Fig. 2

Mreg-cocultured T cells inhibit T-cell proliferation and mo-DC maturation. The functional consequences of exposing CD3⁺ T cells to allogeneic Mregs were investigated in coculture experiments. a Human Mregs generated from CD14⁺ monocytes were cultured together with allogeneic T cells for 5 days. T cells were then analysed by flow cytometry, qPCR, microarray and functional assays. b Proliferation of CFSE-labelled responder CD4⁺ T cells stimulated with plate-bound αCD3 was inhibited by allogeneic Mreg-cocultured T cells to a greater degree than by T cells cultured alone for 5 days (n = 6; mean ± sd). c CD4⁺ T cells flow-sorted from Mreg cocultures antagonised upregulation of CD80 and CD83 by mo-DCs stimulated with 50 ng ml⁻¹ TNFα for 2 days (representative of n = 6). d CD3⁺ T cells cocultured with allogeneic Mregs for 5 days were enriched for FoxP3⁺ T cells that were readily discriminated from CD25⁺ FoxP3^−/low T cells activated by αCD3/αCD28 stimulation for 5 days (representative of n = 4). e Twenty-four-hour secretion of IL-10 by αCD3-stimulated CD4⁺ T cells re-isolated from allogeneic Mreg cocultures (n = 4; KW). f Most IL-10-producing T cells from Mreg cocultures were FoxP3⁺ T cells and vice versa (representative of n = 6 donors). g Enrichment of FoxP3⁺ T cells from naive CD25⁻ CD45RA⁺ CD4⁺ T cells, which were predominantly FoxP3⁻, was significantly more efficient than enrichment from unfractionated CD3⁺ T cells (n = 6; MW). h Coculture with allogeneic Mregs led to a significant enrichment of FoxP3⁺ T cells from a starting population of unfractionated CD3⁺ T cells. By contrast, CD3⁺ T cells that were unstimulated, polyclonally activated or cocultured with IFN-γ Mφ were not enriched for FoxP3⁺ T cells (n = 6; KW). i No increase in TSDR demethylation was observed in CD3⁺ T cells cocultured with Mregs; therefore, enrichment of FoxP3⁺ T cells reflects conversion of non-Tregs (n = 6; KW)