CD4⁺CD25^– cells acquire regulatory function that inhibits naive alloresponses via 8-day MLR culture with IL-10 and TGF-β CD4⁺CD25⁺ T cells are not required for acquisition of in vitro (A) or in vivo (B) inhibitory function. (A) MLR culture contained 10⁵ naive B6 CD4⁺ T-cell responders per well and 10⁵ bm12 splenic stimulators per well plated with or without CD4⁺ T cells (2.5 × 10⁴ per well), derived from a CD4⁺CD25^– culture that had been previously tolerized to bm12 alloantigen by an 8-day culture with IL-10 and TGF-β. Shown is 1 of 2 replicate experiments with similar results. (B) 10⁵ naive B6 CD4⁺ T cells were injected alone (n = 10) or with 10⁵ 8-day MLR IL-10/TGF-β–cultured CD25-depleted CD4⁺ T cells (n = 14) (P < .001). Also shown is long-term survival of all recipients of 10⁵ 8-day MLR IL-10/TGF-β– cultured CD25-depleted CD4⁺ T cells (n = 14) and uniform lethality by day 21 of all recipients (n = 10) of 10⁵ 8-day MLR control-cultured CD4⁺CD25^– T cells. Data represent 2 pooled experiments with similar results.