【作者】 陈伟；

【导师】 王琳；

【作者基本信息】 四川大学 ， 皮肤病与性病学（专业学位）， 2022， 博士

【摘要】 目的:明确EB病毒(Epstein–Barr virus,EBV)相关皮肤T细胞淋巴瘤(cutaneous T-cell lymphoma,CTCL)是否存在免疫逃逸通路的过度激活。利用EBV阳性(EBV~+)B细胞来源的外泌体与EBV阴性(EBV~-)的CTCL细胞系共培养,研究EBV~+B细胞来源的外泌体对CTCL细胞免疫逃逸通路及凋亡的影响。进一步通过对EBV~+B细胞来源的外泌体进行微小RNA(micro RNA,miRNA)测序,明确EBV~+B细胞来源外泌体影响CTCL免疫逃逸及凋亡的关键性ebv-miRNA及其可能的机制。通过以上研究,旨在阐明EBV~+B细胞来源的外泌体在EBV相关CTCL发生发展中的具体作用及潜在机制,为后续开发潜在的治疗靶点提供实验及理论基础。材料和方法:1.验证EBV相关CTCL中是否存在EBV感染及STAT3/IL-10/PD-L1通路激活相关的免疫逃逸效应及EBV感染的情况。(1)对四川大学华西医院皮肤科近5年的32例EBV相关CTCL(20例ENKTCL及12例HVLPD)患者的石蜡组织切片进行免疫组化染色分析,以5例蕈样肉芽肿(mycosis fungoides,MF)及9例炎症性皮肤病作为对照组,进行EBV编码的潜伏膜蛋白-1(latent membrane protein 1,LMP-1)免疫组化染色及EBER 1/2原位杂交,明确EBV相关CTCL中EBV感染的情况;(2)在上述样本中,明确是否存在与STAT3/IL-10/PD-L1通路过度激活相关的免疫逃逸效应。2.EBV~+B细胞来源外泌体对CTCL细胞STAT3/IL-10/PD-L1通路相关免疫逃逸效应及凋亡的影响的研究。研究所用细胞:EBV转化人B淋巴母细胞样细胞系(BL2009细胞),EBV阴性人Burkitt淋巴瘤细胞系(CA46细胞),EBV阴性人CTCL细胞系(Hut78、H9)。实验分为实验组(BL2009细胞来源外泌体与CTCL细胞共培养)及对照组(CA46细胞来源外泌体与CTCL细胞共培养)。(1)在共培养前,首先提取EBV~+B细胞来源的外泌体,并利用蛋白免疫印迹(Western blot,WB)、粒径分析及透射电镜等技术对所提取的外泌体进行鉴定;(2)按上述分组进行共培养后,应用实时定量聚合酶链式反应(real time-quantitative polymerase chain reaction,RT-q PCR)技术检测EBV~+B细胞来源外泌体对CTCL细胞IL-10/PD-L1的m RNA表达水平的影响;(3)利用酶联免疫吸附反应(enzyme-linked immunosorbent assay,ELISA)检测EBV~+B细胞来源外泌体对CTCL细胞分泌IL-10的影响;(4)流式细胞术检测EBV~+B细胞来源外泌体对CTCL细胞膜上PD-L1蛋白表达的影响;(5)WB检测EBV~+B细胞来源外泌体对CTCL细胞PD-L1、t-STAT3、p-STAT3等蛋白表达的影响;(6)在共培养体系建立前加入STAT3磷酸化抑制剂C188-9(依次将浓度设置为0μM、40μM、100μM、200μM)对CTCL细胞进行预处理,随后利用WB技术检测不同浓度C188-9刺激下,EBV~+B细胞来源外泌体对CTCL细胞STAT3磷酸化水平及PD-L1表达量的影响;ELISA检测不同浓度C188-9刺激的情况下,EBV~+B细胞来源外泌体对CTCL细胞IL-10分泌量的影响;(7)流式细胞术检测EBV~+B细胞来源外泌体对CTCL细胞凋亡率的影响;(8)WB检测EBV~+B细胞来源外泌体对CTCL细胞凋亡相关蛋白(Bcl-2、BAX、Cyt C及Caspase3)表达量的影响。3.EBV~+B细胞来源外泌体miRNA测序及EBV编码的miRNA(ebv-miRNA)筛选。(1)提取BL2009细胞来源的外泌体,对外泌体中的总RNA进行分离提纯,随后对外泌体总miRNA进行建库测序;(2)对测序结果进行数据分析,初步筛选ebv-miRNA;(3)查阅文献及数据库,设计ebv-miRNA的模拟物并转染至Hut78及H9细胞内,ELISA检测ebv-miRNA对CTCL细胞分泌IL-10的影响;流式细胞术检测ebv-miRNA对CTCL细胞凋亡的影响;(4)经过上述筛选,得到目标ebv-miRNA,对其进行GO(gene ontology)、KEGG(kyoto encyclopedia of genes and genomes)及PPI(protein-Protein Interaction Networks)分析;(5)WB验证目标ebv-miRNA对CTCL细胞中上述生物信息学分析所预测的相关蛋白质的表达水平的影响。4.对20例EBV相关CTCL(10例ENKTCL及10例HVLPD)的皮损石蜡组织切片行荧光原位杂交(fluorescence in situ hybridization,FISH)检测,以3例MF作为阴性对照。检测目标ebv-miRNA的在EBV相关CTCL中的表达情况。结果:1.EBER 1/2原位杂交:所有的EBV相关CTCL中均表达EBER 1/2,在MF及炎症性皮肤病中均不表达;LMP-1仅在EBV相关CTCL中表达,在MF及炎症性皮肤病中均不表达,差异具有统计学意义(p<0.05)。免疫组化染色结果显示:相较于MF及炎症性皮肤病,EBV相关CTCL的皮损组织中IL-10/PD-L1表达明显升高(p<0.0001),而p-STAT3的表达在几组疾病之间没有差异。对以上几个标记的表达情况进行相关性分析发现,在EBV相关CTCL中,p-STAT3与IL-10的表达量呈正相关(p<0.001;r=0.779)。2.EBV~+B细胞来源外泌体对CTCL细胞STAT3/IL-10/PD-L1通路及凋亡影响的研究。