Extended Data Fig. 5: Phenotypic and transcriptional profiling of antigen specific CD4 T cells generated after Treg depletion in cancer.
From: Differentiation fate of a stem-like CD4 T cell controls immunity to cancer
a-c) Phenotypic analysis of GP66+ and bulk PD1 + CD4 T cells in TDLNs, blood and tumor 5-days after Treg depletion. Median or mean ± s.e.m are shown and analyzed by two-sided unpaired Mann Whitney U or Kruskal-Wallis test with Dunn’s multiple-comparison tests when appropriate. Each individual timepoint represents an independent experiment (n = 2–17 mice per group for each time point). d) Schematic of experimental designs for B16F10-GP or MC38 tumors. e-f) Representative TBET by FOXP3 staining of bulk activated CD4 T cells in B16F10-GP (e) or MC38 (f) TDLNs 5-days after Treg depletion. Data represents 3-4 independent experiments (n = 4–5 mice per group for each experiment). Medians are shown and were analyzed by two sided unpaired Mann-Whitney U test or Kruskal-Wallis test with Dunn’s multiple-comparison tests when appropriate. g) Schematic of experimental design and phenotypic analysis of transferred SMARTAs in B16F10-GP TDLNs 5-days after Treg depletion. Data represents 2 independent experiments (n = 5 mice per group). Medians are represented in each summary plot and were analyzed by two-sided unpaired Mann-Whitney U test. h) ScRNAseq analysis of bulk activated (CD44 + PD1 +) CD4 T cells sorted from TDLNs of Untx or Treg depleted TRAMPC1-GP mice 5-days after depletion (n = 4-12 pooled mice per group). Naïve (CD44- PD1-) CD4 T cells were included as a control. UMAP projection and cluster distribution of activated CD4 T cells in TDLNs for both groups. i-j) Normalized expression of transcription factors and selected genes defining each cluster. k) VISION GSEA analysis using a signature from precursor TCF1+ BCL6low CD4 T cells from chronic LCMV infection for Untx and Treg depleted mice. Mean is represented in each violin and were analyzed by one-way ANOVA with Tukey’s multiple comparison tests. l) Transcriptional comparison of stem-like CD4 T cell cluster between groups. Volcano plots show fold change versus -log(p-value) for each gene. m) UMAP projection of the distribution of all the TCR clonotypes in the TCF1+ stem-like cluster in untreated mice (top). Cluster distribution of the 10 most dominant TCR clonotypes belonging to the Treg cluster in Untx mice. The number of cells sharing the respective TCR clonotype is indicated below (bottom).
