐6 production in macrophages at late stage of infection, which further induces and expands CD11b + Ly6C hi proinflammatory monocytes to inhibit T cell proliferation, function and host anti‐malaria immune responses against N67C infection. Data are representative of three independent experiments and are plotted as the mean ± SD. * p

Techniques Used: Infection, Mouse Assay, Recombinant, Enzyme-linked Immunosorbent Assay, FACS, Activation Assay

19) Product Images from "Combination of DNA Vaccine and Immune Checkpoint Blockades Improves the Immune Response in an Orthotopic Unresectable Glioblastoma Model"

Article Title: Combination of DNA Vaccine and Immune Checkpoint Blockades Improves the Immune Response in an Orthotopic Unresectable Glioblastoma Model

Journal: Pharmaceutics

doi: 10.3390/pharmaceutics14051025

Schematic representation of pTOP vaccine. The pTOP is a plasmid DNA encoding a VSV-G sequence modified by the insertion of tumor epitope sequences in permissive sites. After administration of pTOP, cells will produce VSV-G vesicles incorporating CD4 and/or CD8 epitopes. These immunogenic vesicles will activate both the innate immunity and the adaptive immunity by presentation of the epitopes through the MHC class I or II molecules to the CD8 or CD4 T cells. (TCR = T cell receptor).
Figure Legend Snippet: Schematic representation of pTOP vaccine. The pTOP is a plasmid DNA encoding a VSV-G sequence modified by the insertion of tumor epitope sequences in permissive sites. After administration of pTOP, cells will produce VSV-G vesicles incorporating CD4 and/or CD8 epitopes. These immunogenic vesicles will activate both the innate immunity and the adaptive immunity by presentation of the epitopes through the MHC class I or II molecules to the CD8 or CD4 T cells. (TCR = T cell receptor).

Techniques Used: Plasmid Preparation, Sequencing, Modification

20) Product Images from "Heterogeneity and clonality of kidney-infiltrating T cells in murine lupus nephritis"

Article Title: Heterogeneity and clonality of kidney-infiltrating T cells in murine lupus nephritis

Journal: JCI Insight

doi: 10.1172/jci.insight.156048

CD8 + KITs are clonally expanded, with clones and proliferation spanning the exhausted and transitional compartments. ( A ) UMAP of all 3 scRNA-Seq cohorts (see Figure 1 for cohorts), integrated using Harmony, followed by projection of individual cohorts onto this “combined UMAP.” ( B ) Main-Seq–defined CD8 + clusters labeled by cluster name ( Figure 6 ), mapped onto the combined UMAP. ( C ) Combined UMAP with all putative clusters as outlined in B . ( D ) High-frequency clones (defined as clones representing the top quartile of expressed TCRs) from each cohort are mapped onto the combined UMAP. ( E ) Exhaustion gene set enrichment calculated using Wilcoxon’s test overlaid onto the combined UMAP. ( F ) Dot plots represent exhaustion scores for cells grouped based on clonal frequency among MRL/lpr and FcγR2B –/– . Yaa KITs, with exhaustion scores for B6 naive, early T EX , and terminal T EX shown at left for reference (* P = 0.05, ** P
Figure Legend Snippet: CD8 + KITs are clonally expanded, with clones and proliferation spanning the exhausted and transitional compartments. ( A ) UMAP of all 3 scRNA-Seq cohorts (see Figure 1 for cohorts), integrated using Harmony, followed by projection of individual cohorts onto this “combined UMAP.” ( B ) Main-Seq–defined CD8 + clusters labeled by cluster name ( Figure 6 ), mapped onto the combined UMAP. ( C ) Combined UMAP with all putative clusters as outlined in B . ( D ) High-frequency clones (defined as clones representing the top quartile of expressed TCRs) from each cohort are mapped onto the combined UMAP. ( E ) Exhaustion gene set enrichment calculated using Wilcoxon’s test overlaid onto the combined UMAP. ( F ) Dot plots represent exhaustion scores for cells grouped based on clonal frequency among MRL/lpr and FcγR2B –/– . Yaa KITs, with exhaustion scores for B6 naive, early T EX , and terminal T EX shown at left for reference (* P = 0.05, ** P

Techniques Used: Clone Assay, Labeling

TF and lineage progression analysis of CD8 + KITs. ( A ) Heatmap represents z -scored regulon activity of top TFs inferred by SCENIC (rows) and association with CD8 + T cell clusters (columns). ( B ) Representative TFs were mapped onto CD8 UMAPs; TF selection was based on known regulatory functions or due to identification via SCENIC analysis. Outlines highlight spleen-derived, kidney-infiltrating, and exhausted cells, with dot red color intensity representing log 2 expression. ( C ) CD8 + T cells grouped into 9 distinct clusters and ordered by Slingshot pseudotime trajectory. ( D ) Slingshot lineage overlay on CD8 + T cell UMAP.
Figure Legend Snippet: TF and lineage progression analysis of CD8 + KITs. ( A ) Heatmap represents z -scored regulon activity of top TFs inferred by SCENIC (rows) and association with CD8 + T cell clusters (columns). ( B ) Representative TFs were mapped onto CD8 UMAPs; TF selection was based on known regulatory functions or due to identification via SCENIC analysis. Outlines highlight spleen-derived, kidney-infiltrating, and exhausted cells, with dot red color intensity representing log 2 expression. ( C ) CD8 + T cells grouped into 9 distinct clusters and ordered by Slingshot pseudotime trajectory. ( D ) Slingshot lineage overlay on CD8 + T cell UMAP.

Techniques Used: Activity Assay, Selection, Derivative Assay, Expressing

High-resolution clustering of CD4 + T cells uncovers unique transcriptional programs in KITs. ( A ) UMAP of CD4 + T cells from Main-Seq outlining 13 clusters, with the right panel exhibiting assignment of cell source as determined by HTO. ( B – D ) Gene set enrichment analysis (GSEA) performed in each cell by Wilcoxon’s test (–log 10 [ P value]) using published reference gene signatures ( Supplemental Table 1 ) and related TF expression were overlaid onto the UMAP to identify CD4 phenotypes. This included ( B ) Treg gene signature and Foxp3 expression, ( C ) Th1 signature and Tbx21 /Tbet expression, and ( D ) Th2 signature and Gata3 expression.
Figure Legend Snippet: High-resolution clustering of CD4 + T cells uncovers unique transcriptional programs in KITs. ( A ) UMAP of CD4 + T cells from Main-Seq outlining 13 clusters, with the right panel exhibiting assignment of cell source as determined by HTO. ( B – D ) Gene set enrichment analysis (GSEA) performed in each cell by Wilcoxon’s test (–log 10 [ P value]) using published reference gene signatures ( Supplemental Table 1 ) and related TF expression were overlaid onto the UMAP to identify CD4 phenotypes. This included ( B ) Treg gene signature and Foxp3 expression, ( C ) Th1 signature and Tbx21 /Tbet expression, and ( D ) Th2 signature and Gata3 expression.

Techniques Used: Expressing

TF analysis suggests overarching transcriptional regulation of infiltrating CD4 + T cell clusters. ( A ) Heatmap representing z -scored regulon activity of top TFs inferred by SCENIC and association with CD4 + T cell clusters. ( B ) Expression of selected TFs overlaid onto the CD4 UMAP as depicted in Figure 2.
Figure Legend Snippet: TF analysis suggests overarching transcriptional regulation of infiltrating CD4 + T cell clusters. ( A ) Heatmap representing z -scored regulon activity of top TFs inferred by SCENIC and association with CD4 + T cell clusters. ( B ) Expression of selected TFs overlaid onto the CD4 UMAP as depicted in Figure 2.

Techniques Used: Activity Assay, Expressing

CD4 + KITs exhibit a progressive transcriptional phenotype from cytotoxicity to hypoxia/dysfunction through pseudotime. GSEA performed in each cell by Wilcoxon’s test (–log 10 [ P value]) using published reference genes signature ( Supplemental Table 1 ) overlaid onto the UMAP from Figure 2 A. ( A ) Hypoxia signature, exhaustion signature, and cytotoxic CD4 signature. ( B ) Dot plots show the distribution of exhaustion score in each CD4 + T cell, grouped by cluster number. Dots are colored according to the source of cells they represent. Statistics were calculated by Kruskal-Wallis rank test. ( C ) Monocle trajectory mapping of CD4 + KITs wherein time 0 (dark purple) represents lineage origination with progression to most differentiated (yellow), with cell source mapping. ( D ) Gene signature mapping for the indicated signatures as defined in A .
Figure Legend Snippet: CD4 + KITs exhibit a progressive transcriptional phenotype from cytotoxicity to hypoxia/dysfunction through pseudotime. GSEA performed in each cell by Wilcoxon’s test (–log 10 [ P value]) using published reference genes signature ( Supplemental Table 1 ) overlaid onto the UMAP from Figure 2 A. ( A ) Hypoxia signature, exhaustion signature, and cytotoxic CD4 signature. ( B ) Dot plots show the distribution of exhaustion score in each CD4 + T cell, grouped by cluster number. Dots are colored according to the source of cells they represent. Statistics were calculated by Kruskal-Wallis rank test. ( C ) Monocle trajectory mapping of CD4 + KITs wherein time 0 (dark purple) represents lineage origination with progression to most differentiated (yellow), with cell source mapping. ( D ) Gene signature mapping for the indicated signatures as defined in A .

Techniques Used:

High-resolution clustering of CD8 + T cells identifies unique functional phenotypes. ( A ) UMAP of CD8 + T cells from Main-Seq delineating 9 clusters. ( B ) UMAP with overlay exhibiting assignment of cell source as determined by HTO. ( C ) Heatmap shows top significant (FDR
Figure Legend Snippet: High-resolution clustering of CD8 + T cells identifies unique functional phenotypes. ( A ) UMAP of CD8 + T cells from Main-Seq delineating 9 clusters. ( B ) UMAP with overlay exhibiting assignment of cell source as determined by HTO. ( C ) Heatmap shows top significant (FDR

Techniques Used: Functional Assay

21) Product Images from "miR-15a and miR-15b modulate natural killer and CD8+T-cell activation and anti-tumor immune response by targeting PD-L1 in neuroblastoma"

Article Title: miR-15a and miR-15b modulate natural killer and CD8+T-cell activation and anti-tumor immune response by targeting PD-L1 in neuroblastoma

Journal: Molecular Therapy Oncolytics

doi: 10.1016/j.omto.2022.03.010

Targeting PD-L1 is required for miR-15a- and miR-15b-mediated immune cell activation, and cytokine production (A and D) Representative flow cytometric plots showing the surface PD-L1 expression in SK-N-AS cells that were blocked with anti-PD-L1 or IgG control antibody for 24 h followed by transfection with miR-15a and miR-15b mimics for an additional 24 h (A), and NB-19 cells treated with miR-15a, miR-15b mimics and PD-L1 siRNA for 48 h (D). (B, C, and E) Representative flow cytometric plots showing the expression of intracellular granzyme B in CD8 + T cells (B), and surface CD107a in NK cells (C and E) cocultured (24 h for CD8 + T cells, 5 h for NK cells) with miR-15a and miR-15b expressing SK-N-AS cells (E:T=1:1) treated with anti-PD-L1 antibody for 24 h (B, C) or NB-19 cells (E:T=0.25:1) treated with PD-L1 siRNA for 48 h (E). (F) Representative ELISA assay-based quantification graphs of IFN-γ in the culture medium of NK cells after coculture (E:T=1:1) with miR-15a and miR-15b expressing SK-N-AS cells pretreated with anti-PD-L1 antibody for 24 h. NB cells were treated with anti-PD-L1 antibody (15 μg/mL) for 24 h followed by miRs transfection for another 24 h (SK-N-AS) or PD-L1 siRNA (NB-19) for 48 h and used for coculture experiments with activated CD8 + T (SK-N-AS) or NK (NB-19) cells. Control (ctrl) miR or IgG antibody treated cells served as a control group. Untouched CD8 + T cells and NK cells were isolated, expanded, and activated as previously described, and were used for coculture experiments with NB cells. Cells were permeabilized (intracellular), fixed, stained with antibodies as described in the materials and methods section, and analyzed by the Attune Acoustic Focusing Flow Cytometer. The percentage of surface PD-L1 + (A and D), intracellular granzyme B + (B), and surface CD107a + (C and E) are shown in each of their respective plots. Data represent the mean ± standard error of three to five independent biological experiments. Statistical analyses were performed using a two-sided unpaired t -test. ∗p
Figure Legend Snippet: Targeting PD-L1 is required for miR-15a- and miR-15b-mediated immune cell activation, and cytokine production (A and D) Representative flow cytometric plots showing the surface PD-L1 expression in SK-N-AS cells that were blocked with anti-PD-L1 or IgG control antibody for 24 h followed by transfection with miR-15a and miR-15b mimics for an additional 24 h (A), and NB-19 cells treated with miR-15a, miR-15b mimics and PD-L1 siRNA for 48 h (D). (B, C, and E) Representative flow cytometric plots showing the expression of intracellular granzyme B in CD8 + T cells (B), and surface CD107a in NK cells (C and E) cocultured (24 h for CD8 + T cells, 5 h for NK cells) with miR-15a and miR-15b expressing SK-N-AS cells (E:T=1:1) treated with anti-PD-L1 antibody for 24 h (B, C) or NB-19 cells (E:T=0.25:1) treated with PD-L1 siRNA for 48 h (E). (F) Representative ELISA assay-based quantification graphs of IFN-γ in the culture medium of NK cells after coculture (E:T=1:1) with miR-15a and miR-15b expressing SK-N-AS cells pretreated with anti-PD-L1 antibody for 24 h. NB cells were treated with anti-PD-L1 antibody (15 μg/mL) for 24 h followed by miRs transfection for another 24 h (SK-N-AS) or PD-L1 siRNA (NB-19) for 48 h and used for coculture experiments with activated CD8 + T (SK-N-AS) or NK (NB-19) cells. Control (ctrl) miR or IgG antibody treated cells served as a control group. Untouched CD8 + T cells and NK cells were isolated, expanded, and activated as previously described, and were used for coculture experiments with NB cells. Cells were permeabilized (intracellular), fixed, stained with antibodies as described in the materials and methods section, and analyzed by the Attune Acoustic Focusing Flow Cytometer. The percentage of surface PD-L1 + (A and D), intracellular granzyme B + (B), and surface CD107a + (C and E) are shown in each of their respective plots. Data represent the mean ± standard error of three to five independent biological experiments. Statistical analyses were performed using a two-sided unpaired t -test. ∗p

