【作者】 徐妍；

【导师】 程浩；

【作者基本信息】 浙江大学 ， 内科学， 2008， 博士

【摘要】 尖锐湿疣（Condyloma acuminatum,CA）是我国发病率最高的性传播疾病之一,临床上顽固难治,极易复发。CA的发病率常年居高不下且呈上升趋势,目前的治疗手段主要有通过激光,冷冻,电灼等机械切除去除外生瘤体或加施以免疫治疗来改善症状,但无论何种方法治疗,都有相应的复发率。人乳头瘤病毒（Human papillomavirus,HPV）是其病原体,迄今已有130多种亚型被鉴定,依据不同型别HPV与肿瘤发生的危险性高低分为低危型和高危型,其中HPV6和11型是导致CA的主要型别。HPV呈双链DNA分子,由8000bp组成,其基因组按功能可分为三个部分:早期转录区的E区（E1-E8）,晚期转录区的L区（L1和L2）和无编码作用的非转录区NCR区。高危型HPV的早期蛋白基因E6/E7是致病基因,能影响宿主细胞抑癌基因表达产物的生物学活性,在诱导细胞恶性转化中起了至关重要的作用。因此其具有的免疫表位使E6/E7基因常作为理想的靶位点用于HPV防治的研究。由于HPV具有高度宿主特异性和严格的组织特异性,难以在体外常规细胞培养系统中繁殖以及建立动物模型,给HPV功能研究带来困难。国外关于HPV的研究集中于引起90%以上宫颈癌的HPV16/18型,而对引起CA的HPV6/11型的免疫作用和生物学功能的研究仍极少。反复发作和病程较长的HPV感染患者常有细胞免疫功能异常,感染局部树突状细胞（DCs）数量、功能改变,导致免疫监视系统破坏,不能激发有效的免疫应答,难以清除常规治疗后残存的病毒感染细胞。病毒感染细胞的有效清除主要通过抗原特异性CTL识别细胞表面MHC-Ⅰ类分子限制的抗原多肽,因此筛选并鉴定HPV的CTL表位肽,通过体外多肽抗原负载DCs,诱导抗原特异性CTL产生,对研究病毒感染细胞的有效清除及相关多肽疫苗的研制具有指导意义。目前的多肽疫苗研究侧重于E7抗原及其CTL表位,且由于中国人群中超过50%的人具有等位基因HLA-A2,因此筛选并鉴定E7抗原的HLA-A2限制性CTL表位就显得尤为重要。本实验对HPV11型E7抗原进行HLA-A^*0201限制性CTL表位的预测,并观察表位肽体外诱导的抗原特异性CTL活性。CA患者存在外周血T淋巴细胞亚群异常、单核-巨噬细胞功能障碍以及细胞因子产生失衡所引起的一系列细胞免疫反应抑制效应,与HPV感染长期存在或多次复发有关。CA发病、消退、复发及恶变的机理尚不完全清楚,这其中辅助性T细胞（Th）和细胞毒性T细胞（Tc）在抗病毒免疫中起着重要作用。病毒的持续感染与调节性T细胞密不可分,CD4⁺CD25⁺调节性T细胞通过抑制效应细胞的增殖和细胞因子的分泌从而调控机体对病毒感染的免疫应答。研究证实,转录因子Foxp3（forkhead box p3）特异性表达于调节性T细胞,与调节性T细胞的生长发育和功能维持密切相关。研究Foxp3⁺CD4⁺CD25⁺调节性T细胞及Th,Tc细胞在尖锐湿疣患者外周血中的变化,将为阐明CA发病机制以及从免疫调节的角度治疗CA提供新的思路,同时也为人们进一步开发CD4⁺CD25⁺调节性T细胞疫苗和研究其它自身免疫性疾病的发病机制奠定重要的理论基础。本课题拟进行以下两部分的研究以期了解一些HPV11E7特异性细胞毒反应和尖锐湿疣患者外周血CD4⁺CD25⁺调节性T细胞、Th和Tc细胞的表达,为HPV感染相关疾病的防治提供一些实验依据和参考。第一部分HPV-11E7抗原HLA-A^*0201限制性CTL表位的鉴定HPV是导致CA的主要病原体,其中HPV6和11型占感染的90%以上。特异性的CTL在清除病毒的过程中发挥着很重要的作用。因此筛选并鉴定HPV CTL表位肽,并在体外多肽抗原负载DCs后诱导抗原特异性CTL反应,对研究病毒感染细胞的有效清除及相关多肽疫苗的研制提供实验依据。MHC-肽四聚体染色法是近年来出现的一种高特异性和敏感性的新方法,基于MHC分子和TCR的高特异性结合,采用四聚体技术增强其敏感性,能对特异性CTL进行定性、定量分析,具有高敏感性和高特异性。通过综合几项预测软件结果选择几种表位多肽进行合成,进而多肽负载成熟DCs,激活T淋巴细胞,采用四聚体技术,ELISA及乳酸脱氢酶（LDH）释放试验进行筛选和鉴定预测的表位。1.1 HLA-A^*0201限制性CTL表位的预测,与表位肽和四聚体的合成:综合应用SYFPEITHI,BIMAS,MHC-pred,NetMHC version 2.1,HLALigand/Motif Database,Net Chop 3.0 Server等预测手段,由荷兰Sanquin公司合成并纯化以下HLA-A^*0201限制性多肽及相应四聚体:HPV11E7 7-15（TLKDIVLDL）,HPV11E7 15-23（LQPPDPVGL）,HPV11E7 47-55（PLTQHYQIL）,HPV11E7 81-89（DLLLGTLNI）和HPV11E7 82-90（LLLGTLNIV）。1.2 DCs的体外诱导培养及多肽负载:取HLA-A^*0201志愿者外周血50ml常规密度梯度离心获得PBMC,通过免疫磁珠分选获得CD14⁺细胞,GM-CSF(100 ng·ml^-1)、IL-4(50ng·ml^-1)诱导分化树突状细胞,第5天,实验组分别加入终浓度均为20μg·ml^-1的五组HPV11E7多肽7-15（TLKDIVLDL）,15-23（LQPPDPVGL）,47-55（PLTQHYQIL）（DLLLGTLNI）和82-90（LLLGTLNIV）,对照组不加。第7天收集DCs和上清液。流式细胞仪检测DCs表面标记CD1a、CD83、CD86及HLA-DR;ELISA法检测上清液中IL-12的分泌水平。结果显示我们诱导分化的树突状细胞表面均高表达CD83、CD86及HLA-DR,各多肽组之间无统计学差异。经多肽刺激的DCs分泌IL-12的水平比未经多肽刺激的高30-60倍（P＜0.05）,其中多肽E7 15-23和E7 7-15较之其它多肽能刺激DCs分泌更高水平的IL-12（P＜0.05）,而多肽E7 15-23较多肽E7 7-15刺激DCs分泌更高水平的IL-12（P＜0.05）。1.3多肽负载的DCs诱导抗原特异性T细胞:CD14⁺细胞经GM-CSF(100ng·ml^-1)和IL-4(50ng·ml^-1)诱导培养第5天,加入TNF-α(100ng·ml^-1)和flt-3(50ng·ml^-1)诱导细胞成熟,第7天时收获成熟DCs并调整细胞浓度为2×10⁶·ml^-1,加入终浓度均为20μg·ml^-1的五组多肽,无血清RPMI1640中37℃培养1h。T淋巴细胞来自同一HLA-A^*0201志愿者的PBMC,通过免疫磁珠Pan T Cell Isolation KitⅡ获得。以2×10⁶个T细胞且T∶DCs比为10∶1的比例共培养于24孔板,加IL-2(25 U·ml^-1)。培养第7天和第14天时分别用多肽负载的DCs刺激自身T淋巴细胞,并补加IL-2(25 U·ml^-1),第21天收获悬浮细胞视为抗原特异性T淋巴细胞。分别设单纯DCs刺激的T细胞和未经任何刺激的T细胞为对照组。取上清ELISA法检测IFN-γ浓度,测A₄₅₀值,绘标准曲线,并推算各样本的浓度。结果表明:多肽E7 7-15、82-90和15-23刺激T淋巴细胞分泌较高水平IFN-γ,且明显高于其它多肽和对照组（P＜0.05）,其中多肽E7 7-15刺激T淋巴细胞分泌的IFN-γ浓度最高但无统计学差异（P＞0.05）1.4抗原肽特异性T细胞的tetramer流式细胞技术检测:上述得到的各组抗原特异性T淋巴细胞1×10⁶重悬于PBS,根据各组多肽相应加入PE标记的tetramer室温孵育20min,PBS洗涤一次,加入PE-Cy5和FITC标记的抗CD3和抗CD8抗体孵育15min,流式细胞技术检测CD8⁺tetramer⁺双阳性细胞即抗原特异性CTL细胞的百分比。设只标记抗CD3和抗CD8抗体的T淋巴细胞为阴性对照。结果显示多肽E7 7-15（TLKDIVLDL）负载的树突状细胞刺激同体T细胞后抗原特异性CTL细胞达8%,与对照组和其它多肽实验组相比,差异均有统计学意义（均P＜0.05）。