Expression Of Follicular Helper T Cell Immune Molecules In Peripheral Blood And Lesional Skin Of Patients With Systemic Lupus Erythematosus

Systemic lupus erythematosus（SLE）is a heterogeneous autoimmune disease in which type I interferon-l（IFN-I）drives B cells and helper lymphocyte subsets（T helper lymphocyte,Th）overreacting^[1].Its main characteristics are autoantibody spectrum,differential expression of serum cytokines,significant dysfunction of T and B cells and irreversible destruction of immunologic self^-tolerance^[2].Systemic lupus erythematosus usually exhibits clinical symptoms affecting multiple organ systems such as skin,kidney,musculoskeletal and hematopoietic systems^[3].These effects are usually mediated by the production of autoantibodies and immune complexes.Although its etiology is still unknown,genetic,microbial,environmental and psychological factors are thought to be contributing factors.CD4⁺T cell subsets,such as Th1,Th2,Th17 and Tfh cells,play an important role in the pathogenesis of SLE^[4-6].Follicular helper T cells（Tfh）are primarily located at the germinal center and contribute to B cell differentiation and development^[7].And Tfh cells play an important role in promoting the formation of GC and in the selection of high affinity B cells^[8].It has been reported that Tfh（CD4⁺CXCR5⁺PD-1⁺）cell subpopulation is a cell type that plays an important role in the pathogenesis of SLE^[8].Immune molecule CXCR3 is an important marker of Tfh cell subsets^[9].Studies have shown that CXCR3 is reduced on CD4⁺T cells in patients with lupus erythematosus^[10].However,the expression of CCR4,CCR6 and CXCR3in Tfh-like（CD4⁺CXCR5⁺PD-1⁺）cell subsets in peripheral blood and lesions of SLE patients has not been reported.In this study,the expression of CCR4,CCR6 and CXCR3 in peripheral circulation and skin CD4⁺T lymphocyte subsets,especially Tfh-like cell subsets,in SLE patients and healthy controls were investigated.Furthermore,the distribution frequency of CD4⁺T cell subsets and the expression levels of CCR4,CCR6 and CXCR3 in the secondary lymphoid organs were investigated.To reveal the important role of CD4⁺T cell subsets,especially Tfh cell subsets and immune molecule CCR4,CCR6 and CXCR3 in systemic lupus erythematosus,and to provide necessary theoretical basis for early diagnosis and effective targeted treatment of SLE.Part Ⅰ Changes of CD4⁺T cell subsets in peripheral blood of patients with systemic lupus erythematosusObjective:To investigate the changes of CD4⁺T subsets in peripheral blood mononuclear cells（PBMCs）and the relationship between the expression levels of immune molecules CCR4,CCR6 and CXCR3 in each CD4⁺T cell subset and SLEDAI score,autoantibody level,gender and other important factors associated with systemic lupus erythematosus.Method:（1）A total of 40 patients with systemic lupus erythematosus and 25healthy volunteers were recruited.After asking the medical history and checking the examination results,the disease condition was confirmed,and the disease activity was assessed using the systemic lupus erythematosus disease activity index（SLEDAI score）.Inform the patient of the reason for blood donation,and sign the informed consent,detailed records of all the basic information of the patient and all the examination results,by the nurse after disinfection venous blood 3m L/person.（2）Ficoll density gradient centrifugation was used to separate mononuclear cells from peripheral blood.（3）A small amount of cell suspension was taken and diluted in an appropriate multiple for cell counting.（4）According to the cell count results,the volume of cell suspension corresponding to the appropriate number of cells（at least 2×106cells/tube was required）was calculated and added into the flow tube with the corresponding number.Flowcytometry was used to detect the cells after they were labeled with CD4,CXCR5,PD-1,CCR6,CD62L,CD8,CXCR3,CCR4,CD45RA,CXCR5,CD45RO,and Zombie Aqua^TMby flow cytometry.（5）After the data were analyzed by FlowJo,Graph Pad Prism was used for data statistics,and P<0.05 was considered to be statistically significant.Result:（1）The proportion of Tfh-like（CD4⁺CXCR5⁺PD-1⁺）cells and activated TFH-like（CD4⁺CXCR5⁺PD-1⁺CD45RO⁺）cell subsets in PBMCs of patients with systemic lupus erythematosus is higher than that of healthy volunteers.（2）The proportion of Tfh-like（CD4⁺CXCR5⁺PD-1⁺）cells and activated TFH-like（CD4⁺CXCR5⁺PD-1⁺CD45RO⁺）cell subsets in peripheral blood of patients with systemic lupus erythematosus was positively correlated with SLEDAI score.（3）The expression levels of the immune molecule CXCR3 in Tfh-like and Activated Tfh-like cell subsets negatively correlated with the SLEDAI score but positively correlated with the serum complement C3level.（4）The expression levels of CXCR3 in Tfh-like and Activated Tfh-like cell subsets in peripheral blood of patients with inactive lupus erythematosus are higher than those of patients with active lupus erythematosus.（5）The expression levels of CCR4,CCR6,and CXCR3 in Tfh-like and Activated Tfh-like cell subsets in peripheral blood of female lupus patients are higher than those of male patients.