Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Heatmap showing Pearson’s correlation between hypoxic signature genes expression and immune-related genes expression in basal TNBC samples ( n = 98) in TCGA dataset. b Scatter plots (upper panel) and Pearson’s correlation coefficients (lower panel) showing the expression of hypoxic gene signatures and immune-related genes in breast cancers in TCGA dataset (Basal, n = 98; HER2, n = 58; Luminal A, n = 231; Luminal B, n = 129). Regression lines with a 95% confidence interval (gray fill) are shown in the scatter plots. c Images of fluorescent staining of human TNBC samples. Scale bar, 50 µm. Data were representative of 30 independent experiments. d Quantification of infiltrating IFNγ + CD8 + T cell number in HIF1α − and HIF1α + regions of human TNBC sample ( n = 30). P values were determined with paired two-tailed t -test. e Correlation between infiltrating IFNγ + CD8 + T cell count and HIF1α fluorescent intensity in human TNBC samples ( n = 30). The simple linear regression R 2 and P values (two-tailed) are calculated. Dot plot is shown with regression line and 95% confidence interval. f Representative images of fluorescent staining of mouse 4T1 tumor samples. Scale bar, 50 µm. Data represents three independent experiments. g Flow cytometry (left panel) demonstrating the gating strategy of activated-PIM high (H) and activated-PIM low (L) populations in living cells dissociated from 4T1 tumors. The CD8 + T cell percentage and IFNγ expression in CD8 + T cells was quantified (right panel, n = 6). Data were presented as box and whiskers, with median value and whiskers of minimum and maximum values. P values were determined with an unpaired two-tailed t -test. h Kaplan–Meier overall survival (OS) and distant metastasis-free survival (DMFS) analysis of the indicated gene signatures in TNBC patients. The publicly available data used in Fig. 1a, b are available in the TCGA database under accession code BRCA.exp.547.med.txt [ https://gdc.cancer.gov/about-data/publications/brca_2012 ]. The publicly available data used in h are available in the KM-Plotter-Breast Cancer [ https://kmplot.com/analysis/index.php?p=service&cancer=breast ]. For the remaining data, source data are provided in Source Data file.

Article Snippet: Slides were blocked with 5% normal goat serum in PBS for 20 min. Human TNBC samples were incubated with rabbit anti-human HIF1α antibody (Abcam, CAT# ab51608, dilution 1:100), mouse anti-human CD8 antibody (Cell Signaling Technology, CAT#70309, dilution 1:100), and rat anti-human/mouse IFNγ antibody (Invitrogen, CAT# MM700, dilution 1:50) overnight under 4 °C.

Techniques: Expressing, Staining, Two Tailed Test, Cell Counting, Flow Cytometry

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Schematic graph demonstrating the coculture model. b Representative flow cytograms (upper panel) gated from human pan-T cell culture and quantification (lower panel, n = 3) of differentiated CD8 + T cell subtypes: Tn (naïve T cells), Tcm (central memory T cells), Tem (effector memory T cells), Teff (effector T cells). c Schematic graph demonstrating the normoxia (20% O 2 ) and hypoxia (1% O 2 ) culture condition of T cells coculturing with human TNBC cell line. d Heatmap of the differentially expressed genes (DEGs) in hypoxic cultured human T cells compared to normoxia group. DEGs were identified in edgeR (|logFC| > 1, adjusted P < 0.01). P values were adjusted using Benjamini–Hochberg method in edgeR. DEGs identified in the indicated GO gene clusters are marked in the heatmap. e GSEA analysis of human T cells in hypoxic versus normoxic conditions. Analysis was based on ranked logFC from edgeR. FDR and adjusted p value are shown in the graph. P values were adjusted using Benjamini–Hochberg method in GSEA analysis. f Flow cytometry quantifications of immune effector molecules and exhaustion markers in CD8 + T cells gated from human pan-T cells cultured under the indicated conditions ( n = 4). g Representative flow cytograms of PD-1 and TIM-3 expression in CD8 + T cells gated from human pan-T cells culture. h Flow cytometric quantification of terminally exhausted T cells (PD-1 + TIM-3 + ) in CD8 + T cells gated from human pan-T cells culture ( n = 3). i Flow cytometric quant i fication of proliferating cells (Ki76 + ) in CD8 + and CD4 + T cells gated from human T cells cocultured with TNBC ( n = 3). All flow cytometry data ( b , f , h , and i ) are presented as the mean ± SD of samples from three to four donors. For all flow cytometry data, P values were determined by one-way ANOVA ( f , h ) or two-way ANOVA ( b ) with Turkey’s test, or paired two-tailed t -test ( i ). Raw RNA-seq data i s available in the GEO database with accession number GSE179885 . For the remaining data, source data are provided in Source Data file.