1.5表达HPV11E7的293细胞株建立和LDH法检测CTL活性:按Lipofectamine转染试剂盒将质粒pcDNA3.1/HPV11E7-GFP转入人胚肾（293）细胞,24h后荧光显微镜观察并加G418筛选,挑取单克隆生长的细胞继续培养,逆转录聚合酶链反应（RT-PCR）检测转染细胞mRNA水平的表达。并以此表达HPV11E7的293细胞作为靶细胞,未转染的293细胞为阴性对照。上述得到的抗原特异性T淋巴细胞作为效应细胞,设单纯DCs刺激的T细胞和未经任何刺激的T细胞为对照组。按效靶比例（E∶T ratio）为50∶1、25∶1、10∶1分别接种于圆底96孔板作为实验孔并设3个复孔,操作步骤及计算方法按试剂盒说明进行。特异性杀伤率（%）=（实验样本组释放量—效应细胞组自发释放量—靶细胞组自发释放量）/（靶细胞组最大释放量—靶细胞组自发释放量）×100%。研究结果显示负载HPV11E7多肽的DCs诱导的特异性CTL在各效靶比均能对HPV1IE7/293细胞产生特异性杀伤作用,且在效靶比50∶1时,多肽E7 7-15诱导的CTL杀伤率达（91.48±0.06）%显著高于其它多肽实验组（P＜0.05）。但在效靶比25∶1和10∶1时五组多肽诱导的CTL对靶细胞的杀伤效率无明显差异。单纯DCs刺激的T细胞对HPV11E7/293细胞在各效靶比均无明显杀伤作用。第二部分尖锐湿疣患者外周血Foxp3⁺CD4⁺CD25⁺调节性T细胞和Th1/Th2,Tc1/Tc2细胞的检测及其相关性分析HPV感染的免疫学某些特征提示很可能存在CD4⁺CD25⁺调节性T细胞（Treg）的异常,如HPV病损局部可有Treg细胞高表达,参与其免疫抑制功能和维持无应答表型的膜分子白细胞介素10（IL-10）和转化生长因子β（TGF-β）的过度表达,以及CD4⁺细胞数量的异常改变等。Foxp3是Treg细胞的一个相对特异性标志,并且Foxp3的表达对CD4⁺CD25⁺Treg细胞在胸腺的分化发育和外周的表达是必需的,Foxp3决定了Treg细胞的抑制功能。通过对CA免疫学发病机制的研究发现,在CA免疫状态中存在克隆漂移（clonal diversion）现象,即CA患者在免疫应答过程中存在Th1/Th2细胞分泌细胞因子失衡,Th1细胞及其分泌的细胞因子占弱势,Th2细胞及其分泌的细胞因子占了强势,但是CA患者在Tc1/Tc2动态平衡关系方面的研究甚少。故研究CA患者外周血中Foxp3⁺CD4⁺CD25⁺调节性T细胞的表达水平及Th1/Th2和Tc1/Tc2的分布情况,分析它们之间的关系,为进一步探讨CA发生发展机制提供实验依据。2.1标本来源为2006年10月—2007年9月来我院皮肤科门诊就诊的30例经临床确诊的CA患者。男12例、女18例;年龄19～55岁,平均31岁;CA病程0.1～25月,平均（4.74±5.39）月;根据患者发病情况又分为初发组和复发组,初发组共15例,男7例,女8例,年龄20～51岁,平均32.8岁,病程0.1～6月,平均（1.40±0.89）月;复发组共15例,男5例,女10例,年龄19～55岁,平均34.4岁,病程1.5～25月,平均（8.95±4.56）月。对照组为20例体检健康志愿者,男9例、女11例;年龄22～45岁,平均30.2岁。全部检测对象取血前4周内未使用过任何免疫调节药物并排除患有肝肾疾病及其他免疫性疾病。2.2 Foxp3⁺CD4⁺CD25⁺Treg细胞染色:从4ml肝素抗凝全血中经密度梯度离心法分离外周血单个核细胞（PBMC）,?00μL细胞悬液（1x10⁵细胞）加入抗CD4-PE,CD25-FITC单抗各10μL,室温避光染色20min后,用染色缓冲液洗涤1次,加入新配制的固定/透膜液1mL,4℃暗处孵育30～60 min,2ml透膜缓冲液洗涤2次,离心弃上清,2%正常大鼠血清2μL,4℃避光15 min,以阻断膜表面Fc受体,减少非特异性荧光染色,再加人PE标记抗人Foxp3（克隆号PCH101）抗体20μL,4℃避光孵育30min,洗涤2次,适量的染色缓冲液混匀,流式细胞仪检测,Expo32 flow cytometry sottware软件获取并分析数据,以三色荧光抗体染色阳性细胞占CD4⁺T细胞的百分率记录结果。统计学处理后,外周血中Foxp3⁺CD4⁺CD25⁺Treg细胞在CD4⁺T细胞中的百分率在CA组（3.37±1.03）%和CA复发组（4.68±1.17）%均显著高于正常对照组（1.18±0.53）%（P＜0.001）;在CA初发组（2.06±1.01）%虽高于对照组但差异无统计学意义;CA复发组的Foxp3⁺CD4⁺CD25⁺Treg细胞含量明显高于CA初发组（P＜0.05）。2.2外周血中Th1/Th2及Tc1/Tc2细胞染色200μl新鲜肝素钠抗凝外周血,RPMI 1640培养液按1∶1稀释,分别加入Monensin,PMA,Ionomycin及Monensin,37℃、5%CO₂培养箱中孵育5h,分别加入20μl CD3-TC和20μl CD8-FITC,常温下暗处孵育15min,加入100μl固定液,孵育15min后PBS洗涤一次。加入100μl破膜和溶血液和抗体IFN-γ-PE,IL-4-PE及相应的同型对照,孵育15min,PBS液洗涤并重悬细胞,2h内上机检测。结果显示外周血CD3⁺CD8^-（Th）和CD3⁺CD8⁺（Tc）细胞中,分泌IFN-γ的Th1和Tc1型细胞与健康对照组相比,CA组、CA初发组和CA复发组Th1细胞含量均显著减少（P均＜0.01）,CA组尤其是CA复发组Tcl细胞含量与正常组相比显著降低（P＜0.05）。CA组、CA初发组、CA复发组Th2、Tc2细胞含量较健康对照组降低但是差异均无统计学意义。CA组Th1/Th2比值、Tc1/Tc2比值均较正常组均显著降低（P＜0.05）,尤其是复发组的Th1/Th2（P＜0.01）。CA初发组Th1/Th2、Tc1/Tc2及复发组Tc1/Tc2比值与健康对照组差异均无统计学意义。而Th1及Th1/Th2比值在CA复发组较CA初发组更低（P＜0.05）。2.3尖锐湿疣患者外周血Foxp3⁺CD4⁺CD25⁺ Treg细胞和Th1/Th2及Tc1/Tc2细胞相关性分析通过SPSS13.0统计软件采用Spearman rank行相关性分析。我们发现尖锐湿疣患者外周血中Foxp3⁺CD4⁺CD25⁺调节性T细胞的高表达与Th1细胞（r=—0.798,p＜0.05）,和Tcl细胞（r=—0.746,P＜0.05）的低表达呈负性相关。全文小结1、通过多个数据库筛选得到5个HPV11E7 CTL表位:HPV11E7 7-15（TLKDIVLDL）,15-23（LQPPDPVGL）,47-55（PLTQHYQIL）,81-89（DLLLGTLNI）和82-90（LLLGTLNIV）。2、建立了稳定的免疫磁珠分选及诱导人外周血单个核细胞来源的树突状细胞培养体系。3、通过重组质粒转染细胞株,建立稳定有效表达HPV11 E7基因的293细胞株。4、CTL表位肽能刺激DCs细胞的成熟及分泌IL-12,其中多肽E7 15-23和E7 7-15较之其它多肽能刺激DCs分泌更高水平的IL-12,而且多肽E715-23刺激DCs分泌IL-12的水平较多肽E7 7-15明显增高。5、多肽负载成熟DC后刺激自身T淋巴细胞IFN-γ的分泌;其中多肽E7 7-15刺激T淋巴细胞分泌的IFN-γ浓度最高。6、多肽E7 7-15（TLKDIVLDL）负载的树突状细胞刺激同体T细胞后抗原特异性CTL细胞较对照组和其它多肽实验组均显著增多,且多肽E7 7-15较其它多肽能诱导更强的CTL反应,更强的特异性杀伤表达HPV11E7基因的靶细胞。7、结合tetramer技术和相应的CTL功能实验,HPV11E7多肽7-15（TLKDIVLDL）负载DCs后能诱导较强的抗原特异性CTL,该表位可初步定性为HLA-A^*0201限制性CTL表位。8、三色荧光抗体染色流式细胞术检测,外周血中Foxp3⁺CD4⁺CD25⁺Treg细胞在CD4⁺T细胞中的百分率在CA组和CA复发组均显著高于正常对照组;在CA初发组虽高于正常组但差异无统计学意义,且CA复发组的Foxp3⁺CD4⁺CD25⁺Treg细胞含量明显高于CA初发组。推测Treg细胞可能参与并成为影响机体对HPV的免疫反应的重要原因之一。9、尖锐湿疣患者外周血存在Th1/Th2及Tc1/Tc2淋巴细胞分布失衡情况,Th1和Tc1细胞减少,Th2和Tc2细胞相对占优势,这种失衡在复发患者更趋严重。10、尖锐湿疣患者外周血中Foxp3⁺CD4⁺CD25⁺Tregs的含量增高与Th1和Tc1细胞的减少之间呈负相关。推测这是尖锐湿疣发病及病程反复的机理之一。更多还原

