Figure 6. IL-10 production by CD4⁺ T_conv cells limits anti-tumor immunity upon T_reg cell depletion.

(A) Screen to identify mechanisms of suppression by CD4⁺ T_conv cells from tumors of T_reg-depleted animals. Proliferation of CTV-labeled naïve splenic CD4⁺ T_conv responder cells (T_resp) cultured alone or at a ratio of 8:1 with CD4⁺ Foxp3^EGFP− T_conv suppressor cells (T_supp) isolated at day 16 from B16-F10 tumors of DTx-treated Foxp3^EGFP-DTR animals. Cells were cultured alone (gray) or with suppressors (purple) and with indicated reagents. AG, aminoglutethimide. CD45.2⁺ GFP⁻ T_conv suppressor cells were co-cultured with 1.25×10⁴ suppressor CD4⁺ T_conv cells in the presence of 5.0×10⁴ antigen-presenting cells (APC). Data are representative of 2 independently repeated experiments n > 3. P values show significance of difference between no suppressor and suppressor (Student t test) and are Bonferroni corrected. (B) Representative frequency of dividing T_resp cells incubated with tumor CD4⁺ GFP⁻ T_conv cells in the presence of anti-IL-10R antibodies or vehicle. T_resp cells without tumor T_conv cells were used as a control. (C) Co-correlation between the expression of indicated genes within single cell gene expression profiles of T cells from tumors of PBS- or DTx-treated B16 tumor-bearing Foxp3^EGFP-DTR animals. Pearson correlation co-efficient values are indicated by color scale and genes are hierarchically clustered to identify clusters of co-expressed transcripts within T cell populations. scRNA-Seq data are representative of 3 biological replicates per group. (D) Measurement of Ccr8 and Il10 mRNA expression within CCR8⁻ and CCR8⁺ T_conv cells from PBS- and DTx-treated animals. Data representative of 3-4 biological replicates per group. ordinary one-way ANOVA, Tukey’s multiple comparisons. ns, not significant. (E) Tumor area of heterotopic B16-F10 melanoma tumors at indicated time-points following implantation into Il10^flox/flox Cd4^Cre Foxp3^EGFP-DTR or Il10^+/+ Cd4^Cre Foxp3^EGFP-DTR control mice administered DTx or PBS from day 10-16 post-implantation. Data are representative of 2 independently repeated experiments, n > 7. ordinary one-way ANOVA, Tukey’s multiple comparisons. (F) Representative histograms (top) and replicate measurements (bottom) of the frequency of CD8⁺ CD44⁺ T cells and Foxp3⁻ CD4⁺ CD44⁺ T_conv cells from tumors of animals within indicated treatment groups. (G) Representative frequency and (H) replicate measurements of the frequency (top) and total counts (bottom) of CD8⁺ IFN-γ⁺ TNF⁺ T cells within tumors. Data are representative of 2 independently repeated experiments, n > 4, one-way ANOVA, Kruskal-Wallis, Dunn’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001. Error bars show standard error of the mean (s.e.m.)