S1pr3 is required for egress of Gα13-deficient GC B cells into circulation. (A) Immunohistochemical analysis of mLNs from Gna13 WT (f/+) or KO (f/f mb1-cre) animals stained to detect GC B cells (GL7) and naive follicular B cells (IgD). Arrows indicate subcapsular GCs with greatest degree of dispersal. Black boxes indicate GC shown in enlarged image to the right. Data are representative of at least four animals of each type. (B) Transwell migration of Arhgef1 WT or KO GC B cells to 100 ng/ml CXCL12 in the presence or absence of S1P, or to S1P alone, at the indicated concentrations. Shown are pooled data from four independent experiments. (C) Lymph was collected from Arhgef1 mixed chimeras treated with 1 mg/kg FTY720 or saline i.p. for 3 h and analyzed for the presence of follicular (FoB) or GC B cells. Data are from one experiment representative of two. (D) qPCR of S1pr3 in sorted FoB, total GC B, LZ GC B, and DZ GC B. Shown are pooled data from three independent experiments. (E) mLNs or lymph from Arhgef1 or Gna13 KO mixed chimeras were analyzed by FACS for the presence of LZ phenotype GC B cells. Left panels show example FACS plots, and the right panel summarizes data from three experiments. (F) Transwell migration of Gna13 KO GC B cells that were WT, heterozygous, or deficient for S1pr3. Assay was performed as in B. Shown are pooled data from five independent experiments. (G) Immunohistochemical analysis of mLNs from Gna13 KO S1pr3 WT, heterozygous, or KO animals, stained as in A. Data are representative of three mice of each type. GC dispersal index of individual follicles was calculated as described in Materials and methods and Fig. S1. Bars: (A, whole LNs) 500 µm; (A [enlarged GCs] and G) 100 µm. (H and I) Percentages of GC B cells from mLN (H) lymph or blood (I) from mixed BM chimeras that were reconstituted with ∼60% WT CD45.1 and ∼40% Gna13 KO BM that was also WT, heterozygous, or deficient for S1pr3. Data are pooled from three independent experiments. In dot plots in C–E, H, and I, dots indicate individual mice and lines indicate means. In the dot plot in G, dots indicate the GC dispersal index in individual follicles and lines indicate means. In B and F, error bars show SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001, unpaired two-tailed Student’s t test.