Article Snippet: Slides were blocked with 5% normal goat serum in PBS for 20 min. Human TNBC samples were incubated with rabbit anti-human HIF1α antibody (Abcam, CAT# ab51608, dilution 1:100), mouse anti-human CD8 antibody (Cell Signaling Technology, CAT#70309, dilution 1:100), and rat anti-human/mouse IFNγ antibody (Invitrogen, CAT# MM700, dilution 1:50) overnight under 4 °C.

Techniques: Cell Culture, Flow Cytometry, Expressing, Two Tailed Test, RNA Sequencing Assay

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a RT-qPCR analysis assessing IFNG expression in T/NK cells in an epigenetic-drug screening. Both T cells and NK cells were cultured under 1% O 2 with indicated treatments. Data were presented as the log2 fold change of IFNG mRNA level normalized to vehicle control, mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). b , c Representative histograms (left panel) and flow cytometric quantifications (right panel) of IFNγ expression in human CD8 + T cells ( b n = 4) and NK cells ( c n = 3) with indicated treatments. Quantification data were presented as the mean ± SD of samples from three to four donors. P values were determined by two-way ANOVA with Turkey’s test. d ChIP-qPCR analysis of HDAC1, HDAC2, HDAC3, EZH2, and SUZ12 occupancy on IFNG promoter of human T cells. Four primers were designed to span the promoters of IFNG , with P1 at −1448 to −1354b, P2 at −707 to −628b, P3 at −257 to −171b, P4 at +350 to +461b, relative to TSS. For ChIP analysis of EZH2 and SUZ12 occupancy, RPL30 serves as the negative control and CCND2 as the positive control. e , f ChIP-qPCR analysis of H3K27ac and H3K27me3 enrichment on IFNG promoter of human T cells under indicated conditions. All ChIP-qPCR data ( d – f ) are presented as fold enrichment relative to IgG and expressed as mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). For ChIP-qPCR data of d , e , statistics were performed to analyze bindings of indicated markers across different sites in IFNG promoter ( RPL30 and CCND2 excluded) between hypoxia and normoxia. P values were determined by two-way ANOVA analysis. g RT-qPCR analysis of human T cell with indicated gene knockdown. Data were presented as the fold change of mRNA level normalized to the control group under normoxia (1% O2), mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). Source data are provided as a source data file.

Article Snippet: Slides were blocked with 5% normal goat serum in PBS for 20 min. Human TNBC samples were incubated with rabbit anti-human HIF1α antibody (Abcam, CAT# ab51608, dilution 1:100), mouse anti-human CD8 antibody (Cell Signaling Technology, CAT#70309, dilution 1:100), and rat anti-human/mouse IFNγ antibody (Invitrogen, CAT# MM700, dilution 1:50) overnight under 4 °C.

