Xenotransplantation of human hepatocytic cell line into fully immunocompetent C57BL6 mice using anti-CD4 monoclonal antibody induction therapy

The main goal of anti-CD4 monoclonal antibody therapy is the effective, antigen-specific induction of immunotolerance of allogenic and xenotransplants in fully immunocompetent hosts. The aims of this project were to monitor the cellular immune status of C57BL6 mice treated with temporary CD3+//CD4+ depleting anti-CD4 monoclonal antibody (GK1.5), and to establish and monitor the successful transplantation of human differentiated hepatocytes into the parenchyma of wildtype, immunocompetent C57BL6 mice. Cultured human SV40LT-immortalized hepatocytes were transplanted into immunocompetent C57BL6 mice. The animals were treated with a single i.p. administration of anti-CD4 monoclonal antibody (verum) or PBS (control) in the presence of hepatocyte antigen at day 0 and day 4. Blood T-cell counts were monitored using flow cytometry. Successful transplantation and establishment of human hepatocytes was confirmed by analysis of human albumin expression (ELISA, immunohistochemistry) and the presence of human hepatocytes in the host liver (FISH, in situ hybridization). A significant number of cells functionally expressing human albumin were observed in animals treated with anti-CD4 induction therapy after 14 d. Blood CD4 count was significantly reduced within 2 h of anti-CD4 antibody application, and was normal after 10 d. The successful transplantation of human hepatocytes into immunocompetent mice provides evidence that anti-CD4 induction therapy exclusively downregulates the CD4 count of treated mice, temporarily and reversibly, while showing no effect on other immune cell counts, or immune function. The effective induction of immunotolerance to human differentiated hepatocytes into the liver of C57BL6 mice provides a model for other in vivo evaluations of cell-based therapies in immunocompetent animals. Finally, a transgenic mouse model physiologically expressing human CD4 on T helper cells and HLA-DR3 on antigen-presenting cells while carrying a mouse CD4 knock-out gene is currently being developed to evaluate the potency of clinically relevant anti-human CD4 monoclonal antibody therapies.