(a) Quantitative PCR analysis of S1pr1 and S1pr2 transcript abundance in follicular (FO) and GC B cells, shown relative to HPRT. (b) Number of GC B cells in spleen and mLNs and (c) immunohistochemical staining of mLNs from one year-old S1pr2^+/− or S1pr2^−/− mice. Scale bar, 500 µm. (d) Representative flow cytometric analysis of mLN FO and GC populations from 60% wild-type (CD45.1) and 40% S1pr2^+/+ or S1pr2^−/− (CD45.2) mixed BM chimeras. Numbers indicate frequency of cells within gates. e–f, Contribution of CD45.2 cells to FO and GC populations in indicated tissues of mixed BM chimeras made with 60:40 (e) or 90:10 (f) ratios of CD45.1 and CD45.2 BM. In e, data are pooled from more than 5 experiments (n = 19–23); f, data are representative of 3 experiments (n = 5). (g) Relative contribution of S1pr2^−/− (CD45.2) GC B cells to GC/FO populations in mixed BM chimeras in Mx1-cre⁺Sphk1^{f/− or f/f}Sphk2^−/− hosts. Data are from 3 experiments (n = 6). h–i, Contribution of CD45.2 cells to FO and GC populations in indicated tissues of mixed BM chimeras made with ~60:40 ratios of wild-type CD45.1 cells and CD45.2 cells from either littermate controls or mice deficient in G12 and G13 (h) or p115RhoGEF (Arhgef1) (i). Littermate controls in h are singly deficient in G12. Data are representative of 2 similar experiments. All chimeras in d–i were reconstituted for at least 8 weeks and had been immunized i.p. with SRBCs 6–8 days prior to analysis. * P ≤ .01, ** P ≤ .001, *** P ≤ .0001.

Frequency of GC B cells with activated caspase-3 (a) or fragmented DNA detected by TUNEL assay (b) in chimeras reconstituted with mixtures of S1pr2^+/+ or S1pr2^−/− (CD45.2) and wild-type (CD45.1) BM. The mice were immunized with SRBCs and analyzed after 6–8 days. Data are from 3 experiments (n = 8–9). (c) Flow cytometric analysis of P-Akt T308 in follicular and GC B cells from mixed chimeras. Right panel shows mean fluorescence index (MFI) of P-Akt T308 in the indicated GC B cell populations (n = 7). (d) P-Akt T308 analysis in wild-type GC B cells from spleen suspensions treated with JTE-013 or JTE-013 and wortmannin (WMN) for 30 min immediately ex vivo (n = 3). (e) Graph showing P-Akt T308 MFI of mLN GC B cells from Mx1-cre⁺Sphk1^{f/− or f/f}Sphk2^−/− (S1P-deficient) mice, relative to the average of the controls (n = 12–13, pooled from 6 experiments). (f) P-Akt T308 analysis in GC B cells deficient in p115RhoGEF or both G12 and G13, compared to littermate control cells and wild-type (CD45.1) cells, from mixed BM chimeras. The G12–G13 DKO littermate control was G12-single deficient. Right panels show a summary of P-Akt T308 MFIs in GC B cells from mixed chimeras (data are representative of 2 experiments). (g) Western blot for P-Akt S473 in Ramos cells treated for 5 min with S1P in the presence or absence of JTE-013 (representative of 3 experiments). (h) P-Akt T308 analysis in Ramos cells treated for 5 min with S1P (10nm) alone or in the presence of the indicated inhibitors. Y27632, 10µm; JTE-013, 10µm; bpV(pic), 500nm. (i) Relative P-Akt T308 MFIs in Ramos cells treated with the indicated conditions, compared to untreated (dashed line) (n = 8, pooled from 8 experiments). ^# P ≤ .05, * P ≤ .01, ** P ≤ .001, *** P ≤ .0001.

(a) BM from Cr2-cre transgenic mice was transduced with loxP-stop-loxP Thy-1.1 control retrovirus (Vector) or myristoylated Akt (myr-Akt) Thy-1.1 retrovirus and used to reconstitute irradiated recipient mice. Representative flow cytometric analysis showing representation of myr-AKT–Thy-1.1-expressing cells in IgD^lo Fas⁺ GC B cells relative to IgD^hi FO B cells of mLNs. Right panel shows summary of data as percent contribution of Thy-1.1⁺ cells to FO and GC B cell populations (n = 7, pooled from 2 experiments). (b) TUNEL assay of mLN GC B cells from mice of the type in a. (c) Flow cytometric analysis of P-4E-BP1 in FO and GC B cells from mixed BM chimeras containing wild-type (CD45.1) cells and either S1pr2^+/+ or S1pr2^−/− (CD45.2) cells. Graph on right shows MFI of P-4E-BP1 in the indicated GC populations (n = 4, from 4 experiments). (d) Relative incorporation of ³⁵S-labeled cysteine and methionine in 30 min by Ramos cells removed from S1P (No S1P) or treated with JTE-013, relative to cells cultured with S1P. Control samples were removed from S1P and treated with rapamycin. Triplicate measurements were obtained in each experiment and all data points were divided by the mean of the untreated group. ^# P ≤ .05, * P ≤ .01, ** P ≤ .001, *** P ≤ .0001.

