Fig. 6. scRNA-seq of GC-resident T cells.

Total T cells (Foxp3^RFP+ and Foxp3^RFP– combined) from single photoactivated GCs were sorted either as B220^–/CD4⁺/PA⁺ or as B220^–/TCRβ⁺/PA⁺ (which also includes CD8⁺ T cells) in different samples at days 10 or 20 after primary immunization with NP-OVA. For comparison, we also sorted Treg cells photoactivated in the T-zone of an unimmunized mouse, sorted as B220^–CD4⁺GFP⁺RFP⁺ and Foxp3⁺CXCR5⁺PD-1^hi Tfh cells derived from transferred naïve precursors as in Fig. 5. (A) UMAP plot showing clustering of T cells according to whole transcriptome analysis. (B) Distribution of different subsets of Foxp3-reporter⁺ cells in UMAP space. Cells in color are those that are Foxp3-reporter⁺ in the sample indicated in the graph title. All other analyzed cells are shown in gray. Bar graph shows distribution of T-zone and GC RFP⁺ cells among clusters 1–3. (C) Expression of selected gene signatures by Foxp3^RFP+ and Foxp3^RFP– cells within Tfh Clusters 1 and 2. (D) Expression of selected genes or gene signatures by Foxp3⁺ Tfh cells derived from transferred naïve precursors compared to photoactivated Foxp3^– Tfh cells. Only cells from day 20 post-immunization are included in the Foxp3^– Tfh group. “GSE20366” signatures are from the Broad Institute’s MSigDB database. (E) Distribution of clonal expansions (defined as TCRs detected more than once in the same GC) containing both RFP⁺ and RFP^– cells (“mixed clones”). Cells carrying identical TCRs are linked by lines. (F) Expression of selected genes or gene signatures by Foxp3-RFP⁺ and Foxp3-RFP ^– cells within mixed clones. (A-F) Each symbol represents one cell. P-values are for the Wilcoxon signed-rank test. Numbers in parentheses are Cohen’s d for effect size. The number of GCs included and number of independent experiments are indicated in Table S1.