| Part Ⅰ Expression and significance of Tfh cells and OX40/OX40L molecules in peripheral blood of patients with Graves’ diseaseObjective:To study the changes of the proportion of Tfh cells in peripheral blood of patients with Graves’ disease,the expression of OX40 in Tfh,effector T and Treg cells and the expression of its ligand OX40L in antigen-presenting cells.Methods:In this section,the proportion of CD4+CD25-CD127+CXCR5+PD1+Tfh cells in peripheral blood of healthy volunteers,patients with thyroid adenoma,and patients with Graves’ disease were detected by flow cytometry,and the expression of OX40 on Tfh cells was analyzed at the same time.In addition,the expression of OX40L in CD14+monocytes and CD19+B cells,as well as the expression of OX40 molecules on effector T cells and Treg cells were also analyzed by flow cytometry.Results:(1)The ratio of Tfh cells in peripheral blood of patients Graves’ disease(GD)was significantly higher than that of healthy volunteers(HC)and patients with thyroid adenoma(TA),and the difference was statistically significant(p<0.01).(2)The expression of IL-21 in peripheral CD4+T cells of patients with GD was significantly higher than those of HC and patients with TA(p<0.001);the trend of IL-21 expression in Tfh cells was consistent with this,and all have statistical significance(p<0.01).(3)The expression of OX40 on peripheral Tfh cells in patients with GD was significantly higher than that in patients with TA,the difference was statistically significant(p<0.05).Furthermore,the expression of OX40 on ICOS+Tfh cells was significantly higher than that on ICOS-Tfh cells(p<0.05).(4)OX40 in peripheral effector CD4+ T cells was significantly higher in patients with GD than in patients with TA(p<0.05),and the expression trend of OX40 in Treg cells was consistent with it(p<0.05).(5)The expression of OX40L on peripheral APCs in patients with GD was significantly higher than that in HC and patients with TA,the difference was statistically significant(p<0.05).Conclusion:(1)Compared with HC and patients with TA,The proportion of perpheral Tfh cells in patients with GD increased,and the expression of IL-21 increased too,suggesting that the pathogenesis of GD may related to the abnormal upregulation of Tfh cells.(2)Compared with HC and patients with TA,perpheral OX40+Tfh cells were up-regulated in patients with GD,and OX40 was mainly expressed on activated Tfh cells,suggesting that OX40 molecules may be closely related to abnormal activation of Tfh cells in this disease.(3)The expression of OX40 on peripheral effector CD4+T cells and Treg cells were up-regulated in patients with GD.The expression of OX40 on Treg cells weakens its immunosuppressive properties in vitro.As the function of effector T cells was enlarged and the function of Treg cells was inhibited,the occurrence of GD may be accelerated.(4)OX40L was significantly upregulated on peripheral APCs in patients with GD.Therefore,the expression of OX40L on APCs may interact with O X40 expressed on e ffector CD4+T cells,Tfh cells and Tregs to transduce OX40 signal,thereby promoting Tregs differentiate to effector T cells and affecting the differentiation and function of effector T cells and Tfh cells,which will further promote the development of Graves’ disease..Part Ⅱ Study of Biological Characteristics of OX40+Tfh CellsObjective:To study the expression characteristics of OX40 molecules in tonsil Tfh cells and compare the expression of some related proteins and genes in OX40-Tfh cells from OX40+Tfh cells,exploring the biological characteristics of OX40+Tfh cells.Methods:In this section,the expression of OX40 molecules in Tfh cells at different stages is detected by flow cytometry.Then immunomagnetic beads sorting technology and flow cytometry sorting technology were used to enrich OX40-Tfh cells and OX40+Tfh cells,all genes in OX40-Tfh cells and OX40+Tfh cells were analyzed by transcriptome sequencing.In addition,the expression of related molecules and transcription factors in OX40-Tfh cells and OX40+Tfh cells at protein levels were also detected by flow cytometry.Results:(1)The expresson of OX40 molecules on CXCR5high Tfh cells was significantly higher than that on CXCR5low Tfh cells and CXCR5-non-Tfh cells,and the difference was statistically significant(p<0.001).(2)Compared with OX40-Tfh cells,the expressions of a variety of Tfh cell-related genes and transcription factor genes that promote the differentiation of Tfh cells including CXCR5,CXCL13,PDCD1,IL21,BATF,CD200,BTLA,TNFRSF18,ASCL2,IRF4,STAT1,and TRAF4 were up-regulated in OX40+Tfh cells,the expressions of genes that inhibiting the differentiation,function,and GC localization of Tfh cells including CCR7,ID2,KLF2,PIK3IP1,GPR183 were down-regulated;In addition,the expressions of a vrariety of genes about cyclin-dependent kinases(CDKs),proteins that necessary for DNA replication,and genes related to cell proliferation,including CDK4,CDK6,MCM family genes,CDC45,E2F family genes,etc.increased in OX40+Tfh,while the expressions of a variety of genes that inhibit cell cycling and promote apoptosis,including CDC14A,CDKN1B,GADD45A,SMAD3,BCL2L11,ATM decreased.