(a) Transwell migration of wild-type Bcl2-transgenic FO and GC B cells to CXCL12 (0.3µg/ml) or CXCL13 (1µg/ml) in the presence or absence of S1P as well as JTE-013 (representative of at least 5 experiments). (b) Transwell migration of S1pr2^+/− or S1pr2^−/− Bcl2-transgenic GC B cells to CXCL12 in the presence or absence of S1P (representative of 2 experiments). (c) Immunohistochemical analysis of splenic GL7⁺ GCs in S1pr2^+/− or S1pr2^−/− mice immunized with SRBCs. (d) Lysozyme-specific S1pr2^+/+ or S1pr2^−/−Hy10 (CD45.2) B cells were transferred into recipient (CD45.1) mice along with wild-type Hy10 (CD45.1) B cells and OVA-specific OT-II (CD45.1) T cells. Recipients were DEL-OVA immunized. At d14 LN sections stained to detect CD45.2⁺ Hy10 GC B cells (blue) and FO B cells (IgD, brown). Scale bar, 200µm. (e) Histograms showing velocities of S1pr2^+/+ and S1pr2^−/− GC B cells determined by tracking migration of fluorescently labeled GC B cells using real-time 2-photon microscopy of intact LNs (see also Supplementary Movies 1–2). (f) Confinement of wild-type and S1pr2^−/− GC B cells, displayed as path length/displacement (data in e and f are representative of 4 GCs analyzed in 2 experiments). (g) Example views of GC surface (red) (see also Supplementary Fig. 6) showing tracks of wild-type (CFP⁺, cyan) and S1pr2^−/− GC B cells (GFP⁺, green) (see also Supplementary Movies 3–6). (h) Velocities of GC B cells during migration inside or outside the GC surface. Downward arrows in e and h denote average values of their respective groups. * P ≤ .01, ** P ≤ .001, *** P ≤ .0001, n.s. P = 0.73.

(a) Immunohistochemical analysis of mLN sections from CXCL13-deficient mice reconstituted with BM from either S1pr2^+/− or S1pr2^−/− donors, showing GL7⁺ GC B cell and IgD⁺ B cell distribution. Right panel shows number of GC B cells per mLN in chimeras (n = 8, pooled from 3 experiments). (b) Immunohistochemical analysis of spleen sections from S1pr2^+/− or S1pr2^−/− mice treated with LTβR-Fc for 4 weeks. At the fourth injection, mice were immunized i.p. with SRBCs and analyzed 7 days later. Right panel shows numbers of GC B cells in spleen (n = 8, pooled from 3 experiments). Scale bars, 100µm.

(a) S1P1 surface abundance on follicular B cells from the indicated tissues of control (CTL), S1pr1-deficient (S1P1 KO), or Mx1-cre⁺Sphk1^{f/− or f/f}Sphk2^−/− (S1P-deficient) mice. Data are representative of more than 3 mice of each type. (b) Transcript abundance of S1P lyase (Sgpl), sphingosine-1-phosphate phosphatase-1 (Sgpp1), and lipid phosphate phosphatases (Lpp) 1–3 in B cells and T cells, shown relative to HPRT. (c) Relative amount of S1P remaining in culture supernatants after incubation with B or T cells for the indicated number of minutes, determined by the extent of down-modulation of Flag-S1P1 on a reporter cell line. Data are plotted as MFI of Flag-S1P1 staining relative to reporter cells not exposed to S1P (data are from 3 experiments).

(a) MD4 B cells retrovirally transduced with vectors encoding either S1P2 or control surface receptor (truncated Ngfr) as well as the hCD4 reporter were transferred into day 6 SRBC immunized recipient mice for 24 hours. Immunohistochemical staining of splenic sections shows localization of hCD4⁺ transduced cells in GC-containing follicles. (b) S1P2 or control vector transduced Gpr183^+/− MD4 B cells were transferred into unimmunized recipients. Immunohistochemical staining shows localization of transduced cells in primary follicles. (c) S1P2 transduced Gpr183^+/− MD4 B cells were transferred into either littermate control (Sphk2^−/−) or Mx1-cre⁺Sphk1^{f/− or f/f}Sphk2^−/− (S1P-deficient) recipient mice. Immunohistochemical staining shows localization of transduced cells in primary follicles. Data in a–c are all representative of 3 independent experiments. Scale bars, 200 µm.