DcTs suppress the proliferation of Teffs. CFSE-based proliferation assays and MLR were performed with each population 2.5 × 10⁴ per well in 96-well round-bottom plates. For transwell plate experiments, each population (1 × 10⁵ per well) was incubated in 96-well flat-bottom transwell plates in the presence of beads as stimulators. The proliferation of CFSE-labeled CD4⁺CD25⁻ T cells was analyzed after 3 days (cells with beads) or 6 days (cells with γ-irradiated APCs) on a FACScan cytometer (BD Biosciences). All cultures were evaluated in triplicate. Hypomethylating agent–treated cells suppress the proliferation of anti-CD3/CD28 antibody coated bead-activated Teffs (A-B) and allogeneic APC-activated Teffs (C-D). Treg (1×), (2×), and (4×) indicate the ratios of Treg:Teff = 1:1, 2:1, and 4:1, respectively. azacT (a) and (b) indicate that these azacTs were generated in the presence of AzaC 1μM and 2μM, respectivley. dcTs, azacTs, and pbsTs (right) were generated in the presence of hIL-2 (500 μ/mL; panel A). Only FACS purified GFP⁺ (thus, FOXP3⁺) and not GFP⁻ CD4⁺ cells obtained from Foxp3-ires-GFP KI mice are suppressive (panel B). CD45.1 was used to gate on CFSE-labeled Teffs. dcT: Dec-treated T cells, pbsT: PBS-treated T cells, azacT: AzaC-treated T cells. Neg: negative control, CFSE-labeled Teffs alone; pos: CFSE-labeled Teffs with stimulators, anti-CD3/CD28 antibody coated beads or allogeneic APC; all others contain both CFSE-labeled Teffs and stimulators plus indicated cells such as nTregs, dcTs, pbsTs, or azacTs. GFP⁺ azacT: MoFlo sorted GFP⁺ cells after treatment of AzaC; GFP⁻ azacT: MoFlo sorted GFP⁻ cells after treatment of AzaC.