The modulation of PI-3K/Akt-induced signaling is critical for CD4⁺ Treg homeostasis and stability. Moreover, activation of PI-3K/Akt/mTOR signals within T cell subsets is associated with enhanced Teff cell activity. DEPTOR is a recently discovered mTOR-binding partner and a negative regulator of the Akt/mTOR signaling pathway. However, its expression and function in T cell activation and alloimmunity is unexplored.

By qPCR, Western blot analysis and immunofluorescence, we find high levels of DEPTOR expression in CD4⁺ T cells, and further we observed that its expression levels were reduced at early times (<3h) following mitogen activation. We harvested CD4⁺ T cells from a doxycycline (dox)-inducible DEPTOR transgenic mouse (rtTA^+/+DEPTOR^+/+, iDEP) to evaluate the effect of DEPTOR overexpression on Teff/Treg responses. Treatment of iDEP CD4⁺ T cells with dox (0.3-3 mcg/ml) resulted in a marked induction of DEPTOR, and this effect was associated with reduced expression of pS6K and induced expression of pAkt(S473). We also cultured naive CD4⁺CD25^– iDEP cells in standard iTreg-inducing media (anti-CD3/anti-CD28, TGF-β, IL-2 and retinoic acid) ± dox (3 mcg/ml). We observed that ~80% of the iDEP cells expressed Foxp3 and the addition of dox into cultures (to force DEPTOR expression) had no effect on iTreg differentiation. Nevertheless, in preliminary experiments, dox-treated and DEPTOR-expressing iTregs were more potent to inhibit Teff proliferation in suppression assays in vitro. Also, as expected, dox had no effect on suppressive potential of rapamycin-induced iTreg. We assessed iTreg stability over 7 days by monitoring Foxp3 expression upon restimulation with anti-CD3/anti-CD28. While only 9.9±5.8% of iTreg retained expression of Foxp3 after 7 days of restimulation, the addition of dox (3 mcg/ml to induce DEPTOR) resulted in significantly (p<0.001) higher frequencies of Foxp3⁺ Tregs (46.1±6.0%).

In summary, our findings identify DEPTOR as a cell intrinsic regulator of the mTOR pathway in CD4⁺ T cells and as endogenous immunoregulatory protein that stabilizes Foxp3 expression and enhances Treg function in vitro.

CITATION INFORMATION: Wedel J, Bruneau S, Briscoe D. DEPTOR Inhibits mTORC1 in CD4⁺ T Cells and Increases Treg Function by Stabilizing Foxp3 Expression. Am J Transplant. 2016;16 (suppl 3).

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