Techniques Used: Activation Assay, Expressing, Transfection, Enzyme-linked Immunosorbent Assay, Isolation, Staining, Flow Cytometry

MiR-15a and miR-15b promote CD8 + T cell activation,proliferation and induce CD8 + T cell-mediated NB cytotoxicity (A–D) Representative flow cytometric plots of human peripheral CD8 + T cells showing the expression of intracellular Granzyme B (A), intracellular Perforin (B), surface CD3 and CD8 (C), and intracellular Ki-67 (D) after coculture (E:T=1:1) with miR-15a and miR-15b expressing SK-N-AS (A–D, top panels), and NB-19 (A–D, bottom panels) cells for 24 h. (E) Representative flow cytometric plots of miR-15a and miR-15b expressing SK-N-AS (E, top panel), and NB-19 (E, bottom panel) showing the expression of intracellular active caspase-3 upon coculture (E:T=1:1) with activated human CD8 + T cells for 24 h. (F) A quantification graph showing normalized luciferase activity in SK-N-BE(2) cells expressing miR-15a and miR-15b upon coculture (E:T=5:1) with activated CD8 + T cells for 24 h. Untouched CD8 + T cells were isolated from PBMCs of healthy human blood donors by negative selection using the MojoSort human CD8 + T cell Isolation Kit (BioLegend). For 7 days, CD8 + T cells were activated and expanded using human T-activator CD3/CD28 Dynabeads and used for coculture experiments with NB cells. NB cells were transfected with miRs for 24 h and used for coculture experiments with activated CD8 + T cells for an additional 24 h. Non-targeting control (ctrl) miR cells served as a control group. Cells were permeabilized (intracellular), fixed, and stained for respective antibodies described in the materials and methods section and analyzed by flow cytometry. The percentage of Granzyme B + (A), Perforin + (B), CD3/CD8 + (C), ki67 + (D), and cleaved caspase-3 + cells (E) are shown in each of their respective plots. Data represent the mean ± standard error of three to five independent biological experiments. Statistical analyses were performed using a two-sided unpaired t-test.
Figure Legend Snippet: MiR-15a and miR-15b promote CD8 + T cell activation,proliferation and induce CD8 + T cell-mediated NB cytotoxicity (A–D) Representative flow cytometric plots of human peripheral CD8 + T cells showing the expression of intracellular Granzyme B (A), intracellular Perforin (B), surface CD3 and CD8 (C), and intracellular Ki-67 (D) after coculture (E:T=1:1) with miR-15a and miR-15b expressing SK-N-AS (A–D, top panels), and NB-19 (A–D, bottom panels) cells for 24 h. (E) Representative flow cytometric plots of miR-15a and miR-15b expressing SK-N-AS (E, top panel), and NB-19 (E, bottom panel) showing the expression of intracellular active caspase-3 upon coculture (E:T=1:1) with activated human CD8 + T cells for 24 h. (F) A quantification graph showing normalized luciferase activity in SK-N-BE(2) cells expressing miR-15a and miR-15b upon coculture (E:T=5:1) with activated CD8 + T cells for 24 h. Untouched CD8 + T cells were isolated from PBMCs of healthy human blood donors by negative selection using the MojoSort human CD8 + T cell Isolation Kit (BioLegend). For 7 days, CD8 + T cells were activated and expanded using human T-activator CD3/CD28 Dynabeads and used for coculture experiments with NB cells. NB cells were transfected with miRs for 24 h and used for coculture experiments with activated CD8 + T cells for an additional 24 h. Non-targeting control (ctrl) miR cells served as a control group. Cells were permeabilized (intracellular), fixed, and stained for respective antibodies described in the materials and methods section and analyzed by flow cytometry. The percentage of Granzyme B + (A), Perforin + (B), CD3/CD8 + (C), ki67 + (D), and cleaved caspase-3 + cells (E) are shown in each of their respective plots. Data represent the mean ± standard error of three to five independent biological experiments. Statistical analyses were performed using a two-sided unpaired t-test.

Techniques Used: Activation Assay, Expressing, Luciferase, Activity Assay, Isolation, Selection, Cell Isolation, Transfection, Staining, Flow Cytometry

MiR-15a activates anti-tumor immune response against NB in vivo (A) Photographs showing tumor pictures, and (B) summary graph showing tumor weight of the C57BL/6 mice that received subcutaneous murine NB-975 cells stably expressing GFP-miR-15a or GFP-control (ctrl) miR for 30 days. (C) Representative flow cytometric plots showing the percentage of surface PD-L1 expression on GFP + tumor cells, and (D) quantification, (E) percentage of infiltrated CD8 cells, (F and G) Ki-67, IFN-γ positive infiltrated CD8 + T cells, and (H) perforin positive CD3 − NK1.1 + mouse NK cells analyzed from the single-cell suspension of tumors tissues from C57BL/6 mice that received (subcutaneous) murine NB-975 cells stably expressing miR-15a or Ctrl miR for 30 days. (I) Representative IHC images of CD34 (murine endothelial cells) stained microvessels at 20× magnification. Murine NB975 cells stably expressing GFP-miR-15a or GFP-ctrl miR were injected (subcutaneous) into the left flank of immune-competent C57BL/6 mice. After 30 days, mice were euthanized, tumor tissues were harvested, photographed, and weighed. Tumor tissues were excised, prepared single-cell suspension, and stained for PD-L1 (α-mouse PE-PD-L1), infiltrated CD8 + T cells (α-mouse PE-CD8a), Ki-67 (α-mouse BV421-Ki67), IFN-γ (α-mouse BV42-IFN-γ), CD3 − NK1.1 + mouse NK cells (α-mouse AF700-CD3, and α-mouse APC/FireTM 750-NK1.1), perforin (α-mouse PE-perforin) by flow cytometry using their respective antibodies. (C, and E–H) Representative flow cytometric plots showing an FMO control of cells stained with all fluorochromes except one used to set the background signal for the analysis were given. Bar graphs are shown as the mean ± standard error (n=4 mice per group). Statistical analyses were performed using a two-sided unpaired t -test. (I) A schematic model showing the modulation of anti-tumor immune response in the absence (top panel) or presence (bottom panel) of miR-15 (miR-15a, miR-15b) in NB.
Figure Legend Snippet: MiR-15a activates anti-tumor immune response against NB in vivo (A) Photographs showing tumor pictures, and (B) summary graph showing tumor weight of the C57BL/6 mice that received subcutaneous murine NB-975 cells stably expressing GFP-miR-15a or GFP-control (ctrl) miR for 30 days. (C) Representative flow cytometric plots showing the percentage of surface PD-L1 expression on GFP + tumor cells, and (D) quantification, (E) percentage of infiltrated CD8 cells, (F and G) Ki-67, IFN-γ positive infiltrated CD8 + T cells, and (H) perforin positive CD3 − NK1.1 + mouse NK cells analyzed from the single-cell suspension of tumors tissues from C57BL/6 mice that received (subcutaneous) murine NB-975 cells stably expressing miR-15a or Ctrl miR for 30 days. (I) Representative IHC images of CD34 (murine endothelial cells) stained microvessels at 20× magnification. Murine NB975 cells stably expressing GFP-miR-15a or GFP-ctrl miR were injected (subcutaneous) into the left flank of immune-competent C57BL/6 mice. After 30 days, mice were euthanized, tumor tissues were harvested, photographed, and weighed. Tumor tissues were excised, prepared single-cell suspension, and stained for PD-L1 (α-mouse PE-PD-L1), infiltrated CD8 + T cells (α-mouse PE-CD8a), Ki-67 (α-mouse BV421-Ki67), IFN-γ (α-mouse BV42-IFN-γ), CD3 − NK1.1 + mouse NK cells (α-mouse AF700-CD3, and α-mouse APC/FireTM 750-NK1.1), perforin (α-mouse PE-perforin) by flow cytometry using their respective antibodies. (C, and E–H) Representative flow cytometric plots showing an FMO control of cells stained with all fluorochromes except one used to set the background signal for the analysis were given. Bar graphs are shown as the mean ± standard error (n=4 mice per group). Statistical analyses were performed using a two-sided unpaired t -test. (I) A schematic model showing the modulation of anti-tumor immune response in the absence (top panel) or presence (bottom panel) of miR-15 (miR-15a, miR-15b) in NB.

Techniques Used: In Vivo, Mouse Assay, Stable Transfection, Expressing, Immunohistochemistry, Staining, Injection, Flow Cytometry

22) Product Images from "Generation of Tumor-Specific Cytotoxic T Cells From Blood via In Vitro Expansion Using Autologous Dendritic Cells Pulsed With Neoantigen-Coupled Microbeads"

Article Title: Generation of Tumor-Specific Cytotoxic T Cells From Blood via In Vitro Expansion Using Autologous Dendritic Cells Pulsed With Neoantigen-Coupled Microbeads

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2022.866763

Efficient neoantigen delivery through autologous DC loaded with EpiTCer ® beads. HLA-A2+ healthy donor (HD) blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU derived tumor antigens and further matured into DC. Long-term co-cultures with DC and CD8+ T cells were performed; T cells was harvested and re-stimulated with ANRU tumor cells or 9mer neoantigen peptides. T-cell activation was measured by CD107a expression using flow cytometry. (A, B) HD CD8+ T cells were co-cultured with DC pulsed ANRU tumor lysate (TL), indicated 9mer neoantigen peptide or EpiTCer beads #2 at indicated bead/DC ratio. Tumor specificity or T-cell activation was assessed by re-stimulation with ANRU tumor cells or indicated 9mer neoantigen peptide, respectively. (C–E) HD CD8+ T cells were co-cultured with DC pulsed with indicated 9mer neoantigen peptide (separately or combined/pooled) or EpiTCer beads #2 at indicated bead/DC ratio. Tumor specificity was assessed by re-stimulation with ANRU tumor cells with (E) or without (C, D) the presence of MHC class I blockade (W6/32). Values in panels (D, E) without W6/32 (−) were obtained within the same experiment. Each donor represents one independent experiment.
Figure Legend Snippet: Efficient neoantigen delivery through autologous DC loaded with EpiTCer ® beads. HLA-A2+ healthy donor (HD) blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU derived tumor antigens and further matured into DC. Long-term co-cultures with DC and CD8+ T cells were performed; T cells was harvested and re-stimulated with ANRU tumor cells or 9mer neoantigen peptides. T-cell activation was measured by CD107a expression using flow cytometry. (A, B) HD CD8+ T cells were co-cultured with DC pulsed ANRU tumor lysate (TL), indicated 9mer neoantigen peptide or EpiTCer beads #2 at indicated bead/DC ratio. Tumor specificity or T-cell activation was assessed by re-stimulation with ANRU tumor cells or indicated 9mer neoantigen peptide, respectively. (C–E) HD CD8+ T cells were co-cultured with DC pulsed with indicated 9mer neoantigen peptide (separately or combined/pooled) or EpiTCer beads #2 at indicated bead/DC ratio. Tumor specificity was assessed by re-stimulation with ANRU tumor cells with (E) or without (C, D) the presence of MHC class I blockade (W6/32). Values in panels (D, E) without W6/32 (−) were obtained within the same experiment. Each donor represents one independent experiment.

Techniques Used: Derivative Assay, Isolation, Activation Assay, Expressing, Flow Cytometry, Cell Culture

Efficient stimulation of blood-derived CD8+ T cells using EpiTCer ® bead pulsed DC. HLA-A2+ healthy donor (HD) blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU derived tumor antigens and further matured into DC. Long term co-cultures with DC and CD8+ T cells were performed and T cells harvested and re-stimulated with ANRU tumor cells. Tumor recognition was measured by CD107a expression and IFNγ production using flow cytometry. (A–D) HD CD8+ T cells were co-cultured with DC pulsed ANRU tumor lysate (TL) or EpiTCer beads #1 at indicated bead/DC ratio. Tumor specificity was measured by re-stimulation with ANRU tumor cells and measured by CD107a expression (B) or CD107a expression and IFNγ production (A , C , D) , using flow cytometry. Each donor represents one independent experiment.
Figure Legend Snippet: Efficient stimulation of blood-derived CD8+ T cells using EpiTCer ® bead pulsed DC. HLA-A2+ healthy donor (HD) blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU derived tumor antigens and further matured into DC. Long term co-cultures with DC and CD8+ T cells were performed and T cells harvested and re-stimulated with ANRU tumor cells. Tumor recognition was measured by CD107a expression and IFNγ production using flow cytometry. (A–D) HD CD8+ T cells were co-cultured with DC pulsed ANRU tumor lysate (TL) or EpiTCer beads #1 at indicated bead/DC ratio. Tumor specificity was measured by re-stimulation with ANRU tumor cells and measured by CD107a expression (B) or CD107a expression and IFNγ production (A , C , D) , using flow cytometry. Each donor represents one independent experiment.

