Study On The Epigenetic Regulation Mechanism Of INKT Cell Immunotherapy For RA

ObjectiveStudies have shown that epigenetic regulation affects the development and differentiation of iNKT cells.Our previous study found that both RA patients and RA model mice showed a decrease in the number of iNKT cells in the body,and the subgroup proportion was unbalanced,but the relevant mechanism is still unclear.Cellular immunotherapy provides a new way of thinking and means for the treatment of major diseases and has been widely used in tumors and blood diseases.iNKT cell immunotherapy remodels the immune function of the body through two-way regulation,which will help fundamentally eliminate pathological damage and immune disorder caused by excessive inflammation,and is expected to become a new strategy for the treatment of autoimmune diseases.This study aims to explore the epigenetic mechanism of RA from the perspective of abnormal development and differentiation of iNKT cells.iNKT cells with specific phenotypes and functions were adopted and imported into RA model mice.The mechanism of epigenetic regulation of RA in adoptive immunotherapy with iNKT cells was investigated by observing the immune status of the body and the changes in the phenotype and function of iNKT cells.Methods1.Establishment of rheumatoid arthritis(RA)modelRA models were constructed with hGPI 325-339 and hGPI465-483 peptides.In order to identify the success of RA model construction,the general clinical manifestations of mice were observed.The pathological changes of joints were observed by hematoxylin-eosin staining(HE).The rates and subgroups of iNKT cells were detected by Flow Cytometry(FCM).Cytometric Bead Array(CBA)to detect serum levels of cytokines.2.iNKT cells with specific phenotypes and functions were induced and harvested for identification and safety evaluationMoreover,after 3 days of intraperitoneal injection ofα-Galcer,iNKT cells were sorted out by MACS.The rates and subgroups were detected by FCM.CBA was used to detect the secretion of supernatant cytokines.iNKT cells labeled by DiR were used to track the migration,distribution and metabolism of iNKT cells in RA mice using in IVIS system.3.Efficacyobservationandepigeneticregulationmechanismofadoptive immunotherapy for RA by iNKT cellsTo observe the changes of general clinical manifestations and immune status of mice to evaluate the therapeutic effect.To investigate the epigenetic regulation mechanism of iNKT cell immunotherapy in the treatment of RA,the rates and susets of iNKT and the susets of CD4~+T cell were detected by FCM,protein expression was detected by WB,mRNA level was detected by qRT-PCR,and the modification level of H3K27me3 and H3K4me3 were detected by ChIP-PCR.Results1.The rates of peripheral blood and thymus iNKT in RA mice was significantly decreased,and the subgroup of thymus iNKT1 was significantly increased.PLZF protein and PLZF mRNA expression were significantly decreased in thymus DP T cells,and T-bet protein and T-bet mRNA expression were significantly increased in iNKT cells.H3K27me3 level was significantly increased in the Zbtb16 gene promoter region of thymus DP T cells in RA mice,and H3K4me3 level was significantly increased in the Tbx21 gene promoter region of iNKT cells.The expression of UTX in thymus of RA mice was significantly decreased.The changes of the above indicators were particularly significant in the progressive phase of inflammation(11 days after modeling)and the peak phase of inflammation(14 days after modeling)in RA mice.2.Isolated and purified mouse spleen iNKT cells with a purity of 85%,of which iNKT2accounted for 92%and mainly secreted IL-4.Adoptive infusions of iNKT cells into healthy mice with zero mortality.Adoptive infusions of DiR labeled iNKT cells into RA mice mainly distributed in liver and spleen,and the fluorescence disappeared after 42 days.Both immunotherapy with iNKT cells and treatment withα-Galcer can improve weight loss,relieve joint redness and swelling,reduce the infiltration of foot and ankle arthritis cells,and reduce the level of serum inflammatory cytokines in RA mice,and the effect of immunotherapy with iNKT cells is more obvious.3.Both immunotherapy with iNKT cells and treatment withα-Galcer significantly increased the rates of peripheral blood,spleen and thymus iNKT in RA mice,and corrected the imbalance of thymus,spleen iNKT subsets and spleen CD4~+T cell subsets.Immunotherapy with iNKT cells and treatment withα-Galcer increased the expression of UTX,PLZF and PLZF mRNA,decreased the expression of T-bet and T-bet mRNA in iNKT cells,decreased the H3K27me3 modification level in Zbtb16 gene promoter region,and decreased the H3K4me3 modification level in Tbx21 gene promoter region.Immunotherapy with iNKT cells was more effective.In addition,iNKT cell immunotherapy can up-regulate the expression of GATA3 and Foxp3 and decrease the expression of ROR?t and T-bet in spleen.Conclusions1.Developmental and differentiation defects of iNKT cells in RA mice were associated with abnormal methylation levels of key genes Zbtb16(encoding PLZF)and Tbx21(encoding T-bet)promoters H3K27me3 and H3K4me3.Decreased UTX of thymus histone demethylase resulted in the accumulation of H3K27me3 modification.2.Obtaining iNKT cells(iNKT2)with specific phenotype and function from normal mouse spleen for immunotherapy of RA mice can effectively alleviate clinical symptoms and is superior toα-Galcer treatment.3.iNKT cell immunotherapy can regulate the expression of UTX and the level of histone H3K27 and H3K4 methylation in the promoter regions of Zbtb16 and Tbx21 gene to make iNKT cells develop and differentiate normally,correct the immune disorder,and reconstruct the immune balance,thus effectively alleviating RA.