Human self-reactive T cells are potentially involved in many autoimmune diseases. Although ex vivo detection of self-reactive T cells is possible, exhaustive functional characterization of these cells is impeded by their low frequency. In vitro expansion of antigen (Ag) specific T cells is typically achieved by using autologous (fresh or frozen) irradiated peripheral blood mononuclear cells (PBMCs), EBV-immortalized B cells or dendritic cells in the presence of Ag. These approaches require a large blood volume. We explored a method successfully applied for tumor specific T cells using in vitro expanded autologous B cells. PBMCs were stimulated with irradiated CD40L-expressing fibroblasts and IL-4, resulting in an enriched population of B cell that expressed high levels of MHC and co-stimulatory molecules, essential hallmarks of antigen presenting cells (APCs). Expanded B cells were loaded with Ag, irradiated and then used as APCs to stimulate T cells. The specificity of T cell lines was assessed by comparing their proliferation and IFN-gamma secretion when cultured with antigen-loaded B cells vs. unloaded B cells. T cell lines exhibiting antigen-specific proliferation and/or IFN-gamma secretion were expanded. Using this method, MBP and MOG specific CD4(+) and CD8(+) T cell lines were obtained from multiple donors in comparable numbers to those obtained using the traditional approach (i.e. fresh PBMCs as APCs) and were kept in culture for many weeks. We have shown that myelin specific CD4(+) and CD8(+) T cells can be expanded from a relatively small volume of blood (50-100 ml) from multiple donors using expanded B cells as APCs.