Strong CD28 signal derived from B7 suppresses iTreg generation. (A) CD86 expression is shown on resting and LPS-activated B cells (B220⁺). (B) CD4⁺CD25⁻ cells isolated from Foxp3^gfp reporter mice on a B6 background were stimulated with resting or activated allogeneic B cells from BALB/c mice in the presence of TGFβ and IL-2. Six days after stimulation, cultured cells were harvested and measured for expression of CD4, CD25, and GFP. The data show mean ± SD of percent Tregs (%CD25⁺GFP⁺ on gated CD4⁺ cells) in triplicate wells. (C) The culture condition was the same as in panel B, except that LPS-activated B cells were used and CTLA4-Ig at various concentrations was added into the culture. The data present the fold of increase in the generation of iTregs (CD25⁺GFP⁺ cells among gated CD4⁺ cells) in the culture with CTLA4-Ig relevant to that without CTLA4-Ig. The numbers were presented as the mean ± SD of the fold increase in triplicate wells. The data presented in each panel were reproduced in at least 3 replicate experiments. (D) CD4⁺CD25⁺GFP⁺ nTregs from Foxp3^gfp reporter B6 mice and iTregs generated as in panel B were purified by FACS and used as suppressor cells. Purified T cells from normal B6 mice (50 × 10³/well) were stimulated with irradiated TCD splenocytes from BALB/c mice (250 × 10³/well) without or with various numbers of suppressor cells to achieve the ratios of Treg:Teff as indicated. T-cell proliferation was measured after stimulation for 5 days with a [³H]TdR incorporation assay. Data are presented as percent inhibition compared with no Tregs, and error bars indicate 1 SD in triplicate wells. (E) CD4⁺CD25⁻ cells were purified from Ly5.1⁺ B6 Foxp3^gfp reporter mice and transferred into sublethally irradiated WT or B7.1/7.2 double-knockout syngeneic recipients. PBS or LPS at 50 μg/mouse was injected subcutaneously on day 0 and 7. Two weeks after cell transfer, recipient splenocytes were isolated and stained for expression of CD4, Ly5.1, CD25, and GFP. Percentages of CD25⁺GFP⁺ cells are shown among gated CD4⁺Ly5.1⁺ donor cells, and the data present 6-7 mice per group from 2 pooled experiments.