The induction and maintenance of antigen specific peripheral tolerance is critical for the prevention of autoimmunity and maintenance of immune homeostasis. Antigen presentation by tolerogenic dendritic cells (DCs) initiates the process through the stimulation of regulatory T cells and other mechanisms that may include peripheral deletion and anergy induction of autoreactive T cells. In contrast to DCs that mediate immune priming, tolerogenic DCs have a resting phenotype. They lack expression of co-stimulatory molecules and MHC class II. Recently, a method for identifying murine tolerogenic DCs (lin−, CD45RBhigh, CD11clow) and expanding them ex vivo has been described (

Wakkach et al. Immunity 18:605, 2003
). Directed antigen presentation by viral vector transduction of these cells has the potential to produce antigen specific tolerance for therapeutic purposes. To generate tolerogenic DCs, we cultured lin− murine bone marrow in complete RPMI medium in the presence of 10 ng/ml GM-CSF, 2.5 ng/ml TNF-α and 10–100 ng/ml IL-10 for 6–7 days. Cells were transduced overnight at different time points (day 0, day 3, day 6), with foamy virus vectors and lentivirus vectors containing either the GFP or alkaline phosphatase reporter genes. Transductions were performed both in the presence or absence of fibronectin fragment CH-296. For day 0 transductions, we also evaluated whether stem cell stimulatory cytokines (50 ng/ml TPO, 100 ng/ml SCF, 100 ng/ml FL) improved gene transfer efficiency. On days 6–7 the CD45RBhighCD11clow cell population was flow sorted and then further analyzed. The percentage of CD45RBhighCD11clow cells harvested on days 6–7 varied from 10% to 30% and was increased by IL-10 in a dosage dependent manner. As expected for tolerogenic DCs, these cells were morphologically immature by Giemsa-Wright staining and phenotypically negative to low for the expression of co-stimulatory molecules CD80 and CD86, and MHC class II, even after exposure to lipopolysaccharide. At an MOI of 0.5–1, transduction rates of CD45RbhighCD11clow cells were 30%–37% for foamy virus vectors and 23%–24% for lentivirus vectors. Most importantly, vector transduction did not induce expression of co-stimulatory molecules, suggesting that these two viral vectors do not activate DCs. There was no difference in transduction efficiency at the three different time points tested. Furthermore, transduction efficiencies were not affected by either stem cell stimulatory cytokines or CH-296. In summary, cells with the phenotype of tolerogenic DCs can be efficiently transduced by foamy virus and lentivirus vectors with overnight exposure to vector stocks. In vivo studies in mice are in progress to evaluate if transduced CD45RBhighCD11clow cells are able to induce antigen specific tolerance. These studies have potential clinical implications for the treatment of autoimmune diseases, the induction of tolerance after stem cell transplantation, or the prevention of immune responses triggered by specific transgenes in gene therapy.

Topics:
alkaline phosphatase, antigens, autoimmune diseases, cd80 antigens, cytokine, dendritic cells, fibronectins, gene therapy, gene transfer techniques, giemsa stain

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2005, The American Society of Hematology
2004
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