Techniques: Quantitative RT-PCR, Expressing, Cell Culture, Negative Control, Positive Control

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a ChIP-qPCR analysis of HIF1α and HIF2α occupancy on IFNG promoter in human T cells. VEGFA served as a positive control. b Co-immunoprecipitation shows the physical interaction between HDAC1 and HIF1α, and the interaction between HDAC1 and SUZ12 in human T cells. Data is representative of two independent experiments ( n = 2). c Representative western blot images ( n = 2) to demonstrate knockdown of HIF1α in human T cells. d ChIP-qPCR analysis of HDAC1 occupancy on IFNG promoter in human T cells. e ChIP-qPCR analysis of H3K27ac and H3K27me3 enrichment on IFNG promoter in human T cells with indicated treatments. All ChIP-qPCR data ( a , d , e ) are presented as fold enrichment relative to IgG and expressed as mean ± SD of technical triplicates, representative of two independent experiments ( n = 2). For ChIP-qPCR data of a , statistics were performed to analyze bindings of indicated markers across different sites in IFNG promoter ( VEGFA excluded) between hypoxia and normoxia. P values were determined by two-way ANOVA analysis. f Flow cytometric quantifications of IFNγ in CD8 + T cells gated from human pan-T cells cultured under the indicated conditions. Data were presented as the mean ± SD of three independent experiments ( n = 3). P values were determined by one-way ANOVA with Turkey’s test. g Representative western blot images ( n = 2) to demonstrate the inhibition of HIF1α level by indicated compounds in human T cells. h Representative histograms (left panel) and flow cytometric quantifications (right panel) of IFNγ expression in human CD8 + T cells with indicated treatments. Quantification data were presented as the mean ± SD of samples from four donors ( n = 4). P values were determined by two-way ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: Slides were blocked with 5% normal goat serum in PBS for 20 min. Human TNBC samples were incubated with rabbit anti-human HIF1α antibody (Abcam, CAT# ab51608, dilution 1:100), mouse anti-human CD8 antibody (Cell Signaling Technology, CAT#70309, dilution 1:100), and rat anti-human/mouse IFNγ antibody (Invitrogen, CAT# MM700, dilution 1:50) overnight under 4 °C.

Techniques: Positive Control, Immunoprecipitation, Western Blot, Cell Culture, Inhibition, Expressing

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Cell lysis of TNBC cells cocultured with human T cells from two different healthy donors. Human T cells were stimulated with TNBC cell lysate-primed DC cells. Data were presented as mean ± SD of three independent experiments ( n = 3). P values were determined by two-way ANOVA. b Western blot analysis of IFNγ–regulated proteins in TNBC cells cocultured with human T cells. Data were representative of two independent experiments ( n = 2). c Cell lysis of TNBC cells cocultured with human T cells. Human T cells were stimulated with TNBC cell lysate-primed DC cells and pretreated with indicated compounds. Data presented as mean ± SD of three independent experiments ( n = 3). P values were determined by one-way ANOVA with Dunnett’s test. d Western blot analysis of IFNγ–regulated proteins in TNBC cells cocultured with human T cells. Human T cells were stimulated with TNBC cell lysate-primed DC cells and pretreated with indicated compounds. Data were representative of two independent experiments ( n = 2). e Cell lysis of TNBC cells cocultured with human T cells. Data were presented as mean ± SD of three independent experiments ( n = 3). P values were determined by two-way ANOVA with Dunnett’s test. f Flow cytometric quantifications of immune effector molecules in human CD8 + T cells cultured under the indicated conditions. Data were presented as the mean ± SD of samples from three donors ( n = 3). P values were determined by two-way ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: Slides were blocked with 5% normal goat serum in PBS for 20 min. Human TNBC samples were incubated with rabbit anti-human HIF1α antibody (Abcam, CAT# ab51608, dilution 1:100), mouse anti-human CD8 antibody (Cell Signaling Technology, CAT#70309, dilution 1:100), and rat anti-human/mouse IFNγ antibody (Invitrogen, CAT# MM700, dilution 1:50) overnight under 4 °C.