【Abstract】 Condyloma acuminatum（CA）is one of the most common sexually transmitted diseases,which is difficult to cure because of its high relapse rate. The incidence of CA has a tendency of increase.Currently,we usually eliminate the neoplasm by physical therapy,including laser,microwave,liquid nitrogen,etc, followed by non-specific immunotherapy.Routine therapeutic methods can’t efficiently eradicate CA in that all existing treatment options have an associated failure rate and recurrence of genital warts after treatment.Human papilloma virus（HPV）infection of the genital tract is one of the most common sexually transmitted diseases,including low-risk and high-risk HPV types infection.The low-risk types of HPV 6 and 11 are shown to be responsible for the large majority of condyloma acuminatum（CA）.Cell-mediated immune responses are critical in HPV attenuation however,the antigenic epitopes involved in HPV control have not been successfully characterized.Previous studies have suggested that some HPV-specific CD8⁺ T-cell responses contribute to the control of the virus infection in cervical cancer mainly caused by high-risk types of HPV 16 or 18.However less study was involved in low-risk types of HPV 6 and 11,though it is hypothesized that the weak generation of HPV-specific CTL response could be contributed to poor treatment of CA. Therefore,identification of HPV 6/11 CTL epitopes would be helpful not only in our understanding of the protective immune response,but in the development of immunotherapy strategies against CA as well.Studies on the immune responses to the HPV early genes E6 and E7 have been the central interest in HPV research since they are required for the maintenance of cellular transformation.Both E6 and E7 have immune dominant epitopes that represent ideal targets for vaccine developments against HPV. Among those epitopes,there is a paucity of confirmed human CTL epitopes for HPV,while the majority of these are based on HLA-A^*0201.Successful induction of E7-specific CTLs has been described in mice upon immunization with CTL epitope peptides of E7,recombinant E7 protein,transfected dendritic cells,and recombinant plasmids expressing E7.The CTL could recognize proteolysed fragments of the protein in combination with MHC classⅠmolecules. A single TCR can recognize large numbers of peptides in the context of a single MHC molecule,therefore,identification of CTL epitopes is crucial in understanding the roles of T cell activation.In this context,DCs play a crucial role in the induction of antiviral immune responses through the recruitment and activation of T cells.Endocytosis,processing,and presentation by DCs are required to induce HPV-specific CTLs.However,efficient induction of CTLs is hindered by the poor immunogenicity of antigens and the poor transduction efficiency of DCs.Studies have demonstrated that the HPV 16 E7-specific CTLs can be generated from in vitro-vaccinated PBLs of healthy subjects.This observation indicates that mature dendritic cells can activate E7-specific CTLs from naive precursors in vitro.Previous studies have revealed that CTLs stimulated with endogenously processed HPV-11E7 antigen recognized the synthetic HLA-A2 motif-containing nonamer,HPV-11E7 4-12,and CTLs stimulated with this peptide in vitro recognized targets expressing endogenously processed E7.In the