FIG. 5.

Expression of human adipose tissue-specific markers on induced regulatory T cells (iT_regs) following coculture with ASCs. IFN-γ-treated ASCs were cocultured with naive CD4⁺ T cells for 3 days under low or ambient O₂ culture conditions at ASC:T-cell ratios of 1:10 (10,000 ASCs:100,000 T cells) and 1:1 (100,000 ASCs:100,000 T cells). T cells were collected, incubated with antibodies for FITC-CD25 and PE-CD127, and subjected to flow cytometry to sort for CD127^−/low CD25⁺ iT_regs under (a) ambient and (b) low O₂ culture conditions. Isolated iT_regs were cocultured for 7 days with allogeneic PBMCs isolated from human breast cancer patients at a 10:1 ratio (100,000 PBMCs:10,000 iT_regs) under low and ambient culture conditions. (c) T-cell proliferation was quantified by BrdU analysis in parallel experiments. % T-cell proliferation=(E_Tcell+ASC/E_Tcell)×100 where E_Tcell+ASC=proliferation absorbance measurement for the naive T-cell+ASC group, and E_Tcell=proliferation absorbance measurement for the T-cell group. (d) mRNA was isolated and one-step qRT-PCR was performed following ASC-T-cell coculture using forward and reverse primers and probes for human FoxP3 and TGF-β. Three independent sets of experiments were performed for each treatment. Data are reported as mean (μ)±SE. *P<0.05, **P<0.01, ***P<0.0001.