Foxp3 is subjected to multiple post-translational modifications that govern Treg function. E.g. The histone acetyltransferases, p300 and CBP, acetylate Foxp3, increasing its stability and promoting Treg function. In the absence of acetylation, ubiquitylation and proteasomal degradation of Foxp3 can occur. We reasoned that additional levels of regulation might apply given the vital role of Foxp3 in controlling host immune homeostasis, especially given that protein ubiquitylation can be reversed by DUB enzymes that regulate diverse biologic processes. We now present biochemical and in vivo evidence that a DUB, USP7, is essential for stabilizing Foxp3 expression and controlling Treg function. First, USP7 is highly expressed in both Tcon and Treg cells, and Foxp3 is physically associated with USP7. Second, cotransfection of 293T cells with Foxp3 and USP7 increased Foxp3 production compared to Foxp3 transfection alone. Third, Tregs incubated for 2 hrs with a specific USP7 small molecule inhibitor (USP7i) and washed, showed decreased Foxp3 protein expression and almost complete loss of suppresive function in vitro. Fourth, comparable brief USP7i treatment significantly impaired Treg function in a model of homeostatic proliferation in lymphopenic mice (p<0.01). Fifth, USP7i delivery via Alzet pump revoked Treg-dependent cardiac allograft survival (BALB/c->C57BL/6, p<0.01). Sixth, conditional deletion of USP7 in Tregs, by mating USP7flfl and Foxp3cre mice, led to mice that were born at an expected normal frequency but that died within 4 weeks of birth. These mice had enlarged lymph nodes and spleens, and dense mononuclear cell infiltration of lung and liver tissues. Their analysis showed normal thymic Treg development, but Foxp3+ Treg numbers were markedly decreased in peripheral lymphoid tissues, along with broad Tcon cell expression of activation markers, such as increased CD44 and CD69 and decreased CD62L. Seventh, as with USP7i treatment, and in contrast to WT Tregs, the in vitro suppressive function of USP7-/- Treg was markedly impaired. In conclusion, USP7 has an essential and hitherto unrecognized role in maintaining Foxp3 expression in Treg cells. As a result, therapies that impact USP7 should be avoid in allograft recipients, whereas this strategy may have therapeutic relevance in the management of patients with malignancies.

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