Techniques: Lysis, Western Blot, Cell Culture

Journal: Nature Communications

Article Title: Hypoxia induces HIF1α-dependent epigenetic vulnerability in triple negative breast cancer to confer immune effector dysfunction and resistance to anti-PD-1 immunotherapy

doi: 10.1038/s41467-022-31764-9

Figure Lengend Snippet: a Schematic diagram showing the establishment of humanized mice (humice) with human immune system reconstituted in NIKO mice. The presence of human CD45 + cells, NK cells, CD4 + and CD8 + T cells in the mice’s peripheral system was validated by flow cytometry. b Primary LM2 tumor size in humice (control, n = 14; Keytruda, n = 14; ENT, n = 12; PX478, n = 14; ENT + Keytruda, n = 16; PX478 + Keytruda, n = 16) and NIKO mice (control, n = 10; ENT + Keytruda, n = 10; PX478 + Keytruda, n = 10), at Day 21 of treatments. c Lung metastasis of humice (control, n = 6; Keytruda, n = 6; ENT, n = 6; PX478, n = 6; ENT + Keytruda, n = 7; PX478 + Keytruda, n = 7) and NIKO mice (control, n = 5; ENT + Keytruda, n = 5; PX478 + Keytruda, n = 5) bearing LM2 tumors at Day 35 assessed by bioluminescence (BLI) measurement. d Representative bioluminescence (BLI) images showing the lung metastasis of humice and NIKO mice. e Flow cytometric analysis of LM2 tumors harvested from humanized mice. IFNγ, TNFα, and granzyme B expression was examined in tumor-infiltrating human CD8 + T cells and NK cells. N = 5 for each group. f Flow cytometry analysis of LM2 tumors harvested from humanized mice. Expressions of human PD-L1 and PD-L2 were examined in total living cells dissociated from LM2 tumors. N = 5 for each group. Quantification data of flow cytometry ( e , f ) are presented as a box and whiskers, with median values and whiskers of minimum and maximum values. Data for b and c were presented as mean ± SD . P values were determined by one-way ( e , f ) or two-way ( b , c ) ANOVA with Turkey’s test. Source data are provided as a source data file.

Article Snippet: Slides were blocked with 5% normal goat serum in PBS for 20 min. Human TNBC samples were incubated with rabbit anti-human HIF1α antibody (Abcam, CAT# ab51608, dilution 1:100), mouse anti-human CD8 antibody (Cell Signaling Technology, CAT#70309, dilution 1:100), and rat anti-human/mouse IFNγ antibody (Invitrogen, CAT# MM700, dilution 1:50) overnight under 4 °C.

Techniques: Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2018.02347

Figure Lengend Snippet: Absence of IL-17RA signaling results in increased tissue parasitism and a reduced magnitude of parasite-specific CD8+ T cell responses. (A-C) Relative amount of T. cruzi satellite DNA in spleen (A) , liver (B) and heart (C) of infected WT and IL-17RA KO (KO) mice determined at the indicated dpi. Murine GAPDH was used for normalization. Data are presented as mean ± SD, N = 5 mice. P values calculated with two-tailed T test. (D) Representative plots of CD8 and TSKB20/Kb staining in spleen, liver and blood of WT and KO mice at 22 dpi (WT-I and KO-I, respectively). A representative plot of the staining of splenocytes from non-infected WT mice (WT-N) is shown for comparison. (E) Percentage and cell numbers of TSKB20/Kb+ CD8+ T cells and (F) cell numbers of total CD8+ T cells determined in spleen of WT and IL-17RA KO mice at different dpi. Data shown as mean ± SD, N = 5−8 mice. P values calculated using two-way ANOVA followed by Bonferroni‘s post-test. (G) Experimental layout of IL-17 neutralization in infected WT mice injected with control isotype or anti-IL-17 Abs (WT-cAb and WT-aIL-17, respectively). (H) Representative plots of CD8 and TSKB20/Kb staining in spleen and liver infected control and treated WT mice. (I) Percentage of total and TSKB20/Kb+ CD8+ T cells infected control and treated WT mice. Results from infection-matched WT (WT-I) and KO mice (KO-I) are shown for comparison. Data are representative of five (A–F) , and two (G–I) independent experiments.

