We have previously observed that the cell intrinsic modulatory protein DEPTOR (DEP) stabilizes Foxp3 expression, enhances Treg function and inhibits Treg dedifferentiation. In vivo, sustained expression of DEP within Tregs is potent to prolong allograft survival and promote tolerance following cardiac transplantation. Furthermore, novel therapeutics that target DEP degradation have immunomodulatory effects. To investigate the mechanism of function of DEP in CD4⁺ T cell subsets, we generated CD4⁺ T cell-specific DEP knockout (KO) mice and evaluated T cell responses in vitro and in vivo. Our initial findings were surprising in that CD4-DEPKO mice did not develop autoimmune disease for up to 1 yr. Nevertheless, we find increased phosphorylation of S6 and Akt(S473) in CD4⁺ T cells following mitogen-activation, suggesting that mTOR is hyperactive in the absence of DEP. Using RNA-seq, CD4-DEPKO cells show a deficiency in cytokine signaling. To validate this possibility, we performed in vitro T helper cell differentiation assays and find that CD4-DEPKO cells produce more IL-4 and IL-17A vs. controls. We also generated Foxp3-DEPKO mice to evaluate DEP function in immunoregulation; KO mice develop patchy skin lesions with hyperkeratosis and local inflammatory infiltrates over a 9-12 month period. Phenotyping of T cell subsets from Foxp3-DEPKOs was normal at times preceding dermatitis. In contrast, over time Foxp3-DEPKOs had increased numbers of activated splenic CD4⁺ T cells (CD69⁺: 27% vs. 13%, P<0.05) and expanded populations of memory subsets (CD44^highCD62L^low: 93.1% vs. 32.4%; P<0.05). Notably, there was also a dramatic increase in the number of CD4⁺Foxp3⁺ T cells in Foxp3-DEPKO mice (27% vs. 15%; P<0.05), suggesting that there is a high turnover and/or high dedifferentiation of Tregs in the absence of DEP. We also transplanted MHC class II mismatched B6.C-H-2^bm12 hearts into Foxp3-DEPKO recipients (C57BL/6) or controls. Following transplantation, Treg numbers were dramatically increased in KO recipients (Foxp3⁺: 44.8% vs. 21%, P<0.05) and CD4⁺ Teffs were markedly activated (CD69⁺: 60.2% vs. 17.9%, P<0.05). Thus, DEP regulates cytokine responsiveness, T helper cell differentiation as well as Treg function and stability in vivo. In summary, the potential to target DEP with novel therapeutics provides opportunities to promote immunoregulation post-transplantation.

CITATION INFORMATION: Wedel J., Liu K., Sage P., Laplante M., Briscoe D. Mechanisms of DEPTOR Function in CD4⁺ T Cell Subsets Am J Transplant. 2017;17 (suppl 3).

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