Graft survival without a severe rejection episode and histology of heart grafts from DA hosts that were restored with CD4⁺CD25⁺ T cells cultured with allogeneic APCs and IL-2 or IL-4. (A) Experimental plan to test the ability of IL-2 or IL-4, PVG-alloactivated CD4⁺CD25⁺ T cells from DA rats to suppress severe rejection of heterotopic adult heart grafts from PVG (specific) or Lewis (third-party) donors. All irradiated DA hosts were restored with 5 × 10⁶ naive CD4⁺ T cells. CD4⁺CD25⁺ T cells, either fresh or cultured for 3 to 4 days with allogeneic APC (PVG) and IL-2 or IL-4, were cotransferred with naive CD4⁺ T cells at various ratios. (B) Heart graft survival results showing the ability of naive CD4⁺CD25⁺ T cells cultured with allogeneic APCs and IL-2 (Ts1 cells) or IL-4 (Ts2 cells) to suppress allograft rejection. Irradiated hosts restored with 5 × 10⁶ naive CD4⁺ T cells (●) reject both PVG (n = 12) and Lewis (n = 9) grafts. CD4⁺CD25⁺ T cells cultured with IL-2 (▴) or IL-4 (▵) and PVG allo-Ag, mixed at a ratio of 1:10 with naive CD4⁺ T cells (5 × 10⁵ CD4⁺CD25⁺ with 5 × 10⁶ CD4⁺ cells), suppressed donor specific (PVG) (P = .007 for IL-2, n = 6 and P = .035 for IL-4 cultured, n = 6) but not third-party allograft rejection (NSD, n = 5 for IL-2 and n = 6 for IL-4 cultured). Naive CD4⁺CD25⁺ T cells suppressed severe rejection of PVG allografts at a ratio of 1:1 with naive CD4⁺ T cells (■) (P < .001, n = 9) but not at a ratio of 1:10 (□) (NSD, n = 9). Some control data have previously been published.⁵  Graft survival without a severe rejection episode is only shown to 100 days, but all grafts functioning at that time were monitored to at least 250 days and none had a rejection episode. (C) Photomicrographs (original magnification ×200) of hearts from DA hosts given CD4⁺CD25⁺ T cells cultured with PVG allo-Ag and IL-2 (i,ii) or IL-4 (iii,iv). Comparison of histology of PVG long surviving donor heart (left panels) to the recipient's own heart (right panels) showed similar morphology with preserved muscular structure except for scattered patches of infiltrating mononuclear cells in donor hearts.