present study,we identified five HLA-A^*0201-restricted CTL epitopes for HPV-11E7 using a combination of multiple computational prediction methods.Cultured mature DCs loaded with HLA-A^*0201-restricted HPV-11 E7 peptides were employed to activate the antigen-specific CTLs in vitro.The presence of HPV-11 E7 epitope-specific CTLs was analysed by HLA-peptide tetramerie complexes,IFN-γsecretion and LDH release assays.Specific immune responses against viruses and cancer are controlled by a network of regulatory T cells.The naturally occurring CD4⁺CD25⁺ regulatory T cells（Tregs）is the pivotal cell type that maintains self-tolerance and exerts active immune suppression.The development and function of Tregs is controlled by the forkhead transcription factor Foxp3（forkhead boxp3）.CD4⁺CD25⁺ Treg cells have been shown to suppress virus-specific CD8⁺ T cell responses and delay viral clearance.Immunized mice lacking CD4⁺CD25⁺ Tregs also showed greater resistance to viral challenge.An increased number of CD4⁺CD25⁺ cells were found in patients with autoimmunity,cancer,and chronic infection.The significantly increased frequency of CD4⁺CD25^hiFoxp3⁺ regulatory T cells was found in women who had persistent HPV16 infection.However,little is known of the frequency and distribution of regulatory T cells within PBMC of CA patients and how they are correlated with the development and recurrence of CA.The balance between type 1 and type 2 T-cell subsets in CA patients is thought to modulate antiviral immunity.A polarized Tc1/Tc2 dichotomy as well as an imbalance of Th1/Th2 are thought to be critical determinants of immune responses under certain pathologic conditions.A shift from Th1 to Th2 cytokine profile has been indicated in patients with HPV-associated cervical lesions. However,the balance of Th1/Th2 and Tc1/Tc2 cells in patients with CA is not addressed.Taken together,this project focused on the identification of HLA-A^*0201-restricted CTL epitope of HPV type 11 E7,as well as Foxp3⁺CD4⁺CD25⁺ T cells, Th1/Th2 and Tc1/Tc2 cells expression in patients with CA.It will be useful to the development of immunotherapeutic approaches for low-risk HPV infectious diseases such as CA. The project is composed of two sections.Section one:Mapping of Cytotoxic T Lymphocytes Epitopes in E7 Antigen of Human Papillomavirus Type 11One of the critical steps in the progression to condyloma acuminatum（CA） is the establishment of a persistent human papillomavirus（HPV）infection, majority of HPV type 6 and 11.Cytotoxie T lymphocytes（CTL）,which can be induced by the epitope-based peptides in vitro,are thought to be able to recognize and destroy virus-infected cells.In order to screen and identify HLA-A^*0201 restricted HPV-11E7 CTL epitopes,five epitope peptides and tetramers were selected,including HPV-11E7 7-15（TLKDIVLDL）,15-23 （LQPPDPVGL）,47-55（PLTQHYQIL）,81-89（DLLLGTLNI）and 82-90 （LLLGTLNIV）.Human monocyte-derived dendritic cells（DCs）from HLA-A^*0201 healthy individuals were pulsed with these peptides to assess the expression of CD83,CD86,HLA-DR and the secretion of IL-12.The ability of peptide-loaded mature DCs to activate autologous T cells was evaluated by analyzing the frequency of specific tetramer⁺CD8⁺ T cells using flow cytometry, and the level of IFN-γsecretion by ELISA.The ability of the epitope-specific CTLs to kill the target cells was also analyzed.It was found that the immature DCs could be fully activated by all the five HPV-11E7 peptides and peptide-loaded mature DCs were able to stimulate