The Expression And Biological Effects Of OX40 Molecule On Peripheral Tfh Cells In Graves’ Disease

Part I Expression and biological significance of OX40+ Tfh cells inGraves’ diseaseObjective: To study the expression and significance of Tfh cells,OX40 and OX40 L in thyroid tissue and peripheral blood of Graves’ disease patients,the distribution of Tfh cell subsets in peripheral blood of Graves’ disease patients and relationship between OX40 and different subsets of Tfh cells.Methods: In this study,the expression of CD4,CXCR5 and OX40 in thyroid tissue of Graves’ disease patients were detected by immunohistochemical technique.Then the consistency of expression of each molecule was analyzed.Flow Cytometry was used to detect CD4+CD25-CD127+CXCR5+PD-1+ Tfh cells,the expression of OX40 on Tfh cells,the expression of OX40 L on B cells and monocytes in peripheral blood of Graves’ disease patients.The changes of Tfh1,Tfh2,and Tfh17 in Tfh cells were detected and the relationship between OX40 molecules and Tfh cell subsets was analyzed.Results:(1)CD4,CXCR5 and OX40 molecules were mainly expressed on the cell membrane of thyroid tissue cells in Graves’ disease patients.Compared with the thyroid adenoma group,the positive rates of CD4,CXCR5 and OX40 molecules in the Graves’ disease group increased,with statistical significance(p<0.05).Furthermore,there were statistically significant differences in the expression of CD4,CXCR5 and OX40 in the thyroid tissue of Graves’ disease patients(Kappa=1,p=0.002).(2)Compared with patients with thyroid adenoma and healthy volunteers,the percentage of peripheral Tfh cells in patients with Graves’ disease significantly increased(p= 0.0077),and waspositively correlated with clinical indicators TRAb,FT3 and FT4(p<0.05).The ability of CD4+ T lymphocytes and Tfh cells to secrete IL-21 was significantly enhanced with statistical significance(p<0.01).(3)The proportion of Tfh17 cells in peripheral Tfh cells was the highest in patients with Graves’ disease.Compared with thyroid adenoma patients and healthy volunteers,Tfh1 cells and Tfh17 cells significantly increased,and however,Tfh2 cells significantly decreased,with statistical significance(p<0.05).(4)Compared with patients with thyroid adenoma and healthy volunteers,the expression level of OX40 on peripheral Tfh cells was significantly up-regulated in Graves’ disease patients,and the difference was statistically significant(p=0.014).(5)Compared with other peripheral Tfh cell subsets in patients with Graves’ disease,the expression level of OX40 on Tfh17 cells was the highest.The subpopulation of OX40+ Tfh cells is mainly Tfh17 cells.The proportion of Tfh17 cells in OX40+ Tfh cells was significantly higher than that of OX40-Tfh cells,with a statistically significant difference(p=0.039).(6)The expressions of OX40 L on peripheral blood B cells and monocytes in patients with Graves’ disease was significantly higher than those in thyroid adenoma patients and healthy volunteers.The difference was statistically significant(p<0.05).Conclusion:(1)There may be abnormal increase of CD4+CXCR5+ Tfh and OX40+Tfh in the thyroid tissue of Graves’ disease patients.(2)The percentage of Tfh cells in peripheral blood of patients with Graves’ disease was up-regulated,which was positively correlated with the clinical indicators of Graves’ disease.The secretion of IL-21 increased too.The Tfh cell subsets were mainly Tfh17 cells.(3)The expression level of OX40 on peripheral Tfh cells was significantly up-regulated in Graves’ disease patients,and OX40+ Tfh cells were mainly Tfh17 cell subsets,which may help B cells secrete autoantibodies.(4)The expressions of OX40 L on B cells and monocytes in peripheral blood of patients with Graves’ disease were significantly upregulated.Therefore,OX40 L expressed on antigen-presenting cells may bind to OX40 on Tfh cells to influence Tfh cells,then participating in the occurrence and development of Graves’ disease.Part II Effect of OX40/OX40 L signaling on Tfh cellsObjective: To investigate whether OX40/OX40 L signal can directly affect Tfh cells,promote the differentiation of Tfh cells,maintain the survival of Tfh cells,promote the secretion of cytokines,and provide some theoretical basis for the clinical application of OX40/OX40 L signal in the treatment of autoimmune diseases.Methods: In this study,immunomagnetic beads sorting technology was used to enrich CD4+ T lymphocytes in peripheral blood of healthy volunteers.Stimulated with four types of combination of anti-CD3-m Ab,anti-CD3-m Ab+s OX40 L,anti-CD3-m Ab+rh IL-12,anti-CD3-m Ab+s OX40L+rh IL-12.Flow cytometry and fluorescence quantitative PCR were used to detect the protein and m RNA levels of surface molecules CXCR5,PD-1,ICOS,intracellular factor IL-21,and nuclear transcription factor Bcl6 in CD4+ T lymphocytes.Human tonsillar Tfh cells were sorted by flow cytometry,and were stimulated in the presence or absence of s OX40 L protein.Changes of Tfh cell surface molecules CXCR5,PD-1,ICOS and chemokine receptor CXCR4 were detected by flow cytometry.Results:(1)The purity of CD4+ T lymphocytes was more than 95% by immunomagnetic bead sorting method,which was in accordance with the experimental requirements.(2)After the addition of s OX40 L protein,the expression levels of CXCR5,PD-1,and ICOS were up-regulated in peripheral CD4+ T lymphocytes,which was similar to those of positive control rh IL-12.After co-stimulation with s OX40 L protein and rh IL-12,the expression levels of the three molecules were higher,and the difference was statistically significant(p<0.05).(3)After the addition of s OX40 L protein,the expression levels of Bcl6 and IL-21 in peripheral CD4+ T lymphocytes were up-regulated,and the combination of s OX40 L and rh IL-12 was more significantly up-regulated(p<0.05).(4)s OX40 L protein up-regulated ICOS,Bcl6 and IL-21 m RNA levels in peripheral CD4+ T lymphocytes,and the combination of s OX40 L and rh IL-12 increased more significantly,with statistical significance(p<0.05).(5)Adding s OX40 L protein can maintain the expression of CXCR5,PD-1 and ICOS on the tonsillar Tfhcells,and then maintain the proportion of Tfh cells.The difference was statistically significant(p<0.05).(6)The expression of CXCR4 on Tfh cells was also up-regulated by s OX40 L protein(p<0.05).Conclusion:(1)OX40/OX40 L signal can up-regulate the expression of CXCR5,PD-1,ICOS,Bcl6 and IL-21 on CD4+ T lymphocytes,up-regulate the proportion of Tfh cells and promote the differentiation of Tfh cells.(2)OX40/OX40 L signaling can maintain the expressions of CXCR5,PD-1 and ICOS molecules on Tfh cells,and maintain the survival of Tfh cells.(3)OX40/OX40 L signal can promote the expression of CXCR4 on Tfh cells,and may participate in the migration of Tfh cells into thyroid tissue and the formation of ectopic GC.