S1pr2-high Tfh cells are localized in the GC in an S1PR2-dependent manner. (A–C) OT-II T cells of the indicated genotypes were transferred to congenic recipient mice that were subsequently immunized with OVA in alum. Draining LNs were analyzed 7 d after immunization. Data are pooled from four (A and B) or three (C) independent experiments with 5–7 mice of each type in total. (A) Representative sections stained with anti-GFP antibody to detect Venus, for CD45.2 to identify OT-II T cells, and for IgD to demarcate GCs. OT-II T cells with clearly detectable levels of Venus expression are pointed with arrowheads. Bars, 100 µm. (B) Enumeration of the percentages of OT-II T cells in the GCs out of total OT-II T cells in the follicles. Data are normalized by the area of GCs and follicles, which was not significantly different between three groups, and presented as mean ± SEM of 9 (S1pr2^+/+), 7 (S1pr2^V/+), and 11 (S1pr2^V/V) different follicles. *, P < 0.05; **, P < 0.01 (one-way ANOVA with Bonferroni’s post-test). (C) Flow cytometric enumeration of the percentages of Tfh cells in total OT-II T cell population. Data are presented as mean ± SEM. (D) Flow cytometry of Bcl6, ICOS, and Il21 expression in Tfh cells of draining LN cells from mice of the indicated genotypes on 9 d after immunization. For Il21 detection, mice were crossed with IL-21/hCD2 BAC transgenic mice. Gray- and green-filled histograms show the data of WT CD44^lo CD4⁺ T cells and WT Tfh cells, respectively. The left panel shows gating strategy for Tfh cells. Data are representative of two independent experiments with 3 (Bcl6 and ICOS) or 2 (Il21) mice of each genotype.