(1)提取外泌体后,利用WB检测外泌体中CD9、CD63及CD81蛋白的表达情况,结果示所提取的外泌体表达上述3种外泌体标志蛋白;对外泌体悬液进行纳米粒径分析,结果示悬液中的颗粒直径集中在60-100nm之间;对外泌体悬液进行透射电镜观察可见典型外泌体的“杯状”双层膜结构,以上结果表明外泌体提取成功;(2)对共培养体系进行RT-q PCR检测,发现EBV~+B细胞来源外泌体可上调CTCL细胞IL-10及PD-L1 m RNA的表达水平(p<0.05)(3)对共培养体系进行ELISA检测,发现EBV~+B细胞来源外泌体可上调CTCL细胞培养上清中IL-10的浓度(p<0.001);(4)对共培养体系行流式细胞术检测,结果发现EBV~+B细胞来源外泌体可上调CTCL细胞膜上PD-L1蛋白的表达量;(5)通过对共培养体系进行WB检测,结果发现EBV~+B细胞来源外泌体可上调CTCL细胞中PD-L1及p-STAT3的表达水平,对t-STAT3的表达量无显著影响;(6)应用不同浓度的STAT3磷酸化抑制剂C188-9对CTCL细胞进行预处理(最高浓度为200μM);在经不同浓度的C188-9预处理CTCL后,再加入外泌体悬液进行共培养。WB结果示经不同浓度的C188-9刺激后,EBV~+B细胞来源外泌体上调CTCL细胞PD-L1表达的效应被抑制;ELISA示不同浓度的C188-9预处理后,EBV~+B细胞来源外泌体刺激CTCL细胞分泌IL-10的作用被抑制;(7)应用流式细胞术检测细胞凋亡率,结果示EBV~+B细胞来源外泌体可抑制CTCL细胞的凋亡(p<0.05);(8)经共培养后,利用WB检测CTCL细胞中凋亡相关蛋白的表达变化,结果示EBV~+B细胞来源外泌体对CTCL细胞的凋亡执行蛋白Cyt C及Caspase3的表达均有抑制作用,EBV~+B细胞来源外泌体可上调CTCL细胞中Bcl-2/BAX表达的相对比值。以上结果表明:EBV~+B细胞来源外泌体可通过促进CTCL细胞STAT3的磷酸化来上调IL-10/PD-L1的表达,从而促进CTCL的免疫逃逸效应;EBV~+B细胞来源外泌体可以抑制CTCL细胞凋亡。3.EBV~+B细胞来源外泌体miRNA测序及ebv-miRNA筛选实验。(1)对BL2009细胞来源外泌体的miRNA测序发现,BL2009细胞来源外泌体包含多种EBV编码的miRNA,丰度最高的为:ebv-miR-BHRF1-3、ebv-miR-BART7-3p、ebv-miR-BHRF1-1及ebv-miR-BART8-3p;(2)将这4种ebv-miRNA转染至CTCL细胞后,ELISA示ebv-miR-BHRF1-1可刺激CTCL细胞分泌IL-10(p<0.05);流式细胞术发现ebv-miR-BHRF1-1可抑制CTCL细胞凋亡(p<0.05);(3)对ebv-miR-BHRF1-1进行GO、KEGG、PPI等分析,发现ebv-miR-BHRF1-1可靶向肿瘤细胞PD-1/PD-L1及细胞凋亡等通路,其中PPI分析示ebv-miR-BHRF1-1可能作用于AKT2、Jun或ITPR2来抑制凋亡;(4)将ebv-miR-BHRF1-1转染至CTCL细胞后,WB检测示ebv-miR-BHRF1-1对CTCL细胞p-STAT3及PD-L1蛋白的表达无影响;(5)将ebv-miR-BHRF1-1转染至CTCL细胞后,WB检测示ebv-miR-BHRF1-1可抑制CTCL细胞凋亡执行蛋白Cyt C及Caspase3的表达,而对Bcl-2/BAX/AKT2/p-AKT2/JUN/p-JUN等蛋白表达无明显影响;(6)将ebv-miR-BHRF1-1转染至CTCL细胞后,免疫荧光结果提示ebv-miR-BHRF1-1可以抑制CTCL细胞ITPR2蛋白的表达。以上结果表明:BL2009细胞来源外泌体中包含的ebv-miR-BHRF1-1可刺激CTCL细胞分泌IL-10但不依赖于p-STAT3/PD-L1的表达;BL2009细胞来源外泌体中包含的ebv-miR-BHRF1-1可能通过抑制CTCL细胞的ITPR2蛋白来抑制凋亡。4.对20例EBV相关CTCL(10例ENKTCL及10例HVLPD)皮损的石蜡组织切片行ebv-miR-BHRF1-1的FISH结果示,5例ENKTCL及7例HVLPD,表达ebv-miR-BHRF1-1,而所有MF均不表达该分子。结论:1.本研究首次在EBV相关CTCL患者的皮损组织中发现此类疾病存在与STAT3/IL-10/PD-L1通路过度激活相关的免疫逃逸效应;2.本研究首次发现EBV~+B细胞来源外泌体在EBV促进EBV相关CTCL发展中作为关键桥梁发挥着重要作用,EBV~+B细胞来源外泌体可促进CTCL细胞产生p-STAT3/IL-10/PD-L1相关的免疫逃逸效应,并可抑制CTCL细胞的凋亡,该研究揭示了EBV感染T细胞的可能途径;3.本研究发现EBV~+B细胞来源外泌体中包含大量EBV编码的miRNA,其中,ebv-miR-BHRF1-1可刺激CTCL细胞分泌IL-10还可通过抑制ITPR2蛋白来抑制CTCL细胞凋亡;4.EBV相关CTCL的皮损组织表达ebv-miR-BHRF1-1。EBV可能通过EBV~+B细胞来源的外泌体运送ebv-miR-BHRF1-1促进CTCL的发生发展。更多还原

【Abstract】 Objective:To determine whether there was excessive activation of immune escape pathway in cutaneous T-cell lymphoma(CTCL)associated with Epstein–Barr virus(EBV).To verify the effect of exosomes derived from EBV~+B cells on the immune escape pathway and apoptosis of CTCL cells,the exosomes derived from EBV positive(EBV~+)B lymphoblastic cell line were co-cultured with the EBV negative(EBV~-)CTCL cell lines.Then,we investigated the effect of micro RNA(miRNA)encoded by EBV on immune escape and apoptosis of CTCL through exosomes derived from EBV~+B cells.To