Techniques Used: Derivative Assay, Isolation, Expressing, Flow Cytometry, Cell Culture

EpiTCer-loaded autologous DC enrich for highly tumor-specific CD8+ T cells with limited tumor cross-reactivity. ANRU blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU-derived tumor antigens and further matured into DC. Long-term co-cultures with DC and CD8+ T cells were performed: T cells harvested and (A) re-stimulated with indicated tumor cell line (B) further expanded via rapid expansion (REP). Tumor recognition was measured by CD107a expression. (A) ANRU CD8+ T cells were co-cultured with DC-pulsed ANRU tumor lysate (TL), indicating 9mer neoantigen peptide or EpiTCer beads #2 at indicated bead/DC ratio. Tumor specificity was assessed by re-stimulation with autologous ANRU tumor cells or allogenic KADA (HLA-A2+ melanoma cell line). (B) DC-enriched ANRU CD8+ T cells were further expanded using a REP protocol, harvested and re-stimulated with autologous ANRU tumor cells or allogenic KADA tumor cells. Panel (C) shows the CD8+ mediated ANRU tumor recognition observed before (−) and after REP (REP); same values as in panels (A, B) are displayed.
Figure Legend Snippet: EpiTCer-loaded autologous DC enrich for highly tumor-specific CD8+ T cells with limited tumor cross-reactivity. ANRU blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU-derived tumor antigens and further matured into DC. Long-term co-cultures with DC and CD8+ T cells were performed: T cells harvested and (A) re-stimulated with indicated tumor cell line (B) further expanded via rapid expansion (REP). Tumor recognition was measured by CD107a expression. (A) ANRU CD8+ T cells were co-cultured with DC-pulsed ANRU tumor lysate (TL), indicating 9mer neoantigen peptide or EpiTCer beads #2 at indicated bead/DC ratio. Tumor specificity was assessed by re-stimulation with autologous ANRU tumor cells or allogenic KADA (HLA-A2+ melanoma cell line). (B) DC-enriched ANRU CD8+ T cells were further expanded using a REP protocol, harvested and re-stimulated with autologous ANRU tumor cells or allogenic KADA tumor cells. Panel (C) shows the CD8+ mediated ANRU tumor recognition observed before (−) and after REP (REP); same values as in panels (A, B) are displayed.

Techniques Used: Derivative Assay, Isolation, Expressing, Cell Culture

Efficient expansion of patient blood-derived tumor-specific CD8+ T cells using DC loaded with EpiTCer beads®. ANRU blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU derived tumor antigens and further matured into DC. Long-term co-cultures with DC and CD8+ T cells was performed; T cells were harvested and re-stimulated with ANRU tumor cells or ANRU non-activated monocytes. Tumor recognition or healthy cell reactivity was measured by CD107a expression or IFNγ production using flow cytometry. (A, B) ANRU CD8+ T cells were co-cultured with DC pulsed ANRU tumor lysate (TL), indicating 9mer neoantigen peptide, non-coated EpiTCer beads (empty, E), EpiTCer beads carrying the corresponding wild-type sequence (WT), or EpiTCer beads #2. EpiTCer beads were used at indicated bead/DC ratio. Tumor specificity was assessed by re-stimulation with ANRU tumor cells. (C) ANRU CD8+ T-cell tumor recognition, presenting values from Figures 4A and 5A and Supplementary Figure S2B . (D, E) ANRU CD8+ T cells were co-cultured as described in panels (A, B) , and T-cell activation was measured by re-stimulation with ANRU tumor cells or non-activated ANRU monocytes. Values in panels (A, D) and (B, D) for ANRU tumor reactivity were obtained within the same experiment. (C) Statistical analysis one-way ANOVA, Tukey’s multiple comparison test. Definition of significance: ***p
Figure Legend Snippet: Efficient expansion of patient blood-derived tumor-specific CD8+ T cells using DC loaded with EpiTCer beads®. ANRU blood-derived CD14+ monocytes and CD8+ T cells were isolated; monocytes matured into imDC were loaded with indicated source of ANRU derived tumor antigens and further matured into DC. Long-term co-cultures with DC and CD8+ T cells was performed; T cells were harvested and re-stimulated with ANRU tumor cells or ANRU non-activated monocytes. Tumor recognition or healthy cell reactivity was measured by CD107a expression or IFNγ production using flow cytometry. (A, B) ANRU CD8+ T cells were co-cultured with DC pulsed ANRU tumor lysate (TL), indicating 9mer neoantigen peptide, non-coated EpiTCer beads (empty, E), EpiTCer beads carrying the corresponding wild-type sequence (WT), or EpiTCer beads #2. EpiTCer beads were used at indicated bead/DC ratio. Tumor specificity was assessed by re-stimulation with ANRU tumor cells. (C) ANRU CD8+ T-cell tumor recognition, presenting values from Figures 4A and 5A and Supplementary Figure S2B . (D, E) ANRU CD8+ T cells were co-cultured as described in panels (A, B) , and T-cell activation was measured by re-stimulation with ANRU tumor cells or non-activated ANRU monocytes. Values in panels (A, D) and (B, D) for ANRU tumor reactivity were obtained within the same experiment. (C) Statistical analysis one-way ANOVA, Tukey’s multiple comparison test. Definition of significance: ***p

Techniques Used: Derivative Assay, Isolation, Expressing, Flow Cytometry, Cell Culture, Sequencing, Activation Assay

EpiTCer-pulsed DC efficiently induces functional maturation of blood-derived CD8+ T cells. ANRU (A) or healthy donor (B) blood-derived CD14+ monocytes and CD8+ T cells were isolated, and imDC were loaded with indicated source of ANRU-derived tumor antigens and matured into DC. Long-term co-culture with DC and CD8+ T cells was performed. T cells were harvested and (A) phenotypic analysis was performed or (B) ANRU tumor reactivity was measured. (A) Long-term co-culture with ANRU CD8+ T cells and ANRU DC tumor lysate (TL), non-coated EpiTCer beads (empty, E), EpiTCer wide-type beads (WT) or EpiTCer beads at 40:1 bead/DC ratio was performed. CD8+ T cells were harvested, and phenotypic analysis of maturation status was performed using flow cytometry. Cells were gated on lymphocytes/single cells/live cells/CD3+CD8+ cells, and maturation was investigated via CCR7 and CD45RA. For gating strategy, see Supplementary Figure 3 . (B) HD CD8+ T cells were long-term co-cultured with DC loaded with ANRU tumor lysate. Co-culture was performed in plate (96w plates) or in cell culture flasks; CD8+ T cells were harvested and re-stimulated with ANRU tumor cells with and without MHC class I blocking (W6/32). HD, each donor represents one independent experiment.
Figure Legend Snippet: EpiTCer-pulsed DC efficiently induces functional maturation of blood-derived CD8+ T cells. ANRU (A) or healthy donor (B) blood-derived CD14+ monocytes and CD8+ T cells were isolated, and imDC were loaded with indicated source of ANRU-derived tumor antigens and matured into DC. Long-term co-culture with DC and CD8+ T cells was performed. T cells were harvested and (A) phenotypic analysis was performed or (B) ANRU tumor reactivity was measured. (A) Long-term co-culture with ANRU CD8+ T cells and ANRU DC tumor lysate (TL), non-coated EpiTCer beads (empty, E), EpiTCer wide-type beads (WT) or EpiTCer beads at 40:1 bead/DC ratio was performed. CD8+ T cells were harvested, and phenotypic analysis of maturation status was performed using flow cytometry. Cells were gated on lymphocytes/single cells/live cells/CD3+CD8+ cells, and maturation was investigated via CCR7 and CD45RA. For gating strategy, see Supplementary Figure 3 . (B) HD CD8+ T cells were long-term co-cultured with DC loaded with ANRU tumor lysate. Co-culture was performed in plate (96w plates) or in cell culture flasks; CD8+ T cells were harvested and re-stimulated with ANRU tumor cells with and without MHC class I blocking (W6/32). HD, each donor represents one independent experiment.

Techniques Used: Functional Assay, Derivative Assay, Isolation, Co-Culture Assay, Flow Cytometry, Cell Culture, Blocking Assay

23) Product Images from "Adenosinergic Pathway and Linked Suppression: Two Critical Suppressive Mechanisms of Human Donor Antigen Specific Regulatory T Cell Lines Expanded Post Transplant"

Article Title: Adenosinergic Pathway and Linked Suppression: Two Critical Suppressive Mechanisms of Human Donor Antigen Specific Regulatory T Cell Lines Expanded Post Transplant

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.849939

(A) Phenotypic characterization of ASTRL. Flow cytometry plots show expression of CD25, CD127, and Foxp3 in CD4 + and CD8 + T cells of pre expansion PBMC and ASTRL. (B) Expression of various conventional T regulatory cell markers on CD4 + T cell subsets of pre-expansion PBMC and ASTR L. Histograms and contour plot overlays show PBMC in red and ASTRL in blue. (C) ASTRLs constitute a heterogenous cell population. Figures show a comparison of CD19 + B cell and CD11b + myeoid cell populations in pre-expansion PBMC (top panel) and ASTRL (bottom panel) from five different subjects. (D) ASTRLs constitute a heterogenous cell population. NK cell population in ASTRLs (bottom panel) and pre expansion PBMC (top panel) are shown from five different subjects. (E) ASTRLs upregulate the surface expression of the molecules belonging to canonical (CD39 and CD73) and non-canonical (CD38 and CD203) adenosinergic pathways. Figures on the top panels show the expression of CD39 and CD73 on the CD3 + and CD3 − cell populations of ASTRL in blue and pre-expansion PBMC in red. The bottom panel shows the expression of CD38 and CD203 in CD3 + and CD3 − subsets of ASTRL in blue and pre-expansion PBMC in red. (F) Differentially expressed cytokines in the expansion media of ASTRLs. Post expansion, on days 30–35 ASTRLs were harvested and production of various cytokines in the spent expansion media were measured by Luminex.
Figure Legend Snippet: (A) Phenotypic characterization of ASTRL. Flow cytometry plots show expression of CD25, CD127, and Foxp3 in CD4 + and CD8 + T cells of pre expansion PBMC and ASTRL. (B) Expression of various conventional T regulatory cell markers on CD4 + T cell subsets of pre-expansion PBMC and ASTR L. Histograms and contour plot overlays show PBMC in red and ASTRL in blue. (C) ASTRLs constitute a heterogenous cell population. Figures show a comparison of CD19 + B cell and CD11b + myeoid cell populations in pre-expansion PBMC (top panel) and ASTRL (bottom panel) from five different subjects. (D) ASTRLs constitute a heterogenous cell population. NK cell population in ASTRLs (bottom panel) and pre expansion PBMC (top panel) are shown from five different subjects. (E) ASTRLs upregulate the surface expression of the molecules belonging to canonical (CD39 and CD73) and non-canonical (CD38 and CD203) adenosinergic pathways. Figures on the top panels show the expression of CD39 and CD73 on the CD3 + and CD3 − cell populations of ASTRL in blue and pre-expansion PBMC in red. The bottom panel shows the expression of CD38 and CD203 in CD3 + and CD3 − subsets of ASTRL in blue and pre-expansion PBMC in red. (F) Differentially expressed cytokines in the expansion media of ASTRLs. Post expansion, on days 30–35 ASTRLs were harvested and production of various cytokines in the spent expansion media were measured by Luminex.

