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.

G_αq-deficient B cells exhibit a prosurvival phenotype in vitro. (A–C) WT or Gnaq^−/− splenic B cells were stimulated with 0–25 µg/ml anti-IgM F(ab′)₂ (A), 0–40 µg/ml anti-CD40 (B), or 0–10 µg/ml LPS (C) for 48 h and proliferative responses were measured by ³H-thymidine incorporation after a 5-h pulse. Results represent the mean ± SD of triplicate cultures. (D–F) Purified total splenic B cells (D), sort-purified B220⁺CD93⁺CD23⁻ T1 B cells (E), or sort-purified B220⁺CD93⁺CD23⁺ T2/T3 B cells (F) were cultured in media alone or stimulated with 10–15 µg/ml anti-IgM F(ab′)₂ for 18–24 h. Viability was measured by staining with propidium iodide. Data are shown as the mean ± SD of triplicate cultures. P-values were determined using an unpaired Student’s t test. Data in all panels are representative of three independent experiments.

G_αq-deficient B cells are hyperresponsive to BCR triggering. (A–D) Splenic B cells from Gnaq^−/− and WT chimeras were stimulated with anti-IgM F(ab′)₂ for 0–30 min. Protein lysates were prepared from negatively selected total B cells (A and D) or from enriched FOB and MZB cells (D) and then analyzed by Western blotting. Akt (p-Ser473), PLCγ2, and Erk were probed with phospho-specific Abs and protein-specific Abs. Li-COR quantitative densitometry was used to determine the fold changes in phospho-Akt (B) and phospho-PLCγ2 (C). The ratios of phospho-specific proteins versus total ERK or PLCγ2 were obtained and data were normalized to WT B cells at time 0 (set as 1). Data in all panels are representative of two independent experiments.

G_αq-deficient B cells have a competitive survival advantage over WT B cells in vivo. (A–C) Splenic B cells were purified from WT and Gnaq^−/− chimeras (CD45.2⁺) and transferred i.v. into CD45.1⁺ WT hosts (2.5 × 10⁷ cells/host). (B) Spleen cells from recipient mice were harvested at 1–3 wk after transfer and analyzed by flow cytometry to identify donor B cells. Representative dot plots and the mean percentage ± SD of cells of donor origin are shown. (C) The number of donor B cells recovered per recipient spleen was determined and is shown as the mean ± SD (n = 3–5 mice/group/time point). Data are representative of two independent experiments. (D–G) Lethally irradiated CD45.1⁺ WT mice were reconstituted with a mixture of BM consisting of 50% Gnaq^−/− (CD45.2⁺) BM and 50% WT (CD45.1⁺) BM. (E) Splenic B cells were collected from the 50:50 BM chimeras and flow cytometry was used to identify WT and Gnaq^−/− B cells. Data are presented as the ratio of Gnaq^−/− to WT B cells (percentage of Gnaq^−/− B cells divided by percentage of WT B cells) at each time point and are shown as the mean ± SD from five mice per group. Data are representative of more than three independent experiments. (F) BM and spleen were collected from the 50:50 BM chimeras at 2 mo after reconstitution, and the ratio of Gnaq^−/− to WT B cells was determined as described in E. Splenic B cells were subdivided into total mature (B220⁺CD93⁻), FOB (B220⁺CD21^loCD23^hi), MZB (B220⁺CD21^hiCD23^lo), T1 (B220⁺CD93⁺CD23⁻), T2/T3 (B220⁺CD93⁺CD23⁺), T1 MZB precursors (B220⁺CD93⁺IgM^hiCD21^hi), and T2/FOII MZB precursors (B220⁺CD93⁻CD21^hiCD23^hi). B cells in the BM were subdivided into immature (fraction E) B cells (B220⁺CD93⁺IgM⁺), transitional (B220⁺CD93⁺IgM^hiCD23⁺), and recirculating (B220^hiIgM⁺IgD⁺). Data are shown as the mean ± SD of four to five mice/group. (G) 50:50 BM chimeras (2 mo after reconstitution) were injected with EdU twice over a 12-h period. Spleen cells were isolated at 18 h, stained with Abs to CD19 and CD45.2 and the EdU detection reagent, and then analyzed by flow cytometry. Representative FACS plots are shown and the percentage of B cells of each genotype that incorporated EdU is provided. Data shown are the mean ± SD of five mice. Data in D–G are representative of two independent experiments.

