Figure 6
Figure 6. Early adoptive transfer of CD1dhiCD5+ B cells reduced CD19−/− donor-induced Scl-cGVHD severity. (A) Representative B10-cell purification results from an adoptive transfer experiment. Purified splenic B cells from wild-type male B10.D2 were separated into CD1dhiCD5+CD19+ and CD1dloCD5−CD19+ B-cell populations. The isolated cells were cultured with PMA, ionomycin, LPS, and brefeldin A for 5 hours. IL-10–producing B-cell frequencies in the stimulated cell cultures were determined by intracellular staining with IL-10. (B) CD19−/− donor-transplanted female recipients were also given phosphate-buffered saline, CD1dhiCD5+ B cells, or CD1dloCD5− B cells on the day of BMT or (C) on day 21 after BMT+- Skin scores were monitored every 3 days after BMT. (D) Percentages of host B cells (Ly.9.1+B220+) and (E) percentages and numbers of IL-10–producing B cells in each group are presented in representative histograms and bar graphs. (F-H) B10 cells altered the T-cell IFN-γ production profile but did not affect T-cell proliferation. (F) Representative histograms showing the proliferation of T cells when T cells and irradiated TCD splenocytes were cocultured without the presence of anti-CD3 and anti-CD28 (Unstimulated), or with the presence of anti-CD3 and anti-CD28 (T cells alone), or with either CD1dhiCD5+ (T cells + CD1dhiCD5+) or CD1dloCD5− (T cells + CD1dloCD5−) B cells in the presence of soluble anti-CD3 and anti-CD28. Proliferation was measured by CFSE dilution. LPS-stimulated CD1dhiCD5+ or CD1dloCD5− B cells isolated from wild-type B10.D2 spleens were cocultured with purified T cells from CD19−/− donor-transplanted mice 14 days after BMT in presence of irradiated BALB/c TCD splenocytes and soluble anti-CD3 and anti-CD28 for 72 hours, and with/without IL-10 or anti-IL-10R antibody. (G) Representative histograms and bar graphs showed percentage of IFN-γ–producing T cells. (H) Bar graph indicates concentrations of IFN-γ in supernatant after a 72-hour coculture. The experiments were performed in triplicate. *P < .05; **P < .01.

Early adoptive transfer of CD1dhiCD5+ B cells reduced CD19−/− donor-induced Scl-cGVHD severity. (A) Representative B10-cell purification results from an adoptive transfer experiment. Purified splenic B cells from wild-type male B10.D2 were separated into CD1dhiCD5+CD19+ and CD1dloCD5−CD19+ B-cell populations. The isolated cells were cultured with PMA, ionomycin, LPS, and brefeldin A for 5 hours. IL-10–producing B-cell frequencies in the stimulated cell cultures were determined by intracellular staining with IL-10. (B) CD19−/− donor-transplanted female recipients were also given phosphate-buffered saline, CD1dhiCD5+ B cells, or CD1dloCD5− B cells on the day of BMT or (C) on day 21 after BMT+- Skin scores were monitored every 3 days after BMT. (D) Percentages of host B cells (Ly.9.1+B220+) and (E) percentages and numbers of IL-10–producing B cells in each group are presented in representative histograms and bar graphs. (F-H) B10 cells altered the T-cell IFN-γ production profile but did not affect T-cell proliferation. (F) Representative histograms showing the proliferation of T cells when T cells and irradiated TCD splenocytes were cocultured without the presence of anti-CD3 and anti-CD28 (Unstimulated), or with the presence of anti-CD3 and anti-CD28 (T cells alone), or with either CD1dhiCD5+ (T cells + CD1dhiCD5+) or CD1dloCD5− (T cells + CD1dloCD5−) B cells in the presence of soluble anti-CD3 and anti-CD28. Proliferation was measured by CFSE dilution. LPS-stimulated CD1dhiCD5+ or CD1dloCD5− B cells isolated from wild-type B10.D2 spleens were cocultured with purified T cells from CD19−/− donor-transplanted mice 14 days after BMT in presence of irradiated BALB/c TCD splenocytes and soluble anti-CD3 and anti-CD28 for 72 hours, and with/without IL-10 or anti-IL-10R antibody. (G) Representative histograms and bar graphs showed percentage of IFN-γ–producing T cells. (H) Bar graph indicates concentrations of IFN-γ in supernatant after a 72-hour coculture. The experiments were performed in triplicate. *P < .05; **P < .01.

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