Supplementary Figure 4: Gene signature of CXCR3⁺CXCR5⁺ T_FH cells and the expression of CXCR3 and CXCR5 in IFN-γ⁺ cells

(a) Construction map of the Venus-targeted Ifng locus (Top). The IRES-Venus cassette was inserted immediately after the stop codon of Ifng gene. Splenocytes from Ifng Venus reporter mice were stimulated with anti-TCRβ antibody in the presence of IL-12 and anti-IL-4 antibody (for T_H1), or IL-4 and anti-IFN-γ antibody (for T_H2). Five days after the stimulation, ex-vivo induced T_H1 and T_H2 cells were re-stimulated with anti-TCRβ antibody, and intracellular staining of IFN-γ and IL-4 (bottom) were carried out. (b) Heatmap of T_H1, T_H2, T_H17, and T_FH signature genes, and heatmap of common signature genes of T_H1 and T_FH in CXCR5⁺ and CXCR3⁺CXCR5⁺ cells. CXCR5 single positive and CXCR3 CXCR5 double positive CD4⁺ T cells were sorted from vaccinated C57BL/6 mice at day14 post vaccination. The RNA sequencing analysis of sorted populations was carried out by Hiseq. (c) Comparison analysis of CXCR3⁺CXCR5⁺ and CXCR5⁺ T_FH cells. Fold-change of gene expression in CXCR3⁺CXCR5⁺ versus CXCR5⁺ cells are plotted as histogram. Major T_FH signature genes are shown in red. (d) Flow cytometry analysis of Venus expression was examined in splenic CD4⁺T cells from vaccinated ifng Venus reporter mice at 14 days after immunization. The CD4+ T cells were separated into 3 fractions based on the magnitude of Venus expression (Negative (Neg), intermediated (Med) and High). Each population was analyzed for PD-1, CXCR5, and CXCR3 expression. The bar graph shows cell number of T_FH and CD4⁺T cells in Neg, Med, and High fractions (n=3).