In vivo suppressive function of the K^b-specific CD25⁺ line. (A) In vivo adoptive transfer of T cells and transplant model. T-cell subsets were transferred to T cell–depleted sex-matched syngeneic CBA recipients. For T-cell depletion, mice underwent thymectomy and 2 weeks later were given intraperitoneal injections of anti-CD4– and anti-CD8–depleting mAbs. Fifteen days later, CD4⁺CD25⁻ CD45RB^hi T cells were injected intravenously alone or with CD25⁺ line 1 day before (d −1) recipient mice underwent skin allograft (d 0). (B) In vivo trafficking. Naive CFSE-labeled CD4⁺CD25⁻ T cells (25⁻-CFSE) (1 × 10⁶) were injected intravenously alone or were coinjected with 2 × 10⁶ unlabeled CD25⁺ line or 2 × 10⁶ unlabeled CD25⁺ line injected alone in T cell–depleted CBA mice the day before CBK skin grafts. Trafficking and divisions of the transferred effector T cells were assessed for individual animals by flow cytometric analysis of CFSE dilutions on separate cell suspensions from spleen and graft-draining LNs at various time points after transplantation (here shown at days 8 and 21). The histograms correspond to a minimum of 30 000 acquired events in a mixed lymphocyte gate consisting of live CD4⁺ T cells. Data shown are from 1 of 5 representative mice per group. (C) At day 30 after transplantation, cells were isolated from spleens and graft-draining LNs of recipients in the 3 groups: CD4⁺CD25⁻ T cells injected alone (▪), CD4⁺CD25⁻ T cells coinjected with CD25⁺ line (⊡), or CD25⁺ line injected alone (□). Cells were briefly restimulated in vitro before intracellular staining for the indicated cytokine (IL-2, left panel; IFN-γ, right panel). The frequency of cytokine-positive cells among gated whole CD4⁺ T cells was determined for individual animals. Results shown are mean ± SD from 4 representative animals per group.