Fig. 1

Loss of Gαq limited differentiation of CD19⁺IL-10⁺ Bregs. a–f Splenic cells isolated from Gnaq^−/− mice and WT littermates and subjected to flow cytometry analysis. Splenic cells stained with anti-mouse CD19, CD1d, and CD5 after PMA, ionomycin, and BFA stimulation, followed by intracellular staining with IL-10. CD19-positive cells gated for analysis of CD1d^hiCD5⁺ cells (a, representative images; b, statistical analysis). IL-10-positive cells in CD19⁺ gate also analyzed (d, representative images; e, statistical analysis). Absolute number of CD19⁺CD1d^hiCD5⁺ and CD19⁺IL-10⁺ cells also quantified (c, f). g, h B cells isolated from spleen of WT and Gnaq^−/− mice and stimulated with LPS for 48 h, and then PMA, ionomycin, and BFA added for last 5 h. After culture, cells stained with anti-mouse CD19, followed by intracellular staining with IL-10 and analysis by flow cytometry (g, representative images; h, statistical analysis). Results represent mean ± SD per group (n = 6–8 mice/group). Student’s t test analyzed statistical difference. Data representative of three independent experiments. i–k Purified B cells from spleens of WT and Gnaq^−/− mice stimulated with LPS for 48 h, then culture supernatants harvested and subjected to analysis of IL-10 production by ELISA (i), and cells collected to analyze PD-L1 and FasL expression. j Representative histograms show PD-L1 and FasL expression on Bregs from WT mice (red line), Gnaq^−/− mice (green line), and isotype control (gray line). Mean fluorescence intensity (MFI) of PD-L1 and FasL expression also recorded by flow cytometry (k). Data presented as mean ± SD (n = 6–8), and Student’s t test performed to analyze statistical difference. Data representative of three independent experiments. *p < 0.05, **p < 0.01. IL interleukin, ns not significant, SSC side scatter