Properties of TNFR2, PDL-1 dependent human CD8+ regulatory cells generated ex-vivo that can be strongly protective in vivo (116.4)

Abstract

In addition to CD4+CD25+Foxp3+ Tregs, natural and induced CD8+ Tregs have been described. Here we report our progress in generating human CD8+ Tregs ex-vivo. Naïve CD8+ cells were activated with anti-CD3/28 beads with IL-2 ± TGF-β for 1 to 9 days. As with CD4+ cells, TGF-β markedly enhanced expression of Foxp3. TGF-β also enhanced CD122, CD103, and PD-1, but decreased granzymes, HLA-DR, CD80 and CD86 expression. Sustained Foxp3 expression and suppressive function was IL-2 dependent. Secondarily stimulated CD8+ cells proliferated weakly, and suppressed activation, proliferation and cytokine production by CD4+ cells in vitro. In vivo functional activity was assessed in NOD SCID IL-2R cγ chain-/- mice injected with allogeneic human PBMC. While human CD4+Foxp3+ Tregs doubled survival from GVHD, CD8 Regs, remarkably, quadrupled survival. Only previously activated CD8+ cells had suppressive activity and protection was unrelated to Foxp3 expression. Adding retinoic acid or rapamycin to TGF-β did not improve survival. By contrast, these activated CD8+ cells rapidly expressed TNFR2 and PDL-1 within one day, and at all time points studied only cells expressing these markers had suppressive activity. Suppressive activity in vivo was IL-10 dependent and partially TGF-β dependent. Thus, in agreement with functional CD8regs described in lupus, diabetes and multiple sclerosis, protective CD8 regs can be induced ex-vivo, and the therapeutic potential of TNFR2, PDL-1+ cells should be carefully explored.