Significant increase in transcription frequency and intracellular protein expression of FoxP3 after allo-BMT using Cav-1^−/− T cells. (A) BALB/c BM-derived DCs were irradiated with 40 Gy and cocultured with isolated WT or Cav-1^−/− T cells (n = 4 per group). After 2 days of coculture, cells were harvested and applied to CD11c depletion. The CD11c⁻ T-cell fraction was then further analyzed for differentially transcribed genes by microarray analysis. The RMA was calculated (mean ± SEM). (B) The RMA values for FoxP3 are selectively displayed (n = 4 per group). (C) BALB/c recipient mice were irradiated with a total of 9 Gy and subsequently transplanted with 5 × 10⁶ BM plus 3 × 10⁵ WT or Cav-1^−/− T cells. Recipients were euthanized 7 days post–allo-BMT and analyzed by flow cytometry, using specific staining against CD4 and FoxP3. Relative Treg proportions of all CD4⁺ T cells in the spleen are shown (mean ± SEM; Cav-1^+/+, n = 12 and Cav-1^−/−, n = 14) (left). Absolute Treg numbers in the spleen are displayed (mean ± SEM; Cav-1^+/+, n = 6 and Cav-1^−/−, n = 6) (right). (D) Cav-1 expression in Treg cells was analyzed at day 7 after BMT. Teffs were obtained from sort-purified naive CD4⁺CD25⁻CD127⁺CD45RA⁺ T cells that have been stimulated with anti–CD3-loaded KT64/86 and expanded 7 to 8 days in vitro. The MFI was normalized to the value obtained for Treg cells derived from untreated WT mice. The dotted line indicates the background MFI for Cav-1 that is detected in Cav-1^−/− Treg cells. RMA, robust multi-array average; stim, stimulated. *P < .05; ***P < .001.