Functional expression of S1PR2 and magnitudes of S1pr2 expression in CXCR5^hiPD-1^hi Tfh cells. (A) In vitro chemotaxis assay of CD4⁺ T cells. Splenocytes from mice 10–12 d after sheep red blood cell immunization were cultured in transwell plates and analyzed by flow cytometry. Chemotaxis of CXCR5^hiPD-1^hi and CXCR5⁻ CD4⁺ T cells was measured toward CXCL13 or CXCL12 with or without S1P and/or JTE-013. Data are pooled from three independent experiments and presented as mean ± SEM. n = 8–10. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA with Bonferroni’s post-test). (B) Flow cytometric analysis of Venus expression on B cells (left) and CD4⁺ T cells (right) from PPs of S1pr2^V/+ mice. The gray filled histograms depict B cells or CD4⁺ T cells from S1pr2^+/+ mice. Data are representative of at least two independent experiments. (C) Developmental time course of Tfh cells and Venus^hi Tfh cells. S1pr2^V/+ OT-II T cells and Hy10 B cells (2 × 10⁵ each per head) were cotransferred into recipient mice which were then immunized with HEL-OVA in CFA, and analyzed by flow cytometry on each time point. Total numbers of indicated donor cells in a draining LN are shown. Data are representative of two independent experiments, and presented as mean ± SEM. n = 3–7. (D) Flow cytometry data of PP CD4⁺ T cells from Foxp3^hCD2 S1pr2^V/+ mice. Venus expression on indicated T cell subsets are shown. Data are representative of two independent experiments with five mice in total.