Foxp3+ Tregs are essential for the induction of transplant tolerance and OX40 costimulation is known to break transplant tolerance. The exact mechanism of OX40 in peripheral conversion of conventional T cells to inducible Foxp3⁺ Tregs (iTregs) which can be donor specific, remains poorly defined. Here we demonstrated that OX40 triggers multiple signaling pathways that act in a redundant fashion in suppressing Foxp3 expression, and consequently iTreg induction. We found that OX40 costimulation upregulates Batf3 and Batf expression in activated CD4⁺ T cells and that Batf3 and Batf proteins act as potent repressors of Foxp3 expression. Mechanistically, the Batf proteins bind the Foxp3 locus, and by recruiting the histone deacetylases Sirt1 and Sirt7, they repress Foxp3 expression by inducing a closed chromatin structure. However, OX40 remains capable of suppressing Foxp3 expression in Batf3 and Batf deficient T cells, and in the absence of both Batf proteins, OX40 activates the AKT-mTOR-FOXO1 pathway to suppress the induction of Foxp3⁺ Tregs. Indeed, the suppressive effects of OX40 in iTreg induction can be completely abrogated by targeting the Batf3/Batf and the Akt-mTOR pathway. We also examined whether OX40 costimulation inhibits allogeneic iTreg generation in vivo through Batf proteins and the mTOR pathway in a donor-specific cell transfer model, we adoptively transferred CD45.2⁺ Wt and Batf^-/-Batf3^-/- naïve CD4 T cells into CD45.1⁺ OX40L-Tg mice; some host mice were also injected with allogeneic Balb/c splenocyes and treated with rapamycin for 4 days, and then examined the induction of Foxp3+ T cells within the CD45.2+ fraction. The frequency of induced Foxp3+ T cells from Batf3^-/-Batf^-/- CD4+ T cells following rapamycin treatment in the lymph nodes and the lungs in the OX40L-Tg hosts was significantly higher than those of other groups. Thus, our data uncover new mechanistic insights into OX40-mediated suppression of allogeneic Foxp3⁺ iTregs, and these findings may have important clinical implications in tolerance induction.

CITATION INFORMATION: Zhang X., Xiao X., Lan P., Li J., Dou Y., Chen W., Ishii N., Chen S., Taparowsky E., Li X. OX40 Costimulation Prevents Induction of Foxp3⁺ Tregs through Upregulation of Novel Repressors of Foxp3 Gene Expression Am J Transplant. 2017;17 (suppl 3).

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