Arbour Nathalie, Lapointe Réjean, Saikali Philippe, McCrea Ellie, Regen Tommy, Antel Jack P
Neuroimmunology Unit (Room 111) Montreal Neurological Institute, McGill University 3801 University St. Montreal, QC Canada.
J Immunol Methods. 2006 Mar 20;310(1-2):53-61. doi: 10.1016/j.jim.2005.12.009. Epub 2006 Jan 19.
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.
人类自身反应性T细胞可能参与多种自身免疫性疾病。尽管体外检测自身反应性T细胞是可行的，但由于其频率较低，对这些细胞进行详尽的功能表征受到阻碍。抗原（Ag）特异性T细胞的体外扩增通常是通过在存在Ag的情况下使用自体（新鲜或冷冻）辐照外周血单核细胞（PBMC）、EBV永生化B细胞或树突状细胞来实现的。这些方法需要大量血液。我们探索了一种成功应用于肿瘤特异性T细胞的方法，即使用体外扩增的自体B细胞。用表达CD40L的辐照成纤维细胞和IL-4刺激PBMC，产生富含表达高水平MHC和共刺激分子的B细胞群体，这些是抗原呈递细胞（APC）的基本特征。将扩增的B细胞加载Ag，辐照后用作APC来刺激T细胞。通过比较与加载抗原的B细胞和未加载抗原的B细胞共培养时T细胞系的增殖和IFN-γ分泌来评估T细胞系的特异性。表现出抗原特异性增殖和/或IFN-γ分泌的T细胞系被扩增。使用这种方法，从多个供体获得了与使用传统方法（即新鲜PBMC作为APC）获得的数量相当的髓鞘碱性蛋白（MBP）和髓鞘少突胶质细胞糖蛋白（MOG）特异性CD4(+)和CD8(+) T细胞系，并在培养中保存了数周。我们已经表明，使用扩增的B细胞作为APC，可以从多个供体相对少量的血液（50-100毫升）中扩增髓鞘特异性CD4(+)和CD8(+) T细胞。
