Multiple sclerosis (MS) is an immune-mediated disorder of the central nervous system that causes neuroinflammation and axonal degeneration. The inflammatory symptoms of MS are thought to be mediated by inappropriately activated T cells, but currently, the cause of this inappropriate activation, the antigenic target of the immune response, is still unknown. In an effort to elucidate the antigenic target of MS, we planned on investigating the intrathecal immune responses in MS patients by using individual co-culture systems based on antigen-loaded mature dendritic cells and autologous cerebrospinal fluid (CSF) T cells, where T cell reactivity to the antigen was represented by T cell proliferation. However, before beginningtesting on a large cohort of patient samples, we needed to optimize the experimental conditions of the assay.To do so, we prepared a variety of complex auto-antigens relevant to MS, including whole brain homogenate, myelin, and MO3.13 human oligodendriglial cells exposed to oxidative stress. We also tested environmental antigens, which have been linked to MS by epidemiological studies, such as Epstein-Barr virus and human herpes virus type-6. We then isolated monocytes from peripheral blood mononuclear cells, differentiated them into immature dendritic cells, loaded the dendritic cells with the antigens at different concentrations, and co-cultured them with CFSE-stained autologous T cells. After several days of co-culture, T cell proliferation was determined by flow cytometry. We established the proper protocols for preparation of the auto-antigens and optimized the co-culture conditions. Antigen titrations revealed a dose-dependent effect when tested with peripheral T cells, but as the assay will be used for CSF T cells, we are currently working to determine the optimal antigen concentrations for these cells.
Last updated March 20, 2013