explore preliminarily the potential mechanism of exosomes derived from EBV~+B cells and the ebv-miRNA contained therein in the occurrence and development of EBV related CTCL,and provide experimental and theoretical basis for the subsequent development of potential therapeutic targets.Materials and Methods:1.We verified whether there was immune escape effect related to STAT3/IL-10/PD-L1 pathway activation and EBV infection in EBV related CTCL.The paraffin sections of 32 patients with EBV related CTCL(20 cases of ENKTCL and 12 cases of HVLPD)in the dermatology department of West China Hospital of Sichuan University in recent 5 years were chosen,while 5 cases of mycosis fungoides(MF)and 9 cases of inflammatory dermatosis were used as control.EBV encoded latent membrane protein 1(LMP-1)immunohistochemical staining and EBER 1/2 in situ hybridization were used to determine the EBV infection in EBV related CTCL;(2)We wanted to determin whether there was an immune escape effect related to the over activation of STAT3/IL-10/PD-L1 pathway in EBV related CTCL,so immunohistochemical staining were performed on the above samples.2.The effect of EBV~+B cell derived exosomes on STAT3/IL-10/PD-L1pathway related immune escape effect and apoptosis of CTCL cells.The experiment was an in vitro cell experiment.The cells used in the study are EBV~+B lymphoblastic cell line(BL2009 cell),EBV negative human Burkitt lymphoma cell line(CA46 cell)and EBV negative human CTCL cell line(Hut78,H9).The experiment was divided into experimental group(co-culture of CTCL cells and BL2009 cell derived exosomes)and control group(co-culture of CTCL cells and CA46 cell derived exosomes).(1)Before co-culturing,the extracted exosomes were identified by Western blot(WB),particle size analysis and transmission electron microscopy;(2)RT-q PCR(real time quantitative polymerase chain reaction)was used to detect the effect of EBV~+B cell derived exosomes on the m RNA expression of IL-10/PD-L1 in CTCL cells;(3)Enzyme linked immunosorbent assay(ELISA)was used to detect the effect of EBV~+B cell derived exosomes on the secretion of IL-10 by CTCL cells;(4)The effect of EBV~+B cell derived exosome on the expression of PD-L1 protein on CTCL cell membrane was detected by flow cytometry;(5)WB was used to detect the effect of EBV~+B cell derived exosomes on the expression of PD-L1,t-STAT3,p-STAT3 and other proteins in CTCL cells;(6)Add STAT3 phosphorylation inhibitor C188-9 before the establishment of co-culture system(set the concentration to 0μM in turn 40μM,100μM and 200μM).The phosphorylation of STAT3 in CTCL cells was inhibited by pretreatment.Then WB detected the effects of EBV~+B cell derived