Decreased splenic regulatory B cells in CD19^−/− donor-transplanted GVHD mice. Female wild-type BALB/c recipients were irradiated (800 cGy) and transplanted with TCD BM and splenic T and B cells from male wild-type B10.D2 or CD19^−/− B10.D2 mice. (A) Frequencies of host B cells were identified by Ly9.1. Numbers of (B) splenic B cells (B220⁺), (C) marginal zone (MZ) B cells (CD1d⁺CD21⁺B220⁺), and (D) CD1d^highCD5⁺ B cells (CD1d^hiCD5⁺B220⁺) were analyzed every 7 days after BMT. Splenic single-cell suspensions from CD19^+/+ and CD19^−/− donor-transplanted GVHD groups were cultured with PMA, ionomycin, LPS, and brefeldin A for 5 hours. (E) The number of splenic IL-10–producing B220⁺ B cells (Breg cells) was determined by intracellular staining every 7 days after BMT; 4 to 5 mice per group. *P < .05; **P < .01; ***P < .001.