Investigation On The Influence Of The Cross Species Incompatibility Of Complement Regulatory Protein For Modulating Mouse Complement C3 Opsonization On Human Red Blood Cells

Background and Objectives:Red blood cells(RBCs)are the most abundant cell population in blood which play crucial roles in a series of physiological process including oxygen transportation,carbon dioxide exchanges.Abnormalities in RBCs differentiation lead to a variety of severe hematological diseases such as sickle cell anemia,thalassemia,etc.Therefore,the development of an in vivo model able to mimick human erythrocyte differentiation and physiological function is urgently needed for relevant human disease mechanism research and drug discovery.Humanized mice are defined as the mice hosting human genes/cells/tissues,which are considered as important tools to investigate human diseases,such as infectious diseases,hematological malignancies.However,there is still lack of a humanized mouse model with stable high levels of human RBC reconstitution.Previous studies by our group showed that immunodeficient mouse phagocytes mediating rejection is the key reason causing human RBC deficiency in humanized mice in which mouse complement C3 play critical roles in the direct opsonization of human RBCs.We developed a Cobra Venom Factor plus macrophage depletion strategy to improve human RBC ratios in humanized mice.However,it is still unclear why mouse complement C3 could opsonize human RBCs without antibody binding and how it mediates mouse phagocytes rejection.Therefore,our study aims to further investigate the underlined mechanism that how mouse complement C3 involves in human erythrocyte rejection through species incompatibility of complementary regulatory proteins in humanized mice which may contribute to further improvement of the construction of humanized mouse models with human RBCs that could be applied in human relevant hematological disease investigation.Methods:1.Construction of human erythrocytes and hematopoietic stem cells(HSCs)overexpressing mouse complement regulatory protein based on lentiviral vector:lentiviral transfection technology was used to introduce mouse complement regulatory protein into human erythroid cell lines,human HSCs or human HSCs-differentiated RBCs through lentiviral vector,to prepare human HSCs or human RBCs overexpressing mouse complement regulatory protein,further testing effects on complement deposition and adhesion of human RBCs and immunodeficient mouse macrophages on C3 conditioning.2.Mouse complement C3 deposition experiment:human RBCs or leukocytes were co-incubated with mouse fresh serum or heated complement-inactivated serum,and the rate of complement C3 deposition on the surface of human RBCs or leukocytes was analyzed by flow cytometry.3.Adhesion experiment:activated macrophages collected from mouse peritoneal cavity were used as effector cells,and human RBCs were added after adhesion.The adhesion of human RBCs and immunodeficient mouse macrophages in fresh or heated complement-inactivated serum of mice was observed by optical microscope and fluorescence microscope,and the adhesion ratio was analyzed by Image-Pro plus software.4.In vitro human erythrocyte differentiation experiment:using in vitro hematological stem cell culture/differentiation technology,human HSCs were differentiated into erythroid progenitor cells(day 1 to day 14)and terminal mature RBCs(day 15 to day 23).5.Construction of humanized mouse model:human HSCs that transfected with the lentiviral vectors containing mouse complement regulatory protein CD55 were injected intravenously into immunodeficient mice pretreated with Busulfan to construct a humanized mouse model;Chimerism rates and phenotypes of human hematopoietic cells in blood and bone marrow were monitored at indicated time points after transplantation.Results:1.Overexpression of mouse complement regulatory protein CD59a cannot prevent the opsonization of mouse complement C3 on human erythroid cells.2.Overexpression of mouse complement regulatory protein CD55 efficiently prevents the opsonization of mouse complement C3 on human erythrocytes and inhibits the adhesion of human erythrocytes to mouse macrophages in the presence of mouse complement.3.Simultaneous overexpression of mouse complement regulatory proteins CD55and CD59a do not further enhance the anti-opsonization effect for human erythrocytes compared with the CD55 incorporation strategy.4.Overexpression of mouse complement regulatory protein CD55 efficiently prevents the opsonization of mouse complement C3 on human HSCs.5.Mouse complement C3 also opsonizes human CD45~+CD19~+B lymphocytes.6.Mouse CD55 positive human RBCs can be developed into the mature RBCs in the humanized mice made by the transplantation of human HSCs that transducted with the lentiviral vectors containing mouse CD55 gene,showing resistance to mouse complement C3 opsonization in vivo.Conclusion:This thesis demonstrates that incompatibility between human and mouse complement regulatory protein CD59a is not likely to be the key reason causing the opsonization of human erythrocytes by mouse complement C3.Instead,we firstly confirmed that the incompatibility of human and mouse complement regulatory protein CD55 is the key reason causing the opsonization and indicated that overexpression of mouse complement regulatory protein CD55 on either human erythrocyte cell lines or human HSCs markedly prevent the opsonization by mouse complement C3 either in vitro or in vivo and inhibit the immunological rejection mediated by mouse phagocytes in the presence of mouse complement.In sum,our study firstly reveals that the incompatibility of complement regulatory protein between murine and human facilitates the rejection of human erythrocytes by mouse macrophages in mouse complement C3 dependent manner which provides a novel approach to improve the survivals of human RBCs in immunodeficient mice for their in vivo investigation.