the epitope-specific T cells in vitro.There was an increased frequency of CD8⁺ T cells specific for the E7 7-15 epitope compared to other four predicted epitopes of HPV-11E7（p＜0.05）.The epitope-specific CTLs for E7 7-15 induced the strongest cytotoxicity to HPV-11E7 expressing cell line at an E:T ratio of 50:1（p＜0.05）.Taken together, these findings demonstrate that E7 7-15（TLKDIVLDL）is an HLA-A^*0201-restricted CTL epitope of HPV type 11.We propose that this epitope could be more helpful in the characterization of HPV control mechanism and be useful to the development of immunotherapeutic approaches for low-risk HPV infectious diseases such as CA.1.1 Epitope selectionEpitopes were selected and analysed by a computer-assisted algorithm. Consecutive overlapping 9-amino-acid peptides that possessed the specific primary and secondary anchoring amino acids for HLA-A2 were identified. Based on the presence of favorable and unfavorable amino acids,the peptides were scored as indeterminate,low,medium,or high binding probability to HLA-A2 molecules.From the set of possible combinations of nanomer or decamer peptides in the HPV-11E7 proteins,five peptides were predicted as having a high binding ability to HLA-A2 molecules based on favorable amino acid combinations.These HLA-A^*0201-restricted HPV-11E7 peptides were selected for the further immunogenicity studies.1.2 Tetrameric peptide-MHC classⅠcomplexes and synthetic peptideTetramers of HPV-11E7,including the following HLA-A^*0201-binding peptides:TLKDIVLDL,LQPPDPVGL,PLTQI-IYQIL,DLLLGTLNI and LLLGTLNIV,were synthesized by Sanquin.These tetramers corresponded to the peptides of HPV-11 E7 7-15,15-23,47-55,81-89 and 82-90 respectively.The default panel of antibodies used for these studies was TRI-COLOR conjugate anti-CD3 and FITC-labeled anti-CD8.Isotype controls were used in all flow cytometry experiments.Individual peptides were dissolved in dimethyl sulphoxide to provide stock solutions of 40-100 mg/ml.The purity of monocytes was determined by FACS analysis of CD14 expression on the cell surface.More than 90%of the isolated cells were CD14⁺.FACS analysis of differentiated DCs co-cultured with five HPV-11E7 peptides for 48 hours showed high expression of HLA-DR,CD83 and CD86 on DCs with no significant difference between each group.To determine the IL-12 secretion of peptides pulsed DCs,supernatants were obtained at 48h from cultures.IL-12 concentration in DCs cultured with the selected peptides was 30 to 60-fold higher than that of DCs cultured in medium alone（P＜0.05）.IL-12 concentration in DCs cultured with E7 15-23 peptides was higher than in DCs cultured with E7 7-15 peptides（P＜0.05）,and IL-12 concentration in these two groups was remarkably higher than in DCs stimulated with the other three peptides and controls（P＜0.05）.1.3 Cell isolationPBMC were isolated from HLA^*0201 healthy donors（young women with no sexual experience）by Lympholyte-H density gradient centrifugation as recommended by the manufacturer.T cells for one experimental setting were isolated from the same donor.1.4 Generation of DCs and peptides pulsingCD14 cells were isolated from PBMC by positive selection using CD14 microbeads and their purity was determined by FACS with anti-CD14 Ab.DCs were generated by culturing monocytes in RPMI1640 medium containing 10% FCS,1%L-glutamine,1%streptomycin,1%penicillin,IL-4（50ng/ml）and GM-CSF（100 ng/ml）for 7 days in 6-well plates at a density of 2×10⁶ cells per well.Final maturation of monocyte-derived DCs was induced