αcd3 okt3

CD14+monocytes and CD4+CD127low T-cells were isolated from PBMCs of healthy blood donors using RosetteSep^™ Human Monocyte Enrichment Kits and CD4+CD127low T-cell Enrichment Kits, respectively (Stem Cell Technologies). CD14+monocytes were further purified to >97% purity by magnetic separation with anti-CD14 conjugated microbeads (Miltenyi Biotec). Memory CD4+ Th (T helper) and Tregs were obtained by sorting pre-enriched T-cells with fluorescently labeled mAbs against CD4, CD25, CD127, CD45RA, CD45RO and lineage markers along with propidium iodide (PI; Life Technologies). Th were defined as PI-Lin-CD4+CD45RA-CD45RO+CD127+CD25-/low, and Tregs were defined as PI-Lin-CD4+CD127low CD25+. Cells were sorted with a BD FACSAria IITM after staining, as above. Monocytes (1×10⁶) were cultured with allogeneic Th and Tregs at a 10:1:1 in 12-well plates with 50ng/ml αCD3 (OKT3, BioLegend). HLA-DR+CD2- DCreg were sorted out by flow cytometry on day 4 of culture. DCreg were stimulated with 1μg/ml LPS. After 18 hr, cells were washed 3x in PBS and HLA-DR+CD2- DC were purified by FACS and incubated with allogeneic naïve CD4+ T-cells (10⁵/well) at a 1:2 DCs to T-cell ratio in the presence or absence of 2μg/ml anti-TGFβ. After 6 days, responder CD4+ T-cells were analyzed for CD25 and Foxp3 expression.