Study On The Mechanism Of Adoptive Treatment Of RA By Thymus-derived INKT Cells

ObjectiveIn order to explore the immunological mechanism of thymus derived iNKT cells adoptive therapy for RA mice,(1)we established a mouse model of rheumatoid arthritis(RA)and detect the frequency of iNKT cell and subsets,Th cell subsets and cytokines.(2)The proliferation of iNKT cells of mice induced by thymus throughα-Galcer injection and obtain the specific iNKT cells.(3)Adoptive infusion of iNKT cells into RA mice to observe the therapeutic effecte.(4)Explore the mechanism of effect of iNKT cells on RA mice in vivo,provide a new theoretical basis for RA cell immunotherapy treatment.Methods1.To establish a mouse model of RA and identify it.HGPI325-339,hGPI469-483 peptide fragments of 1:1 mixed,emulsified with complete Freund’s adjuvant.DBA/1 mice were given subcutaneous injection of the immunogens and pertussis toxin was injected into the abdominal cavity to strengthen the immunity at 0h and48h.In healthy mice as the control group,observe the changes of body weight,ankle joint swelling of ankle joint.The joint tissues stained with hematoxylin and eosin(HE)was used to evaluate the inflammatory cells.Flow Cytometry(FCM)was used to detect the frequency of iNKT cells in peripheral blood,the frequency of iNKT cells and subsets in spleen and thymus,The frequency of Th1/Th2/Th17 subsets in spleen.Cytometric bead array(CBA)was used to analyze the changes of serum cytokines IL-4、IL-10、TNF-α、IFN-γ、IL-17A、IL-2、IL-6.2.The directional induction of iNKT cells derived from thymus and the evaluation of immuno biological function and safetyDBA/1 mice were injected subcutaneously withα-GalCer(2μg/mouse)forα-GalCerⅠgroup.α-GalCerⅡgroup was repeated subcutaneous injection of alpha-GalCer(2μg/mouse,48h/time),the mice were injection subcutaneously with the same volume of PBS at the base of tail for the control group.The changes of the frequency of iNKT and subsets in thymus were detected by FCM.The iNKT cells were isolated and purified by magnetic cell sorting(MACS).Stimulated the secretion of cytokines in the supernatant and detect by the CBA kit.Labeled the cells with fluorescent probe(DiR)and transfered the tail vein to the normal mice.The changes of cell colonization and migration were observed by the live animal imaging system.The body weight,,activities and postures are evaluated to determine the safety of their infusions.3.To observe the effect of thymus derived iNKT cells on RA mice and discuss its influence mechanismRA mice were treated with thymus derived iNKT cells.Withα-GalCer as positive control.To observe the changes of body weight and ankle swelling changes.The joint tissues stained with HE was used to evaluate the changes of inflammatory cells.FCM was used to detect the frequency of iNKT cells,iNKT subsets and the changes of Th subsets.The levels of cytokines in the serum of each group were detected by CBA.Wester blot was used to detect the expression of protein inthymus and spleen at mouse.Result1.Compared with the control group,the body weight of RA mice increased slowly(p<0.05).On the 6th day of modeling,the toes appeared red and swollen,the swelling reached the peak on the 14th day after the model establishment and then gradually eased.A large number of inflammatory cells were found in the ankle joint of RA mice after fourteenth days of HE staining.The frequency of iNKT cells in peripheral blood,thymus and spleen of RA mice decreased significantly at the peak of inflammation(p<0.05)by FCM.The frequencies of iNKT1 subsets in thymus and spleen of RA mice were significantly increased at the early stage of inflammation,while those of Th1 and Th17(p<0.05).Th2 subpopulation increased significantly in the middle and late stages of inflammation(p<0.05).The levels of TNF-α,IFN-γand IL-6 in the serum increased significantly at the peak of inflammation(p<0.05).2.After single subcutaneous injection of alpha-GalCer,The frequency of iNKT in the thymus of mice reached its peak in eighth days,and the frequency was about 16%.After multiple subcutaneous injection of alpha-GalCer,the iNKT frequency of thymus was reached peak in second days,the frequency was about 10%,and then decreased gradually.On the eighth day after subcutaneous injection of alpha-GalCer,the frequency of iNKT1 subgroup reached the minimum value,the frequency was about 0.31%,and the frequency of iNKT2subgroup reached the peak,with a frequency of 50%.Purification of iNKT from mice thymus on the eighth day after subcutaneous injection of alpha-GalCer by MACS,about 70%purity,CBA detection of the cytokine in the supernatant of iNKT cell stimulated by PMA/IO was mainly IL-4,adoptive transfer of cells to normal mice,the mice were normal,small animal imaging tracer cells located in the liver and spleen.3.Both iNKT cell adoptive transfusion andα-GalCer injection could significantly improve the body weight and swelling of joints in RA mice(p<0.05),reduce the infiltration of foot and ankle cells,and significantly increase the levels of peripheral blood,thymus and spleen iNKT cell frequency;The disorder of iNKT subgroup,Th subgroup in spleen and serum cytokine was corrected.Compared with the control group,the expression of T-bet protein in RA group was significantly increased at the early stage of inflammation(p<0.05).Compared with RA group,the expression of GATA-3 protein in cell group andα-GalCer group was significantly increased(p<0.05),there was no significant difference in T-bet expression.Conclusion1.The GPI mixed peptide-induced RA mouse model was more similar to RA patients in immunopathology of CD4~+T cells and iNKT cells.The onset of RA is not only associated with a decrease in the frequency of iNKT,but also has a close association with changes in iNKT cell subsets.In the early stages of RA inflammation,restoring the body’s iNKT frequency and adjusting the imbalance of iNKT subgroups may be beneficial to regulating the balance of Th subsets and cytokines in RA mice,and to play a role in the treatment of RA.2.On the 8th day after single subcutaneous injection ofα-Galcer tail,a large number of iNKT2 type cells can be harvested from the mouse thymus.After adoptive infusion in mice can settle in the spleen and liver,and the safety has been verified.3.Infusion of iNKT cells into RA mice increased iNKT frequency,altered iNKT subsets,Th cell subsets and related cytokines in RA and had a positive effect on RA...