Phenotype and in vitro reactivity of CD4⁺CD25⁺ T cells from hosts with long surviving grafts given CD4⁺CD25⁺ T cells that had been cultured with allogeneic APCs and IL-2 or IL-4. (A) Fluorescence-activated cell sorter profiles showing normal proportions of CD4⁺CD25⁺ T cells in tolerant hosts and Foxp3 expression by the majority of CD4⁺CD25^+high T cells. Expression of surface CD4 (Cy5), CD25 (PE), and intracellular Foxp3 (FITC) was analyzed by 3-color staining. Isotype control mAb staining showed specificity of Foxp3 Ab. (B) Cytokine and cytokine receptor expression profiles of fresh naive CD4⁺CD25⁺ T cells from normal DA rats (□) or CD4⁺CD25⁺ T cells from DA hosts with long surviving PVG allograft after being restored with IL-2–cultured Ts1 (▨) or IL-4-cultured Ts2 (■) cells. CD4⁺CD25⁺ T cells from Ts1 restored hosts had higher Ifnγr and Il-5 but no Il-5Rα mRNA expression. CD4⁺CD25⁺ T cells from hosts that had been restored with Ts2 cells had higher Il-5rα and low Il-5 mRNA. Thus, tolerant hosts' CD4⁺CD25⁺ T cells had a cytokine and cytokine receptor profile similar to the alloactivated CD4⁺CD25⁺ T-cell population given to induce tolerance. (C) In vitro reactivity of CD4⁺CD25⁺ T cells from DA hosts with long surviving PVG allografts after being restored with IL-4/PVG–activated (Ts2) CD4⁺CD25⁺ Tregs. CD4⁺CD25⁺ T cells from these tolerant hosts were cultured either without stimulator cells (□) or against self- (DA), specific donor (PVG), or third-party (Lewis) stimulator cells. Control cultures not supplemented with cytokine/mAb (▨) were compared with those supplemented with IFN-γ (▩), IL-5 (■), or a combination of IL-5 and anti-IL-5 mAb (▩). These cells had higher proliferation to specific donor allo-Ag in the presence of IL-5 that was blocked by anti-IL-5 mAb, consistent with the IL-5Rα expression on tolerant CD4⁺CD25⁺ T cells having a functional role.