Gnaq^−/− B cells are more resistant to BAFF neutralization in vivo and are more sensitive to BAFF stimulation in vitro. (A–D) 50:50 mixed BM chimeras were generated using Gnaq^−/− BM (CD45.2⁺) and WT (CD45.1⁺) BM. 8 wk after reconstitution, chimeric animals were injected i.p. with a blocking Ab to BAFF/BLyS (clone 10F4) or an isotype control Ab. (B) Peripheral blood samples were collected from Ab-treated chimeric animals between 7 and 21 d and flow cytometry was used to identify the percentage of B cells that were of WT or Gnaq^−/− origin. Data are shown as the ratio (±SEM) of Gnaq^−/− to WT B cells (percentage of Gnaq^−/− B cells divided by percentage of WT B cells; n = 5–10 mice). P-values were determined using an unpaired Student’s t test. (C) Spleen cells from Ab-treated chimeras were isolated, enumerated, and analyzed by flow cytometry at 21 d after treatment. The numbers of mature splenic B cells (B220⁺CD24^lo) of WT and Gnaq^−/− origin were determined and are reported as the mean ± SD of five mice/group. The fold depletion caused by BAFF neutralization is shown above the bar graphs. (D) The ratio of Gnaq^−/− to WT B cells in the spleen was calculated (percentage of Gnaq^−/− B cells divided by percentage of WT B cells) and is shown as the mean ± SD. P-values were determined using an unpaired Student’s t test. Data in A–D are representative of three independent experiments. (E) Splenic CD43^neg naive B cells were isolated from Gnaq^−/− and WT chimeras and enriched for CD21^lo FOB and CD21^hi MZB cells. The total or enriched B cell subsets were cultured in BAFF-free media overnight. Live cells were recovered from the cultures and stimulated with recombinant BAFF for 0–10 min. Protein lysates were prepared and analyzed by Western blotting. Phospho-Akt (p-Ser473), total Akt, and phospho-S6 ribosomal protein were detected using phospho- and protein-specific Abs. Data in E is representative of two experiments.

