Extended Data Fig. 5: Pathogen-specific transcriptional programs separate by cell type and infection and establishment of T_FH and T_FR core signatures.

RNA-seq of CD4⁺CD44⁺PD-1⁺CXCR5⁺FoxP3-RFP⁻IL-21–GFP⁺ T_FH, CD4⁺CD44⁺PD-1⁺CXCR5⁺FoxP3-RFP⁻IL-21–GFP⁻ T_FH, CD4⁺CD44⁺PD-1⁺CXCR5⁺FoxP3-RFP⁺IL-21–GFP⁺ T_FR, and CD4⁺CD44⁺PD-1⁻CXCR5⁻FoxP3-RFP⁻IL-21–GFP⁻ T_EFF cells from draining lymph nodes of wild-type mice infected with indicated pathogens at early peak GC response. a, Gating strategy of sorted populations. b, Principal Component Analysis (PCA) plots of sorted IL-21⁺ T_FH, IL-21⁻ T_FH, T_FR, and T_EFF cell transcriptomes stratified by either (top) cell type or (bottom) infection type. Data show independent samples of 2–3 per cell type per infection. c, Analysis of draining lymph node cells from wild-type mice infected with indicated pathogens. gMFI of upregulated (CD200 and BTLA) and downregulated (CCR7) core T_FH signature markers for CD4⁺CD44⁺CXCR5⁺PD-1⁺Ly6C⁻CD162⁻ T_FH cells. Data show experiments of 5–7 mice per group and mean ± SEM. d, UpSet plot of T_FR signature showing intersections of downregulated genes expressed differentially between T_FR vs. T_FH contrast and T_FR vs. T_EFF contrast for five infections. False discovery rate (FDR) < 0.05. e,f, MA plot visualizing log fold change against the mean expression of genes expressed differentially between (e) T_FR and T_FH cells and (f) T_FR and T_EFF cells for five infections. g, GSEA of differentially expressed genes in T_FH (T_FH vs. T_EFF contrast) and T_FR cells (T_FR vs. T_EFF contrast) combined for five infections. Barcode and ES plots displaying enrichment of genes (ES and NES) upregulated in core T_FH signature (T_FH Core Up), T_FH genes, genes indirectly regulated by Bcl-6 through repressor-of-repressor circuits (Indirect Bcl-6-rr), genes directly repressed by Bcl-6 (Direct Bcl-6-r), and Bcl-6 target genes (Bcl-6 targets) in T_FH cells (T_FH vs. T_EFF) contrast^{53,54,59,78,90}. Statistical tests: one-way ANOVA of multiple comparisons and Pearson correlation with two-tailed P value. **** P values are <0.0001 or otherwise indicated.

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