PMC:2806624 / 42890-44551 JSONTXT

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Positive and negative control gates were made according to T cells cultured only in the presence of IL-2 without anti-CD3/28 stimulation.\nHuman naive CD4+ T cells were isolated by negative selection by MACS from PBMCs and either transfected with a scrambled siRNA or with a combination of RUNX1 and RUNX3 siRNA. Cells were then cultured under iT reg cell differentiating conditions and mixed with 2 × 105 autologous irradiated PBMCs that were used as APCs and autologous CFSE-labeled CD4+ T cells. T reg cell to responder cell ratio was 1:20, 1:10, and 1:5. To check the proliferation of the CD4+ T cells without suppression, no T reg cells were added in a control group. Cells were stimulated with 2.5 µg/ml anti-CD3 mAb, cultured in a 96-well plate and the proliferation of the effector cells was determined by analyzing the CFSE dilution by flow cytometry after 5 d of culture. 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