OBJECTIVE: CD4+CD25+ regulatory T cells (Tregs) have been found to have a decreased effector function in patients with multiple sclerosis (MS). In this study, we co-cultured naïve CD4+ T cells of MS patients with myelin basic protein (MBP)85-99 peptide as specific antigen and allogenic B cells as antigen-presenting cells, in an attempt to generate adequate antigen-specific CD4+CD25+ Tregs with normal or improved immune function. PATIENTS AND METHODS: Naïve CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) from patients with MS (n=5) and healthy controls (HC, n=5). Furthermore, these purified naive CD4+ T cells were co-cultured with the CD40-activated B cells and MBP85-99 peptide to induce MBP-reactive CD4+CD25highCD127low Tregs. After harvesting these Tregs via a flow sorter, real-time PCR and mixed lymphocyte reaction (MLR) assay were performed to characterize cellular immune function. Supernatant interleukin (IL)-10 and transforming growth factor (TGF)-ß1 protein levels were detected by an enzyme-linked immunosorbent assay (ELISA). RESULTS: With this method, the frequency of CD4+CD25highCD127low Tregs in CD4+ T cells was 3.5%-6%. In both MS and HC groups, there were relatively lower proliferation indices (PI) of MLR assay but higher supernatant IL-10 and TGF-ß1 levels in the presence of MBP than those in the presence of other control antigens, where no significant differences were found. CONCLUSIONS: Via the ex vivo culture, adequate MBP-reactive CD4+CD25+ Tregsderived from autologous naïve CD4+ T cells of MS patients, were obtained and returned to normal without immune defects, and even upregulated their immunosuppressive function mostly through the elevated release of IL-10 and TGF-ß1.