Development Of A Humanized Animal Model For Human Thymic Involution And Evaluation Of Human T Cell Differentiation In Porcine Thymus

Objective:Thymus,as the central immune organ,plays a pivotal role in generating T cells endowed with self-tolerance and immune functionality.Research has demonstrated that the aging process is associated with a decline in thymic function,leading to compromised immunity.Notably,the thymus is the foremost organ to undergo aging within the body.As aging progresses,the structural integrity and composition of the thymic matrix microenvironment undergo deterioration.Consequently,there is a gradual reduction in the population of thymocytes within the thymus.This reduction,in turn,results in a diminished pool of naive T cells and a decline in the diversity of the T cell receptor repertoire.These alterations collectively contribute to an impairment of the overall immune function of the body.Part 1:With the advancements in modern medicine,there has been a progressive extension in the average lifespan of individuals.However,the aging process,particularly in the thymus,exerts substantial negative impacts on the immune function of the body as age increases.These repercussions manifest in diminished anti-infection capabilities,elevated susceptibility to autoimmune diseases,compromised tumor immune surveillance,and a reduction in immune response to vaccines.These negative effects will increase the medical and social burden.Addressing this societal challenge involves delving into the mechanisms underpinning human thymus aging with the aim of elucidating pathways for its delay and correction.It is noteworthy that a majority of contemporary studies investigating thymus senescence and degeneration rely heavily on murine models.Given the inherent species differences between mice and humans,the translational fidelity of research findings becomes a critical concern,hindering the seamless application of numerous outcomes.To overcome this limitation,the construction of aging animal models that more closely mimic the human physiological environment is imperative.Such models are instrumental in providing a nuanced understanding of the intricate relationship between human thymus aging and immune dysfunction.This knowledge,in turn,facilitates a comprehensive exploration of the onset and progression of immune dysfunction-related diseases during thymus aging,ultimately offering valuable insights for the prevention and reversal of thymic aging.Part 2:In comparison to conventional murine models,the humanized mouse model,wherein human embryonic thymus and hematopoietic stem cells are transplanted,offers a closer approximation to the human physiological environment.This model stands as an optimal platform for investigating the intricacies of human thymus aging,T cell differentiation,and development.However,the construction of this model necessitates the availability of human hematopoietic stem cells and embryonic thymus tissue,the latter being a resource constrained by its limited availability.Recent studies have demonstrated that the pig thymus can support the differentiation and development of human T cells.Nevertheless,there exist discernible defects in the human T cells produced within the pig thymic environment,and the underlying mechanisms of these deficiencies remain elusive.Unraveling this mechanism is pivotal,as it lays the groundwork for the conceptualization and development of gene-edited pigs.Such genetically modified pigs aim to enhance the functional similarity between pig and human thymus,thereby facilitating the establishment of a humanized animal model for thymus aging.This advancement not only contributes to refining our understanding of thymus-related immune responses but also holds promise in establishing immune tolerance for xenotransplantation applications.Research Methods:1.Development of a humanized animal model for human thymic involution(1)Systematically analyzing age-associated variations in the proportions of CD4~+naive T cells,CD4~+RTEs,and TRECs in peripheral blood.(2)Coordinating the creation of humanized mice through the simultaneous transplantation of human embryonic thymus and human CD34~+HSCs.(3)Implementing a biweekly sampling protocol,collecting blood from the tail vein from week 10 to week 22 to assess the proportion of human CD4~+naive T cells,CD4~+RTEs,and changes in human TRECs in peripheral blood.(4)Sacrificing mice at predetermined intervals(10,16 and 22 weeks post-construction)and meticulously scrutinizing the human embryonic thymus.This examination includes the determination of thymocyte proportions,quantification of thymic epithelial cells,and assessment of the expression levels of Ep CAM,Foxn1,and Aire.Additionally,evaluating the extent of thymic fat infiltration in embryos.2.Evaluation of human T cell differentiation in porcine thymus(1)Acquiring mouse thymus sections via vibration sectioning and isolating DP and SP cells,followed by their purification.(2)Cultivating mouse DP or SP cells on mouse thymus slices in vitro and conducting a comparative analysis of apoptosis,differentiation,CCR7 expression,and CD69 expression of pre-DP in the presence or absence of mouse slices.(3)Employing confocal microscopy to delineate the localization of mouse DP and SP cell subsets in mouse thymic sections.Furthermore,ascertaining the localization and distribution dynamics of mouse pre-DP in thymic sections over the course of culture time.(4)Obtaining human and porcine embryonic thymus sections through vibration sectioning,isolating and purifying human pre-DP thymocytes,and subsequently subjecting them to in vitro culture within human or porcine embryonic thymus sections.(5)Comparing the differentiation patterns of human pre-DP,along with the expression profiles of CCR7 and CD69,and the spatiotemporal distribution and localization dynamics of pre-DP between human and porcine thymus sections.(6)Constructing humanized mice through the co-transplantation of human CD34~+HSCs and human embryonic thymus(H/H group),as well as through the co-transplantation of human CD34~+HSCs and porcine embryonic thymus(H/P group).Subsequently,assessing the expressions of CCR7 and CD69 in human thymocyte subsets within the thymus of both groups of mice.Conclusion1.In the context of constructed humanized murine models,the initial phase following transplantation of the human embryonic thymus is marked by an acute and reversible degeneration,ostensibly attributed to surgical stress.Subsequently,there is an observable progression of age-related degeneration.Notably,the expression levels of crucial regulatory elements,namely Foxn1 and Aire,exhibit a significant reduction in transplanted human embryonic thymic epithelial cells by the tenth week post-humanization.This evidences the persistence of intrinsic mechanisms governing stress-induced and aging-induced degeneration within the transplanted human embryonic thymus in immunodeficient murine hosts.The establishment of such a model underscores its potential utility as an in vivo platform for investigating human thymus degeneration dynamics and evaluating the efficacy of therapeutic interventions.2.Within the thymic microenvironment of porcine origin,the developmental trajectory of human thymocytes has been discerned to be contingent upon the chemokine ligand receptor axis,as elucidated through the application of the thymic vibration-slice culture system.In this porcine thymic environment,a noteworthy diminution in the expression of CCR7 within human thymocytes is observed,suggesting it plays the role as a pivotal determinant influencing the aberrant development of human thymocytes.This investigation furnishes a theoretical foundation,thereby paving the way for the prospective refinement of porcine thymic environments conducive to optimal human T cell development.Such optimization endeavors,in turn,hold promise for augmenting the scope and efficacy of constructing humanized animal models characterized by a functional human immune system.