exosomes on STAT3 phosphorylation and PD-L1 expression in CTCL cells stimulated by different concentrations of C188-9;ELISA was used to detect the effect of EBV~+B cell derived exosomes on the secretion of IL-10 in CTCL cells stimulated by different concentrations of C188-9;(7)The effect of EBV~+B cell derived exosomes on the apoptosis rate of CTCL cells was detected by flow cytometry;(8)WB was used to detect the effect of EBV~+B cell derived exosomes on the expression of apoptosis related proteins(Bcl-2,BAX,Cyt C and Caspase3)in CTCL cells.3.MiRNA sequencing of EBV~+B cell derived exosomes and screening of EBV encoded miRNA(ebv-miRNA).(1)Extract the exosomes from BL2009 cells,isolate and purify the total miRNA in exosomes,and then build a library for sequencing the total miRNA in exosomes;(2)The sequencing results were analyzed to screen ebv-miRNA preliminarily;(3)After consulting literature and database,ebv-miRNA mimics were designed and transfected into Hut78 and H9 cells,and the effect of ebv-miRNA on the secretion of IL-10 by CTCL cells was detected by ELISA;The effect of ebv-miRNA on apoptosis of CTCL cells was detected by flow cytometry;(4)After the above experiments,the target ebv-miRNA was obtained and analyzed by GO(gene ontology),KEGG(kyoto encyclopedia of genes and genomes)and PPI(protein protein interaction networks);(5)WB verified the effect of target ebv-miRNA on the expression level of related proteins predicted by the above bioinformatics analysis in CTCL cells.4.To determine the expression of target ebv-miRNA in EBV related CTCL,paraffin sections of 20 cases of EBV related CTCL(10 cases of ENKTCL and 10cases of HVLPD)were examined by fluorescence in situ hybridization(FISH)while3 cases of MF were used as negative controls.Results:1.EBER 1/2 in situ hybridization was expressed in all EBV related CTCL,but not in MF and inflammatory dermatosis;LMP-1 was only expressed in EBV related CTCL,but not in MF and inflammatory dermatosis,with a statistically significant difference(p<0.05).Immunohistochemical staining showed that compared with MF and inflammatory dermatosis,the expression of IL-10/PD-L1 in the skin lesions of EBV related CTCL was significantly increased(p<0.0001),while the expression of p-STAT3 did not show difference among the several groups of diseases;Correlation analysis of the expression of the above markers showed that p-STAT3 was positively correlated with the expression of IL-10 in EBV related CTCL(p<0.001;r=0.779).2.The study on the effect of EBV~+B cell derived exosomes on STAT3/IL-10/PD-L1 pathway and apoptosis of CTCL cells.