Extended Data Fig. 5: Characterization of murine conventional T_FH and T_FH17^Der cells, and gating strategy of the T_FH-B cell doublet.

(a) GSEA of scRNA-seq data of ZsGreen+ compared to ZsGreen− T_FH cells. The significant 5 pathway clusters with gene sets (nodes, p < 0.01) upregulated in ZsGreen+ T_FH cells were visualized with Cytoscape and EnrichmentMap. The graph represents the annotated results for clustered gene set comparisons. Sets of genes are organized in a network, where each set is a node and edges connect similar sets. Automated networks layout groups of related sets of genes into a network of pathway clusters. (b) GSEA of scRNA-seq data of B cells conjugated with ZsGreen+ T_FH cells compared to B cells conjugated with ZsGreen− T_FH cells. The significant 5 pathway clusters with 3 nodes (gene sets, p < 0.01) upregulated in B cell conjugated with ZsGreen+ T_FH cells were visualized with Cytoscape and EnrichmentMap. (c) Heatmap of clustered genes in the leading-edge subsets of gene sets related to cell adhesion (Extended Data Fig. 5b). Red and blue colors indicate positively and negatively regulated transcripts, respectively, upon comparing B cells conjugated with ZsGreen+ T_FH cells to the B cells conjugated with ZsGreen− T_FH cells. Darker the color, the greater the fold difference. (d) (I) Gating scheme for Fig. 4f is shown, illustrating the gating strategy of the indicated T cell type for T-B conjugate analysis, without excluding doublets. Additionally, the gating strategy in (II) that excludes doublets is shown as a comparison. Doublets were excluded by FSC-A and FSC-H gating in (II) for single-cell analysis. Note, the extra PD-1^loCXCR5⁺ cells in gate (I), but not gate (II), indicate the B cells that are bound to T cells and are typically excluded using the single-cell analysis. In the Extended Data Fig. 5, ANOVA test is used in scRNA-seq analysis in a-c.