(3)Compared with OX40-Tfh cells,the proteins of Bcl6,IL-21,CXCR4,Ki67 were also highly expressed in OX40+Tfh cells,with statistical significance(p<0.05).(4)The expressions of OX40L on B-cells and CD11c+HLADR+myeloid APCs of tonsil tissue were relatively high.Conclusion:(1)OX40 molecule was mainly expressed on mature GC Tfh cells,but was lower in pre-Tfh cells and non-Tfh cells.(2)The expressions of a variety of Tfh cell-related genes were up-regulated in OX40+Tfh cells,while the expressions of genes that inhibit the differentiation,function,and GC localization of Tfh cell were down-regulated in OX40+Tfh cells,suggesting that OX40 signal can help Tfh cells to differentiate and migrate to GC;The expressions of multiple genes promoting cell cycling and cell proliferation also increased,while the expressions of many genes that inhibit cell cycling and promote apoptosis decreased,suggesting that OX40 signaling maybe promote cell proliferation and inhibit cell apoptosis.(3)Higher levels of Bcl6,IL-21,CXCR4,and Ki67 are expressed in OX40+Tfh,which further proves that OX40+Tfh cells maybe a kind of cells with strong ability of proliferation,differentiaion and migration.(4)OX40L is highly expressed in tonsil APCs,suggesting that OX40L can bind to OX40 on Tfh cells and affect Tfh cells through OX40 signal.Part Ⅲ Effects of OX40/OX40L Signal on the Differentiation and Function of Tfh CellsObjective:To explore the effects of OX40 signal on Tfh cells at different stages of differentiation,and to study the role of OX40 signal in promoting Tfh cells to assist B cell differentiaion and the secretion of antibodies,as well as the mechanism by which OX40/OX40L signal works.Methods:In this section,CD4+T cells were stimulated by sOX40Lprotein,and the expression of Tfh cell-related molecules were detected by qPCR experiments.After CXCR5high Tfh cells and CXCR5low Tfh cells were enriched by flow cytometry sorting technology,they were stimulated by sOX40L to study the roles of OX40/OX40L signal on the differentiation of Tfh cells.The effects of OX40 signal on the function of Tfh cells were analyzed by co-incubating B cells with OX40-Tfh cells or OX40+Tfh cells under different conditions.Finally,flow cytometry was used to explore the signal pathways regulatied by OX40/OX40L involved in Tfh cells.Results:(1)Compared with the control group,the expressions of Bcl6,CXCR5,PD-1,and IL-21 mRNA in CD4+T cells were significantly increased after the addition of sOX40L protein,and the expression of Blimp-1 mRNA was decreased(p<0.05).(2)After the addition of sOX40L protein,the percentages of CD4+CXCR5highPD-1high GC Tfh cells in CXCR5high Tfh cells and CXCR5low Tfh cells were significantly higher than the control group(p<0.05).However,regardless of whether it was CXCR5high or CXCR5low group,the proportion of CD4+CXCR5low PD-1low pre-Tfh cells was not significantly different from the control group.(3)Compared with the control group,the addition of sOX40L protein significantly reduced the proportion of apoptotic cells in CXCR5high Tfh cells(p<0.01),but there was no significant difference in CXCR5low Tfh cells.(4)When OX40-Tfh cells were co-incubated with B cells,the proportion of CD19+CD27+CD38+ plasma cells were increased after adding sOX40L protein(p<0.001);With sOX40L protein added,the proportion of plasma cells was also increased after OX40+Tfh cells co-incubated with B cells(p<0.01),and was higher than that of OX40-Tfh cells and B cells co-cultured group(p<0.0001).In addition,with the addition of blocking Anti-OX40L monoclonal antibody,the plasma cell ratio was significantly reduced(p<0.01).The ELISA results were consistent with the flow cytometry results.(5)After adding sOX40L protein,the expression of pPI3K and p Akt in Tfh cells were p-regulated,and the difference was statistically significant(p<0.05).Conclusion:(1)OX40/OX40L signal can up-regulate the mRNA expressions of Bcl6,CXCR5,PD-1 and IL-21,and down-regulate the expressions of Blimp-1 mRNA,suggesting that OX40/OX40L signal can promote the differentiation of CD4+T cells into Tfh cells.(2)The OX40/OX40L signal may help promote the differentiation of pre-Tfh cells to GC Tfh cells,and maintain the proportion of GC Tfh cells,but not the proportion of pre-Tfh cells.(3)OX40/OX40L signal mainly inhibits GC Tfh cell apoptosis,but has no obvious inhibitory effect on pre-Tfh cell apoptosis.(4)Tfh cells stimulated by OX40 signal can significantly promote B cells to differentiate into plasma cells and secrete IgG antibodies.Moreover,the OX40 signal blocking experiment further proves the important role of OX40/OX40L signal in the assistance of Tfh to B cells,that is,OX40/OX40L signal can enhance the function of Tfh cells to assist B cells.(5)The effects of OX40/OX40L on Tfh cells depends in part on the PI3K/Akt signaling pathway. |
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