Exploration Of A Humanized Mouse Model In The Study Of Human Hematopoietic And Lymphoid Systems

Humanized mice provide not only a powerful tool to understand human physiology and pathology in vivo, but also a preclinical model to test clinical therapies. The primary objective of my thesis project is to explore the potential of humanized mice as a model for the study of human hematopoietic and lymphoid systems, which is composed of two parts:1) Human red blood cell (RBC) and platelet development in humanized mice; 2) Establishment of a TCR transgenic humanized mouse model for anti-tumor gene therapy1. Human RBC and platelet development in humanized miceAn animal model supporting human erythropoiesis will be highly valuable for assessing the biological function of human RBCs under physiological and disease settings, and for evaluating protocols of in vitro RBC differentiation from human embryonic stem cells. Although immunodeficient mice on the NOD background have been widely used to study human hematopoietic stem cell function in vivo, the successful use of these mice in the study of human erythropoiesis and RBC function has not been reported. We have previously shown that co-transplantation of human fetal thymic tissue (under renal capsule) and CD34+fetal liver cells (FLCs; i.v.) in NOD/SCID or NOD/SCID/γc-/- mice results in the development of multilineage human hematopoietic cells. Here, we analyzed human RBC reconstitution in these humanized mice. Although a large number of human erythrocytes, which consisted predominantly of immature nucleated erythrocytes, were detected in the bone marrow of human fetal thymus/CD34+ FLC-grafted mice, human RBCs were undetectable in blood of these mice, even in those with nearly full human chimerism in peripheral blood mononuclear cells (PBMCs). Recipient mouse macrophage-mediated rejection is, at least, one of the major mechanisms responsible for the lack of human RBCs in these mice, as human RBCs became detectable in blood following macrophage depletion and disappeared again after withdrawal of treatment. Furthermore, treatment with human erythropoietin (EPO) and human IL-3 significantly increased human RBC reconstitution in mice that were depleted of macrophages. Like the human RBCs developed in the humanized mice, exogenously injected normal human RBCs were also rapidly rejected by macrophages in NOD/SCID mice. And we found rejection of human RBCs in NOD/SCID mice is significantly faster than that of mouse CD47KO RBCs, which suggest rejection of human RBCs by mouse macrophages could be independent of CD47-SIRPαsignaling.Like the human RBCs shown above, we also analyzed human platelet reconstitution in these humanized mice. Although high ratio of human leukocytes, which consisted predominantly of human T and B cells, were detected in these animals, the chimeric level of human platelet was very low in these mice. We find recipient mouse macrophage-mediated rejection is the key mechanism responsible for the low constitution of human platelets in these mice, as the ratio of human platelets became almost as same as the one of human leukocytes in blood soon after macrophage depletion and disappeared again after withdrawal of treatment. Like the human platelets developed in the humanized mice, exogenously injected normal human platelets were also rapidly rejected by macrophages in NOD/SCID mice. And mouse CD47KO platelets were rejected much slower than human platelets in NOD/SCID mice which suggests rejection of human platelets by mouse macrophages could be independent of CD47-SIRPαsignaling and some xeno-antigen might induce the activation of human platelets.2. Establishment of TCR-transgenic humanized mouse model for anti-tumor studyInduction of tumor antigen-specific immune responses is considered as a potential anti-cancer immunotherapy. Transduction of patient autologous peripheral blood mononuclear cells (PBMCs) with lentiviral vectors containing tumor antigen specific TCR has been used to generate tumor-specific T cells and injection of these T cells have resulted in significant cancer regression in some patients in pre-clinical trials. Mouse studies have shown that engineering hematopoietic stem cells (HSCs) with tumor antigen-specific TCR genes provides a more efficient cancer therapy, in which tumor antigen-specific T cells developing from the virally-transduced HSCs mediated strong anti-tumor responses leading to elimination of established tumors. In this study, I explored the potential of this approach to achieve anti-tumor responses within a human immune system in humanized mice. HLA-A*0201-restricted Mart-1 (Melanoma Antigen Recognized by T cells) specific TCR transgenic humanized mice were established in immunodeficient mice by transplantion of human fetal thymus (under renal capsule) and autologous human CD34+ fetal liver cells (i.v.), which have been transduced with lentiviral vector containing the TCR gene. FACS analysis using Mart-1-specific tetramers demonstrated the development of human CD8+ T cells expressing Mart-1 specific TCR in these humanized mice. The ratio of Mart-1 TCR+ T cells which developed from virally-transduced CD34+ cells was further increased by depleting the preexisting human T cells in the thymic graft. Importantly, Mart-1 TCR+ T cells in humanized mice were able to response to Mart-1 antigens and produce IFN-y after immunization with Mart-1 peptides. These humanized mice are, to our knowledge, the first TCR transgenic humanized mouse model with functional T cells expressing an antigen-specific TCR, which provide a powerful platform for developing anti-tumor immunotherapies through engineering human HSCs with tumor antigen specific TCR genes.In conclusion, humanized mice provide an excellent small animal for the study of human hematopoietic and lymphoid systems. The development of strategies to overcome macrophage-mediated rejection will further permit the investigation of development, differentiation and function of human erythroid cells and megakaryocytes in humanized mice. Moreover, not only a functional human immune system, but also the development of functional tumor-antigen-specific T cells can be established in the humanized mice, making these mice a novel and valuable preclinical model for exploring cancer immunotherapy.