rapamycin, and TGFb on human Treg cell generation ex vivo. Normal donor CD4 T cells were isolated by CD4 positive selection and CD41CD127-cells were isolated by CD127 negative selection and CD4 positive selection. Relative to total CD4 T cells, CD41CD127-cells were enriched for the Treg transcription factor, Foxp3 (n 5 8 experiments; P 5 0.042); and also had reduced secretion of IL-2 (P 5 0.05) and IFN-g (P 5 0.03). Both subsets were expanded using anti-CD3, anti-CD28 co-stimulation and IL-2 6

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... apamycin and 6 TGF-b. Relative to culture input of total CD4 cells, input of CD41CD127-cells were potent suppressors at day 12, as measured by inhibition of responder T cell proliferation in response to allogeneic dendritic cell (DC) stimulation. The enhanced capacity of CD127 negative selection to generate Tregs occurred whether expansion was performed in rapamycin (P 5 0.02), TGFb (P 5 0.02), or combination of both (P 5 0.01). CD127 negative selection alone (without rapamycin or TGF-b) was not sufficient to generate Treg cells, as the resultant product secreted high levels of IL-2 and IFN-g. In contrast, addition of rapamycin or TGF-b, and in particular the combination, resulted in a Treg phenotype as defined by: (1) high Foxp3 expression (50% CD41Foxp31); (2) reduced Th1 cytokine secretion; and (3) suppressor function in the allo-MLR. Transwell showed that expanded Treg cell suppression was contact dependent. We hypothesized that such suppression was mediated via modulation of DC function; to address this, Treg cells and DC were co-cultured, and purified "conditioned" DC were utilized as the APC source in allo-MLR. Indeed, DC conditioned by Treg cells generated from CD41CD127-enriched cells and expanded in rapamycin and TGF-b had: (1) significantly reduced secretion of IL-6 and TNF-a; (2) increased expression of the inhibitory molecule PDL1; and (3) greatly reduced allostimulatory function; importantly, antibody blockade of DC expression of PDL1 partially reversed the suppressive DC phenotype. In conclusion, CD127-selction combined with ex vivo expansion in rapamycin and TGF-b generates human Treg cells that inhibit alloreactivity by modulating DC function. Adotive transfer of such Tregs has implications in preventing GVHD after haematopoietic stem cell transplantation.