336: Relative Contribution of CD127 Negative Selection, Rapamycin, and TGF-β to the Generation of Human Regulatory T Cells that Inhibit Alloreactivity via Dendritic Cell Modulation

Regulatory T cells (Tregs) express reduced IL-7Rα (CD127) and preferentially expand in rapamycin or TGF-β. Here, we evaluated the independent and combined effect of CD127 negative selection, rapamycin, and TGF- β on human Treg cell generation ex vivo. Normal donor CD4 T cells were isolated by CD4 positive selection and CD4+CD127- cells were isolated by CD127 negative selection and CD4 positive selection. Relative to total CD4 T cells, CD4+CD127- cells were enriched for the Treg transcription factor, Foxp3 (n = 8 experiments; P = 0.042); and also had reduced secretion of IL-2 (P = 0.05) and IFN-γ (P = 0.03). Both subsets were expanded using anti-CD3, anti-CD28 co-stimulation and IL-2 ± rapamycin and ± TGF-β. Relative to culture input of total CD4 cells, input of CD4+CD127- 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 = 0.02), TGF-β (P = 0.02), or combination of both (P = 0.01). CD127 negative selection alone (without rapamycin or TGF-β) was not sufficient to generate Treg cells, as the resultant product secreted high levels of IL-2 and IFN-γ. In contrast, addition of rapamycin or TGF-β, and in particular the combination, resulted in a Treg phenotype as defined by: (1) high Foxp3 expression (∼50% CD4+Foxp3+); (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 CD4+CD127- enriched cells and expanded in rapamycin and TGF-β had: (1) significantly reduced secretion of IL-6 and TNF-α; (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-β 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. Regulatory T cells (Tregs) express reduced IL-7Rα (CD127) and preferentially expand in rapamycin or TGF-β. Here, we evaluated the independent and combined effect of CD127 negative selection, rapamycin, and TGF- β on human Treg cell generation ex vivo. Normal donor CD4 T cells were isolated by CD4 positive selection and CD4+CD127- cells were isolated by CD127 negative selection and CD4 positive selection. Relative to total CD4 T cells, CD4+CD127- cells were enriched for the Treg transcription factor, Foxp3 (n = 8 experiments; P = 0.042); and also had reduced secretion of IL-2 (P = 0.05) and IFN-γ (P = 0.03). Both subsets were expanded using anti-CD3, anti-CD28 co-stimulation and IL-2 ± rapamycin and ± TGF-β. Relative to culture input of total CD4 cells, input of CD4+CD127- 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 = 0.02), TGF-β (P = 0.02), or combination of both (P = 0.01). CD127 negative selection alone (without rapamycin or TGF-β) was not sufficient to generate Treg cells, as the resultant product secreted high levels of IL-2 and IFN-γ. In contrast, addition of rapamycin or TGF-β, and in particular the combination, resulted in a Treg phenotype as defined by: (1) high Foxp3 expression (∼50% CD4+Foxp3+); (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 CD4+CD127- enriched cells and expanded in rapamycin and TGF-β had: (1) significantly reduced secretion of IL-6 and TNF-α; (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-β 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.