Changes in splenic immune cells during Scl-cGVHD development. Female BALB/c recipients were irradiated (800 cGy) and transplanted with 10 × 10⁶ TCD BM cells and 10 × 10⁶ unfractionated splenocytes from either male B10.D2 (allogeneic) or male BALB/c (syngeneic) donors. Spleen samples were collected every 7 days after BMT. Single-cell suspensions were stained for surface markers and to differentiate live cells from dead cells. (A) Percentage and number of Thy1.2⁺ T cells among total live splenocytes, (B) percentages and numbers of CD11b⁺ monocytes/macrophages among total live splenocytes, (C) B220⁺ B cells among total live splenocytes, (D) ratio of CD8⁺:CD4⁺ T cells, and (E) percentage of CD1d^hiCD5⁺ among (F) CD19⁺ B cells analyzed every 7 days after BMT by flow cytometry. Splenic single-cell suspensions were cultured with PMA, ionomycin, LPS, and brefeldin A for 5 hours. B10 cell (splenic IL-10–producing B cells) frequencies were determined by intracellular staining with IL-10; 3 to 5 mice per group. *P < .05; **P < .01; ***P < .001.