The Potential Role And Its Mechanism Of Mammalian Target Of Rapamycin Complex 2 In Interleukin-35 Mediated T Lymphocyte Proliferative Suppression

Objective:The present study was performed to investigate:(1) the effect of interleukin-35 (IL-35), an immunomodulatory factor, on proliferation of T lymphocytes; (2) the potential role of mammalian target of rapamycin complex 2 (mTORC2) in the process of IL-35 mediated T lymphocyte proliferative suppression; and (3) the regulatory mechanism underlying mTORC2 in IL-35 mediated T lymphocyte proliferative response, particularly with mTORC2-SGK1-p27 signal pathway. Thus, it may provide new clues for development of novel immunomodulatory strategy for sepsis.Methods:Jurkat cells used as a cell model of human T lymphocytes were suspended in RPMI-1640 with 10% FBS at 1×105 cell/ml on 12-well cell culture plates. Activated by PMA (50 ng/ml) and Ionomycin (1μM) for 6 h, then cells were stimulated with IL-35 at different time points of 12,24,48 h, and different doses of 10, 50,250 ng/ml, and subjected to the following experiments.1. Proliferative activity of T lymphocytes stimulated by IL-35 was determined by CCK method. Then, the time/dose effect of IL-35 on proliferative activity of T cells was analyzed. By use of flow cytometry, cell cycle distributions of T cells stimulated by IL-35 were examined. 2. By use of Western blot (WB) and confocal immunofluorescence, expression of mTORC2 (in particular, its core component:mTOR and Rictor) and its downstream signal molecules including SGK1 and p27 were measured. Then, the potential role of mTORC2 in IL-35 mediated T cell proliferative suppression were analysed.3. In order to identify the regulatory pathway of mTORC2 and its downstream signal molecules including SGK1 and p27 in the process of IL-35 mediated T cell proliferative ersponse, we used lentiviral transfection to regulate the key component of mTORC2-Rictor, and constructed cell model. Cell models were stimulated with IL-35 as we did before. With methods of CCK and WB, proliferative activity of T lymphocytes and protein expressions of mTOR, SGK1 and p27were determined in order to clarify the key point of mTORC2 and its downstream signal molecules including SGK1 and p27 in the process of IL-35 mediated T cell proliferative suppression.Results:1. IL-35 could inhibit the proliferation of T cells in a time-and does-dependent manner. In comparison with the control group, it was noted that the proliferative rates of T cells were significantly inhibted at 12 h in the test group treated with IL-35 in 50 ng/ml and 250 ng/ml (P<0.05). In the test group with the dose of 10 ng/ml, suppressive proliferation of T cells was also found at 24nh and 48 h (P<0.05). Results from flow cytometry showed that more T cells were arrested in G0/G1 phase in the presence of IL-35 (50 ng/ml and 250 ng/ml) compared with the control group (P<0.05).2. Role of mTORC2 in IL-35 mediated T cell proliferative suppression:results from WB showed that phosphorylation of protein mTOR, Rictor, SGK1 and p27 was markedly enhanced in the group stimulated by P/I (activation control group) compared with negative control group which T cells were stimulated by PBS (p-mTOR Ser2481, p-Rictor Thr1135, p-SGK1 Ser422, p-p27 T157; P<0.05); Phosphorylation of protein mTOR, Rictor, SGK1 and p27 in the test group which T cells were stimulated with various doses of IL-35 was significantly down-regulated in comparison to the activated control group (P<0.05), however, no statistical difference was noticed in expression of whole protein levels of signal molecules in various groups. In addition, results from confocal immunofluorescence examination confirmed the changes in p-mTOR Ser2 481, p-Rictor Thr1135, p-SGK1 Ser422 and p-p27 T157in T cells stimulated by IL-35.3. Cells were transfected with lentiviruses which designed to regulate phosphorylation site Thr1135 of Rictor. Then the cells were stimulated with IL-35 again to determine changes in proliferative rates. Results showed that interference by lentiviruses on site Thr1135 of Rictor could obviously reverse the effect of IL-35 mediated T cell proliferative suppression. Additionally, results from WB showed that in cells interfered by lentiviruses on site Thr1135 of Rictor stimulated by IL-35, no statistical differences in phosphorylation of protein mTOR, SGK1, and p27 could be noted compared with the control group which cells were expressed normal Rictor.Conclusions:IL-35 can suppress the proliferative response of T lymphocytes in a time- and dose- dependent manner. Cell cycle arrest appears to be the important way involved in IL-35 mediated proliferative suppression of T cells. mTORC2 whose core components are mTOR and Rictor plays a key role in the regulation of IL-35 mediated T cell proliferative response, and mTORC2-SGK1-p27 seems to be a major signal pathway in this process. Therefore, interference of protein Rictor might markedly regulate the effect of IL-35 on proliferation of T cells.