The presence of Tregs in the graft is crucial for the improved survival of mice receiving Cav-1^−/− T cells. (A) Splenocytes isolated from untreated WT and Cav-1^−/− mice were stained for CD4 and Foxp3, and analyzed by flow cytometry (mean ± SEM; WT, n = 10 and CAV-1^−/−, n = 10; pooled from 2 experiments). (B) CD4⁺CD25⁻ were isolated from C57BL/6 WT or Cav-1^−/− donor splenocytes. The purity of CD4⁺CD25⁻ fraction is shown (1 of 4 comparable results). (C) Irradiated BALB/c recipient mice received 5 × 10⁶ BM cells isolated from C57BL/6 WT mice and 2 days later received 3 × 10⁵ CD4⁺CD25⁻ conventional T cells from either WT or Cav-1^−/− mice. Survival was monitored for 100 days. Data were pooled from 2 independent experiments (WT control group, n = 17 and Cav-1^−/− group, n = 18). (D) Donor cells were purified as in (B) and the enrichment of the CD4⁺CD25⁺ fraction is shown (1 of 4 comparable results). (E) Irradiated BALB/c recipient mice were transplanted with 5 × 10⁶ BM cells isolated from C57BL/6 WT mice plus 3 × 10⁵ CD4⁺ CD25⁺ Tregs from either WT C57BL/6 or Cav-1^−/− mice. Two days later, recipients received 3 × 10⁵ CD4⁺ and CD8⁺ T cells from C57BL/6 mice. Survival was monitored for 100 days. Data were pooled from 2 independent experiments (P = .0078; WT control group, n = 18 and Cav-1^−/− group, n = 19). (F) Irradiated BALB/c recipient mice were transplanted with 5 × 10⁶ BM cells isolated from C57BL/6 WT mice. In addition, 2 × 10⁵ CD4⁺ WT cells and 1 × 10⁵ CD8⁺ T cells from either WT or Cav-1^−/− mice (C57BL/6) were also injected. The experiment was performed once (n = 10 per group). n.s., not significant.