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Figure S4. Continued provision of TGF-β and RA is not required for the maintenance of Foxp3⁺ T cells in culture. CD4⁺ T cells from DO11.10 SCID mice were cultured with 0.5 x 10⁵ CD103⁺ MLN DCs, 0.2 µg/ml OVA peptide, 2 ng/ml rhTGF-β, and 0.03 µg/ml RA. At day 3 of culture, cultures were split and maintained in either IL-2 alone or a combination of IL-2, rhTGF-β, and RA. Cultures continued to be split at 3-d intervals. At the indicated time points, T cells were stained for CD4 and Foxp3, and the absolute number of Foxp3⁺ T cells in culture was determined.