(1)WB detection of CD9,CD63 and CD81 proteins in exosomal suspension showed that the above three marker proteins had positive expression;The diameter of nanoparticles in the exosomal suspension was analyzed,and the results showed that the diameter of particles in the suspension was concentrated between 60-100 nm;The typical"cup-like"double membrane structure of exosome was observed by transmission electron microscopy;The above results indicated that the exosomes were successfully extracted;(2)RT-q PCR was used to detect the co-culture system.The results showed that the EBV~+B cell derived exosomes could up regulate the expression of IL-10 and PD-L1 m RNA in CTCL cells(p<0.05);(3)The results of ELISA showed that the EBV~+B cell derived exosomes could up regulate the concentration of IL-10 in the supernatant of CTCL cells(p<0.001);(4)Flow cytometry was used to detect the PD-L1 expression in co-culture system.The results showed that EBV~+B cell derived exosomes could up regulate the expression of PD-L1 protein on the CTCL cell membrane;(5)The results of WB showed that the EBV~+B cell derived exosomes could up regulate the expression of PD-L1 and p-STAT3 in CTCL cells,but had little effect on the expression of t-STAT3;(6)Different concentrations of STAT3 phosphorylation inhibitor C188-9 could inhibit the phosphorylation of STAT3 in CTCL cells in various degrees,among them200μM is the strongest;CTCL was pretreated with different concentrations of C188-9,and then co-cultured with exosome suspension.WB results showed that after stimulation with different concentrations of C188-9,the effect of EBV~+B cell derived exosomes on up regulating PD-L1 expression in CTCL cells was inhibited;ELISA showed that after stimulation with different concentrations of C188-9,the effect of EBV~+B cell derived exosomes on up regulating the secretion of IL-10 by CTCL cells was inhibited;(7)Flow cytometry was used to detect the apoptosis rate of the co culture system.The results showed that EBV~+B cell derived exosomes could inhibit the apoptosis of CTCL cells(p<0.05);(8)WB showed that EBV~+B cell derived exosomes could inhibit the expression of apoptosis executive protein Cyt C and Caspase3 protein in CTCL cells,and EBV~+B cell derived exosomes could up regulate the relative ratio of Bcl-2/BAX expression in CTCL cells.The above results showed that EBV~+B cell derived exosomes could up regulate the expression of IL-10/PD-L1 by stimulating the phosphorylation of STAT3 in CTCL cells;Exosomes derived from EBV~+B cells could inhibit the apoptosis of CTCL cells.3.Sequencing of EBV~+B cell derived exosomal miRNA and screening of ebv-miRNA.