Techniques Used: Flow Cytometry, Expressing, Luminex

24) Product Images from "Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma"

Article Title: Targeting HNRNPM Inhibits Cancer Stemness and Enhances Antitumor Immunity in Wnt-activated Hepatocellular Carcinoma

Journal: Cellular and Molecular Gastroenterology and Hepatology

doi: 10.1016/j.jcmgh.2022.02.006

HNRNPM inhibition curbs immune escape and enhances PD-1 blockade by promoting CD8+ T cells activation phenotype. A , Schematic diagram of Hep1-6-OVA cells co-cultured with OTI cells. B , The flow cytometry analysis of IFN-γ+ or granzyme B+ CD8+ T cells between control and shHNRNPM groups. Data were from 3 independent experiments. ∗∗∗ P
Figure Legend Snippet: HNRNPM inhibition curbs immune escape and enhances PD-1 blockade by promoting CD8+ T cells activation phenotype. A , Schematic diagram of Hep1-6-OVA cells co-cultured with OTI cells. B , The flow cytometry analysis of IFN-γ+ or granzyme B+ CD8+ T cells between control and shHNRNPM groups. Data were from 3 independent experiments. ∗∗∗ P

Techniques Used: Inhibition, Activation Assay, Cell Culture, Flow Cytometry

25) Product Images from "Self-Adjuvanting Lipoprotein Conjugate αGalCer-RBD Induces Potent Immunity against SARS-CoV-2 and its Variants of Concern"

Article Title: Self-Adjuvanting Lipoprotein Conjugate αGalCer-RBD Induces Potent Immunity against SARS-CoV-2 and its Variants of Concern

Journal: Journal of Medicinal Chemistry

doi: 10.1021/acs.jmedchem.1c02000

Adjuvant–protein conjugate induced potent RBD-specific cellular immune responses. Specific cytokine-producing T cell immune responses were assessed using splenocytes from immunized mice on day 35. (A) IFN-γ ELISpot assay. (B) Representative ELISpot wells. (C) Flow cytometry assay of IFN-γ and TNF-α double-positive cells in CD8+ T cells. (D) CD8+ TNF-α+ T cells, as a percentage of CD8+ T cells. (E) CD8+ IFN-γ+ T cells, as a percentage of CD8+ T cells. (F) Flow cytometry assay of IFN-γ and TNF-α double-positive cells in CD4+ T cells. (G) CD4+ TNF-α+ T cells, as a percentage of CD4+ T cells. (H) CD4+ IFN-γ+ T cells, as a percentage of CD4+ T cells. Data are shown as the mean ± SEM of five mice per group, each sample being characterized in triplicate. Statistical significance was determined using one-way ANOVA with Dunn’s multiple comparison test. No significant difference: ns, p
Figure Legend Snippet: Adjuvant–protein conjugate induced potent RBD-specific cellular immune responses. Specific cytokine-producing T cell immune responses were assessed using splenocytes from immunized mice on day 35. (A) IFN-γ ELISpot assay. (B) Representative ELISpot wells. (C) Flow cytometry assay of IFN-γ and TNF-α double-positive cells in CD8+ T cells. (D) CD8+ TNF-α+ T cells, as a percentage of CD8+ T cells. (E) CD8+ IFN-γ+ T cells, as a percentage of CD8+ T cells. (F) Flow cytometry assay of IFN-γ and TNF-α double-positive cells in CD4+ T cells. (G) CD4+ TNF-α+ T cells, as a percentage of CD4+ T cells. (H) CD4+ IFN-γ+ T cells, as a percentage of CD4+ T cells. Data are shown as the mean ± SEM of five mice per group, each sample being characterized in triplicate. Statistical significance was determined using one-way ANOVA with Dunn’s multiple comparison test. No significant difference: ns, p

Techniques Used: Mouse Assay, Enzyme-linked Immunospot, Flow Cytometry

26) Product Images from "Rational construction of controllable autoimmune diabetes model depicting clinical features"

Article Title: Rational construction of controllable autoimmune diabetes model depicting clinical features

Journal: PLoS ONE

doi: 10.1371/journal.pone.0260100

CD8 + T cell infiltration and severe ketoacidosis in STZ+DT mice. a-b . H E staining of islets from STZ+DT mice and control groups at day 6 ( a ), and prediabetic NOD mice at 10-week old ( b ). Arrow pointed at inflammatory infiltrates. c . Immunofluorescence of CD3 (red), insulin (green) and DAPI (blue) of islets. Scale bars represent 50μm. d . T cells subsets in pancreatic infiltrates. CD8 + and CD4 + T cells were identified by flow cytometry. Mice age as in 3a and 3b. e. Statistical analysis of 3d. Difference was calculated between STZ+DT and control groups (top left, horizontal comparisons) or between STZ+DT and NOD model (top right, horizontal comparisons). Both CD4 + T cells (gray) and CD8 + T cells (white) were analyzed. Statistical significance was also calculated with CD8 + versus CD4 + T cells within STZ+DT or prediabetic NOD (vertical comparisons). n = 5. N = 3. f . Blood glucose fluctuation after CD8 + T cell depletion. Anti-CD8 neutralizing antibody (Orange) or isotype control (Black) were i . p . injected at day 3, 6, 9, 12. n = 4. N = 3. g . Auto-antigen specific IFN-γ response in CD8 + infiltrates. IFN-γ response in CD8 + infiltrates in STZ+DT mice (left) and statistical analysis (right). PMA+Ionomycin (PAM+IONO) was added as positive control. OVA 257-264 was added as negative control. GAD65 114-122 and IAPP 5-13 stimulated responses were analyzed. Mice age as in 3a and 3b. n = 3. N = 3. h. Ketoacidosis in STZ+DT mice (Day 30) and NOD mice (2-week old for pre-diabetic and after diabetes onset). Plasma β-hydroxybutyric acid (BHA) was analyzed. n = 5. N = 3. *, p
Figure Legend Snippet: CD8 + T cell infiltration and severe ketoacidosis in STZ+DT mice. a-b . H E staining of islets from STZ+DT mice and control groups at day 6 ( a ), and prediabetic NOD mice at 10-week old ( b ). Arrow pointed at inflammatory infiltrates. c . Immunofluorescence of CD3 (red), insulin (green) and DAPI (blue) of islets. Scale bars represent 50μm. d . T cells subsets in pancreatic infiltrates. CD8 + and CD4 + T cells were identified by flow cytometry. Mice age as in 3a and 3b. e. Statistical analysis of 3d. Difference was calculated between STZ+DT and control groups (top left, horizontal comparisons) or between STZ+DT and NOD model (top right, horizontal comparisons). Both CD4 + T cells (gray) and CD8 + T cells (white) were analyzed. Statistical significance was also calculated with CD8 + versus CD4 + T cells within STZ+DT or prediabetic NOD (vertical comparisons). n = 5. N = 3. f . Blood glucose fluctuation after CD8 + T cell depletion. Anti-CD8 neutralizing antibody (Orange) or isotype control (Black) were i . p . injected at day 3, 6, 9, 12. n = 4. N = 3. g . Auto-antigen specific IFN-γ response in CD8 + infiltrates. IFN-γ response in CD8 + infiltrates in STZ+DT mice (left) and statistical analysis (right). PMA+Ionomycin (PAM+IONO) was added as positive control. OVA 257-264 was added as negative control. GAD65 114-122 and IAPP 5-13 stimulated responses were analyzed. Mice age as in 3a and 3b. n = 3. N = 3. h. Ketoacidosis in STZ+DT mice (Day 30) and NOD mice (2-week old for pre-diabetic and after diabetes onset). Plasma β-hydroxybutyric acid (BHA) was analyzed. n = 5. N = 3. *, p

Techniques Used: Mouse Assay, Staining, Immunofluorescence, Flow Cytometry, Injection, Positive Control, Negative Control

27) Product Images from "Proinflammatory Matrix Metalloproteinase-1 Associates With Mitral Valve Leaflet Disruption Following Percutaneous Mitral Valvuloplasty"

Article Title: Proinflammatory Matrix Metalloproteinase-1 Associates With Mitral Valve Leaflet Disruption Following Percutaneous Mitral Valvuloplasty

Journal: Frontiers in Cardiovascular Medicine

doi: 10.3389/fcvm.2021.804111

Expression of MMP-1 in peripheral blood mononuclear cells. (A) Representative dot plots illustrating the selection of MMP-1 positive gate on non-stimulated and rIFN-γ stimulated cells (bottom panel) using as references non stained and PE-isotype-stained cells (upper panel). Graph shows the individual frequency of total MMP-1 expression in PBMCs ( n = 5). (B) Unsupervised high dimensional analysis of flow cytometry data tSNE showing MMP-1 positive clusters (expression level plot, upper left panel) and matching areas on phonograph grid plot (bottom left). Heat map shows the hierarchical clustering of all identified clusters, according CD4, CD8, CD14 and MMP-1 expression (right panel). *means statistically significant.
Figure Legend Snippet: Expression of MMP-1 in peripheral blood mononuclear cells. (A) Representative dot plots illustrating the selection of MMP-1 positive gate on non-stimulated and rIFN-γ stimulated cells (bottom panel) using as references non stained and PE-isotype-stained cells (upper panel). Graph shows the individual frequency of total MMP-1 expression in PBMCs ( n = 5). (B) Unsupervised high dimensional analysis of flow cytometry data tSNE showing MMP-1 positive clusters (expression level plot, upper left panel) and matching areas on phonograph grid plot (bottom left). Heat map shows the hierarchical clustering of all identified clusters, according CD4, CD8, CD14 and MMP-1 expression (right panel). *means statistically significant.

Techniques Used: Expressing, Selection, Staining, Flow Cytometry

Inflammatory infiltrate in stenotic mitral valves. (A) Representative Masson's trichrome (upper panel) and hematoxylin and eosin (bottom panels) staining showing clusters of mononuclear inflammatory cells localized in low-density collagen areas (left). Representative images for immunohistochemistry staining for CD68, CD8, and CD4 stenotic mitral valves (right). Graph shows the frequency of each cell subset from patients in no leaflet tear group (blue, n=8) and in leaflet tear group (red, n = 8). Scale bar = 200 mm. (B) Correlation analysis between the frequency of CD68, CD8, and CD4 and leaflet thickness in rheumatic mitral valves in no leaflet tear group (blue, n = 8) and in leaflet tear group (red, n = 7).
Figure Legend Snippet: Inflammatory infiltrate in stenotic mitral valves. (A) Representative Masson's trichrome (upper panel) and hematoxylin and eosin (bottom panels) staining showing clusters of mononuclear inflammatory cells localized in low-density collagen areas (left). Representative images for immunohistochemistry staining for CD68, CD8, and CD4 stenotic mitral valves (right). Graph shows the frequency of each cell subset from patients in no leaflet tear group (blue, n=8) and in leaflet tear group (red, n = 8). Scale bar = 200 mm. (B) Correlation analysis between the frequency of CD68, CD8, and CD4 and leaflet thickness in rheumatic mitral valves in no leaflet tear group (blue, n = 8) and in leaflet tear group (red, n = 7).

Techniques Used: Staining, Immunohistochemistry

28) Product Images from "Identification of Claudin 6-specific HLA class I- and HLA class II-restricted T cell receptors for cellular immunotherapy in ovarian cancer"

Article Title: Identification of Claudin 6-specific HLA class I- and HLA class II-restricted T cell receptors for cellular immunotherapy in ovarian cancer

Journal: Oncoimmunology

doi: 10.1080/2162402X.2021.2020983

Analysis of CLDN6-specific T-cell response in ovarian cancer patients. (a) CLDN6- or CEFT-specific T-cell responses from 17 patients (P01–P17) were determined by ELISPOT assays. PBMC were cultured with CLDN6 or CEFT peptides for 13–15 d and IFN-γ spots in the presence (pep) or absence (-) of the respective peptide were enumerated. (b) IFN-γ and TNF-α production from CD8 + or CD4 + T cells from P01 against CLDN6 peptide-pulsed or -unpulsed autologous P01 EBV-B cells was analyzed by intracellular cytokine staining.
Figure Legend Snippet: Analysis of CLDN6-specific T-cell response in ovarian cancer patients. (a) CLDN6- or CEFT-specific T-cell responses from 17 patients (P01–P17) were determined by ELISPOT assays. PBMC were cultured with CLDN6 or CEFT peptides for 13–15 d and IFN-γ spots in the presence (pep) or absence (-) of the respective peptide were enumerated. (b) IFN-γ and TNF-α production from CD8 + or CD4 + T cells from P01 against CLDN6 peptide-pulsed or -unpulsed autologous P01 EBV-B cells was analyzed by intracellular cytokine staining.

Techniques Used: Enzyme-linked Immunospot, Cell Culture, Staining

Cancer cell recognition by CLDN6-specific TCR gene-engineered T cells. (a) CLDN6 and HLA-A2 expression on ovarian cancer cells with or without IFN-γ treatment was determined by flow cytometry. Expression was shown as quadrant gating based on the unstained control (left) and histogram including unstained controls (right). PA-1/A2: PA-1 transduced with HLA-A2. (b, c) CLDN6-specific CD8 + T-cell line (b), or CD8-TCR- or mock-transduced T cells (c) were cocultured with or without IFN-γ-treated or -untreated cancer cells and IFN-γ level in the culture supernatant was measured by ELISA. Error bars indicate standard deviation of technical duplicates.
Figure Legend Snippet: Cancer cell recognition by CLDN6-specific TCR gene-engineered T cells. (a) CLDN6 and HLA-A2 expression on ovarian cancer cells with or without IFN-γ treatment was determined by flow cytometry. Expression was shown as quadrant gating based on the unstained control (left) and histogram including unstained controls (right). PA-1/A2: PA-1 transduced with HLA-A2. (b, c) CLDN6-specific CD8 + T-cell line (b), or CD8-TCR- or mock-transduced T cells (c) were cocultured with or without IFN-γ-treated or -untreated cancer cells and IFN-γ level in the culture supernatant was measured by ELISA. Error bars indicate standard deviation of technical duplicates.

