Elevated level of Interleukin-35 in colorectal cancer induces conversion of T cells into iTr35 by activating STAT1/STAT3

IL-35 is a novel heterodimeric and inhibitory cytokine, composed of interleukin-12 subunit alpha (P35) and Epstein-Barr virus -induced gene 3 (EBI3). IL-35 has been reported to be produced by a range of cell types, especially regulatory T cells, and to exert immunosuppressive effects via the STATx signaling pathway. In this study, we demonstrated that IL-35 expression was elevated in both serum and tumors in patients with colorectal cancer. IL-35 mainly expressed in CD4⁺ T cells in human colorectal cancer tumors and adjacent tissues. Increased IL-35 expression in tumoradjacent tissues was significantly associated with tumor metastasis. IL-35 inhibited the proliferation of CD4⁺CD25^- T effector cells in vitro in a dose-dependent manner, and its suppression was partially reversed by applying IL-35-neutralizing antibodies. IL-35 treatment activated the phosphorylation of both STAT1 and STAT3 in human CD4⁺ T cells. Meanwhile, IL-35 induced a positive feedback loop to promote its own production. We observed that Tregs obtained from colorectal cancer patients were capable of inducing more IL-35 production. In addition, EBI3 promoter-driven luciferase activity was higher than that of the mock plasmid after IL-35stimulation. Thus, our study indicates that the high level of IL-35 in colorectal cancer promotes the production of IL-35 via STAT1 and STAT3, which suppresses T cell proliferation and may participate in tumor immunotolerance.