Function of expanded Tregs. (A) CD4⁺ T cells were assessed for cytokine production before and after treatment by intracellular staining with fluorescence-labeled antibodies. PBMCs from before and after treatment were cultured with or without anti-CD3 and anti-CD28 antibodies for 8 hours. The cells were stained with anti-CD4 followed by intracellular staining for FoxP3, IFN-γ, and IL-2. For analysis, the PBMCs were gated on CD4⁺ T cells. Numbers on plots represent the percentages for each quadrant. Data shown are derived from 1 subject and are representative experiments from 3 different subjects. (B) PBMCs from baseline and at week 8 were stained for CD4, CD25, and CD127 followed by intracellular staining for FoxP3. Gating for FoxP3⁺ CD4⁺ cells was set with results from isotype-matched control IgG staining. Tregs were sorted from PBMCs derived from a study subject (C) in dose level 5 at week 8 and from a healthy control individual (D) based on the expression of CD4, CD127, and CD25. Thirty thousand autologous CD4⁺CD127⁺CD25⁻ T cells (responders) were cocultured with 100 000 irradiated allogeneic PBMCs and anti-CD3 antibody. Where indicated, CD4⁺CD127⁻CD25⁺ Tregs were added at the indicated ratios of responders/Tregs. ³H-thymidine incorporation was assessed after 5 days of culture. Results are representative experiments from 3 different subjects. Error bars represent SD.