Techniques Used: Expressing, Flow Cytometry, Transduction, Enzyme-linked Immunosorbent Assay, Standard Deviation

Characterization of CLDN6-specific TCR gene-engineered T cells. (a) TCR gene-engineered T cells were cocultured with 20-mer CLDN6 overlapping peptides-pulsed or -unpulsed P01 EBV-B cells for 24 h. IFN-γ level in the culture supernatant was measured by ELISA. (b) Cytokine production from CD8-TCR-engineered CD8 + T cells against CLDN6 peptide-pulsed K562 cells transduced with indicated HLA genes was analyzed by intracellular cytokine staining. (c) CD8-TCR- or mock-transduced T cells were stained with or without HLA-A2/CLDN6 132–140 tetramer along with anti-Vβ8 antibody. (d) IFN-γ production on CD8-TCR-transduced CD8 + T cells against different concentration of CLDN6 132–140 was analyzed by intracellular cytokine staining. Error bars indicate standard deviation of technical duplicates. (e) IFN-γ production on CD4-TCR-transduced CD4 + T cells stimulated with CLDN6 1–20 -pulsed or -unpulsed partially HLA-matched EBV-B cells ( Table 1 ) was analyzed by intracellular cytokine staining.
Figure Legend Snippet: Characterization of CLDN6-specific TCR gene-engineered T cells. (a) TCR gene-engineered T cells were cocultured with 20-mer CLDN6 overlapping peptides-pulsed or -unpulsed P01 EBV-B cells for 24 h. IFN-γ level in the culture supernatant was measured by ELISA. (b) Cytokine production from CD8-TCR-engineered CD8 + T cells against CLDN6 peptide-pulsed K562 cells transduced with indicated HLA genes was analyzed by intracellular cytokine staining. (c) CD8-TCR- or mock-transduced T cells were stained with or without HLA-A2/CLDN6 132–140 tetramer along with anti-Vβ8 antibody. (d) IFN-γ production on CD8-TCR-transduced CD8 + T cells against different concentration of CLDN6 132–140 was analyzed by intracellular cytokine staining. Error bars indicate standard deviation of technical duplicates. (e) IFN-γ production on CD4-TCR-transduced CD4 + T cells stimulated with CLDN6 1–20 -pulsed or -unpulsed partially HLA-matched EBV-B cells ( Table 1 ) was analyzed by intracellular cytokine staining.

Techniques Used: Enzyme-linked Immunosorbent Assay, Transduction, Staining, Concentration Assay, Standard Deviation

Recognition of CLDN6-overexpressing cancer cells by CD8-TCR- and CD4-TCR-transduced T cells. (a) Expression of CLDN6, HLA-A2, and HLA-class II on the parental, A2/CLDN6-overexpressing, and DR4/CLDN6-overexpressing A2780 ovarian cancer cell line was determined by flow cytometry. (b) IFN-γ production from CD8-TCR or CD4-TCR-transduced T cells against indicated A2780 cell lines was determined by intracellular cytokine staining. (c) Cytotoxicity of CD8-TCR or mock-transduced T cells against indicated A2780 cell lines was determined by a 4-h calcein-AM release assay. (d) Induction of apoptotic cell death in cancer cells by CD8-TCR-transduced T cells was determined by annexin V and PI staining after 18-h coculture.
Figure Legend Snippet: Recognition of CLDN6-overexpressing cancer cells by CD8-TCR- and CD4-TCR-transduced T cells. (a) Expression of CLDN6, HLA-A2, and HLA-class II on the parental, A2/CLDN6-overexpressing, and DR4/CLDN6-overexpressing A2780 ovarian cancer cell line was determined by flow cytometry. (b) IFN-γ production from CD8-TCR or CD4-TCR-transduced T cells against indicated A2780 cell lines was determined by intracellular cytokine staining. (c) Cytotoxicity of CD8-TCR or mock-transduced T cells against indicated A2780 cell lines was determined by a 4-h calcein-AM release assay. (d) Induction of apoptotic cell death in cancer cells by CD8-TCR-transduced T cells was determined by annexin V and PI staining after 18-h coculture.

Techniques Used: Expressing, Flow Cytometry, Staining, Release Assay

Establishment of CLDN6-specific TCR gene-engineered T cells. Healthy donor PBMC were retrovirally transduced with TCR genes from CLDN6-specific CD8 + T cells (CD8-TCR) or CD4 + T cells (CD4-TCR) derived from the patient P01 ( Figure 2b ). (a) Transduction efficiency of TCR- or mock-transduced T cells were analyzed by TCR-Vβ8 or -Vβ7.1 antibody. (b, c) IFN-γ production against CLDN6 pooled peptide-pulsed or -unpulsed P01 EBV-B cells were analyzed by intracellular cytokine staining. IFN-γ production from Vβ8 or Vβ7.1 positive/negative cells (b), and CD8 positive/negative cells (c) is shown.
Figure Legend Snippet: Establishment of CLDN6-specific TCR gene-engineered T cells. Healthy donor PBMC were retrovirally transduced with TCR genes from CLDN6-specific CD8 + T cells (CD8-TCR) or CD4 + T cells (CD4-TCR) derived from the patient P01 ( Figure 2b ). (a) Transduction efficiency of TCR- or mock-transduced T cells were analyzed by TCR-Vβ8 or -Vβ7.1 antibody. (b, c) IFN-γ production against CLDN6 pooled peptide-pulsed or -unpulsed P01 EBV-B cells were analyzed by intracellular cytokine staining. IFN-γ production from Vβ8 or Vβ7.1 positive/negative cells (b), and CD8 positive/negative cells (c) is shown.

Techniques Used: Transduction, Derivative Assay, Staining

Multi-cytokine production from TCR-transduced CD4 + or CD8 + T cells. (a) CD4-TCR-transduced T cells was stimulated with a CLDN6 1–20 peptide pulsed on the patient P01-derived EBV-B cells and cytokine production was analyzed on Vβ7.1 + CD4 + or Vβ7.1 + CD8 + T cells. (b) CD8-TCR-transduced T cells was stimulated with a CLDN6 132–140 peptide pulsed on the patient P01-derived EBV-B cells and cytokine production was analyzed on Vβ8 + CD4 + or Vβ8 + CD8 + T cells. Pie charts indicate percentages of cells producing 4/3/2/1/0 cytokines.
Figure Legend Snippet: Multi-cytokine production from TCR-transduced CD4 + or CD8 + T cells. (a) CD4-TCR-transduced T cells was stimulated with a CLDN6 1–20 peptide pulsed on the patient P01-derived EBV-B cells and cytokine production was analyzed on Vβ7.1 + CD4 + or Vβ7.1 + CD8 + T cells. (b) CD8-TCR-transduced T cells was stimulated with a CLDN6 132–140 peptide pulsed on the patient P01-derived EBV-B cells and cytokine production was analyzed on Vβ8 + CD4 + or Vβ8 + CD8 + T cells. Pie charts indicate percentages of cells producing 4/3/2/1/0 cytokines.

Techniques Used: Derivative Assay

29) Product Images from "Anti‐PD‐L1/TGF‐βR fusion protein (SHR‐1701) overcomes disrupted lymphocyte recovery‐induced resistance to PD‐1/PD‐L1 inhibitors in lung cancer, et al. Anti‐PD‐L1/TGF‐βR fusion protein (SHR‐1701) overcomes disrupted lymphocyte recovery‐induced resistance to PD‐1/PD‐L1 inhibitors in lung cancer"

Article Title: Anti‐PD‐L1/TGF‐βR fusion protein (SHR‐1701) overcomes disrupted lymphocyte recovery‐induced resistance to PD‐1/PD‐L1 inhibitors in lung cancer, et al. Anti‐PD‐L1/TGF‐βR fusion protein (SHR‐1701) overcomes disrupted lymphocyte recovery‐induced resistance to PD‐1/PD‐L1 inhibitors in lung cancer

Journal: Cancer Communications

doi: 10.1002/cac2.12244

Treatment response of 6 different groups (control, DDP, anti‐PD‐1, DDP + anti‐PD‐1, DDP + anti‐PD‐1 + anti‐TGF‐β, DDP + SHR‐1701) in an orthotopic mouse model. (A) CMT167 cells were transplanted into the lungs of C57BL/6 mice. Follow‐up treatment (injected with DDP, anti‐PD‐1, anti‐TGF‐β, SHR‐1701, or isotype control antibody) was then performed in six groups as shown. (B) Images and quantification of in vivo bioluminescence imaging of the six groups (control, DDP, anti‐PD‐1, DDP + anti‐PD‐1, DDP + anti‐PD‐1 + anti‐TGF‐β, DDP + SHR‐1701) at the indicated time points. The P ‐value represents a comparison of bioluminescence intensity on the 15th day. (C) Effect of different treatments (control, DDP, anti‐PD‐1, DDP + anti‐PD‐1, DDP + anti‐PD‐1 + anti‐TGF‐β, DDP + SHR‐1701) on survival of CMT167 orthotopic tumor‐bearing mice. (D‐E) Representative images of IHC staining for tumor‐infiltrating Foxp3 + Treg cells (D) and CD8 + T cells (E). Scale bars: 100 μm, upper panel; 40 μm, lower panel. (F) Orthotopic tumors were analyzed by flow cytometry for GITR (left panel), IFN‐γ (middle panel), and CD69 (right panel) expression on CD8 + T cells after stimulation with CD3 and CD28 (both 1 mg/mL) for 48 h. (G) IFN‐γ (left panel) and CD69 (right panel) expression on CD8 + T cells in spleens were analyzed. Bar graphs represent the means ± SEM for each group. B‐G: n = 5 for each group. One‐way ANOVA statistical tests were adopted for more than two groups (B, D, E, F, and G). Log‐rank tests were used in C. *, P
Figure Legend Snippet: Treatment response of 6 different groups (control, DDP, anti‐PD‐1, DDP + anti‐PD‐1, DDP + anti‐PD‐1 + anti‐TGF‐β, DDP + SHR‐1701) in an orthotopic mouse model. (A) CMT167 cells were transplanted into the lungs of C57BL/6 mice. Follow‐up treatment (injected with DDP, anti‐PD‐1, anti‐TGF‐β, SHR‐1701, or isotype control antibody) was then performed in six groups as shown. (B) Images and quantification of in vivo bioluminescence imaging of the six groups (control, DDP, anti‐PD‐1, DDP + anti‐PD‐1, DDP + anti‐PD‐1 + anti‐TGF‐β, DDP + SHR‐1701) at the indicated time points. The P ‐value represents a comparison of bioluminescence intensity on the 15th day. (C) Effect of different treatments (control, DDP, anti‐PD‐1, DDP + anti‐PD‐1, DDP + anti‐PD‐1 + anti‐TGF‐β, DDP + SHR‐1701) on survival of CMT167 orthotopic tumor‐bearing mice. (D‐E) Representative images of IHC staining for tumor‐infiltrating Foxp3 + Treg cells (D) and CD8 + T cells (E). Scale bars: 100 μm, upper panel; 40 μm, lower panel. (F) Orthotopic tumors were analyzed by flow cytometry for GITR (left panel), IFN‐γ (middle panel), and CD69 (right panel) expression on CD8 + T cells after stimulation with CD3 and CD28 (both 1 mg/mL) for 48 h. (G) IFN‐γ (left panel) and CD69 (right panel) expression on CD8 + T cells in spleens were analyzed. Bar graphs represent the means ± SEM for each group. B‐G: n = 5 for each group. One‐way ANOVA statistical tests were adopted for more than two groups (B, D, E, F, and G). Log‐rank tests were used in C. *, P

Techniques Used: Mouse Assay, Injection, In Vivo, Imaging, Immunohistochemistry, Staining, Flow Cytometry, Expressing

Treatment efficacy of PD‐1 blockade immunotherapy was compromised in DDP‐treated mice. (A) In vivo bioluminescent images and quantification of five groups (control, DDP, anti‐PD‐1, concurrent DDP + anti‐PD‐1, sequential DDP + anti‐PD‐1) at the indicated time points. P‐ value represents a comparison of bioluminescence intensity on the 15th day after injection of luciferase‐expressing CMT167 cells into the lungs. (B) Kaplan‐Meier survival analysis of five groups (control, DDP, anti‐PD‐1, concurrent DDP + anti‐PD‐1, sequential DDP + anti‐PD‐1). (C‐F) Spleens and tumors of two groups were stained for TGF‐β (C and D) and IL‐2 (E and F). Scale bars: 100 μm, upper panel; 40 μm, lower panel. (G‐H) The serum levels of TGF‐β (G) and IL‐2 (H) in lung cancer patients with or without satisfied recovery were detected by ELISA. (I‐J) The heatmap shows the gene expression in the TGF‐β pathway of CD8 + T (I) and Treg cells (J) in control as well as DDP‐treated tumors, respectively. As indicated in the scale bar, the values of gene expression are marked in red for high expression and green for low expression. One‐way ANOVA statistical tests and log‐rank tests were used respectively in A and B. Unpaired t‐tests were used to compare two groups (C, D, E, F, G, and H). *, P
Figure Legend Snippet: Treatment efficacy of PD‐1 blockade immunotherapy was compromised in DDP‐treated mice. (A) In vivo bioluminescent images and quantification of five groups (control, DDP, anti‐PD‐1, concurrent DDP + anti‐PD‐1, sequential DDP + anti‐PD‐1) at the indicated time points. P‐ value represents a comparison of bioluminescence intensity on the 15th day after injection of luciferase‐expressing CMT167 cells into the lungs. (B) Kaplan‐Meier survival analysis of five groups (control, DDP, anti‐PD‐1, concurrent DDP + anti‐PD‐1, sequential DDP + anti‐PD‐1). (C‐F) Spleens and tumors of two groups were stained for TGF‐β (C and D) and IL‐2 (E and F). Scale bars: 100 μm, upper panel; 40 μm, lower panel. (G‐H) The serum levels of TGF‐β (G) and IL‐2 (H) in lung cancer patients with or without satisfied recovery were detected by ELISA. (I‐J) The heatmap shows the gene expression in the TGF‐β pathway of CD8 + T (I) and Treg cells (J) in control as well as DDP‐treated tumors, respectively. As indicated in the scale bar, the values of gene expression are marked in red for high expression and green for low expression. One‐way ANOVA statistical tests and log‐rank tests were used respectively in A and B. Unpaired t‐tests were used to compare two groups (C, D, E, F, G, and H). *, P

