Splenic cytotoxic CD8⁺ T cells, IL-6–producing CD11b⁺ cells, and TNF-α– and IFN-γ–producing CD4⁺ T cells are increased in CD19^−/− donor-transplanted GVHD mice. (A) Percentages of CD11b⁺ and Thy1.2⁺ cells within a live gate in CD19^+/+ and CD19^−/− donor-transplanted groups. (B) The number of splenic T cells (Thy1.2⁺) and (C) the ratio of splenic CD8⁺:CD4⁺ T cells was analyzed 14 days after BMT. Splenic single-cell suspensions from CD19^+/+ donor and CD19^−/− donor-transplanted groups were cultured with PMA, ionomycin, LPS, and brefeldin A for 5 hours for detection of IL-6–producing CD11b⁺ cells, or with PMA, ionomycin, and brefeldin A for 4 hours for detecting granzyme B–producing CD8⁺ T cells and TNF-α– and IFN-γ–producing CD4⁺ T cells. (D) Percentages of cytotoxic CD8⁺ T cells (Granzyme B–producing CD8⁺ T cells) among T cells (Thy1.2⁺). (E) From within a splenic live gate, percentages of CD11b⁺ and IL-6⁺ cells are shown in CD19^+/+ and CD19^−/− donor-transplanted groups. (F) Percentage of IL-6–producing CD11b⁺ cells among total live cells. (G) Percentage of TNF-α–producing CD4⁺ T cells and (H) percentage of IFN-γ–producing CD4⁺ T cells among CD4⁺ T cells; 4 to 5 mice per group. *P < .05; **P < .01. LPS-stimulated B cells purified from wild-type or CD19^−/− B10.D2 spleens were cocultured with purified CD11b⁺ cells from CD19^−/− donor-transplanted mice 14 days after BMT with/without IL-10 or anti–IL-10R antibody. (I) Representative histograms and bar graphs showed percentage of IL-6– or TNF-α–producing CD11b⁺ cells from CD11b⁺ cells. (J) Bar graphs indicate concentrations of IL-6 and TNF-α in supernatant after a 24-hour coculture. Experiments were performed in triplicate. *P < .05; **P < .01.