Characterization of dendritic cells for in vitro induction of T cell tolerance

A major barrier to effective allogeneic bone marrow transplantation is Graft versus host disease (GVHD). Donor T cells are a recognized prerequisite for generation of GVHD. Dendritic cells (DCs) are the most powerful antigen-presenting cell (APC) in the immune system. The ability of DCs to stimulate primary T lymphocytes and T cell dependant immune responses many provide opportunities for therapeutic intervention in bone marrow transplantation. According to recent reports, existing myeloid and plasmacytoid DC subsets, both under specific in vitro culture conditions are capable of secreting specific cytokines responsible for the polarization T cells to a T helper 2 (Th2 response). Generation of Th2 cells in turn results in donor specific T cell tolerance to recipient DC presented antigen. DCs have great potential in preventing GVHD. The first goal of this study was to isolate DC subset from human peripheral blood are apheresis product using newly developed markers for blood dendritic cell antigens (Miltenyi Biotech) and characterize them. The second goal was to determine the culture conditions that would support the maintenance and/or development of specific recipient DC subset capable of inducing donor T cell tolerance. DCs were isolated by immunomagnetic cell sorting method from a population of mononuclear cells (MNCs). MNCs were labeled with recently developed antiBDCA-1 (blood dendrite cell antigen-1) and BDCA-4 immunomagnetic beads to isolate myeloid (mDCs) and plasmacytoid dendritic cells (pDCs) respectively, on a magnetic separation column. Positively selected dendritic cells were characterized by evaluating the presence of absence of specific makers by flow cytometry with simultaneous three-color staining and a two-step acquisition procedure. A total of 12 isolations for BDCA-1 positive cells and three for BDCA-4 positive cells were done. Staining with fluorochrome-labeled antibodies to CD11c, Il-3R? (CD123), and HLA-DR confirmed the identity of mDC (CD11c+, IL-3R?low/HLA-DR+/Lineage¯) and pDC (CD11c¯/IL-3R?high/HLA-DR+/Lineage¯) subsets. A simultaneous staining with antilineage cocktail which contained a mixture of monoclonal antibodies) specific for individual lineage markers, was done to exclude lineage positive cells during the flow cytometry analysis. Flow cytometry data confirmed successful isolation of both myeloid and plasmacytoid dendritic cell subsets. Dendritic cells are otherwise generated by culturing MNCs and CD34+ cells in the presence of cytokines such as GM-CSF and IL-4. It is concluded that recently developed antibodies to BDCA can be used for the isolation of dendritic cells directly from peripheral blood, instead of culturing MNCs or CD34+ cells over prolonged period for generation cells.