Article Snippet: Flow cytometry and/or cell sorting was performed with a combination of the following Abs (BD Biosciences, Biolegend, eBioscience, Life Technologies, Santa Cruz Biotechnology or Cell Signaling): FITC/AF488-labeled anti-mouse: CD8 (53-6.7), anti-CD25 (7D4), CD69 (H1.2F3), CD44 (IM7), Bcl-2 (10C4), IgG (polyclonal goat), and anti-rat IgG (polyclonal goat); PE-labeled anti-mouse: Fas (Jo2), CTLA-4 (UC10-4B9), PD-1 (RMP1-30), Ki-67 (SolA15), IL-17RA (PAJ17R), CD120b (TR75-89), Bim (rabbit.

Techniques: Infection, Two Tailed Test, Staining, Neutralization, Injection

Journal: Frontiers in Immunology

Article Title: IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2018.02347

Figure Lengend Snippet: IL-17RA signaling during T. cruzi infection promotes survival of CD8+ T cells. (A) Representative dot plots of CD8 and TSKB20/Kb staining and representative histograms of BrDU in cell suspensions from the spleen of WT and KO mice at 0 dpi (WT-N) and 20 dpi (WT-I and KO-I) and within the indicated total and TSKB20/Kb+ CD8+ T cell gates. Numbers in the histograms indicate the frequency of BrDU+ cells from the corresponding colored group. Histograms are representative of one out of five mice. Bars in the statistical analysis represent data as mean ± SD, N = 5 mice. P values were calculated with two-tailed T test. (B) Representative histograms of TMRE staining in total (left) and TSKB20/Kb+ (right) CD8+ T cells from the spleen of WT and IL17RA KO mice at 10 dpi (top) and 20 dpi (bottom). Numbers indicate the frequency of TMRE- (apoptotic) cells in total and TSKB20/Kb+ CD8+ T cells from the corresponding colored group. Gray tinted histogram show staining in CD8+ T cells from non-infected WT mice (WT-N). Histograms are representative of one out of five mice. Bars in the statistical analysis represent data as mean ± SD, N = 5 mice. P values were calculated with two-tailed T test. (C, D) Representative histograms of TMRE (C) and BAD (D) stainings in cultures of purified CD8+ T cells activated with coated anti-CD3 and anti-CD28 in the presence of the indicated cytokines. Statistical analysis in (C) shows the frequency of TMRE- (apoptotic) cells in each biological replicate ( N = 4 mice) and the mean ± SD . P values calculated with paired two-tailed T test (ns, not significant). Statistical analysis in (D) represent protein expression as the geometric mean of fluorescence intensity in each replicate ( N = 4 mice). Lines link paired samples. P values calculated with paired two-tailed T test. Data in (A-D) are representative of three and two independent experiments, respectively.

Article Snippet: Flow cytometry and/or cell sorting was performed with a combination of the following Abs (BD Biosciences, Biolegend, eBioscience, Life Technologies, Santa Cruz Biotechnology or Cell Signaling): FITC/AF488-labeled anti-mouse: CD8 (53-6.7), anti-CD25 (7D4), CD69 (H1.2F3), CD44 (IM7), Bcl-2 (10C4), IgG (polyclonal goat), and anti-rat IgG (polyclonal goat); PE-labeled anti-mouse: Fas (Jo2), CTLA-4 (UC10-4B9), PD-1 (RMP1-30), Ki-67 (SolA15), IL-17RA (PAJ17R), CD120b (TR75-89), Bim (rabbit.

