A specific combination of Cy conditioning, IL-2 treatment, and Treg transfer rescues scurfy syndrome. (A) Rescue of scurfy mice. Scurfy male mice (X^Sf/Y.Rag1^+/− on a CD45.2 background) were conditioned by an intraperitoneal injection of 50 mg/kg Cy on day 10 and then received 5 × 10⁵ congenic CD45.1 WT Tregs on day 14. Next, 1000 IU/g IL-2 was injected intraperitoneally once a day for 5 days and then once a week. In an initial experiment, all mice were euthanized on day 50 for flow cytometry analysis. Survival was analyzed in a second experiment (n = 3 mice per group). (B) The scurfy disease score was rated (as described in “Materials and methods”) in Treg-treated mice and vehicle (PBS)-treated mice. Differences were apparent after day 31 (P = .003 in a Mann-Whitney test) and after day 38 (P < .001). (C) Flow cytometry analysis of CD45.1 chimerism, gated on CD4⁺ T cells in the lymph nodes, spleen, blood, liver, and lung. IL (n = 3 mice per group). (D) Survival of scurfy mice untreated (PBS), treated with Cy only (Cy), Cy and IL-2 (Cy+IL-2+PBS or Cy, IL-2, and 5 × 10⁵ Tregs (Cy+IL-2+Treg). There was a significant difference (P = .0004 in a log-rank test) between Cy+IL-2+PBS and Cy+IL-2+Treg groups (n = 6 mice per group). (E) A representative flow cytometry histogram of CD62L expression, gated on CD4+ T cells in mice that had received Treg or vehicle (n = 3 mice per group). ***P < .001; ****P < .0001.