Long-term in vitro culture of CD49d⁻CD127⁻ Treg cells. Untouched Treg cells were obtained by CD49d/CD127 MACS depletion as described in Figure 4. (A) Expansion of Treg cells. Total CD4⁺ T cells (□) and CD49d⁻CD127⁻ Treg cells (■) were expanded in vitro by stimulation with α-CD3/CD28 beads in the presence of IL-2. Cells were counted on days 21 and 33; absolute cell numbers are shown. (B) FACS analysis. Dot plots show the expression of CD25 and Foxp3 for the cultures on days 0 (top row) and 33 (bottom row). Numbers represent percentages of cells per quadrant. (C) Summarized purity of Foxp3⁺ cells of 8 independent experiments after the indicated cultivation period. The linked data points refer to purity of the culture at the beginning of cultivation and on days 33 to 36 based on Foxp3 expression (day 0, 91.01% ± 3.27%; days 33-36, 74.28% ± 12.74%).