Techniques: Infection, Staining, Two Tailed Test, Purification, Expressing, Fluorescence

Journal: Frontiers in Immunology

Article Title: IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2018.02347

Figure Lengend Snippet: Substantial differences in the gene-expression profile of CD8+ T cells from T. cruzi -infected IL-17RA KO and WT mice. (A–F) Microarray analysis of purified CD8+ T cells from infected WT (WT-I) and IL-17RA KO (KO-I) mice (22 dpi) and non-infected controls (WT-N and KO-N). N = 3 mice per group. (A) Dot plot displaying the number of genes that show a significant ≥1.2-fold difference in fold change expression of WT-I and KO-I relative to non-infected counterparts. Colors indicate sets of genes with different expression patterns. (B) Top two enrichment plots in KO-I ( p < 0.001) determined by supervised analysis of all infection induced genes in WT-I and KO-I using GSEA26495 and GSEA41867 (MSigDB C5BP) signature gene sets. (C) Heat maps of expression of selected genes according to categories relevant to CD8+ T cell biology. The gene expression values of each sample were normalized by division to the average values of the corresponding samples from non-infected counterparts. (D) Heat map of expression of genes encoding molecules associated to IL-17RA signaling. Gene expression values represent non-normalized data from 3 mice. (E–G) IPA of genes induced by the infection but showing significantly higher expression in IL-17RA KO mice (red genes in A ). Top 20 “Diseases and Bio-functions” (E) , “Canonical pathways” (F) , and “Up-stream regulators” (G) that were most significantly altered ( P < 0.05 with Fischer's exact test) in KO mice are shown. The activation Z-score was calculated to predict activation (red), inhibition (blue), categories where no predictions can be made and unknown results (with Z-score close to 0). Categories significantly activated or inhibited according to Z-score are marked with a star.

Article Snippet: Flow cytometry and/or cell sorting was performed with a combination of the following Abs (BD Biosciences, Biolegend, eBioscience, Life Technologies, Santa Cruz Biotechnology or Cell Signaling): FITC/AF488-labeled anti-mouse: CD8 (53-6.7), anti-CD25 (7D4), CD69 (H1.2F3), CD44 (IM7), Bcl-2 (10C4), IgG (polyclonal goat), and anti-rat IgG (polyclonal goat); PE-labeled anti-mouse: Fas (Jo2), CTLA-4 (UC10-4B9), PD-1 (RMP1-30), Ki-67 (SolA15), IL-17RA (PAJ17R), CD120b (TR75-89), Bim (rabbit.

Techniques: Expressing, Infection, Microarray, Purification, Activation Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2018.02347

Figure Lengend Snippet: CD8+ T cells from T. cruzi infected IL-17RA KO mice show phenotypic and functional features compatible with exhausted cells. Representative flow cytometry data that show the phenotype of total and TSKB20/Kb+ CD8+ T cells from the spleen of infected WT (WT-I) and IL-17RA (KO-I) mice (22 dpi). Stainings of non-infected WT (WT-N) and KO (KO-N) mice are showed in plots or gray tinted histograms for comparison. (A) Plots of CD62L vs. CD44 expression. Gates indicate the frequency of naïve, memory and effector subsets. (B–D) Representative histograms and statistical analysis of the geometric mean of expression or percentage of cells that express activation markers (B) , inhibitory receptors (C) , and death receptors (D) in total and TSKB20/Kb+ CD8+ T cells from the spleen of infected WT (WT-I) and IL-17RA (KO-I) mice (22 dpi). Data in statistical analysis (B–D) are presented as mean ± SD, N = 4−6 mice. (E) Percentage of CD8+ T cells from the spleen of non-infected (N) and 22-day infected (I) WT and KO mice expressing Granzyme A (GzmA), Perforin (Prf) or Granzime B (GzmB) after 5 h of PMA/Ionomycin stimulation. Data shown as mean ± SD, N = 2−5 mice. (F) Percentage of CD8+ T cells from the spleen of 22-day infected WT and KO mice that exhibit different combinations of effector functions including CD107a mobilization and/or IFNγ and/or TNF production upon 5 h of the indicated stimulation. Data shown as mean ± SD, N = 5 mice. Data were background subtracted. All P values were calculated using two-tailed T test. Data are representative of two-three independent experiments.