（6）The expression level of CXCR3 in CD4⁺T cell subsets of peripheral blood in menopausal women with lupus erythematosus is increased.Conclusion:（1）The increasing proportion of Tfh-like（CD4⁺CXCR5⁺PD-1⁺）cell subsets and the decreasing expression level of immune molecule CXCR3in human peripheral blood CD4⁺T cell subsets are closely related to the occurrence of systemic lupus erythematosus.Patients with systemic lupus erythematosus have significant increases in the proportion of Tfh-like（CD4⁺CXCR5⁺PD-1⁺）cell subsets in CD4⁺T cell subsets in peripheral blood and decreases in the expression level of the immune molecule CXCR3.（2）The prevalence rate of SLE in women is higher than that in men,which may be related to the increase of the expression levels of immune molecules CCR4,CCR6 and CXCR3 in Tfh-like（CD4⁺CXCR5⁺PD-1⁺）cell subsets.（3）The expression level of Tfh-like cell subgroup CXCR3 in female patients may be related to the estrogen level.Part Ⅱ Changes of CD4⁺T cell subsets in skin lesions of patients with systemic lupus erythematosusObjective:To investigate the changes of CD4⁺T cell subsets in skin tissue and the relationship between the expression levels of immune molecules CCR4,CCR6 and CXCR3 in CD4⁺T cell subsets and systemic lupus erythematosus.Method:（1）Four patients with systemic lupus erythematosus and three healthy volunteers were recruited.Disease activity was assessed using the SLEDAI score after history questions and examination results to confirm the patient’s medical condition.Informed the patient of the cause of the skin lesion sampling,and signed an informed consent form,after recording all the basic information of the patient,medication and all the examination results in detail,the nurse disinfected and used a disposable skin drill sampler to collect skin tissue samples at the lesion.（2）After the skin tissue was embedded in paraffin,the pathological tissue was sectioned.（3）The sections of pathological tissues were stained with Hematoxylin-eosin staining,HE),and the infiltration of inflammatory cells was observed under the microscope.（4）After immunohistochemical staining of the pathological sections,the distribution of CD4⁺T cells was observed under the microscope.（5）Separating the remaining skin tissues and preparing a single cell suspension for cell counting.（6）According to the counting result,the volume of the cell suspension corresponding to the required number of cells in the sample tube was calculated and added into the flow tubes with corresponding numbers,and the flow antibody was stained and detected on a computer.（7）After analyzing the streaming data with FlowJo software,Graph Pad Prism software was used for data statistics,and P<0.05 was considered to be statistically significant.Result:（1）Compared with healthy volunteers,there is significant inflammatory cell infiltration in the skin tissue of patients with systemic lupus erythematosus and the inflammatory cells are CD4⁺T cells.（2）Compared with healthy volunteers,the proportion of CD4⁺T cell subsets（Th1 cell subsets and Tfh-like cell subsets）in the skin tissue of patients with systemic lupus erythematosus is higher.（3）The expression level of the immune molecule CXCR3 in CD4⁺T cell subsets（including total CD4⁺T cells,Na（?）ve T cells,effector T cells and Tfh-like cells）of the diseased skin tissue of lupus patients is higher than that of the healthy control group.Conclusion:（1）CD4⁺T cell infiltration increases in diseased skin tissues of patients with systemic lupus erythematosus.（2）In the diseased skin tissues of patients with systemic lupus erythematosus,the proportion of CD4⁺T cell subsets and the expression level of the immune molecule CXCR3 are increased.Part Ⅲ Changes of CD4⁺T cell subsets in human tonsil tissueObjective:to investigate the changes of CD4⁺T cell subsets in human tonsil tissue and the expression levels of immune molecules CCR4,CCR6 and CXCR3 in CD4⁺T cells.Method:（1）Nine volunteers with tonsil hypertrophy/adenoid hypertrophy were recruited.The patient was confirmed not to have immunodeficiency,severe persistent infection（such as hepatitis B,C or HIV infection）,malignant tumors of the lymphatic system,cytogenetic diseases,cancer,etc.after medical history was asked and examination results were reviewed.The patient was informed of the reason for the tonsillectomy,and an informed consent form was signed.After all the basic data of the patient and all the examination results were recorded in detail,the tonsillectomy was performed and the tonsillar tissue was collected.（2）Tonsil tissues are treated by a mechanical grinding method and single cell suspension liquid is prepared for cell counting.（3）According to the counting result,the volume of the cell suspension corresponding to the required cell number of each sample was calculated and added into a flow tube with a corresponding number,and the flow antibody was stained and detected on a computer.（4）After the data were analyzed with FlowJo software,the data were statistically analyzed with Graph Pad Prism software,and P<0.05 was considered to be statistically significant.Result:（1）In human tonsil tissue,the expression levels of immune molecules CCR4,CCR6 and CXCR3 in Tfh cell subsets are higher than those of other CD4⁺T cell subsets.（2）The expression levels of immune molecules CXCR3,CCR6 and CCR4 in unactivated Tfh cell subsets are higher than those in other Tfh cell subsets.Conclusion:（1）Tfh cell subsets have important immune function in human tonsil tissue.（2）In human tonsil tissue,the high expression of immune molecules CCR4,CCR6 and CXCR3 contributes to the function of Tfh cell subsets.