Foxp3Interacting Protein UXT Regulates Tregs Function

CD4+CD25+T regulatory cells (Tregs) are known as a subset of CD4+T cells which has the immune suppressive function and play a crucial role in keeping immune homeostasis. Forkhead transcriptional factor box P3(Foxp3) mainly expresses in Treg cells and is important for the negative immune regulation effect of Tregs. Foxp3was described as the master control gene for Treg cells development and differentiation. Subsequent studies have confirmed that Foxp3is the capital regulatory factor of Tregs biological function. The researches around Foxp3is the current hot spot, however, the specific mechanisms regulating its action remain unknown. In the previous work, our group used Foxp3as the bait to screen for its interactive proteins in a human leukocyte yeast two-hybrid library and obtained several molecules. Among these molecules, ubiquitously expressed transcript (UXT) could interact with Foxp3both in yeast and in mammalian cells. We used2recombinant plasmid pEGFP-UXT and pDsRed2-Foxp3to co-transfect HEK293cells. The con-focal fluorescence microscopy result indicated that both Foxp3and UXT were localized in the nucleus. This result was more persuasive for these two proteins interaction. To explore the UXT-binding region within Foxp3, we constructed5deletion mutants of Foxp3.Then the β-galactosidase assays were performed and the results showed that UXT directly interacts with proline-rich domain of Foxp3. This domain of Foxp3was both essential and sufficient to mediate interaction with UXT. In addition, our preliminary result indicated that the forced over-expression of UXT could enhance the Foxp3protein level in gastric carcinoma cell line SGC-7901. However, the mechanism of action is still needed to be further studied. Therefore, the purpose of this study is to explain the biological function and mechanism of UXT in Tregs and to provide a new explanation for Tregs tolerance-inducing modality. The elucidation of the mechanisms of in vivo interaction and regulation of Foxp3will help us to develop new drugs and find therapy to treat cancers.In order to confirm these questions, this study contains3aspects of experiments:1. To certify the co-localization of Foxp3and UXT protein in Treg cells and CD4+T cell lymphoma Jurkat cell line;2. To detect the influence of UXT expression level on the Tregs function changes;3. To explore the molecule mechanism of how UXT affects Tregs and its key element Foxp3.In part one, our data confirmed that Foxp3and UXT were constitutive expressed in freshly isolated human Treg cells. The confocal fluorescence microscopy result demonstrated that both Foxp3and UXT were mainly co-localized in the nucleus and perinuclear area of Tregs and Jurkat lymphoma cells. Tregs from cancer patients expressed a highly significant increase in levels of Foxp3and UXT compared to healthy age-matched controls. These results implied that the Foxp3high expression in colon cancer patients Tregs may has a tightly correlation with UXT high expression and the interaction between these two molecules is likely to affect Tregs function and significant for tumor development.In the second part of our experiments, the BrdU incorporation assay showed that treatment of CD4+C25+Treg cells with SiRNA targeting UXT weakened Tregs suppressive function on wild-type autoallergic responder T cells. Furthermore, electrotransformation mediated transfer of UXT in CD4+CD25-T cells induced a suppressive function like Tregs. Upon stimulation with immobilized anti-CD3, UXT-transduced CD4+CD25-T cells were proliferated signicantly less than control, this hyporesponsiveness phenotype was similar with the Tregs after TCR stimulation. Thus, we suppose UXT plays critical roles in inducing and maintenance of suppressive and hyporesponsiveness phenotype of Tregs. UXT is a possible regulatory factor toward Tregs function.Besides, in part three, we also provide evidence for the functional mechanism of UXT. Though several approaches, including Real-time PCR, flow cytometry and ELISA, we found that UXT up-regulated Treg-associated cell surface markers such as CD25, CD103, CTLA-4and GITR, and promoted the secretion of suppressive cytokines IL-10and TGF-β. These results demonstrate that UXT contributes to Tregs suppressive properties. In addition, UXT transduction effect on the cell cycle progression in T lymphocytes induced hyporesponsiveness to TCR stimulation and suppressed cellular proliferation. As a molecular chaperone, UXT contributed to the sustained presence of Foxp3inside the nucleus. ChIP analysis showed that Foxp3binding to IL-2, CTLA-4, and CD25promoters was increased when UXT was over-expressed, this binding was markedly diminished when UXT was interferenced. The most interesting finding is that UXT can directly regulate the expression of Foxp3. Over-expression of UXT in Treg cells, CD4+T lymphocytes and Jurkat cell line resulted in the up-regulating of Foxp3both in mRNA and protein levels. Luciferase reporter assay proved that UXT executed this promote function in transcriptional level. UXT has the ability to activate the Foxp3promoter. More evidence of molecular mechanism needs to be confirmed in future research.In summary, we performed different experiments to verify the co-expression of UXT and Foxp3in Tregs. The protein expression level changes of UXT altered the suppressive function and hyporesponsiveness of Tregs. UXT may carry out its function through regulating the expression of Treg-associated cell surface markers and the secretion of suppressive cytokines, regulating the cell cycle, promoting the presence of Foxp3inside the nucleus, promoting the transcriptional activity of Foxp3, and directly up-regulating the Foxp3expression. These finding will provide a firenew explanation for the regulation of Tregs and its master control gene Foxp3, and will lay a basis for the elucidation of more deepgoing molecular mechanism.