Impaired BM B-cell development in cGVHD. B10.BR mice were transplanted with BM only or BM and T cells from B6 donor mice, and BM cells from tibiae and femurs were harvested on day 30. (A) Representative flow cytometry plots demonstrating frequency of donor-derived pre-pro–B, pro-B, pre-B, and immature B cells in the BM of transplanted animals. The numbers indicate the frequency of cells found in the gated (not total) population. (B-C) Frequency of cells found from all cells evaluated in the BM (B) and total number (C) of donor-derived pre-pro–B (B220⁺CD43⁺CD19⁻IgM⁻), pro-B (B220⁺CD43⁺CD19⁺IgM⁻), pre-B (B220⁺CD43⁻CD19⁺IgM⁻), and immature B cells (B220⁺CD43⁻CD19⁺IgM⁺) isolated from the BM of transplanted mice. Combined data from 5 independent experiments are shown. *P < .05, ***P < .001, ****P < .0001 (Mann-Whitney U test); n = 24 (16 for overall number of cells) and n = 28 (18 for overall number of cells) for the BM only and BM plus T cells groups, respectively. (D) Expression of Ebf1 in sorted pre-pro–B and pro-B progenitors isolated from the BM-only control group (open bars) and cGVHD group (filled bars). Pooled data from 2 independent experiments are shown. ****P < .0001 (unpaired Student t test); n = 6 for each group; sorting purity was >95% and >90% for pre-pro–B and pro-B progenitors, respectively. (E) Expression of Pax5 gene in sorted pro-B and pre-B progenitors isolated from the BM-only control group (open bars) and cGVHD group (filled bars); n = 3 for each group, >90% and >95% for pro-B and pre-B progenitors. Bars represent standard error of the mean. NS, not significant.