(1)The miRNA sequencing of BL2009 cell derived exosomes showed that BL2009 cell derived exosomes contained a variety of EBV encoded miRNA,with the highest abundance of ebv-miR-BHRF1-3,ebv-miR-BART7-3p,ebv-miR-BHRF1-1 and ebv-miR-BART8-3p;(2)After these four ebv-miRNA were transfected into CTCL cells,ELISA showed that ebv-miR-BHRF1-1 could stimulated CTCL cells to secrete IL-10(p<0.05);Flow cytometry showed that ebv-miR-BHRF1-1 could inhibit the apoptosis of CTCL cells(p<0.05);(3)Through GO,KEGG and PPI analysis of ebv-miR-BHRF1-1,it was found that ebv-miR-BHRF1-1 could target tumor cell PD-1/PD-L1 immune checkpoint and apoptosis pathway,among which PPI analysis showed that ebv-miR-BHRF1-1might act on AKT2,Jun or ITPR2 to inhibit apoptosis;(4)When ebv-miR-BHRF1-1was transfected into CTCL cells,WB showed that ebv-miR-BHRF1-1 had no effect on the expression of p-STAT3 and PD-L1 proteins in CTCL cells;(5)When ebv-miR-BHRF1-1 was transfected into CTCL cells,WB showed that ebv-miR-BHRF1-1 could inhibit the expression of Cyt C and caspase3,but had no effect on the expression of Bcl-2/BAX/AKT2/p-AKT2/JUN/p-JUN;(6)After transfecting ebv-miR-BHRF1-1 into CTCL cells,immunofluorescence results indicated that ebv-miR-BHRF1-1 could inhibit the expression of ITPR2 protein in CTCL cells.The above results showed that ebv-miR-BHRF1-1 contained in BL2009cell derived exosomes could stimulate CTCL cells to secrete IL-10 but did not depend on the activation of p-STAT3/PD-L1;the ebv-miR-BHRF1-1 contained in BL2009 cell derived exosomes might inhibit apoptosis by inhibiting the ITPR2protein of CTCL cells.4.The FISH results of ebv-miR-BHRF1-1 on paraffin sections of 20 cases of EBV related CTCL(10 cases of ENKTCL and 10 cases of HVLPD)showed that ebv-miR-BHRF1-1 was expressed in 5 cases of ENKTCL and 7 cases of HVLPD,but not in MF.Conclusions:1.In this study,for the first time we found that there was an immune escape effect related to the activation of STAT3/IL-10/PD-L1 pathway in the skin lesions of patients with EBV related CTCL.2.In this study,for the first time we found that EBV~+B cell derived exosomes played as a key bridge in EBV promoting the development of EBV related CTCL.EBV~+B cell derived exosomes could promote the immune escape effect related to p-STAT3/IL-10/PD-L1 in CTCL cells,and inhibit the apoptosis of CTCL cells.3.In this study,we found that EBV~+B cell derived exosomes contained a large number of EBV-encoded miRNA.Among them,ebv-miR-BHRF1-1 could stimulate CTCL cells to secrete IL-10;and ebv-miR-BHRF1-1 could inhibit the apoptosis of CTCL cells by inhibiting ITPR2 protein.4.EBV related CTCL expressed ebv-miR-BHRF1-1.EBV might transport ebv-miR-BHRF1-1 through exosomes derived from EBV~+B cells to promote the occurrence and development of CTCL.更多还原