Pathogen primed memory T cells can cross-react with allogeneic antigen and mediate graft rejection, a process termed allogeneic heterologous immunity. Th17 cells are pro-inflammatory CD4⁺ T cells that provide immunity to pathogens and are pathogenic in autoimmune disease. Previous work demonstrated that Th17 memory cells are uniquely resistant to the CD28/CTLA-4 blocker belatacept and associated with acute rejection in renal transplant recipients, but the mechanisms of this resistance remain unclear. We found that Th17 cells express higher levels of CTLA-4 and are more sensitive to CTLA-4 checkpoint blockade, suggested that their resistance to belatacept may be related to belatacept-mediated inhibition of CTLA-4 coinhibitory signals. As such, we hypothesized that selective CD28 blockade with a domain antibody (dAb) would effectively suppress Th17 cells by leaving coinhibitory CTLA-4 signaling intact. We stimulated human peripheral blood T cells in the presence of CD28 dAb or control dAb and endogenous cosignaling receptors. Although CD4⁺ Th1 and Th17 memory cells both constitutively express the costimulatory receptor CD28, selective CD28 blockade inhibited Th1 cells but surprisingly augmented Th17 responses. Conversely, CD28 ligation augmented Th1 cells but inhibited Th17 populations. CD28 ligation of purified CD4⁺ T cells induced similar levels of the activation markers CD69 and CD25 on Th1 and Th17 populations, but significantly induced CTLA-4 surface expression on Th17 memory cells, demonstrating that CD28 costimulation results in a selective upregulation of high CTLA-4 expression on Th17 cells. On CD4⁺ T cells, the loss of expression of FOXO3, but not FOXO1, correlated with higher expression of CTLA-4, suggesting a repressive role for this transcription factor in Ctla4 expression. Relative to Th1 cells, Th17 cells also expressed lower levels of the transcription factor FOXO3, suggesting that FOXO3 may specifically regulate high CTLA-4 expression on Th17 memory cells. Thus, high CTLA-4 expression on human memory Th17 cells is dependent on CD28 costimulatory signals, and these cells selectively lose expression of the transcription factor FOXO3 relative to Th1 cells. These results demonstrate that intact CTLA-4 coinhibitory signaling is essential for the suppression of Th17 responses.

CITATION INFORMATION: Krummey S, Suchard S, Nadler S, Larsen C, Ford M. High CTLA-4 Expression and Diminished FOXO3 Expression Underlie Differential CD28 Signaling on Human Memory Th17 Cells. Am J Transplant. 2017;17 (suppl 3).

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