Altered B cell homeostasis in G_αq-deficient chimeras. (A) Frozen sections of WT and Gnaq^−/− BM chimera spleens were stained with anti-CD90.2 to identify T cell zones (blue), anti-B220 to identify B cell follicles (green), and anti-CD21 (red) to identify follicular DC networks in the B cell follicle and CD21^hi B cells within the MZ. (B) Total B cells were isolated from WT and Gnaq^−/− chimera spleens and analyzed in in vitro chemotaxis assays using 300 ng/ml CXCL12, 500 ng/ml CXCL13, 400 ng/ml CCL19, or 10 nM S1P as chemoattractants. Data are presented as the mean ± SD of triplicate wells. (C) Splenic B cells from CD45.1⁺ WT and CD45.2⁺ Gnaq^−/− chimeras were purified. WT B cells were labeled with biotin and equivalent numbers of WT and Gnaq^−/− B cells were transferred into CD45.1⁺ hosts. Frozen sections were prepared from spleens 18 h after transfer and stained with SA-488 to visualize the transferred WT B cells (green), anti-CD45.2 to visualize transferred Gnaq^−/− B cells (red), and anti–MOMA-1 (blue) to visualize the MZ. Bars, 50 µm. (D–F) BM and spleens were harvested from Gnaq^−/− and WT chimeras, stained with the indicated Abs to identify B cell subsets, and then analyzed by flow cytometry. (D) The absolute numbers of total BM B cells, pro–B, pre–B (large and small), and immature B cells were determined. (E) The numbers of T1, T2, and T3 transitional B cells and the numbers of total splenic B cells, FOB cells, and MZB cells were determined. (F) The numbers of splenic T2/FOII MZB precursors and T1 MZB precursors were determined. Data shown are the mean ± SD of five mice/group. P-values were determined using an unpaired Student’s t test. Data in all panels are representative of three or more independent experiments.