G_αq-deficient B cells display an activated phenotype and are intrinsically prone to develop autoreactive specificities. (A and B) 50:50 mixed BM chimeras were generated using Gnaq^−/− BM (CD45.2⁺) and WT (CD45.1⁺) BM. 11 wk after reconstitution, cells from BM, spleen, and lungs were harvested and B cells were analyzed by flow cytometry for expression of PNA and FAS. Representative FACs plots are shown (A) and the percentages of WT or Gnaq^−/− B cells expressing a GC phenotype (PNA^hiFAS⁺) were determined (B). Data are shown as the mean ± SD of four to five mice per time point. P-values were determined using an unpaired Student’s t test. All data shown are representative of two independent experiments. (C) WT and Gnaq^−/− BM chimeras were generated and aged for 5 mo. Serum samples were collected monthly. The presence of ANAs (ANA reactivity) in the serum (diluted 1:100) was then determined by fluorescence microscopy using fixed HepG2 cells. The data are shown as the percentage of animals with detectable ANAs in the serum (n = 10 mice/group). (D) Serum samples were collected from female WT and Gnaq^−/− chimeras at 9 mo after reconstitution. Samples were evaluated for reactivity to dsDNA by ELISA. Data shown are the mean OD ± SD from a 1/200 dilution of the serum samples. n = 4–5 mice/group. P-values were determined using an unpaired t test. Data in panels C and D are representative of two or more independent experiments. (E–I) 50:50 mixed BM chimeras were made by reconstituting irradiated Igh^a hosts with 50% WT BM (Igh^a allotype) and 50% Gnaq^−/− BM (Igh^b allotype; referred to as Gnaq^−/−:WT chimeras) or with 50% WT BM (Igh^a allotype) plus 50% WT BM (Igh^b allotype; referred to as WT:WT chimeras). (F and G) Peripheral blood from the WT:WT (F) and Gnaq^−/−:WT (G) chimeras was analyzed by flow cytometry between 2 and 10 mo after reconstitution using allotype-specific Abs to identify the B cells of either WT or Gnaq^−/− origin. Data are shown as the mean percentage of B cells of the different genotypes ± SD. n = 10 mice/group. (H) Serum, isolated from WT:WT and Gnaq^−/−:WT chimeras at 5 mo after reconstitution, was incubated (1:100 dilution) with fixed HepG2 cells. ANA-reactive Abs were detected using allotype-specific Abs. Representative fluorescent images are shown. Bars, 50 µm. (I) The fluorescence signal detected from each serum sample was determined and shown as the mean fluorescent signal ± SD (n = 9–10 mice/group). Data in E–I are representative of two independent experiments. P-values were determined using an unpaired Student’s t test.

G_αq-deficient chimeras develop B cell–dependent systemic autoimmunity. (A and B) Arterial blood from WT and Gnaq^−/− BM chimeras (8 mo after reconstitution) was isolated and complete blood cell counts were performed. Mean red blood cell counts (A) and hemoglobin concentration (B) are reported. Data shown are the mean ± SD of four mice/group and are representative of two independent experiments. P-values were determined using an unpaired Student’s t test. (C) The diameters of ankle and wrist joints were measured in WT and Gnaq^−/− chimeras at 5 mo after reconstitution. Data are represented as the mean ± SD of 8–10 mice/group. Similar results were seen in three experiments. P-values were determined using an unpaired Student’s t test. (D–K) Histological analysis (hematoxylin and eosin [H&E]) of knee and ankle joints from WT and Gnaq^−/− chimeras was performed at 5 mo after reconstitution. (D) Normal knee joint from a WT chimera showing femur (F) and Tibia (T). (E) Knee joint from a Gnaq^−/− chimera with focal soft tissue inflammation (arrow) adjacent to a joint space (S) where the synovial lining is disrupted. A higher power view (F) demonstrates a focus of mixed inflammatory cells (arrow) and necrosis of the overlying synovium. (G) The articular surface of the distal femur of a Gnaq^−/− chimera is irregular, thinned, and covered by fibrous pannus (arrows). (H) Normal ankle joint from a WT chimera. (I) Ankle from a Gnaq^−/− chimera with marked inflammation of soft tissues accompanied by exostotic bone (arrows) and accumulation of fibrin within the joint space (S). (J) Bone from the foot of a Gnaq^−/− chimera with proliferative periostitis overlying an area of bone lysis represented as scalloping of the bone surface (arrows). (K) Higher power of bone from the foot shows multinucleated osteoclasts within resorptive foci (arrows). Images are from one representative animal out of 5–10 mice/group. Bars: (D–J) 100 µm; (K) 20 µm. Two independent experiments were performed. (L) Survival of Gnaq^−/− and WT chimeras was tracked for 6 mo after BM reconstitution. Data are shown as the percentage of survival of each group (n = 10 mice/group). P-values were determined using the Wilcoxon test. Data are representative of three independent experiments. (M) G_αq-deficient and WT chimeras were treated with anti-CD20 (clone 18B12) or control Ab at 2 mo after reconstitution and survival was tracked over the next 11 mo. The experiment was performed once with 10 mice/group. P-values were determined using a Log-rank test.