by exposure of cells to TNF-α（100ng/ml）and flt-3（50ng/ml）for 48 hours on day 5.On day 5,2×10⁶ differentiated DCs were incubated with each of the five HPV11E7 peptides（TLKDIVLDL,LQPPDPVGL,PLTQHYQIL,DLLLGTLNI or LLLGTLNIV）in 6-well plates（20μg/ml）.After 48 hours of incubation,CD1a, CD86,CD83,HLA-DR expressions were analyzed by flow cytometry to determine the DCs maturation.IL-12 concentration in the supernatant was determined by ELISA.1.5 Mixed lymphocyte-DC culture and CD8⁺ responder T cellsDCs generated from PBMC of healthy HLA-A^*0201-positive donors were induced by GM-CSF,IL-4,TNF-αand flt-3 for maturation.Mature DCs were loaded with the aforementioned peptides at a density of 2×10⁶ cells/ml by placing the DCs in a serum-free medium containing the peptides（20μg/ml）for 1h at 37℃.Autologous T lymphocytes were isolated from PBMC by negative selection with the Pan T Cell Isolation KitⅡand mixed with the peptide-loaded mature DCs in a 24-well plate at a lymphocyte:DC ratio of 10:1.On day 7 and 14, additional DCs loaded with 20μg/ml peptides and T lymphocytes were added to maintain the ratio of lymphocytes to DCs at 10:1 with at least 2×10⁶ T cells/ml in the presence of 25 U/ml IL-2.After 3 weeks,induction of the antigen-specific T cells（suspension cells）was ready to be assessed by their capacities of IFN-γsecretion using ELISA,the tetramer staining,and a LDH release assay.Secretion of IFN-γincreased significantly after 3 weeks of incubation in the groups containing T cells cocultured with peptide-loaded DCs,indicating a strong activation and stimulation of T cells.However,only a small increase of IFN-γsecretion was found both in the nonloaded DCs alone,and the T cells cocultured with nonloaded DCs.Secretion of IFN-γincreased 5 to 10-fold in T cells cocultured with peptide-loaded DCs,especially with peptides E7 7-15 （TLKDIVLDL）,15-23（LQPPDPVGL）and 82-90（LLLGTLNIV）（P＜0.05）.T cells cocultured with E7 7-15 peptide-loaded DCs showed the highest level of IFN-γsecretion. 1.6 Tetramers staining and flow cytometryPeptide-loaded DCs and T cells were cocultured according to the above mentioned protocol.Seven days after the last round of restimulation,suspension cells（antigen-specific T cells）were harvested from the cultures.The antigen-specific T cells（1×10⁶）were resuspended in 50μl of PBS,and incubated with the tetramers（PE）for 20 min at room temperature.After a single wash,the TRI-COLOR conjugate and FITC-labeled antibodies directed against CD3 and CD8 were added for 15 min.The cells were then washed with PBS again and analyzed by flow cytometry.Less than 0.03%tetramer⁺ cells detected in circulating CD8⁺ T cells represented the maximum staining observed in the controls.Specific tetramer staining was observed in all cultures of autologous T cells stimulated with each of the five HPV-11E7 peptide-loaded DCs.On the average,a 100-fold increase in the frequency of specific tetramer⁺ CD8⁺ cells was observed when cocultured with the HPV-11E7 peptides loaded DCs.Again, autologous T cells stimulated with E7 7-15（TLKDIVLDL）peptide-loaded DCs showed the highest frequency of specific tetramer⁺ CD8⁺ cells as compared to other peptides（P＜0.05）.1.7 CTL assayIn addition to using peptides loaded autologous DCs for assessing HPV11E7 epitope specific CTL reactivity,a HPV-11E7 expressing cell line was established as target cell in cytolytic assay.The 293 cells were transfected with pcDNA3.1-HPV11E7-GFP by the Lipofectamine kit.With a selective medium containing G418 at a concentration of 