Optimization Of The "Skin-Immune" Humanized Mouse Model And Study On The Immunological Characteristics Of Human Skin Trm

Aims:Memory T cells in the skin are mainly divided into three types: central memory T cells(Tcm),effector memory T cells(Tem),and tissue-resident memory T cells(Trm).They exhibit different phenotypes and play important roles in regional immune protection of the skin.Trm,a recently discovered group of memory T cells,can reside in the skin for extended periods.Previous research has revealed their "dual role" in providing local skin immunity and promoting certain diseases.However,the mechanisms underlying Trm settlement and migration in the skin remain unclear,making the immunological characteristics of Trm in skin regional immunity a current research hotspot.Nevertheless,the study of human skin memory T cells is challenging due to sample limitations,especially the tracking of skin Trm in the human body.Therefore,establishing an ideal animal model is an urgent and powerful research tool for investigating regional skin immunity.Currently,most relevant skin immune animal models are based on the mouse immune system,which limits the study of human skin regional immunity.In recent years,some models have been constructed by reinfusing human lymphocytes to simulate the human immune system,but due to the limited types of infused cells and their short survival times,the simulation and study of skin regional T cells still do not yield satisfactory results.In our previous research,we established a "Skin-Immune" dual-system humanized mouse model that can simulate the human immune system and transplant human skin,making it a breakthrough in skin regional immunity research.However,due to the high technical difficulty of constructing this model,as well as the challenges in intraoperative and postoperative management and low graft survival rates,large-scale implementation is difficult,and it is even more challenging to apply it as a basis for further research.In our current study,we aim to optimize the "Skin-Immune" dual-system humanized mouse model to improve the success rate of skin transplantation and the applicability of this model.We seek to fully simulate human skin from its structure to its immune function and establish a complete human T cell immune system.We will use this model to explore the immunological characteristics of the three types of memory T cells in the skin,particularly Trm.Additionally,we will construct an HIS-SKIN(humanized immune system and skin)secondary transplantation model to study the entire process of Trm settlement and secondary migration.Finally,to investigate the differences in Trm under pathological and physiological conditions,we will build an HIS-SKIN humanized vitiligo model.Through transcriptomic analysis,we will explore the gene differences between Trm and the other two types of memory T cells and the signaling pathways of Trm in vitiligo.This will provide important insights for future Trm research.Methods:(1)Construction of the "Skin-Immune" dual-system humanized mouse model.NCG mice were subjected to busulfan myeloablation,and fetal liver-derived CD34+hematopoietic stem cells and homologous thymus were transplanted to establish the humanized mice model.Three weeks after humanization,adjustments were made to the skin transplantation procedure,intraoperative and perioperative management,including improvements in bandaging techniques,intraoperative bandaging time,postoperative dressing change time,and postoperative feeding conditions.Graft survival rates were observed,and flow cytometry was used to assess the reconstruction of the skin immune system.Immunofluorescence techniques were employed to identify tissue structure,T cell composition,and melanocyte distribution in the transplanted skin.(2)Application of flow cytometry to assess the reconstitution of memory T cells in the "Skin-Immune" dual-system humanized mouse model,including the transplanted human skin,mouse skin,liver,lung,spleen,peripheral blood,lymph nodes,and bone marrow.Expression of memory T cell-related markers in the transplanted skin and the expression of Trm-related chemokine receptor in the model mice were examined.An anti-CD122 antibody was used to block CD122 on skin CD122+ cells,and flow cytometry was employed to observe immunological changes in the transplanted human skin and systemic immunity in the model mice.(3)Construction of the HIS-SKIN secondary transplantation model: after 15 weeks of reconstruction of the humanized immune system in the "Skin-Immune" dual-system humanized mouse model(donor),lymphocytes from the human skin of the transplanted human skin-only transplantation mice(recipient)were transplanted.Flow cytometry was used to assess the T cell immunological changes in donor and recipient mice before transplantation,3 weeks after transplantation,6 weeks after transplantation,and 12 weeks after transplantation.Emphasis was placed on observing the phenotype of Trm and the expression of key factors.Immunofluorescence was used to observe the migration and chemokine expression of skin Trm at various time points,reconstructing the entire process of Trm secondary migration and settlement.(4)Construction of the HIS-SKIN humanized vitiligo model: Fetal liver-derived CD34+ hematopoietic stem cells and homologous human skin tissue were transplanted after infection with Mart-1-TCR lentivirus in vitro,creating a Mart-1-TCR transgenic humanized skin transplantation mouse model.Subsequently,Mart-1 peptide and imiquimod were used in combination for immune induction to stimulate local inflammation in the skin,leading to vitiligo caused by Mart-1-specific CD8+ T cell attack.After immune induction,flow cytometry was used to detect the expression of Mart-1-specific T cells in the skin of the model mice,immunohistochemistry was employed to assess melanocyte loss,and immunofluorescence was used to identify the role of Mart-1-specific T cells in the skin with respect to melanocytes.