Article Snippet: Flow cytometry and/or cell sorting was performed with a combination of the following Abs (BD Biosciences, Biolegend, eBioscience, Life Technologies, Santa Cruz Biotechnology or Cell Signaling): FITC/AF488-labeled anti-mouse: CD8 (53-6.7), anti-CD25 (7D4), CD69 (H1.2F3), CD44 (IM7), Bcl-2 (10C4), IgG (polyclonal goat), and anti-rat IgG (polyclonal goat); PE-labeled anti-mouse: Fas (Jo2), CTLA-4 (UC10-4B9), PD-1 (RMP1-30), Ki-67 (SolA15), IL-17RA (PAJ17R), CD120b (TR75-89), Bim (rabbit.

Techniques: Infection, Functional Assay, Flow Cytometry, Expressing, Activation Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2018.02347

Figure Lengend Snippet: Checkpoint blockade reinvigorates parasite-specific CD8+ T cell immunity and enhances parasite control in infected IL-17RA KO mice. (A) Layout of the checkpoint blockade experiment. (B) Representative plots and statistical analysis of CD8 and TSKB20/Kb staining in spleen and liver of the indicated experimental groups. Numbers within plots indicate the frequency of total CD8+ cells (up) and TSKB20-specific CD8+ T cells (down) at 21 dpi. Bar graphs represent data as mean ± SD, N = 7 mice. (C) Relative amount of T. cruzi satellite DNA in the spleens of the indicated experimental groups at 21 dpi. Murine GAPDH was used for normalization. Data are presented as mean ± SD relative to WT-cAb group, N = 7 mice. (D) Enzymatic activity (UI/L) of alanine transaminase (ALT), aspartate transaminase (AST), creatinine kinase-MB and creatinine phosphokinase (CPK) on plasma of the indicated experimental groups at 21 dpi. N = 7 mice. All P values were calculated with two-tailed T test. Data are representative of two independent experiments.

Article Snippet: Flow cytometry and/or cell sorting was performed with a combination of the following Abs (BD Biosciences, Biolegend, eBioscience, Life Technologies, Santa Cruz Biotechnology or Cell Signaling): FITC/AF488-labeled anti-mouse: CD8 (53-6.7), anti-CD25 (7D4), CD69 (H1.2F3), CD44 (IM7), Bcl-2 (10C4), IgG (polyclonal goat), and anti-rat IgG (polyclonal goat); PE-labeled anti-mouse: Fas (Jo2), CTLA-4 (UC10-4B9), PD-1 (RMP1-30), Ki-67 (SolA15), IL-17RA (PAJ17R), CD120b (TR75-89), Bim (rabbit.

Techniques: Infection, Staining, Activity Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: IL-17RA-Signaling Modulates CD8+ T Cell Survival and Exhaustion During Trypanosoma cruzi Infection