500μg/ml,the G418-resistant positive cell clones stably expressing HPV-11E7（HPV-11E7/293）were isolated under the fluorescent microscope and identified by RT-PCR.The HPV11E7 positive 293 cells（1×10⁴ per well）as target cells were incubated with the antigen-specific T cells,which served as effector cells at various effector-to-target cell（E/T）ratios of 10:1,25:1,50:1.The untransfected cells served as a negative target cell control and autologous DCs without loaded peptides were incubated with T cells and served as an effect cell control.After 6 hours of co-incubation at 37℃,the supernatant was collected to assess the lactate dehydrogenase concentration using CytoTox 96 nonradioactive cytotoxicity assay kits with accordance to the manufacturer’s protocol.The cytotoxicity activity of T cells was assessed based on the following formula with the mean values from triplicated wells:Percent Cytotoxicity=（experimental value-effector spontaneous value-target spontaneous value）/（target maximum-target spontaneous value）×100.After the 293 cells were transfected with pcDNA3.1/HPV11E7 plasmid,green fluorescence located in the cellular nucleus and plasma could be seen in positive clones.HPV-11E7 mRNA expression in selective cell line was also confirmed by RT-PCR.The results showed that the established cells positive for HPV-11E7 could further be used as a HPV-11E7-expressing cell line.Autologous T ceils were eocultured with HPV-11E7 peptide-loaded DCs as described above.Again seven days after the last round of restimulation,cells from each culture were collected and analyzed for the specific CTL responses.Induction of cytotoxicity in T cells in response to peptide-loaded DCs was investigated. When the T lymphocytes acted as effector cells adhering to HPV11E7-expressing 293 cells at a 50:1 E/T ratio,T cells cocultured with E7 7-15 peptide-loaded DCs had a stronger cytotoxicity（91.48±0.06%）than others（P＜0.05）.However,this difference was not observed at the E/T ratio 25:1 or 10:1.The untransfected 293 cells did not show obvious cytolytic activity in all the groups at any E/T ratio. Section two:Foxp3⁺CD4⁺CD25⁺ regulatory T cells expression and Th1/Th2, Tc1/Tc2 profiles in peripheral blood of patients with condyloma acuminatumCondyloma acuminatum（CA）is a disease with proliferative lesions of genital epithelium caused by human papillomavirus（HPV）infection.The balance between type 1 and type 2 T-cell subsets in CA patients is thought to modulate antiviral immunity.CD4⁺CD25⁺ Regulatory T cells（Tregs）inhibit proliferation and cytokine production by both Th1 and Th2 cells and reversibly suppress CTL-mediated immunity.A better understanding of the mechanisms of T-cell regulation in CA might help in developing more effective therapeutic strategies.2.1 PatientsA total of 30 patients（12 males and 18 females）and 20 healthy controls（9 males and 11 females）included in this study were recruited from Sir Run Run Shaw Hospital,School of Medicine,Zhejiang University,Hangzhou,People’s Republic of China from October 2006 to September 2007.The clinical diagnosis of CA was made according to the criteria of China National Center for STD.None of the patients had a history of chronic systemic diseases or disorders of immunity. All of them were HIV negative at the time of the study.Fifteen patients were first onset cases and another 15 were relapsed cases of CA.Disease course ranged from 1 month to 25 months with an average of 4.7 months.The mean age of the patients was 31±3.86 years（ranged from 19 to 55 years）and