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Objective  The benefits of IL-35 treatment have been verified in multiple animal models of diseases, while its influence on T cells immunity under normal condition still needs to be elucidated. The present study was designed to investigate the effects modulating IL-35 levels  in vivo  and  in vitro  on T cells, response and also the effects on T cells subsets in normal mice.    Methods  A plasmid pMSCV-IL-35-GFP carrying mouse linear IL-35 fragment with two subunits joint together was constructed and the heterodimer expression was confirmed. Normal mice were randomly divided into three groups and received an intravenous injection of PBS, pMSCV-GFP and pMSCV-IL-35-GFP respectively. After 72 h, spleen tissues and peripheral blood were harvested for following analysis. Meanwhile, splenic T cells were isolated and incubated with 10, 30, or 50 ng/mL recombinant IL-35 factor for 24 h with the addition of anti-CD3/CD28  in vitro . T-cell subsets were assessed by Fluorescence activated cell sorting (FACS) and related cytokines together with effector molecules were determined by real time PCR.    Results  Western blotting confirmed a 52 kDa band in the cell lysate of HEK 293T transducted with pMSCV-IL-35-GFP plasmid, indicating a successful expression of IL-35. Ebi3 and IL-12A, two subunits of IL-35, could be identified 72 h post DNA injection. IL-35 upregulation  in vivo  effectively inhibit CD4 +  and CD8 +  T cell proliferation and Th1 cytokine secretion. Effector molecules of CD8 +  T cells were also remarkably suppressed. On the contrary, high level of IL-35 significantly induced CD4 +  CD25 +  Tregs and Th2 enhancement. The  in vitro  study provided similar results.    Conclusion  The results indicated Th1 and CD8 +  T cell inhibition and Th2 and Tregs bias in the presence of IL-35 under a normal state which partly contributed to its therapeutic potential.

Interleukin 35 induced Th2 and Tregs bias under normal conditions in mice