doi: 10.3389/fimmu.2018.02347

Figure Lengend Snippet: Intrinsic IL-17RA-signaling modulates the maintenance, phenotype and function of CD8+ T cells during T. cruzi infection. (A) Concentration of IL-21 in plasma of infected (20 dpi) WT and IL-17RA KO mice. Data are presented as mean ± SD, N = 4 mice. (B) Amounts of Il17ra, Il17rc , and Il17rd transcript determined in CD8+ T cells purified from the spleen of non-infected WT mice, normalized to 18S RNA. Data are presented as mean ± SD, N = 4 mice (C) Representative histograms of the expression of IL-17RA (protein) on total, memory and effector CD8+ T cell subsets defined according to CD44 and CD62L staining in spleen cell suspensions from non-infected (N) and infected (I) WT mice (14 dpi). Staining of IL-17RA on spleen CD8+ T cells from IL-17RA KO mice is showed as negative control. (D) Kinetic evaluation of the upregulation of IL-17RA (MFI: mean of fluorescence intensity) induced by T. cruzi expression on bulk effector and memory CD8+ T cells and TSKB20-specific CD8+ T cells. Data in statistical analysis are presented as mean ± SD, N = 4. (E) Representative plots and statistical analysis of CD8 and TSKB20/Kb staining and of CD45.1 and CD45.2 staining within Total and TSKB20/Kb+ CD8+ T cells in the blood and spleen of infected CD45.1/CD45.2 WT recipient mice (20 dpi) non-injected (control) or injected with equal numbers of CD45.1+ WT and CD45.2+ KO CD8+ T cells. Numbers in the plots indicate the frequency of the correspondent cell subset. Bar graphs display the frequency of CD45.1+ WT cells and CD45.2+ KO cells within the indicated populations upon gating only in the injected cells. (F) Representative plots and statistical analysis of CD8 and TSKB20/Kb staining and of CD45.1 and CD45.2 staining within Total and TSKB20/Kb+ CD8+ T cells in the spleen of infected CD8α-/- recipients (17 dpi) injected with equal numbers of CD45.1+ WT and CD45.2+ KO CD8+ T cells. Pie charts display the frequency of CD45.1+ WT and CD45.2+ KO cells within the indicated gates. Bar graph shows the ratio between the frequencies of TSKB20/Kb+ CD8+ T cells and the total CD8+ T cells within CD45.1 WT and CD45.2 IL-17RA KO populations. (G) Representative histograms and statistical analysis of the geometric mean of PD-1 expression in total and TSKB20/Kb+ CD8+ T cells within CD45.1+ WT and CD45.2+ IL-17RA KO CD8+ T cells from the spleen of CD8α-/- recipient mice. Data in statistical analysis are presented as mean ± SD, N = 4−6 mice. (H) Percentage of polyfunctional effector cells denoted by expression CD107a, IFNγ and/or TNF upon 5 h of the indicated stimulation on CD45.1+ WT and CD45.2+ IL-17RA KO CD8+ T cells from the spleen of CD8α-/- recipient mice. Data shown as mean ± SD, N = 4 mice. Data were background subtracted. All P values in (E–G) were calculated using two-tailed T test. Data are representative of two independent experiments. (I) Experimental layout of the adoptive transfer of equal number of CD8+ T cells purified from WT mice or IL-17RA KO mice into CD8α knockout mice immediately followed by T. cruzi infection. N = 3 mice/group. (J) Representative dot plots of the frequency of CD8+ T cells determined 1 day after injection in blood of the different experimental groups. (K) Parasitemia (15 dpi) in the different experimental groups. Infected non-transferred CD8α knockout mice were used as control. P value calculated using Gehan-Breslow-Wilcoxon Test. Data in (A–J) are representative of two independent experiments.

Article Snippet: Flow cytometry and/or cell sorting was performed with a combination of the following Abs (BD Biosciences, Biolegend, eBioscience, Life Technologies, Santa Cruz Biotechnology or Cell Signaling): FITC/AF488-labeled anti-mouse: CD8 (53-6.7), anti-CD25 (7D4), CD69 (H1.2F3), CD44 (IM7), Bcl-2 (10C4), IgG (polyclonal goat), and anti-rat IgG (polyclonal goat); PE-labeled anti-mouse: Fas (Jo2), CTLA-4 (UC10-4B9), PD-1 (RMP1-30), Ki-67 (SolA15), IL-17RA (PAJ17R), CD120b (TR75-89), Bim (rabbit.

Techniques: Infection, Concentration Assay, Purification, Expressing, Staining, Negative Control, Fluorescence, Injection, Two Tailed Test, Adoptive Transfer Assay, Knock-Out