Increased potential of Cav-1^−/− T cells to develop into Tregs. (A) 1 × 10⁵ Treg (CD4⁺CD25⁺) were cocultured with 1 × 10⁵ carboxyfluorescein diacetate succinimidyl ester-labeled conventional T cells (CD4⁺CD25⁻) stimulated with CD3/CD28 beads for 48 hours. T-cell proliferation was assayed by flow cytometry (mean ± SEM, pooled from 5 experiments). (B) BMDCs from BALB/c mice were cocultured with C57BL/6 WT or Cav-1^−/− T cells at a 1:2 ratio (2.5 × 10⁶ DC + 5 × 10⁶ T cells) and the level of Foxp3 expression was analyzed by flow cytometry. Data are pooled from 5 independent repetitions (mean ± SEM, n = 5). (C) Purified CD4⁺ T cells were stimulated with 1 μg/mL (low) or 10 μg/mL (high) anti-TCR (145-2C11) antibody for 5 minutes in the presence of 1 μg/mL TGF-β for 30 minutes. After cell lysis, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by WB for anti–phospho-Smad2 (p-Smad2) and anti-GAPDH (1 of 3 comparable experiments is shown). (D) Purified CD4⁺CD25⁻ T cells were incubated for 72 hours in the presence of increasing amounts of plate-bound anti-CD3 antibodies under Treg-generating conditions (IL-2 and blocking antibodies against IL-4 and interferon-γ). Cells were harvested and stained with cell viability dye and antibodies against CD4, CD25, and Foxp3 (1 of 3 comparable experiments is shown). (E) Cells were treated as in (D) with the addition of exogenous TGF-β (1 ng/mL). The pooled results of at least 3 independently performed experiments are shown. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. BMDC, bone marrow derived dendritic cells; CSFE, carboxyfluorescein diacetate succinimidyl ester; WB, western blot.