CD74 (Invariant Chain) Regulates Cellularity And Maturation Of Medullary Thymic Epithelial Cells

BackgroundThe thymus is the primary lymphoid organ responsible for the generation and maturation of T cells.Thymic epithelial cells(TECs)account for the majority of thymic stromal components and are indispensable for T-cell development,T-cell receptor(TCR)repertoire selection and specific lineage differentiation.They are further divided into cortical(cTECs)and medullary TECs(mTECs)based on their localization within the thymus.mTECs account for more than 90%of adult thymic epithelial cells and play an important role in the negative selection and self-tolerance of thymic cells,while cTECs are critical for the positive selection.CD74,a nonpolymorphic transmembrane glycoprotein,is a molecular chaperone of major histocompatibility complex class?(MHC?).CD74 is mainly expressed in antigen-presenting cells,such as B cells,dendritic cells and TECs and is a useful marker for the classification of thymic epithelial neoplasms.However,the exact role of CD74 in TECs is poorly understood.Here,we focused on the effect of CD74 on TECs to elucidate the role of CD74 in thymopoiesis and thymic epithelial cell development,which will provide a theoretical basis for the classification,diagnosis and targeted therapy of thymic epithelial neoplasms.Methods1.By downloading the known public single-cell RNA-sequencing results,we intended to explore the expression level of CD74 in thymic epithelial cells by bioinformatics analysis.2.CD74 gene knockout mouse model and flow cytometry were used to analyze the phenotype of CD74 deficiency on thymic epithelial cells.3.The mTECs from CD74 deficient mice and control mice were sorted and RNA were extracted and analyzed by RNA-sequencing.Bioinformatics analysis was performed by using GO and KEGG databases.Flow cytometry and in vitro cultured thymic epithelial cells technology combined with western blotting were used to verify the results of bioinformatics analysis.4.Flow cytometry was used to analyze the effect of CD74 deficiency on thymocytes and peripheral T cells.Results1.We have demonstrated that CD74 mainly expressed in mature population of medullary thymic epithelial cells and cortical thymic epithelial cells.The expression level of CD74 was nearly consistent with MHCII molecules.2.We have found that in the absence of CD74 leads to decreased medulla and mTECs number,reduced expression level of MHCII and CD80,leading to the defected mTECs maturation.3.We have found that down-regulated canonical NF-?B signaling in mTECs after CD74 deletion.We have demonstrated that the total protein level and phosphorylation of NF?B-p65 are decreased and its nuclear translocation,especially after RANKL stimulation,is decreased in CD74 deficient primary cultured TECs.4.CD74 deficiency results in impaired CD4+T cell development and decreased naive CD4+T cells in the peripheral,while leading to the increased peripheral CD8+cell abundance.Moreover,the number of regulatory T cells also reduced both in the thymus and spleen.ConclusionOur results suggested that CD74 is essential for the mTECs maturation and cellularity maintenance and CD4+T cell differentiation.Dramatically down-regulated canonical NF-?B signaling pathway may partially lead to a diminished medulla in the thymic architecture and decreased maturation of mTECs in CD74 deficiency mice.