Genealogy Analysis Of Peripheral T Helper Cell In Rheumatoid Arthritis And Its Functional Study

Objective:1.Comparative of rheumatoid arthritis（RA）patients’s Peripheral Blood Mononuclear Cells（PBMCs）,Synovial Fluid Mononuclear Cells（SFMCs）and healthy control Peripheral Blood（HCPB）with the proportion of T cell subpopulations,looking for new pathogenic T cell subpopulations related to RA pathogenic mechanism.2.analyze the activation T cells in RA patients,and analyze the correlation between them with clinical indicators.3.To classify the new pathogenic T cells of RA patients into subgroups,and analyze the different subgroups in the Rheumatoid Arthritis Peripheral Blood（RAPB）and Rheumatoid Arthritis Synovial Fluid（RASF）in order to infer a new type of pathogenic T cell subpopulation that plays an important role in the inflammation tissues.4.Observe the influence of new pathogenic T cells on disease activity and the influence of B cells in the same inflammation tissus of new pathogenic T cells in order to study their functions.Methods:1.Through data analysis of single^-cell sequencing data in the cell database,the composition ratio of different clustered cells in the joint cavity effusion of patients with rheumatoid arthritis and the expression of transcription factors were observed,and the subsets were classified accordingly.The abnormally increased proportion of Tph subsets and their specific expression molecules are classified and integrated,and further detected by flow cytometry.2.Collect 153 samples of patients with rheumatoid arthritis who meet the clinical diagnostic criteria from the Southwest Hospital of the Army Military Medical University as the rheumatoid arthritis test group.Select 17 cases from the physical examination center as a healthy control group.3.The density gradient centrifugation method was used to extract PBMCs and SFMCs from patients with rheumatoid arthritis.The extracted cells were study with flow cytometry to detect their composition,including:Follicular helper T cell（Tfh）(CD4⁺CD45RO⁺PD^-1^HiCXCR5⁺),Peripheral helper T cell（Tph）(CD4⁺CD45RO⁺PD^-1^HiCXCR5^-),and detect the expression of related surface markers and cytokines,including:HLA-DR,ICOS,CXCR3,CCR6,IFNγ,I-17A,IL-4,IL-10,IL-21.4.The Tfh and Tph in the RAPB and RASF are classified into subsets based on the differences in the expression of chemokine receptors and cytokine secretion,The same subset was compared statistically to find the cell subpopulation enriched in the RASF,and then study its role in the inflammation site.5.Analyze the correlation between the IL-21⁺T cells and IL-10⁺T cells enriched in the inflammation site and clinical inflammation indicators,and through the correlation analysis of the cell subgroup frequence and different inflammation indicators.The cell subgroups that are closely related to the disease can be used as a marker for clinical diagnosis and treatment.6.Compare the frequence of Tph and Tfh and its activated cell subsets with the frequence of each subgroup of B cells present in the same sample,and carry out correlation analysis,so as to research fuction of Tph and Tfh.Results:1.Compared with the HCPB,the frequence of Tph and Tfh in RAPB was significantly higher（P<0.001）,and the frequence of Tph and Tfh in RASF was significantly higher than that in RAPB（P<0.0001）.The proportion of Tph in RAPB was significantly higher than that of Tfh（P<0.0001）,and the proportion of Tph in RASF was also significantly higher than Tfh（P<0.0001）.2.In RAPB,Tph has a positive correlation with C-reactive protein（CRP）level（P<0.001）;HLA-DR⁺Tfh has a positive correlation with erythrocyte sedimentation rate（ESR）（P<0.01）;ICOS⁺Tph has a positive correlation with ESR（P<0.01）,ICOS⁺Tfh has a positive correlation with ESR（P<0.01）;HLA^-DR⁺Tfh has a positive correlation with CRP（P<0.05）;ICOS⁺Tph has a positive correlation with DAS28 score（P<0.05）.3.The proportion of IL-21⁺Tph in RASF is significantly higher than that in RAPB（P<0.05）,the proportion of IL-10⁺Tph in RASF is significantly higher than that in RAPB（P<0.0001）,and the proportion of IL-10⁺Tfh in RASF is significantly higher.The frequence in RAPB was significantly higher than that in RAPB（P<0.0001）,and the frequence of IL-10⁺Tph in RASF was positively correlated with the concentration of IL^-10 cytokine in RASF（P<0.05）.4.In RAPB,IL-21⁺Tfh has a positive correlation with ESR（P<0.01）,and IL-21⁺Tfh has a positive correlation with CRP（P<0.01）;in RASF,IL-21⁺Tfh has a Negative correlation with ESR and CRP（P<0.05）.Conclusion:1.There is a PD^-1^HiCXCR5^-Tph cell subset in the RAPB and RASF,which is significantly higher than that in the healthy control,suggesting that Tph is a sensitive index for disease diagnosis and can be used as RA diagnostic markers of disease.2.Activated Tph has a significant correlation with RA disease activity indicators,suggesting that activated Tph can promote the development of disease inflammation,and activated Tph can be used as one of the biomarkers of disease progression.3.Tph can be classified into subsets.IL-10⁺Tph subsets are significantly higher in RASF than in RAPB,suggesting that IL-10⁺Tph cells play an important role in inflammation.4.The frequence of IL-21⁺Tph and IL-21⁺Tfh in RAPB and RASF has a significant positive correlation with disease activity,suggesting that IL-21⁺Tph and IL-21⁺Tfh have a direct relationship Pro-inflammatory effect.