Figure 4.

Unique gene expression profile of the ex–T-bet Tfh cells reveals cell surface markers for identifying this population. (A) RNA-Seq analysis was performed using CD4⁺CD44^highCD25⁻Tetramer⁺-CXCR5^highPD-1^hightdTomato⁺, CXCR5^highPD-1^hightdTomato⁻, CXCR5^lowPD-1^lowtdTomato⁺, and CXCR5^lowPD-1^lowtdTomato⁻ antigen-specific cell populations from dLN of AS15/CFA-immunized T-bet-Cre-tdTomato mice. Differentially expressed genes were clustered. Cell samples are in biological duplicates. (B) Overlapping of the 67 and 113 genes that were expressed at higher levels in tdTomato⁺ cells compared with tdTomato⁻ counterparts in Tfh and non-Tfh compartment, respectively. (C) tdTomato⁺ Tfh cell–specific genes (compared with the tdTomato⁻ Tfh cells) that encode cell surface molecules. (D) Antigen-specific Tfh cells from the AS15/CFA-immunized T-bet-Cre-tdTomato mice were stained with anti-NKG2D, anti-NKG2A/B, or anti-CXCR3. Data are representative of three independent experiments. (E) NKG2D and IFN-γ-eYFP expression in antigen-specific Tfh cells from immunized IFN-γ-eYFP mice were measured. Data are representative of two independent experiments with five animals in each experiment.