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.