Techniques Used: Mouse Assay, In Vivo, Injection, Luciferase, Expressing, Staining, Enzyme-linked Immunosorbent Assay

SHR‐1701 inhibited TGF‐β signaling pathway, activated PI3K/Akt/Erk signaling pathway and rescued the anti‐tumor function of peripheral CD8 + T cells isolated from lung cancer patients with impaired lymphocyte recovery. (A‐B) Flow analysis of Ki‐67 expression (A) and IFN‐γ secretion (B) of peripheral CD8 + T cells isolated from patients with satisfied/poor lymphocyte recovery after pre‐treatment of SHR‐1701 or anti‐PD‐1 Abs. (C‐D) Western blotting analysis of the protein levels including TGF‐βR, pSmad2, and total Smad2 in peripheral CD8 + (C) or CD4 + (D) T cells from patients with poor/satisfied lymphocyte recovery. (E) Western blotting analysis of the activation of pAkt and pErk after treatment of anti‐PD‐1 blocking Abs/SHR‐1701/anti‐PD‐1 blocking Abs + TGF‐βR inhibitors/PBS in peripheral CD8 + T cells sorted from patients. (F) Western blotting analysis of the expression of Foxp3 after treatment of SHR‐1701/TGF‐βR inhibitors/PBS in peripheral CD4 + T cells sorted from patients. (G) SHR‐1701 reverses the tumor's response to PD‐1 blockade immunotherapy, from resistant to sensitive, by regulating the balance between CD8 + T cells and Tregs. A‐B: n = 6 for each group. C‐D: n = 5 for each group. E, F: The examination was repeated in 3 patients with poor recovery and 3 patients with satisfied recovery. One‐way ANOVA statistical tests were adopted for A and B. *, P
Figure Legend Snippet: SHR‐1701 inhibited TGF‐β signaling pathway, activated PI3K/Akt/Erk signaling pathway and rescued the anti‐tumor function of peripheral CD8 + T cells isolated from lung cancer patients with impaired lymphocyte recovery. (A‐B) Flow analysis of Ki‐67 expression (A) and IFN‐γ secretion (B) of peripheral CD8 + T cells isolated from patients with satisfied/poor lymphocyte recovery after pre‐treatment of SHR‐1701 or anti‐PD‐1 Abs. (C‐D) Western blotting analysis of the protein levels including TGF‐βR, pSmad2, and total Smad2 in peripheral CD8 + (C) or CD4 + (D) T cells from patients with poor/satisfied lymphocyte recovery. (E) Western blotting analysis of the activation of pAkt and pErk after treatment of anti‐PD‐1 blocking Abs/SHR‐1701/anti‐PD‐1 blocking Abs + TGF‐βR inhibitors/PBS in peripheral CD8 + T cells sorted from patients. (F) Western blotting analysis of the expression of Foxp3 after treatment of SHR‐1701/TGF‐βR inhibitors/PBS in peripheral CD4 + T cells sorted from patients. (G) SHR‐1701 reverses the tumor's response to PD‐1 blockade immunotherapy, from resistant to sensitive, by regulating the balance between CD8 + T cells and Tregs. A‐B: n = 6 for each group. C‐D: n = 5 for each group. E, F: The examination was repeated in 3 patients with poor recovery and 3 patients with satisfied recovery. One‐way ANOVA statistical tests were adopted for A and B. *, P

Techniques Used: Isolation, Expressing, Western Blot, Activation Assay, Blocking Assay

Immunohistochemistry staining of CD8 + T cells and Foxp3 + Tregs in the thymus, spleen and tumor of mice models with impaired lymphocyte recovery. (A‐F) CD8 + T cells (A, C, E) decreased in specimens of DDP‐treated mice, while Foxp3 + Treg cells (B, D, F) increased. Scores for immunohistochemistry staining are presented in bar graphs. Five random fields from each section were counted. Scale bars: 100 μm, upper panel; 40 μm, lower panel. (G‐I) Ratio of CD8 + /Foxp3 + in the thymus (G), spleen (H), and tumor (I). (J‐K) GSEA plot showing NESs for DDP‐induced differentiation of CD4 + T cells to Tregs using RNA‐seq data from tumor‐infiltrated CD4 + cells. A‐I: n = 5 for each group. J, K: n = 2 for each group. Data are presented as the mean ± SEM for each group. *, P
Figure Legend Snippet: Immunohistochemistry staining of CD8 + T cells and Foxp3 + Tregs in the thymus, spleen and tumor of mice models with impaired lymphocyte recovery. (A‐F) CD8 + T cells (A, C, E) decreased in specimens of DDP‐treated mice, while Foxp3 + Treg cells (B, D, F) increased. Scores for immunohistochemistry staining are presented in bar graphs. Five random fields from each section were counted. Scale bars: 100 μm, upper panel; 40 μm, lower panel. (G‐I) Ratio of CD8 + /Foxp3 + in the thymus (G), spleen (H), and tumor (I). (J‐K) GSEA plot showing NESs for DDP‐induced differentiation of CD4 + T cells to Tregs using RNA‐seq data from tumor‐infiltrated CD4 + cells. A‐I: n = 5 for each group. J, K: n = 2 for each group. Data are presented as the mean ± SEM for each group. *, P

Techniques Used: Immunohistochemistry, Staining, Mouse Assay, RNA Sequencing Assay

Flow cytometry analysis of peripheral CD8 + T and Treg cells in patients and mouse models lacking lymphocyte recovery. (A‐B) Flow cytometry analysis of CD4 + CD25 + Tregs (A) and CD3 + CD8 + T cells (B) and from fresh heparinized peripheral blood of patients with satisfactory recovery and poor recovery. CD4 + CD25 + Tregs were gated from CD3 + lymphocytes. (C) Comparison of the CD8 + T cell‐to‐Treg ratio in patients with satisfactory recovery versus poor recovery. (D) Absolute counts of total lymphocytes in DDP‐treated mice. The mice treated with PBS were used as control (Ctrl). (E‐F) Percentages, and absolute counts of CD3 + CD8 + T cells (E) and CD4 + Foxp3 + Tregs (F) in the peripheral blood of control and DDP‐treated mice. The mice treated with PBS were used as control (Ctrl). Left panel: Flow cytometric plots showing the percentages of CD3 + CD8 + T cells (E) and CD4 + Foxp3 + Tregs (F). Middle and right panel: Columns of percentages (Middle) and absolute counts (Right) of CD3 + CD8 + T cells (E) and CD4 + Foxp3 + Tregs (F). (G) The CD8 + T cell/Treg ratio in DDP and control (Ctrl) groups. A‐C: satisfied recovery group, n = 18; poor recovery group, n = 16. D‐G: n = 5 for each group. For all panels, data are presented as the mean ± SEM for each group. Each point represents an individual animal. *, P
Figure Legend Snippet: Flow cytometry analysis of peripheral CD8 + T and Treg cells in patients and mouse models lacking lymphocyte recovery. (A‐B) Flow cytometry analysis of CD4 + CD25 + Tregs (A) and CD3 + CD8 + T cells (B) and from fresh heparinized peripheral blood of patients with satisfactory recovery and poor recovery. CD4 + CD25 + Tregs were gated from CD3 + lymphocytes. (C) Comparison of the CD8 + T cell‐to‐Treg ratio in patients with satisfactory recovery versus poor recovery. (D) Absolute counts of total lymphocytes in DDP‐treated mice. The mice treated with PBS were used as control (Ctrl). (E‐F) Percentages, and absolute counts of CD3 + CD8 + T cells (E) and CD4 + Foxp3 + Tregs (F) in the peripheral blood of control and DDP‐treated mice. The mice treated with PBS were used as control (Ctrl). Left panel: Flow cytometric plots showing the percentages of CD3 + CD8 + T cells (E) and CD4 + Foxp3 + Tregs (F). Middle and right panel: Columns of percentages (Middle) and absolute counts (Right) of CD3 + CD8 + T cells (E) and CD4 + Foxp3 + Tregs (F). (G) The CD8 + T cell/Treg ratio in DDP and control (Ctrl) groups. A‐C: satisfied recovery group, n = 18; poor recovery group, n = 16. D‐G: n = 5 for each group. For all panels, data are presented as the mean ± SEM for each group. Each point represents an individual animal. *, P

Techniques Used: Flow Cytometry, Mouse Assay

The activation of peripheral, splenic and tumor‐infiltrated CD8 + T cells accessed by GSEA and flow cytometry from DDP‐treated and control mice. (A) Percentage of Ki‐67 + CD8 + T cells in the peripheral blood are shown for the control (Ctrl) and DDP‐treated mice. (B‐C) Changes in the percentage of Ki‐67 + CD8 + T cells after CD3 and CD28 (both 1 mg/mL) co‐stimulation in tumors (B) and spleens (C) harvested from DDP‐treated and control (Ctrl) mice. (D‐E) Proportions of PD‐1 + CD8 + T cells and PD‐1 + CD4 + Foxp3 + T cells in tumors harvested from DDP‐treated and control (Ctrl) mice. (F) The ratio of PD‐1 + CD8 + T cells to PD‐1 + Treg cells in TME of DDP‐treated and control (Ctrl) mice. (G) GSEA plot showing NESs for DDP‐inhibited activation status, as well as the function of CD8 + T cells using RNA‐seq data from tumor infiltrated CD8 + T cells. For all panels, bar graphs represent the means ± SEM for each group. Statistically significant differences are indicated as the results of unpaired Student's t‐test (A, D, E, and F) or one‐way ANOVA (B and C). A‐F: n = 5; G: n = 2. **, P
Figure Legend Snippet: The activation of peripheral, splenic and tumor‐infiltrated CD8 + T cells accessed by GSEA and flow cytometry from DDP‐treated and control mice. (A) Percentage of Ki‐67 + CD8 + T cells in the peripheral blood are shown for the control (Ctrl) and DDP‐treated mice. (B‐C) Changes in the percentage of Ki‐67 + CD8 + T cells after CD3 and CD28 (both 1 mg/mL) co‐stimulation in tumors (B) and spleens (C) harvested from DDP‐treated and control (Ctrl) mice. (D‐E) Proportions of PD‐1 + CD8 + T cells and PD‐1 + CD4 + Foxp3 + T cells in tumors harvested from DDP‐treated and control (Ctrl) mice. (F) The ratio of PD‐1 + CD8 + T cells to PD‐1 + Treg cells in TME of DDP‐treated and control (Ctrl) mice. (G) GSEA plot showing NESs for DDP‐inhibited activation status, as well as the function of CD8 + T cells using RNA‐seq data from tumor infiltrated CD8 + T cells. For all panels, bar graphs represent the means ± SEM for each group. Statistically significant differences are indicated as the results of unpaired Student's t‐test (A, D, E, and F) or one‐way ANOVA (B and C). A‐F: n = 5; G: n = 2. **, P

Techniques Used: Activation Assay, Flow Cytometry, Mouse Assay, RNA Sequencing 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 "Platelets are dispensable for the ability of CD8+ T cells to accumulate, patrol, kill and reside in the liver"

Article Title: Platelets are dispensable for the ability of CD8+ T cells to accumulate, patrol, kill and reside in the liver

Journal: bioRxiv

doi: 10.1101/2021.11.09.467964

In vivo generation of tissue resident memory T cells is not affected by the absence of platelets in the spleen and liver. (A) C57BL/6 and Mpl -/- recipients received 1×10 4 OT-I cells prior to immunisation with 5×10 4 P . berghei CS 5M RAS 24h later to generate CD8 tissue resident memory (TRM) populations in vivo after 28 days. Total transferred cells recovered from the spleen and liver were assessed at day 28 post immunisation from both C57BL/6 and Mpl -/- recipients. (B) Representative flow cytometry plots and (C) summary data pooled from 2 independent experiments each with 5 mice per group; bars are mean ± S.D; analyzed via LMM.
Figure Legend Snippet: In vivo generation of tissue resident memory T cells is not affected by the absence of platelets in the spleen and liver. (A) C57BL/6 and Mpl -/- recipients received 1×10 4 OT-I cells prior to immunisation with 5×10 4 P . berghei CS 5M RAS 24h later to generate CD8 tissue resident memory (TRM) populations in vivo after 28 days. Total transferred cells recovered from the spleen and liver were assessed at day 28 post immunisation from both C57BL/6 and Mpl -/- recipients. (B) Representative flow cytometry plots and (C) summary data pooled from 2 independent experiments each with 5 mice per group; bars are mean ± S.D; analyzed via LMM.

