Figure 5.
Figure 5. S1PR2 and CXCR5 expression in Tfh cells are required for supporting GC responses. (A and B) S1pr2+/+ or S1pr2V/+ (control), or S1pr2V/V CD4+ T cells were transferred to CD28−/− mice, and mice were immunized with NP-CGG in alum or CFA and analyzed 2 wk after immunization. Data are pooled from three independent experiments. (A) Flow cytometric enumeration of the percentages of GC cells in lymphocytes. n = 6 (control), 6 (S1pr2V/V), and 4 (no transfer control). Data are presented as mean ± SEM. (B) Enumeration of CD4+ T cell number in GC sections. 22 GCs from 5 mice (control) and 24 GCs from 5 mice (S1pr2V/V) were analyzed. Data are presented as mean ± SEM. (C) ELISA data of sera collected from mixed BM chimeras of indicated genotypes. Sublethally irradiated Rag1-deficient mice were reconstituted with S1pr2+/+ μMT BM cells or S1pr2V/V μMT BM cells mixed with Tcrα−/− BM cells, and immunized i.p. with NP-CGG in alum. Ratios of anti-NP1 IgG1 concentration to anti-NP24 IgG1 concentration are shown. Data are representative of three independent experiments, and are presented as mean ± SEM. n = 4 (S1pr2+/+ μMT) and 4 (S1pr2V/V μMT). (D) CD4+ T cells of the indicated genotypes were transferred to Cd28−/− mice. On the next day, the mice were immunized i.p. with NP-OVA in alum, and GC B cell frequencies were analyzed 3 wk after immunization by flow cytometry. Data are pooled from three independent experiments, and are presented as mean ± SEM. n = 6 (Cxcr5−/−), 7 (Cxcr5−/− S1pr2V/V), 5 (WT), and 3 (no transfer control). (E) CD4+ T cells were mixed in 1: 1 ratio, and transferred to Cd28−/− mice. On the next day, the mice were immunized i.p. with NP-OVA in alum, and analyzed 3 wk after immunization. Data are representative of two experiments, and are presented as mean ± SEM. Shown is the number of CD45.2 CD4+ T cells in GC sections normalized by the number of CD45.1 CD4+ T cells in GC sections. 9 different GCs from 2 mice were analyzed for each donor type. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (A, B, D, and E). One-way ANOVA with Bonferroni’s post-test (A, D, and E) and Student’s t test (B) were used for statistical analysis.

S1PR2 and CXCR5 expression in Tfh cells are required for supporting GC responses. (A and B) S1pr2+/+ or S1pr2V/+ (control), or S1pr2V/V CD4+ T cells were transferred to CD28−/− mice, and mice were immunized with NP-CGG in alum or CFA and analyzed 2 wk after immunization. Data are pooled from three independent experiments. (A) Flow cytometric enumeration of the percentages of GC cells in lymphocytes. n = 6 (control), 6 (S1pr2V/V), and 4 (no transfer control). Data are presented as mean ± SEM. (B) Enumeration of CD4+ T cell number in GC sections. 22 GCs from 5 mice (control) and 24 GCs from 5 mice (S1pr2V/V) were analyzed. Data are presented as mean ± SEM. (C) ELISA data of sera collected from mixed BM chimeras of indicated genotypes. Sublethally irradiated Rag1-deficient mice were reconstituted with S1pr2+/+ μMT BM cells or S1pr2V/V μMT BM cells mixed with Tcrα−/− BM cells, and immunized i.p. with NP-CGG in alum. Ratios of anti-NP1 IgG1 concentration to anti-NP24 IgG1 concentration are shown. Data are representative of three independent experiments, and are presented as mean ± SEM. n = 4 (S1pr2+/+ μMT) and 4 (S1pr2V/V μMT). (D) CD4+ T cells of the indicated genotypes were transferred to Cd28−/− mice. On the next day, the mice were immunized i.p. with NP-OVA in alum, and GC B cell frequencies were analyzed 3 wk after immunization by flow cytometry. Data are pooled from three independent experiments, and are presented as mean ± SEM. n = 6 (Cxcr5−/−), 7 (Cxcr5−/− S1pr2V/V), 5 (WT), and 3 (no transfer control). (E) CD4+ T cells were mixed in 1: 1 ratio, and transferred to Cd28−/− mice. On the next day, the mice were immunized i.p. with NP-OVA in alum, and analyzed 3 wk after immunization. Data are representative of two experiments, and are presented as mean ± SEM. Shown is the number of CD45.2 CD4+ T cells in GC sections normalized by the number of CD45.1 CD4+ T cells in GC sections. 9 different GCs from 2 mice were analyzed for each donor type. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (A, B, D, and E). One-way ANOVA with Bonferroni’s post-test (A, D, and E) and Student’s t test (B) were used for statistical analysis.

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