337: The Predictive Value of Gene Expression Profiles for Acute Graft-Versus-Host Disease after Hematopoietic Cell Transplantation with Nonmyeloablative Conditioning for Hematological Malignancy
Biology of Blood and Marrow Transplantation
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
... 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.