Techniques Used: In Vivo, Flow Cytometry, Mouse Assay

Forced expression of ASGPs reduces accumulation of effector CD8+ T cells in the spleen. (A) Graphical representation of the normal glycoprotein pathway of glycosylation modifications from a single Threonine/Serine residue to a Core-1 residue. Enzymatic modification via St3GalI adds a sialic acid residue to the terminal core 1 residue, whereas C2GlcNAcT-I adds a N-acetylglucosamine residue resulting in a core-2 residue with each residue having its own distinct, functional properties. (B) Rationale behind the generation and desired conditional knockout model of CD8+ effector cell loss of ST3GalI enzyme ultimately resulting in effector cells with desialylated residues on core-1 molecules, resulting in greater PNA binding. (C) WT mice received equal ratios of both activated WT OT-I CD8+ lymphocytes (Ly5AB) and either St3GalI heterozygous conditional knockouts (GzmB Crex St3GalI +/- ), or homozygous mutant conditional knockouts (GzmB Crex St3GalI -/- ). All mice were then immunised as per previous experiments using 5×10 3 RAS and transferred cells quantified at both day 7 and day 28 post immunisation from the spleen and liver. (D) Flow cytometry analysis of WT OT-I cells (AB) and both homozygous and heterozygous St3GalI KO cells (BB) in the spleen and liver at day 7, and day 28 post immunisation. (E) Ratio of WT (AB) to GzmB Cre x St3GalI (BB) transgenic cells at day 7 and day 28 post immunisation in the spleen and liver. TEff, TEM, TRM and TCM cell phenotypes as a percentage of total OT-I cells and their WT to transgenic ratios recovered in the spleen (F-G) and liver (H-I) at day 28 post immunisation. Data are from a single experiment with 5 mice per group analyzed via 2-way ANOVA with Tukey post-test; bars are mean ± S.D; analyzed via LMM; * p
Figure Legend Snippet: Forced expression of ASGPs reduces accumulation of effector CD8+ T cells in the spleen. (A) Graphical representation of the normal glycoprotein pathway of glycosylation modifications from a single Threonine/Serine residue to a Core-1 residue. Enzymatic modification via St3GalI adds a sialic acid residue to the terminal core 1 residue, whereas C2GlcNAcT-I adds a N-acetylglucosamine residue resulting in a core-2 residue with each residue having its own distinct, functional properties. (B) Rationale behind the generation and desired conditional knockout model of CD8+ effector cell loss of ST3GalI enzyme ultimately resulting in effector cells with desialylated residues on core-1 molecules, resulting in greater PNA binding. (C) WT mice received equal ratios of both activated WT OT-I CD8+ lymphocytes (Ly5AB) and either St3GalI heterozygous conditional knockouts (GzmB Crex St3GalI +/- ), or homozygous mutant conditional knockouts (GzmB Crex St3GalI -/- ). All mice were then immunised as per previous experiments using 5×10 3 RAS and transferred cells quantified at both day 7 and day 28 post immunisation from the spleen and liver. (D) Flow cytometry analysis of WT OT-I cells (AB) and both homozygous and heterozygous St3GalI KO cells (BB) in the spleen and liver at day 7, and day 28 post immunisation. (E) Ratio of WT (AB) to GzmB Cre x St3GalI (BB) transgenic cells at day 7 and day 28 post immunisation in the spleen and liver. TEff, TEM, TRM and TCM cell phenotypes as a percentage of total OT-I cells and their WT to transgenic ratios recovered in the spleen (F-G) and liver (H-I) at day 28 post immunisation. Data are from a single experiment with 5 mice per group analyzed via 2-way ANOVA with Tukey post-test; bars are mean ± S.D; analyzed via LMM; * p

Techniques Used: Expressing, Modification, Functional Assay, Knock-Out, Binding Assay, Mouse Assay, Mutagenesis, Flow Cytometry, Transgenic Assay, Transmission Electron Microscopy

In vitro activated CD8 T-lymphocytes demonstrate enhanced migration to organs such as the liver and develop an effector phenotype with patrolling behaviour. (A) 2×10 6 SIINFEKL pulsed OT-I T-cells (CTV) and 2×10 6 naïve GFP + OT-I cells were transferred to C57BL/6 mice. 4 hours post adoptive transfer, flow cytometry analysis was conducted on axillary lymph nodes, spleen, bone marrow, lung and liver from recipients. (B-C) Proportion of donor cells isolated from each organ after co-transfer of equal amounts of activated (blue) and naïve (green) OT-I cells; data in B-C from 5 mice per group in one of two independent experiments analyzed via one-sample t test; bars are mean ± S.D; *** p
Figure Legend Snippet: In vitro activated CD8 T-lymphocytes demonstrate enhanced migration to organs such as the liver and develop an effector phenotype with patrolling behaviour. (A) 2×10 6 SIINFEKL pulsed OT-I T-cells (CTV) and 2×10 6 naïve GFP + OT-I cells were transferred to C57BL/6 mice. 4 hours post adoptive transfer, flow cytometry analysis was conducted on axillary lymph nodes, spleen, bone marrow, lung and liver from recipients. (B-C) Proportion of donor cells isolated from each organ after co-transfer of equal amounts of activated (blue) and naïve (green) OT-I cells; data in B-C from 5 mice per group in one of two independent experiments analyzed via one-sample t test; bars are mean ± S.D; *** p

Techniques Used: In Vitro, Migration, Mouse Assay, Adoptive Transfer Assay, Flow Cytometry, Isolation

32) Product Images from "Recombinant BCG-Prime and DNA-Boost Immunization Confers Mice with Enhanced Protection against Mycobacterium kansasii"

Article Title: Recombinant BCG-Prime and DNA-Boost Immunization Confers Mice with Enhanced Protection against Mycobacterium kansasii

Journal: Vaccines

doi: 10.3390/vaccines9111260

M. kansasii -specific polyfunctional CD4 + and CD8 + T cells in rBCG-Mkan85B/DNA-Mkan85B-prime–boost-vaccinated CB6F1 mice. ( A ) A representative fluorogram of epitope-specific polyfunctional CD4 + and CD8 + T cell inductions in splenocytes from unvaccinated and vaccinated CB6F1 mice infected with M. kansasii ; TNF is shown on the x -axis and IFN-γ is shown on the y -axis. ( B ) Polyfunctional CD4 + and CD8 + T cells from the unvaccinated and vaccinated CB6F1 mice infected with M. kansasii . Polyfunctional CD4 + T cell induction by stimulation with PPD (left panel), polyfunctional CD4+ T cell induction by stimulation with peptide 25 (middle panel), and polyfunctional CD8 + T cell induction by stimulation with Pep8 (right panel) in unvaccinated, BCG-, and rBCG-Mkan85B/DNA-Mkan85B-vaccinated mice are shown. The data represent two independent experiments with five to six mice per group. The error bars represent the SD. * p
Figure Legend Snippet: M. kansasii -specific polyfunctional CD4 + and CD8 + T cells in rBCG-Mkan85B/DNA-Mkan85B-prime–boost-vaccinated CB6F1 mice. ( A ) A representative fluorogram of epitope-specific polyfunctional CD4 + and CD8 + T cell inductions in splenocytes from unvaccinated and vaccinated CB6F1 mice infected with M. kansasii ; TNF is shown on the x -axis and IFN-γ is shown on the y -axis. ( B ) Polyfunctional CD4 + and CD8 + T cells from the unvaccinated and vaccinated CB6F1 mice infected with M. kansasii . Polyfunctional CD4 + T cell induction by stimulation with PPD (left panel), polyfunctional CD4+ T cell induction by stimulation with peptide 25 (middle panel), and polyfunctional CD8 + T cell induction by stimulation with Pep8 (right panel) in unvaccinated, BCG-, and rBCG-Mkan85B/DNA-Mkan85B-vaccinated mice are shown. The data represent two independent experiments with five to six mice per group. The error bars represent the SD. * p

Techniques Used: Mouse Assay, Infection

M. kansasii -specific polyfunctional CD4 + and CD8 + T cells in BCG- or rBCG-Mkan85B-vaccinated CB6F1 mice. ( A ) A representative fluorogram of epitope-specific polyfunctional CD4 + and CD8 + T cell inductions in splenocytes from unvaccinated and vaccinated mice infected with M. kansasii ; TNF is shown on the x -axis and IFN-γ is shown on the y -axis. ( B ) Polyfunctional CD4 + and CD8 + T cells from unvaccinated and vaccinated CB6F1 mice infected with M. kansasii . Polyfunctional CD4 + T cell induction by stimulation with PPD (left panel), polyfunctional CD4 + T cell induction by stimulation with peptide 25 (middle panel), and polyfunctional CD8 + T cell induction by stimulation with Pep8 (right panel) in unvaccinated, BCG-, and rBCG-Mkan85B-vaccinated mice are shown. The data represent two independent experiments with three to four mice per group. The error bars represent the SD. * p
Figure Legend Snippet: M. kansasii -specific polyfunctional CD4 + and CD8 + T cells in BCG- or rBCG-Mkan85B-vaccinated CB6F1 mice. ( A ) A representative fluorogram of epitope-specific polyfunctional CD4 + and CD8 + T cell inductions in splenocytes from unvaccinated and vaccinated mice infected with M. kansasii ; TNF is shown on the x -axis and IFN-γ is shown on the y -axis. ( B ) Polyfunctional CD4 + and CD8 + T cells from unvaccinated and vaccinated CB6F1 mice infected with M. kansasii . Polyfunctional CD4 + T cell induction by stimulation with PPD (left panel), polyfunctional CD4 + T cell induction by stimulation with peptide 25 (middle panel), and polyfunctional CD8 + T cell induction by stimulation with Pep8 (right panel) in unvaccinated, BCG-, and rBCG-Mkan85B-vaccinated mice are shown. The data represent two independent experiments with three to four mice per group. The error bars represent the SD. * p

Techniques Used: Mouse Assay, Infection

33) Product Images from "Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells"

Article Title: Antigen Presenting Cells from Tumor and Colon of Colorectal Cancer Patients Are Distinct in Activation and Functional Status, but Comparably Responsive to Activated T Cells

Journal: Cancers

doi: 10.3390/cancers13205247

Phenotypic assessment of TILs and colonic T cells. ( A ) Gating strategy on tumor suspension where (CD11c/CD15/CD19)- CD3+ TILs are separated into CD4+ vs. CD8+ T cells and from which, four T cell subsets are subsequently defined by CD103 and CD39 expression. ( B ) Histogram overlays of CD69 and PD-1 staining on CD4 vs. CD8 T cells at specified sites. ( C ) Percent of total CD3+ T cells and CD4 or CD8 T cells within indicated population. ( D ) Percent of T cell subsets defined by CD103 and CD39 within CD3+ T cells. ( E ) MFI of CD69 and PD-1 staining on indicated T cell subsets and sites. Bars and bold horizontal line show the mean. Connecting lines indicate autologous samples. (* p
Figure Legend Snippet: Phenotypic assessment of TILs and colonic T cells. ( A ) Gating strategy on tumor suspension where (CD11c/CD15/CD19)- CD3+ TILs are separated into CD4+ vs. CD8+ T cells and from which, four T cell subsets are subsequently defined by CD103 and CD39 expression. ( B ) Histogram overlays of CD69 and PD-1 staining on CD4 vs. CD8 T cells at specified sites. ( C ) Percent of total CD3+ T cells and CD4 or CD8 T cells within indicated population. ( D ) Percent of T cell subsets defined by CD103 and CD39 within CD3+ T cells. ( E ) MFI of CD69 and PD-1 staining on indicated T cell subsets and sites. Bars and bold horizontal line show the mean. Connecting lines indicate autologous samples. (* p

Techniques Used: Expressing, Staining

Characterization of APCs in tumor and adjacent colon from patients with CRC. ( A ) Tumor section showing CD11c+ APCs, CD8+ TILs and EpCAM+ epithelial cells. Arrows denote CD11c+CD64+CD163+ MPs ( B ) and CD11c+ CD64-CD163- DCs ( C ). Cell nuclei are DAPI+. Scale bars: ( A ) 500 μm (left) and 100 μm (right), ( B ) 10 μm and ( C ) 5 μm. ( D ) Gating strategy on tumor suspension showing CD45+ lineage (CD3/CD19/CD56)- HLA–DR+CD15-CD11c+ APCs divided into total CD64+ MPs containing CD14+ MPs (lower left panel), and total CD64- DCs comprising CD141+ CDC1 and CD1c+ CDC2 (lower right panel). Total MPs and DCs in tumor (T) and colon (C) by numbers per 1 million cells ( E ), or percent of HLA–DR+ APCs ( F ). ( G ) Percent and proportion of APC subsets within HLA–DR+ APCs. Bars show the mean. Connecting lines indicate autologous samples. (* p
Figure Legend Snippet: Characterization of APCs in tumor and adjacent colon from patients with CRC. ( A ) Tumor section showing CD11c+ APCs, CD8+ TILs and EpCAM+ epithelial cells. Arrows denote CD11c+CD64+CD163+ MPs ( B ) and CD11c+ CD64-CD163- DCs ( C ). Cell nuclei are DAPI+. Scale bars: ( A ) 500 μm (left) and 100 μm (right), ( B ) 10 μm and ( C ) 5 μm. ( D ) Gating strategy on tumor suspension showing CD45+ lineage (CD3/CD19/CD56)- HLA–DR+CD15-CD11c+ APCs divided into total CD64+ MPs containing CD14+ MPs (lower left panel), and total CD64- DCs comprising CD141+ CDC1 and CD1c+ CDC2 (lower right panel). Total MPs and DCs in tumor (T) and colon (C) by numbers per 1 million cells ( E ), or percent of HLA–DR+ APCs ( F ). ( G ) Percent and proportion of APC subsets within HLA–DR+ APCs. Bars show the mean. Connecting lines indicate autologous samples. (* p

