Abstract
The CD4+CD25+FOXP3+ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy. Forkhead box P3 (FOXP3) is a key transcription factor governing Treg lineage differentiation and their immune-suppressive function. Here, using Treg cells, as well as HEK-293T and Jurkat T cells, we report that the stability of FOXP3 is directly and positively regulated by the E3 ubiquitin ligase ring finger protein 31 (RNF31), which catalyzes the conjugation of atypical ubiquitin chains to the FOXP3 protein. We observed that shRNA-mediated RNF31 knockdown in human Treg cells decreases FOXP3 protein levels and increases levels of interferon-γ, resulting in a Th1 helper cell-like phenotype. Human Treg cells that ectopically expressed RNF31 displayed stronger immune-suppressive capacity, suggesting that RNF31 positively regulates both FOXP3 stability and Treg cell function. Moreover, we found that RNF31 is up-regulated in Treg cells that infiltrate human gastric tumor tissues compared with their counterparts residing in peripheral and normal tissue. We also found that elevated RNF31 expression in intratumoral Treg cells is associated with poor survival of gastric cancer patients, suggesting that RNF31 supports the immune-suppressive functions of Treg cells. Our results suggest that RNF31 could be a potential therapeutic target in immunity-based interventions against human gastric cancer.
Highlights
The CD4؉CD25؉Forkhead box P3 (FOXP3)؉ regulatory T (Treg) cells are critical for maintaining immune tolerance in healthy individuals and are reported to restrict anti-inflammatory responses and thereby promote tumor progression, suggesting them as a target in the development of antitumor immunotherapy
We examined the activity of Treg cells using an in vitro suppression assay, and found that Treg cells after ring finger protein 31 (RNF31) knockdown had significantly impaired suppressive capacity toward Tconv cell proliferation (Fig. 1, F and G), revealing that RNF31 is indispensable for human Treg cell function
Our work reports that RNF31 is required for maintaining human Treg lineage stability and cell function via affecting FOXP3 protein stability directly
Summary
Regulatory T cell; LUBAC, linear ubiquitin chain assembly complex; IL-2, interleukin 2; shRNA, short hairpin RNA; INF␥, interferon ␥; MFI, mean fluorescence intensity; Ab, antibody; TNF␣, tumor necrosis factor ␣; IP, immunoprecipitation; IF, immunofluorescence; IB, immunoblot; DAPI, 4Ј,6-diamidino-2-phenylindole; PBMC, peripheral blood mononuclear cell; HRP, horseradish peroxidase; NEMO, NF-B essential modulator. It has been reported to be involved in innate and adaptive immune responses downstream of TLR, NLRP3, T cell receptor, B cell receptor, NOD2, and TNFR ligation [17,18,19,20,21,22] These signals involve the linear ubiquitination of ASC to facilitate NLPR3 inflammasome assembly and NEMO to strengthen canonical NF-B activation [19, 23]. It has been demonstrated that Treg cell–specific ablation of RNF31 in mice causes severe Treg cell deficiency and lethal immune pathology, revealing an ongoing requirement of LUBAC activity for Treg cell homeostasis [24] Whether it is critical for human Treg function and the underlying mechanism requires further exploration. Here we characterized a unique regulatory pathway for FOXP3 stability, which could be a potential drug target for anti-tumor immunotherapy
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