Extended Data Fig. 7: CD4 T cell help is required for effector CD8 differentiation in TDLNs.
From: Differentiation fate of a stem-like CD4 T cell controls immunity to cancer
a-c) Th1 differentiation and tumor growth kinetics shown as mean ± s.d. for a) TRAMPC1-GP (Untx n = 8, aCD8 n = 6), b) B16F10-GP (n = 3 mice per group) and c) MC38 tumors (n = 4 mice per group) for Untx mice, Treg depleted mice, total CD4-depleted mice (GK1.5 clone), and combination of Treg and CD8 T cell (Lyt3.2 clone) depleted mice. Medians shown in summary plots. Statistical comparisons were performed using two-sided unpaired Mann-Whitney U test or Kruskal-Wallis test with Dunn’s multiple-comparison tests when appropriate. d-e) Phenotypic analysis of d) GP33+ and e) SPAS1 + CD8 T cells in TRAMPC1-GP TDLNs 5-days after Treg or total CD4 T cell depletions. f) Phenotype of GP33 + CD8 T cells in tumor 5-days after Treg or total CD4 T cell depletion. Every time point represents an independent experiment (n = 5–17 mice per group). Medians or mean ± s.e.m are represented in each summary plot and were analyzed by Kruskal-Wallis test with Dunn’s multiple-comparison tests. g) Schematic of experimental designs. h-i) Phenotypic analysis of bulk activated CD8 T cells in h) B16F10-GP i) or MC38 TDLNs after Treg or total CD4 depletion. Summary plots show total number or frequency of activated CD8 T cells expressing the indicated marker at the respective time points (n = 3–7 mice per group per timepoint for each independent experiment). Medians are represented in each summary plot and were analyzed by Kruskal-Wallis test with Dunn’s multiple-comparison tests. j) Experimental design. ScRNAseq of sorted naïve and activated (CD44 + PD1 +) CD8 T cells from TDLNs of Untx, Treg depleted, and total CD4 depleted TRAMPC1-GP bearing mice 5-days after depletion (n = 4–12 pooled mice per group). k-m) Cluster distributions and UMAP projections of normalized expression of genes defining the naïve, LN-stem, and effector CD8 clusters in all conditions. n) Transcriptional comparisons of LN-stem and effector CD8 T cells between groups. Volcano plots show fold change versus -log(p-value) for each gene within each respective comparison. o) Transcriptional comparison of selected genes across LN-stem (green) and effector (red) clusters between Untx, Treg depleted, and CD4 depleted groups. The color and size of the circles represent the normalized expression and proportion of cells expressing that gene, respectively. p) VISION GSEA using an IFN signaling signature. Signature enrichment score is represented as violin plots for LN-stem (green) and effector (red) clusters in the respective groups. Mean is represented in each violin and were analyzed by one-way ANOVA with Tukey’s multiple comparison tests (n = 4–12 pooled mice per group).
