Almasi et al. Induction of Treg cell development in the presence of silymarin was performed on isolated na?ve CD4+ T cells. Effect of silymarin-induced Tregs on T cell suppression was determined by CFSE labeling method. Results of this part showed that silymarin significantly decreased IFN, RORt and IL-17 gene expressions and upregulated Foxp3, TGF- and IL-10 mRNA. More silymarin-enhanced na?ve CD4+ T cells differentiated to Tregs (67%) than the control (47%). Silymarin-induced Tregs reduced proliferation of na?ve activated T cells ( 50%). For in vivo study, mice were immunized with ovalbumin (Ova) on days 1 and 14. Silymarin (100?mg/Kg) was intraperitoneally administered two days before the first Ova challenge followed by on every day for two weeks. Splenocytes were then isolated for assessment of CD4+ T cell subsets and ex vivo analysis using flow cytometry. Treatment of Ova-immunized mice with silymarin increased Tregs (11.24??1.2%, L. Gaertn. (Asteraceae). It consists of one flavonoid (taxifolin) and a family of flavolignans including silybin A, silybin B, isosilybin A, isosilybin B, silychristin, isosilychristin, silydianin and also contains a small amount of fatty acids and polyphenolic compounds [13]. Silymarin has anti-inflammatory, anti-fibrotic and antioxidant properties, therefore, this extract is used to reduce inflammatory reactions and fibrogenesis in chronic liver diseases [14C17]. In several clinical trials the protective effects of silymarin in patients with cirrhosis as well as cancer, hepatitis C, diabetes and hypercholesterolemia have been shown [18]. Previous studies investigated the immunomodulatory function of silymarin. The results indicated that this extract prevented the production of proinflammatory cytokines from CD4+ T Micafungin cells and attenuated the proliferation of these cells in response to specific antigens such as those of hepatitis C virus, candida and tetanus, mitogenic stimulation and anti-CD3 antibody [19, 20]. Silibinin, the main compound of silymarin, inhibited the expression levels of the cytokines TNF, IFN, IL-4, IL-2 and inducible nitric oxide synthase (iNOS) in the liver [21]. Mice received intraperitoneal injections of silymarin had evidence of suppressed T cell function [13]. Almasi et al. in their study showed that silymarin inhibited the proliferation of mitogen-stimulated T cells. The authors suggested that silymarin had significantly stronger suppressive activity on T cell proliferation compared to FK506 and rapamycin [22]. Gharagozloo et al. reported that silymarin repressed in vitro cell proliferation by inducing arrest Micafungin in the G1 FNDC3A phase of the cell cycle and inhibited mTOR signaling pathway in human stimulated T cells. Their research showed the capability of silymarin to reduce T lymphocyte activation and proliferation in mice by inhibition of nuclear Micafungin factor (NF)-B activation and preventing its translocation to the nucleus [23]. These data have indicated that silymarin has various significant anti-inflammatory and immunomodulatory effects which are especially noted Micafungin by inhibition of CD4+ T cell proliferation and function. However, to the best of our knowledge there is a lack of adequate data on the influence of silymarin on T cell subsets particularly Treg cells as the central cells of immunoregulation and Th17 cells. Th17 cells similar to Th1 cells are the major cells that contribute to T cell-mediated inflammation and autoimmune disease. Therefore, in the present study we aim to investigate the in vivo and in vitro effects of silymarin on induction and differentiation of Treg cells. Inhibition of immune responses due to suppressive activities attributed Treg cells is closely related to the presence of the inflammatory T cell subsets, Th1 and Th17 and their balance with Treg cells. Therefore, we have also assessed the effects of silymarin on Th1 and Th17 responses. Materials and methods Reagents Dimethyl sulfoxide (DMSO), trypan blue, propidium iodide, Roswell Park Memorial Institute 1640 (RPMI 1640) culture medium and silymarin were obtained from Sigma St. Louis, MO. The silymarin was free of endotoxin as described before [24]. Fetal bovine serum (FBS) was obtained.