Fig. S1PR2 and CXCR5 cooperatively regulate localization of Tfh cells in GCs to support GC reactions. Follicular helper T (Tfh) cells are the helper T (Th) cell subset specialized in providing help to cognate antigen-specific B cells in the secondary lymphoid organs. Tfh cells develop in a manner dependent on the transcription element Bcl6, and they communicate important molecules for shaping B cell reactions such as IL-4, IL-21, CD40L, and PD-1 (Good-Jacobson et al., 2010; Kitano et al., 2011). Tfh cells are particularly important for the germinal center (GC) reaction that is essential for high affinity antibody production (Vinuesa et al., 2010) and is also thought to be important for the generation of immunological memory space (McHeyzer-Williams et al., 2012). Tfh cells access the B cell follicle by up-regulating CXCR5 and by down-regulating CCR7 (Haynes et al., 2007). In GC-containing follicles, Tfh cells are found both in the GC and the follicular mantle (FM), the outer follicle region surrounding the GC. Although some Tfh cells migrate between the GC and FM and between neighboring GCs, Tfh cells with the highest manifestation of PD-1 and CXCR5 look like preferentially accumulated in GCs (Linterman et al., 2012; Shulman et al., 2013). However, the mechanism of GC Tfh cell localization is definitely incompletely recognized. Because CXCR5 deficiency in T cells only mildly reduces the number of Th cells in the GC (Junt et al., 2005; Arnold et al., 2007; Haynes et al., 2007), additional homing receptors will also be likely to be involved in the GC Tfh cell localization. Recently, it has been found that sphingosine-1-phosphate receptor 2 (S1PR2), a G12/13-coupled receptor, is highly indicated in GC B cells and is involved in their clustering in the inner region of follicles (Green et al., 2011). Our earlier microarray analysis showed that CXCR5hiPD-1hi Tfh cells communicate modestly more transcripts than CXCR5loPD-1lo Th cells (Kitano et al., 2011). In this study, using the is definitely expressed at numerous levels in Tfh cells and that Tfh cells with high manifestation of are retained in the GC in an S1PR2-dependent manner. Furthermore, we have shown that double deficiency of S1PR2 and CXCR5 in T cells seriously impairs their localization to GCs and ability to support GC B cells, suggesting that S1PR2 takes on a cooperative part with CXCR5 in Tfh cell biology. RESULTS and Conversation Regulatory effect of S1PR2 on Tfh cell migration in vitro First, we tested for functional manifestation of S1PR2 in CXCR5hiPD-1hi Tfh cells by carrying out transwell migration analysis (Fig. 1 A). Migration of these cells toward CXCL13 and CXCL12 (Ansel et al., 1999) was suppressed by S1P. This suppression by S1P was reversed by treatment with the S1PR2 antagonist JTE-013, suggesting LY2603618 (IC-83) the suppression was mediated by S1PR2. These results are consistent with the previously explained function of S1PR2 that inhibits Rac-mediated chemotaxis by Rho activation (Skoura and LY2603618 (IC-83) Hla, 2009). In contrast, S1P rather induced migration of CXCR5?CD4+ T cells, which was most likely mediated by Gi signaling-coupled S1P receptors, particularly S1PR1 (Matloubian et al., 2004). JTE-013 did not impact S1P- or CXCL12-induced migration of CXCR5?CD4+ T cells, suggesting that S1PR2 expression is usually minimal in Proc these cells. Open in a separate window Number 1. Practical manifestation of S1PR2 and magnitudes of manifestation in CXCR5hiPD-1hi Tfh cells. (A) In LY2603618 (IC-83) vitro chemotaxis assay of CD4+ T cells. Splenocytes from mice 10C12 d after sheep reddish blood cell immunization were cultured in transwell plates and analyzed by circulation cytometry. Chemotaxis of CXCR5hiPD-1hi and CXCR5? CD4+ T cells was measured toward CXCL13 or CXCL12 with or without S1P and/or JTE-013. Data are pooled from three self-employed experiments and offered as mean SEM. = 8C10. *, P 0.05; **, P 0.01; ***, P 0.001; ****, P 0.0001 (one-way ANOVA with Bonferronis post-test). (B) Circulation cytometric analysis of Venus manifestation on B cells (left) and CD4+ T cells (ideal) from PPs of mice. The gray packed histograms depict B cells or CD4+ T cells from mice. Data are representative of at least two self-employed experiments. (C) Developmental time course of Tfh cells and Venushi Tfh cells. OT-II.