Mechanism Of Human T Cells Thymic Selection And Tolerance In TCR Transgenic Humanized Mice

Background:Human autoimmune diseases are increasingly prevalent,partly because of retention of many susceptibility genes in the overall gene pool and partly because of new environmental factors that have surged in the past century.These diseases are contributed by uncontrolled autoreactive T cells,whose presence in the blood circulation of patients as well as many asymptomatic individuals,can be ascribed to an imperfect thymic selection process.T cells acquire a T cell receptor(TCR)during their development in the thymus,and then become exposed to self-MHC for positive selection,and to MHC/self-peptide complexes for negative selection,leading to a purge of most autoreactive T cells before the remaining mature T cells can be released into the periphery.Most of what we know regarding mechanisms of thymic selection comes from animal models.Some studies performed on human thymic tissue,usually removed during cardiac surgery,have offered snapshots on the phenotype and distribution of human thymocytes,with limited insights on their function.However,longitudinal studies of human immune cells and tissues are not feasible beyond blood samples,and using human thymic tissue to assess the development and fate of thymocytes in relation to their antigen specificity is challenging.It is now possible to generate multiple‘identical'humanized mice from a single human donor that allow longitudinal studies as well as various manipulations such as introduction of specific TCRs and/or antigens into the hematopoietic stem cells(HSCs)that will reconstitute the human immune system.This in vivo model opens new possibilities for studying the thymic development of human autoreactive T cells,the contribution of specific subsets of APCs to central tolerance and the implications of dual TCR expression in autoimmunity and tumor immunity.Objective:The purpose of this study was to establish a humanized mouse model of T cells thymic selection,to fully understand the developmental process and phenotypic changes of human autoreactive T cells in the thymus,and to better understand the function of various antigen presenting cells in thymus selection.Methods:(1)A lentiviral vector carrying the MART1 TCR gene and the MART1antigen peptide-GFP was prepared by a genetic engineering method.The lentivirus was packaged to transduce HLA-A2+MART1~-tumor cells to identify the function of the MART1 peptide.(2)Establishment of humanized mouse model with human immune system:NSG mice were conditioned with sublethal(1.5 Gy)toatal body irradiation,and transplanted fetal thymus tissue graft under the kidney capsule.MART1 peptide group mice were simultaneously infused with1×10~5 MART1 TCR transduced hematopoietic stem cells and 1×10~5MART1 peptide transduced hematopoietic stem cells.Another group of control mice were simultaneously infused with 1×10~5 Mart-1 TCR transduced hematopoietic stem cells and 1×10~5 control lentivirus transduced hematopoietic stem cells.The level of human hematopoietic cell reconstitution in humanized mice was determined by flow cytometry(FCM).(3)Detection of human antigen presenting cell subsets in each tissue of a humanized mouse model.(4)Analyzing the phenotypic changes and costimulatory molecule alteration of MART1 reactive TCR T cells and Treg cells development influenced by absent or present autoantigen.Results:We developed a humanized mouse model involving transduction of autoreactive TCRs and cognate autoantigens on human hematopoietic stem cells,which allows analysis of thymic selection of human T cells in vivo.This study found that hematopoietic stem cells developed into T cells and a variety of antigen-presenting cells(APC)subpopulations after entering the thymus.The effect of thymus deletion of autoreactive T cell clones was significant.TCR-specific T cells pass through thymic selection behaved as CCR7~+PD-1~-,while the thymic deleted T cells showed CCR7~-PD-1~+.MART1 TCR T cells down-regulated TCR,CD3,CD8 and CD4 after they encountered autoantigen in the thymus.This study also provide evidence that secondary TCR?influence MHC class I-restricted T cells to develop as CD4+,particularly regulatory T cells.Conclusions:The humanized mouse model described created a powerful tool to study the thymic development of human antigen-specific T cells side-by-side with the general polyclonal thymocyte population,allowing a more accurate dissection of T cell phenotypes in the presence or absence of a specific self-antigens.It may be leveraged to address the extent to which specific autoreactive T cell clones identified in human autoimmune diseases undergo central tolerance,or absent complete thymic deletion.The establishment of this model has important application value for the study of T cell immune tolerance,the occurrence of autoimmune diseases caused by autoreactive T cells,and the study of thymus selection escape mechanism.