DEPTOR is an evolutionarily conserved, first-in-kind cell intrinsic factor with potential to regulate the activity of multiple intracellular signaling pathways. However, its biology in T cell-dependent immunity is not yet known. By Western blot, we find high levels of DEPTOR in unactivated murine CD4⁺ T cells, but its expression decreases rapidly (3-24 hrs) following stimulation with mitogen (anti-CD3). We isolated CD4⁺ T cells from doxycycline (dox)-inducible DEPTOR (iDEP) transgenic mice (C57BL/6, H-2^b), and treated the cells with dox (0.3-3 μg/ml). By Western blot, dox markedly induced DEPTOR within unactivated and mitogen-activated CD4⁺ cells. Forced overexpression was associated with reduced (P<0.05) proliferative responses to anti-CD3/anti-CD28 (³H-thymidine), and reduced IL-2, IL-4 and IFNγ production (P<0.05) by quantitative multianalyte profiling, ELISPOT and qPCR vs. no dox iDEP cells and WT cells with/without dox. By FACS, CD25 expression on iDEP and WT CD4⁺ T cells were similar, and addition of IL-2 did not reverse the anti-proliferative effect of cell intrinsic DEPTOR overexpression. By Western blot, DEPTOR overexpression resulted in a marked inhibition of STAT1 (Y701/S727) activity/signaling, but had minimal effects on PI-3K/Akt/mTOR and MAPK signaling, suggesting that its primary function is to modulate cytokine receptor-mediated activation. To determine function in vivo, we performed fully MHC mismatched BALB/c (H-2^d) cardiac transplants in iDEP or WT C57BL/6 (H-2^b) recipients. Recipient mice were fed dox chow pre- and post-transplant. We found that induced DEPTOR in iDEP recipients resulted in significant prolongation of graft survival (MST 35 days; n=5; P<0.001) vs. WT mice + dox (MST 8 days; n=7) vs. WT mice no dox (MST 7 days; n=14). To further determine if CD4⁺ T cell DEPTOR modulates alloimmunity, in preliminary studies we performed BALB/c (H-2^d) heterotopic cardiac transplants in Rag2^-/- IL2Rγ^-/- (H-2^b) mice. On day 2 post-transplant, adoptive transfer of alloprimed iDEP CD4⁺ T cells was performed in dox-treated or no dox recipients. Again, induced DEPTOR resulted in prolonged graft survival (MST >24 days; n=2) vs. controls (MST 14 days; n=2). Collectively, these findings for the first time identify DEPTOR as a novel cell intrinsic immunomodulator that functions in effector CD4⁺ T cell activation in vitro and in vivo. Our observations indicate that DEPTOR activity is critical in CD4⁺ T cell homeostasis and alloimmunity.

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