The Study Of Tissue Resident Memory T Cells And Regional Immune Tolerance Of Human Skin Using Humanized Mouse Model

Aims:As the main interface area between the body and the external environment,skin has unique regional immune characteristics.Tissue resident memory T cells(T_RM)is a special T cell subpopulation of the skin,which play an important role in the onset and recurrence of many autoimmune skin diseases（such as vitiligo）,but its specific mechanism and migration process are not clear.Melanoma-associated antigen recognized by T cells（MART-1）is one of the differentiation antigens of melanocytes.Clinical studies have found that the peripheral blood of vitiligo patients contains a high proportion of MART-1 specific T cells,suggesting that these cells are closely related to the development of the disease.Some healthy people also have a high proportion of MART-1 specific T cells,but they do not attack melanocytes in the skin,suggesting that these cells are in a state of immune tolerance under normal conditions.In order to study the immune characteristics and tolerance mechanism of the skin region,it is a powerful research method to establish an ideal disease animal model.However,due to the differences between mice and human skin in anatomical level,cell composition and many other aspects,the traditional mouse model can not be used as an ideal research platform.Therefore,in this study,based on the humanized mice closest to the physiological and pathological characteristics of human body,the skin/thy/HSCs humanized mice（STH hu-mice）were constructed by transplanting the homologous human skin,in order to study the basic immunological characteristics of the skin.The HLA-A2restricted MART-1 TCR tg humanized mice was constructed,and by simulating the process of breaking the local immune tolerance of skin,to induce the onset of vitiligo,and study the production process and immune characteristics of specific TRM.Methods:（1）The fetal skin was cryopreserved with 0.5mol/L trehalose tissue cryopreservation solution in liquid nitrogen.After resuscitation,it was transplanted to NCG mice to observe the development process of skin after transplantation.Histopathology and immunohistochemistry were used to identify whether the grafted skin has complete tissue structure,and to study the local immune cell composition,the number and distribution of melanocytes.（2）NCG mice were irradiated with 1.75Gy sublethal dose and then constructed the STH humanized mice via transplantation of homologous thymus（under renal capsule）,fetal liver derived CD34⁺hematopoietic stem cells（caudal vein）and fetal human skin（dorsal skin）tissue.To detected the reconstruction of human immune system in peripheral blood of mice on time.The mice were sacrificed after 18 weeks,and the grafted human skin was detected by flow cytometry,histopathology and immunohistochemistry to see the reconstruction of immune system and distribution of immune cells in mouse skin,peripheral blood,spleen,bone marrow,and grafted human skin and thymus.（3）NCG mice were irradiated with 1.75Gy sublethal dose and then constructed MART-1-TCR tg humanized mice via transplantation of HLA-A2⁺homologous thymus,HLA-A2 restricted MART-1-TCR lentivirus infected fetal liver derived CD34⁺hematopoietic stem cells and human skin.The human immune system reconstruction and the expression of MART-1-TCR specific T cells were detected on time.The mice were sacrificed after 16 weeks to detect the immune cell reconstitution in mouse skin,peripheral blood,spleen,bone marrow,and grafted human skin and thymus,the expression of T_RM and MART-1 specific T cells in skin were detected by flow cytometry.（4）Re-constructed MART-1-TCR tg humanized mice,the human immune system reconstruction and the expression of MART-1-TCR specific T cells were detected on time.Some of the mice were sacrificed after 16 weeks to detect the immune cell reconstitution in mouse skin,peripheral blood,spleen,bone marrow and grafted human skin and thymus,the expression of T_RM and MART-1 specific T cells in skin were detected by flow cytometry.The rest of the mice were divided into three groups:a)MART-1 peptide+imiquimod,b)DMSO+imiquimod,c)DMSO,once a week,for 4 weeks.The activation of MART-1-specific T cells in peripheral blood was detected every week.The mice were sacrificed in the 5th week,and the expression of the above cells was detected by flow cytometry.The destruction of melanocytes was identified by immunohistochemistry.Results:（1）Histopathology showed that the cryopreserved fetal skin had the basic skin tissue structure of healthy adults after transplantation,and the new black hair was seen 6-7 weeks after transplantation,able to continued growing.IHC showed few human CD45⁺cells infiltrate in the dermis.The results of MART-1 IHC showed that the density and distribution of melanocytes in the basal layer of the transplanted skin were close to that of the healthy adult skin.（2）The infiltration of human CD45⁺cells in the grafted skin of the STH humanized mice was significantly higher than that in the mouse skin,most of them were CD3⁺T cells,only a few CD11c⁺dendritic cells（DCs）,CD19⁺B cells were barely found.Compared with the peripheral blood and spleen of mice,a high proportion of CD4⁺CD69⁺CD103^-T_RM,CD4⁺CD69⁺CD103⁺T_RM and CD8⁺CD69⁺CD103⁺T_RM were detected in the grafted skin.（3）In the peripheral blood,spleen and other tissues of MART-1-TCR tg humanized mice,different levels of MART-1-specific T cells were detected,the phenotype of which was mainly CD45RA⁺,but MART-1-specific T cells were not detected in the grafted skin.（4）High level expression of tetramer⁺CD8⁺T cells was detected in peripheral blood,spleen and other tissues of MART-1-TCR tg humanized mice.No MART-1specific T cells were detected in the grafted skin without local treatment.After continuous treatment with imiquimod or imiquimod+peptide,different levels of MART-1 specific T cells were infiltrated in the grafted skin,but IHC staining showed that only 2 mice with most MART-1 specific T cells infiltrated in grafted skin have no MART-1⁺melanocyte within the grafted skin.Non-humanized skin-NCG mice showed no melanocytes destruction within grafted skin.Conclusions:（1）Grafted fetal skin can survive and mature after cryopreservation in liquid nitrogen under the protection of trehalose.（2）The composition of immune cells in the grafted skin of STH humanized mice is similar with that of healthy adult skin,human T_RM can only form in grafted human skin.It is an ideal model for studying the regional immunity of skin,the formation mechanism of T_RM and the migration process.（3）Under the condition that the local immune tolerance of skin is not broken,the MART-1 specific T cells in the peripheral blood of MART-1-TCR tg humanized mice are not activated and cannot enter the skin to kill the target cells.（4）When the skin region immune tolerance is broken,the peripheral MART-1specific T cells of the MART-1-TCR tg mice can enter the grafted human skin and mouse skin,but they can only form a specific resident phenotype in human skin.When MART-1 specific T cells reach to a higher level,they have a killing effect on melanocytes.This investigation provides an ideal model which is closest to human physiological and pathological characteristics for describing skin regional immunity,and studying the formation mechanism and migration process of T_RM in autoimmune skin diseases.It is of great significance for the development and update of skin regional immunology,the research and development of new targets of immunotherapy,and the transformation and application of skin regional immunology discovery.