Figure 1.

Tfh cells in GCs do not express T-bet but some have a history of T-bet expression. (A) ZTCE-tdTomato mice were treated (i.p.) with TMX on days 0, 2, and 5. After 10 d, the expression of ZsGreen and tdTomato by the splenic naive CD4 T cells (CD4⁺CD44^lowCD25⁻) and memory-like CD4 T effector cells (CD4⁺CD44^hiCD25⁻) cells was assessed by flow cytometry. The plot represents a typical profile from more than five experiments. (B) Splenic CD4⁺CD44^highCD25⁻tdTomato⁺ZsGreen⁻ (T⁺G⁻) and CD4⁺CD44^highCD25⁻tdTomato⁺ZsGreen⁺ (T⁺G⁺) cells sorted from TMX-treated naive ZTCE-tdTomato mice were used for RNA-Seq analysis. Samples are in biological duplicates. (C) Relative gene expression levels of Tfh-related genes in CD4⁺CD44^highCD25⁻tdTomato⁺ZsGreen⁻ (T⁺G⁻) and CD4⁺CD44^highCD25⁻tdTomato⁺ZsGreen⁺ (T⁺G⁺) populations were measured by qRT-PCR (n = 4). Data are representative of three independent experiments. (D) Experimental procedure of immunizing ZTCE-tdTomato mice with AS15/CFA. (E and F) ZTCE-tdTomato mice were immunized with AS15/CFA for 16 d and treated with TMX as shown in D. dLN cells were analyzed by flow cytometry. The percentage of ZsGreen⁺ cells among the tdTomato⁺ cells was calculated for the PD-1^lowCXCR5^low non-Tfh and PD-1^hiCXCR5^hi Tfh populations (mean ± SD; n = 7; *, P < 0.0001). Data are representative of three independent experiments. (G) ZTCE-tdTomato mice were treated as in D. Different cell types in GC were analyzed by multicolor tissue imaging. GC (yellow, BCL-6); ex–T-bet-Tfh cell (pink, CD4-blue and tdTomato-red); T-bet-ZsGreen-green. Arrows indicate ex–T-bet-Tfh cells in GC. Data are representative of two independent experiments with two animals in each experiment.