ALBERT

Description: Abstract 1008 Regulatory T cells (Tregs) have been shown to be potent inhibitors of autoimmunity, and to be capable of suppressing alloimmune responses that occur during both allograft rejection and graft-versus host disease. However, they have yet to gain widespread use clinically, due in part to the fact that it remains extremely costly and difficult to produce them in sufficient numbers and with sufficient suppressive capacity to significantly impact the alloimmune response. Here we have used our established non-human primate model to demonstrate that significant Treg expansion (up to 600-fold in 21 days) can be maintained, and suppressive capacity enhanced by exposing Treg cultures to a short burst of sirolimus at the end of the culture period. Using a highly sensitive and specific in vitro CFSE-MLR assay we show that Tregs significantly inhibit allo-proliferation of multiple T cell subpopulations including both CD4+ and CD8+ T cells (3.2 and 2.7-fold inhibition of proliferation, respectively), as well as their CD28+CD95+ and CD28-CD95+ subpopulations (2.2 and 2.1 and 1.9 and 2.7-fold inhibition of CD4+ and CD8+ subpopulation proliferation, respectively). Tregs were able to combine in vitro with the newly FDA-approved CTLA4-Ig analog belatacept to enhance the inhibition of alloproliferation that occurred with either agent alone (4.8-fold inhibition of CD8 T cell proliferation with Tregs + belatacept, compared to 3.0-fold or 1.9-fold inhibition of CD8 T cell proliferation with Tregs or belatacept alone, respectively). Importantly, we have found that the suppressive activity of ex-vivo expanded Tregs could be further enhanced by pulsing with sirolimus. Thus, while long-term culture of Tregs in the presence of sirolimus (1–1000 nM) profoundly inhibited Treg expansion (50–800 fold inhibition of expansion when cultured in the presence of 1–1000 nM sirolimus), a 48 hour pulse of sirolimus (100 nM) on days 20–21 of culture completely preserved Treg yields while doubling their suppressive function against CD8 proliferation when compared to unpulsed Tregs, p