(5)Utilizing single-cell RNA sequencing(scRNA-seq)to analyze the transcriptional characteristics of human skin memory T cells in the "Skin-Immune" dual-system humanized mouse model and the HIS-SKIN humanized vitiligo model:Dimensionality reduction clustering of skin lymphocytes and annotation of differentially expressed genes at the single-cell level.Identification of differential genes and functionally enriched pathways within and between memory T cell groups.Analysis of cell communication to identify the communication levels between Trm and other memory T cells,as well as the strength of ligand-receptor pairs between different memory T cell types.Selection and identification of key genes related to vitiligo-associated Trm,GO and KEGG enrichment analysis of vitiligo-associated Trm,and construction of signal regulation networks for these key genes to explore the pathogenic mechanisms of Trm in vitiligo.Results:(1)Successful optimization of the "Skin-Immune" dual-system humanized mouse model resulted in significantly improved graft survival rates and reduced mouse mortality.The humanized mouse T cells exhibited distinct CD4+ and CD8+phenotypes.The transplanted human skin showed intact structure and the potential to develop mature melanocytes.Both CD4+ and CD8+ T cells were abundantly present in the dermis and epidermis of the transplanted skin.(2)In the "Skin-Immune" dual-system humanized mouse model,memory T cells in the transplanted human skin were predominantly Trm,consistent with previous studies.CD49 a and CLA were highly expressed in skin Trm in terms of surface markers.CCR4 and CCR6 were highly expressed in skin Trm with regard to chemokine receptors.Blocking the T cell-specific target CD122 led to a comprehensive decrease in chimerism and T cell proportion in the model mice.CD122 levels in the skin significantly decreased,and CD69+CD103+ Trm were reduced.(3)The HIS-SKIN secondary transplantation model was successfully constructed.From 3 to 6 weeks after transplantation,CD4+ memory T cells transitioned from systemic distribution in recipient mice to skin-specific distribution,all of which exhibited the Trm phenotype.Until 12 weeks,they gradually disappeared.During migration,both Trm phenotypes were enriched in the dermis at 3 weeks,present in both the dermis and epidermis at 6 weeks,and concentrated near the basement membrane.CCR4 was co-localized during migration.At 12 weeks after transplantation,only a small amount of γ δ TCR was expressed in the skin and spleen.(4)The human-induced vitiligo model of HIS-SKIN was successfully constructed.Immunopathological findings indicating a significant loss of melanocytes,with Mart-1-specific T cells attacking melanocytes within hair follicles.Single-cell sequencing showed that the expression of various memory T cell characteristic genes was consistent with previous research.In this study,it was found that under physiological conditions,the MAPK signaling pathway,T cell receptor pathway,and cytokine pathways were upregulated in Trm,while ribosomal pathway genes were downregulated.Under vitiligo conditions,CD8+ Trm had the strongest communication with Tcm.The most significant regulatory pathway in vitiligo may involve CD69—TNFAIP3—RGS family—JUN—ZFP36—NR4A family,with key genes in vitiligo Trm being RGS2,NR4A1,NR4A2,and NR4A3.Conclusions:(1)The optimized "Skin-Immune" dual-system humanized mouse model can effectively simulate healthy adult skin in terms of both structure and immune function,particularly with respect to T cell reconstruction.It is suitable for research on T cell immunity in skin regions.(2)In the "Skin-Immune" dual-system humanized mouse model,the skin region is predominantly populated by memory T cells,with Trm being the main subset.Their proportion and phenotype closely resemble those of healthy adults.Trm-related surface markers may assist in maintaining Trm retention in the skin,promoting the formation of Trm precursors.Several chemokine receptor receptors,including CCR4 and CCR6,may play crucial roles in recruiting and homing T cells to the skin,contributing to Trm generation and enhancing their residence in the skin.(3)The application of a CD122 inhibitor successfully blocked human CD122 targets on mouse transplanted skin,effectively regulating T cell generation and the expression of Trm surface markers.This approach has the potential to advance clinical drug research and validates the functionality of this model.(4)Only CD4+ Trm in the skin could redistribute to various organ tissues after entering the peripheral blood circulation and eventually migrate to secondary skin,where they could reestablish residency.However,these CD4+ Trm with the ability to resettle have a lifespan of less than 3 months.The migration of CD4+ Trm from the circulation to the skin followed an "inside-out" pattern,gradually crossing from the dermis to the epidermis and ultimately transforming into undifferentiated T cells.During this process,CCR4 may play a critical role in homing Trm to the skin.(5)Under physiological conditions,the transcriptional regulation of human skin Trm is influenced by TCR signaling-related pathways,with upregulation of the MAPK cascade pathway,T cell receptor pathway,and cytokine pathway promoting Trm formation.Downregulation of the ribosomal pathway significantly enhances the residency of memory T cells,leading to their long-term retention in local tissues,a feature not shared by other types of memory T cells in the skin.(6)In individuals with vitiligo,the skin Trm cells are regulated by various key genes and metabolic pathways.In vitiligo,the interaction between CD8+ Trm cells and other memory T cells may lead to the occurrence,recurrence,and spread of vitiligo.During this pro cess,vitiligo-related Trm cells exhibit a unique gene regulatory network,providing new targets for understanding the pathogenesis of vitiligo and clinical gene therapy research.In summary,this study describes the regional skin immunity,investigates the formation mechanisms and migration processes of Trm cells in pathological and physiological states,and provides an ideal model that closely resembles the physiological,pathological,and immunological characteristics of the human body.It also offers new research targets for the study of skin memory T cell immunity.This research holds significant importance in advancing the translation of regional skin basic immunity into clinical applications.