Techniques Used:

Correlation analyses of ex vivo surface activation markers of APC and T cell subsets. Heat maps from Spearman correlation between CD80 MFI of APC subsets vs. CD69 MFI of CD4 ( A ), or CD8 ( B ) T cell subsets in tumor and adjacent colon. Similar analyses on PD-L1 MFI of APC subsets vs. PD-1 MFI on CD4+ ( C ), or CD8+ ( D ) T cell subsets. (* p
Figure Legend Snippet: Correlation analyses of ex vivo surface activation markers of APC and T cell subsets. Heat maps from Spearman correlation between CD80 MFI of APC subsets vs. CD69 MFI of CD4 ( A ), or CD8 ( B ) T cell subsets in tumor and adjacent colon. Similar analyses on PD-L1 MFI of APC subsets vs. PD-1 MFI on CD4+ ( C ), or CD8+ ( D ) T cell subsets. (* p

Techniques Used: Ex Vivo, Activation Assay

34) Product Images from "Prevention of Cyclophosphamide-Induced Immunosuppression in Mice With Traditional Chinese Medicine Xuanfei Baidu Decoction"

Article Title: Prevention of Cyclophosphamide-Induced Immunosuppression in Mice With Traditional Chinese Medicine Xuanfei Baidu Decoction

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2021.730567

Effects of XFBD on LPS-induced splenic lymphocyte proliferation and T lymphocyte subsets in CY-treated mice. (A) LPS-induced splenic lymphocyte proliferation; (B) representative flow cytometry analysis result of CD4 + subset; (C) representative flow cytometry analysis result of CD8 + subset; and (D) representative images of Splenic T lymphocyte subsets detected by flow cytometry. Normal, administered with saline; Model: intraperitoneally administered with cyclophosphamide; XFBD: intraperitoneally administered with cyclophosphamide first and followed by XFBD at 3.9 g/kg/day; and LH: intraperitoneally administered with cyclophosphamide first and followed by LH at 20 mg/kg. Data are expressed as mean ± SD ( n = 3).* p
Figure Legend Snippet: Effects of XFBD on LPS-induced splenic lymphocyte proliferation and T lymphocyte subsets in CY-treated mice. (A) LPS-induced splenic lymphocyte proliferation; (B) representative flow cytometry analysis result of CD4 + subset; (C) representative flow cytometry analysis result of CD8 + subset; and (D) representative images of Splenic T lymphocyte subsets detected by flow cytometry. Normal, administered with saline; Model: intraperitoneally administered with cyclophosphamide; XFBD: intraperitoneally administered with cyclophosphamide first and followed by XFBD at 3.9 g/kg/day; and LH: intraperitoneally administered with cyclophosphamide first and followed by LH at 20 mg/kg. Data are expressed as mean ± SD ( n = 3).* p

Techniques Used: Mouse Assay, Flow Cytometry

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    RAD21 inversely correlates with IFN signaling activity in OV. ( A ) Normalized enrichment scores correlated with RAD21 expression using Hallmark gene sets from TCGA-OV database (high vs. low RAD21 expression, top vs. bottom 10%; n = 25 per group). ( B and C ) GSEA analysis ( B ) and heatmaps ( C ) showing the inverse correlation between RAD21 expression and the IFN signaling pathways and genes in the TCGA-OV database. ( D – I ) Comparative analysis showing the association of high expression of RAD21 with low expression of IFN activity ( D ), immune score ( E ), cytotoxicity score ( F ), infiltration levels of CD8 + T cells ( G ), and expression of T cell marker genes <t>CD8A</t> , CD3E ( H ), GZMA , and GZMB ( I ) in patients with OV from the TCGA database. ( J and K ) Representative IHC staining ( J ) and quantification ( K ) showing the inverse correlation between RAD21 expression and CD8A expression in ovarian tumors (SYSUCC cohort). ( L ) Correlation of RAD21 mRNA levels with response to ICB in patients with melanoma treated with PD-1, PD-L1, or CTLA4 mAbs (GSE91061 and GSE168204). ( M and N ) Representative IHC images ( M ) and quantification ( N ) for RAD21 expression in OV responders ( n = 6) versus nonresponders ( n = 12) to immune checkpoint inhibitors. Data in D – I , K , L , and N are shown as mean ± SD (2-tailed t test).
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    RAD21 inversely correlates with IFN signaling activity in OV. ( A ) Normalized enrichment scores correlated with RAD21 expression using Hallmark gene sets from TCGA-OV database (high vs. low RAD21 expression, top vs. bottom 10%; n = 25 per group). ( B and C ) GSEA analysis ( B ) and heatmaps ( C ) showing the inverse correlation between RAD21 expression and the IFN signaling pathways and genes in the TCGA-OV database. ( D – I ) Comparative analysis showing the association of high expression of RAD21 with low expression of IFN activity ( D ), immune score ( E ), cytotoxicity score ( F ), infiltration levels of CD8 + T cells ( G ), and expression of T cell marker genes CD8A , CD3E ( H ), GZMA , and GZMB ( I ) in patients with OV from the TCGA database. ( J and K ) Representative IHC staining ( J ) and quantification ( K ) showing the inverse correlation between RAD21 expression and CD8A expression in ovarian tumors (SYSUCC cohort). ( L ) Correlation of RAD21 mRNA levels with response to ICB in patients with melanoma treated with PD-1, PD-L1, or CTLA4 mAbs (GSE91061 and GSE168204). ( M and N ) Representative IHC images ( M ) and quantification ( N ) for RAD21 expression in OV responders ( n = 6) versus nonresponders ( n = 12) to immune checkpoint inhibitors. Data in D – I , K , L , and N are shown as mean ± SD (2-tailed t test).

    Journal: The Journal of Clinical Investigation

    Article Title: RAD21 amplification epigenetically suppresses interferon signaling to promote immune evasion in ovarian cancer

    doi: 10.1172/JCI159628

    Figure Lengend Snippet: RAD21 inversely correlates with IFN signaling activity in OV. ( A ) Normalized enrichment scores correlated with RAD21 expression using Hallmark gene sets from TCGA-OV database (high vs. low RAD21 expression, top vs. bottom 10%; n = 25 per group). ( B and C ) GSEA analysis ( B ) and heatmaps ( C ) showing the inverse correlation between RAD21 expression and the IFN signaling pathways and genes in the TCGA-OV database. ( D – I ) Comparative analysis showing the association of high expression of RAD21 with low expression of IFN activity ( D ), immune score ( E ), cytotoxicity score ( F ), infiltration levels of CD8 + T cells ( G ), and expression of T cell marker genes CD8A , CD3E ( H ), GZMA , and GZMB ( I ) in patients with OV from the TCGA database. ( J and K ) Representative IHC staining ( J ) and quantification ( K ) showing the inverse correlation between RAD21 expression and CD8A expression in ovarian tumors (SYSUCC cohort). ( L ) Correlation of RAD21 mRNA levels with response to ICB in patients with melanoma treated with PD-1, PD-L1, or CTLA4 mAbs (GSE91061 and GSE168204). ( M and N ) Representative IHC images ( M ) and quantification ( N ) for RAD21 expression in OV responders ( n = 6) versus nonresponders ( n = 12) to immune checkpoint inhibitors. Data in D – I , K , L , and N are shown as mean ± SD (2-tailed t test).

    Article Snippet: First, OT-I cells were stained with fluorescence-labeled antibodies against CD8 (BioLegend, catalog 100706) for 1 hour at 4°C.

    Techniques: Activity Assay, Expressing, Marker, Immunohistochemistry, Staining

    RAD21 suppresses antitumor immunity in vivo. ( A ) Tumor volume and tumor weight over time in C57BL/6 mice implanted with Rad21 -KO and control B16-OVA mouse cells. Data are shown as mean ± SEM (2-way ANOVA) for tumor volume and as mean ± SD (2-tailed t test) for tumor weight ( n = 6 mice per group). ( B and C ) Tumor volume in nude mice ( n = 8 mice per group) ( B ) and C57BL/6 mice ( n = 6 mice per group) ( C ) implanted with Rad21 -KO and control B16-OVA mouse cells. Mice were pretreated with CD8-depleting antibodies at –1, 2, and 5 days. Data are shown as mean ± SEM (2-way ANOVA). ( D and E ) Flow cytometry analysis showing the numbers of tumor-infiltrating CD8 + T cells ( D ) and expression of activation marker CD69 and effector molecules IFN-γ and GZMB ( E ) in CD8 + T cells. Data are shown as mean ± SD ( n = 5, 2-tailed t test). ( F ) Mice with established Rad21 -KO and control B16-OVA tumors were treated with anti–PD-1 at indicated time points. Tumor volume and survival rates are shown. Data are shown as mean ± SEM (2-way ANOVA). ( G ) Representative bioluminescence images of mice with established Rad21 -KO and control ID8 tumors treated with anti–PD-1 formed by intraperitoneal injection at day 9 and day 12. ( H ) The bar graph shows the change in bioluminescence in mice. Data are shown as mean ± SEM (1-way ANOVA). ( I and J ) Flow cytometry analysis showing the numbers of tumor-infiltrating CD8 + T cells ( I ) and expression of CD69 and effector molecules IFN-γ and GZMB ( J ). Data are shown as mean ± SD (2-tailed t test). ** P

    Journal: The Journal of Clinical Investigation

    Article Title: RAD21 amplification epigenetically suppresses interferon signaling to promote immune evasion in ovarian cancer

    doi: 10.1172/JCI159628

    Figure Lengend Snippet: RAD21 suppresses antitumor immunity in vivo. ( A ) Tumor volume and tumor weight over time in C57BL/6 mice implanted with Rad21 -KO and control B16-OVA mouse cells. Data are shown as mean ± SEM (2-way ANOVA) for tumor volume and as mean ± SD (2-tailed t test) for tumor weight ( n = 6 mice per group). ( B and C ) Tumor volume in nude mice ( n = 8 mice per group) ( B ) and C57BL/6 mice ( n = 6 mice per group) ( C ) implanted with Rad21 -KO and control B16-OVA mouse cells. Mice were pretreated with CD8-depleting antibodies at –1, 2, and 5 days. Data are shown as mean ± SEM (2-way ANOVA). ( D and E ) Flow cytometry analysis showing the numbers of tumor-infiltrating CD8 + T cells ( D ) and expression of activation marker CD69 and effector molecules IFN-γ and GZMB ( E ) in CD8 + T cells. Data are shown as mean ± SD ( n = 5, 2-tailed t test). ( F ) Mice with established Rad21 -KO and control B16-OVA tumors were treated with anti–PD-1 at indicated time points. Tumor volume and survival rates are shown. Data are shown as mean ± SEM (2-way ANOVA). ( G ) Representative bioluminescence images of mice with established Rad21 -KO and control ID8 tumors treated with anti–PD-1 formed by intraperitoneal injection at day 9 and day 12. ( H ) The bar graph shows the change in bioluminescence in mice. Data are shown as mean ± SEM (1-way ANOVA). ( I and J ) Flow cytometry analysis showing the numbers of tumor-infiltrating CD8 + T cells ( I ) and expression of CD69 and effector molecules IFN-γ and GZMB ( J ). Data are shown as mean ± SD (2-tailed t test). ** P

    Article Snippet: First, OT-I cells were stained with fluorescence-labeled antibodies against CD8 (BioLegend, catalog 100706) for 1 hour at 4°C.

    Techniques: In Vivo, Mouse Assay, Flow Cytometry, Expressing, Activation Assay, Marker, Injection

    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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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.

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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.

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: Injection, Mouse Assay, Whisker Assay

    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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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.

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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.

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: 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

    Journal: Nature

    Article Title: Nociceptor neurons affect cancer immunosurveillance

    doi: 10.1038/s41586-022-05374-w

    Figure Lengend 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

    Article Snippet: Finally, the cells were stained (30 min, 4 °C) with one of anti-CD45–BV421 (1:100, BioLegend, 103134), anti-CD45.1–BV421 (1:100, BioLegend, 110732), anti-CD45.2–BV650 (1:100, BioLegend, 109836), anti-CD45-Alexa Fluor 700 (1:100, BioLegend, 103128), anti-CD11b-APC/Cy7 (1:100, BioLegend, 101226), anti-CD8-AF700 (1:100, BioLegend, 100730), anti-CD8–BV421 (1:100, BioLegend, 100753), anti-CD8–PerCP/Cyanine5.5 (1:100, BioLegend, 100734), anti-CD8–Pacific Blue (1:100, BioLegend, 100725), anti-CD4–PerCP/Cyanine5.5 (1:100, BioLegend, 100540), anti-CD4-FITC (1:100, BioLegend, 100406), anti-PD-1–PE-Cy7 (1:100, BioLegend, 109110), anti-LAG3–PE (1:100, BioLegend, 125208), anti-LAG3–PerCP/Cyanine5.5 (1:100, BioLegend, 125212) or anti-TIM3–APC (1:100, BioLegend, 119706), washed and analysed using a LSRFortessa or FACSCanto II (Becton Dickinson).

    Techniques: Injection, Mouse Assay, Whisker Assay