The Treg percentages were significantly higher in all the experiment groups compared to the control groups. These changes were deduced by applying TGF-β1 neutralizing antibody into the co-culture system. Our results indicated that the

CD4+ T cells can be induced into CD4+CD25+FoxP3+ T cells by BMMCs via TGF-β1. Regulatory T cells (Tregs) can suppress immune responses to donor alloantigens, and have the potential to play an important role in both inducing and maintaining transplant tolerance in vivo[1]. The transcription factor forkhead box P3 (FoxP3) is the recognized master gene governing the development and function of both natural and induced Tregs, especially in mice [2–4]. Mast cells (MCs) have long been recognized as major players in allergy [5], but EPZ-6438 solubility dmso in recent years MCs have been identified as being responsible for a far more complex range of functions in the innate and adaptive immune responses [6–9]. However, the role of mast cells www.selleckchem.com/products/bmn-673.html in the generation of adaptive immune responses, especially in transplant immune responses, is far from being resolved [10]. Recently,

Lu et al. found that mast cells may be essential intermediaries in Treg-mediated transplant tolerance [11]. While the mechanisms involved are still not well understood, some previous studies have shown that MCs can serve as a source of transforming growth factor (TGF)-β1 [12], which is required for introduction and maintenance of Treg cells both in vitro and in vivo[13–16]. Therefore, this study was designed to test the hypothesis that bone marrow-derived mast cells (BMMCs) can induce CD4+ T cells to CD4+CD25+FoxP3+ Tregs via TGF-β1 Protein kinase N1 in vitro. C57BL/6 (H-2b) mice were maintained and housed at the animal facilities of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Bone marrow cells were obtained from C57BL/6 mice. The cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 10 mM Hepes, 50 µM 2-mercaptoethanol, penicillin/streptomycin/L-glutamine, 10 ng/ml mouse interleukin (IL)-3 (Peprotech, Rocky Hill, NJ, USA) and

10 ng/ml mouse stem cell factor (SCF) (Peprotech) at 37°C in a humidified atmosphere containing 5% CO2. Every 7 days, the non-adherent cells were transferred into fresh enriched medium. After 4 weeks, the purity of the mast cells was assessed by flow cytometry. Spleen cells were obtained from C57BL/6 mice. T cells were isolated from the spleen cells with CD3 T cell isolation kit (Miltenyi, Bergisch Gladbach, Germany). Purity of CD3+ T cells typically exceeded 95%. To determine the purity and the characteristic of BMMCs, BMMCs were collected after 4 weeks’ culture. They were dropped onto a slide and stained with toluidine blue (1%, pH = 1) for 10–20 s. The slide was then washed with distilled water for about 2 min. The cells were observed under a microscope.