Extended Data Fig. 3: RNA-seq of TFH and non-TFH populations from multiple genetic backgrounds.

Related to Fig. 3 a, Schematic of the SMARTA cell transfer system used for RNA-seq analysis in KLH-gp₆₁ immunization. Non-T_FH (CXCR5^loPSGL1^hi) populations from WT, Bcl6^f/f Cre^Cd4, Prdm1^f/f Cre^CD4, or Bcl6^f/fPrdm1^f/f Cre^Cd4 SMARTA cells and T_FH (CXCR5^hiPSGL1^lo) populations from WT or Prdm1^f/f Cre^CD4 SMARTA cells were sorted from C57BL/6 host mice given WT, Bcl6^f/f, Prdm1^f/f, or Bcl6^f/fPrdm1^f/f Cre^CD4 SMARTA CD4 T cells, followed by infection of the host mice with KLH-gp₆₁ in alum + cGAMP, and analyzed 8 days later. Naive SMARTA cells were isolated as CD44^loCD62L^hiCD45.1⁺ from uninfected mice. Representative flow cytometry of T_FH and T_H1 subsets from four independent experiments. b, Heatmap of gene expression of curated T_FH-associated genes. Scale, row z-score. DKO, Bcl6^f/fPrdm1^f/f Cre^CD4. As a first analysis the effect of Bcl6/Prdm1 double-deficiency on the T_FH biology, we assessed expression of a broad curated set of T_FH-associated genes across all samples from RNA-seq gene expression profiling. Bcl6^f/fPrdm1^f/fCre^CD4 T_FH-like cells lost expression of positively T_FH-associated genes in comparison to WT T_FH cells or Prdm1^f/fCre^CD4 T_FH cells. Conversely, Bcl6^f/fPrdm1^f/fCre^CD4 T_H1-like cells had a gene expression profile different from WT T_H1 or Bcl6^f/fCre^CD4 T_H1 cells. c, Principal component analysis of differential gene expression from RNA-seq of LCMV_Arm infection. Principal component analysis provided similar findings as (b), supporting the overall hypothesis that T_FH is not a default differentiation pathway of CD4⁺ T cells and Bcl-6 has important activities beyond inhibition of Prdm1. d, Gene expression changes were clustered by k-means clustering analysis (k=10) of LCMV_Arm infection. e, Hierarchical clustering analysis of genes upregulated or downregulated in T_FH cells relative to their expression in T_H1 cells (1.4-fold cut off, Adj. P <0.05) of LCMV_Arm infection shown in Fig. 3c. Scale, row z-score. f, GSEA of BCL-6 bound genes from human tonsillar GC-T_FH compared to Cluster 2 genes (left) or Cluster 3 genes (right) differentially expressed between Bcl6^f/fPrdm1^f/f T_FH-like cells and Prdm1^f/f T_FH cells. NES, normalized enrichment score; FDR, false discovery rate. g, GSEA of Blimp-1 bound genes from CD8 and T_H17 cells in comparison of Cluster 2 and Cluster 3 genes differentially expressed between Bcl6^f/fPrdm1^f/f T_H1-like cells and Bcl6^f/f T_H1 cells.