controls 30.2±2.72 years （ranged from 22 to 45 years）.The study was approved by the ethics committees of Sir Run Run Shaw Hospital.Informed consents were obtained from all patients.2.2 Foxp3⁺CD4⁺CD25⁺ T cells expression in peripheral blood from CA patientsIn order to identify Tregs,4 ml heparinized peripheral blood from patients or controls were obtained.PBMC were isolated by Lympholyte-H density gradient centrifugation as recommended by the manufacturer.To analyze CD4,CD25 and Foxp3 expression,PBMC were stained with anti-human CD4-PE-Cy5 and anti-human CD25-FITC monoclonal antibodies,followed by intracellular staining with anti-Foxp3 using anti-human Foxp3-PE Staining Set. Isotype-matched antibodies were used as controls.Treg cells were identified as CD25-positive and Foxp3-positive cells among CD4 cells with triple-color flow cytometry analysis.The raw data were processed with Expo32 software.The results showed that the number of Foxp3-expressing cells within the CD4⁺CD25⁺ T and CD4⁺ T lymphocyte population of CA patients were higher than health controls.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells within the total CD4⁺ population was significantly increased in the blood of total CA patients （3.37±1.03）%and relapsed patients（4.68±1.17）%compared to health controls （1.18±0.53）%（p＜0.001）.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was also increased in the blood of first onset CA patients（2.06±1.01）%compared to health controls but the difference was not statistically significant（P＞0.05）.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was significantly increased in the blood of relapsed CA patients compared to first onset patients（p＜0.001）.2.3 Type 1/ Type 2 immune response in peripheral blood from CA Patients and the health controlsHeparinized peripheral whole blood（200ul）diluted with 1:1 RPMI 1640 medium were incubated with 2μmol/L Monensin,50 ng/mL PMA and 1μmol/L Ionomycin at 37℃in 5%CO₂ for 5 h and then stained with CD3-TC and CD8-FITC MoAb for 15 min at room temperature in dark.Following two washes with PBS,the cells were resuspended and fixed with 100μl Reagent A of the Fix & Perm kit for 15 min.The cells were washed again and suspended in 100μl Reagent B of the Fix & Perm kit and incubated with IFN-γ-PE or IL-4-PE at 4℃in dark for 15 min.The cell population was detected by triple-color flow cytometry and analyzed by Expo32 software.Percentages of Th1 in PBMC of CA patients,including relapsed patients and first onset patients were all significantly lower than normal control group（P＜0.01）.Percentages of Tc1 in CA patients were lower than that in normal control group,especially in relapsed patients（P＜0.05）.Although percentages of Th2 and Tc2 in CA patients were lower than normal control,the difference was not significant（P＞0.05）.Meanwhile,Th1 /Th2 and Tc1/Tc2 ratios of the CA patients were lower than normal control group （P＜0.05）,especially Th1/Th2 ratios in relapsed patients（P＜0.01）.Th1/Th2 ratio of the first onset CA patients and Tc1/Tc2 ratio all CA patients were lower than normal control group but the difference was not statistically significant（P＞0.05）. Percentages of Th1 and Th1/Th2 ratio in PBMC of the relapsed CA patients were significantly lower compared to the first onset group（P＜0.05）.2.4 Correlation between the frequency of Foxp3⁺CD4⁺CD25⁺ T cells and Th1/Tc1 in the peripheral blood from CA patientsCorrelations between two variables were calculated by the Spearman rank correlation test used SPSS statistical software.P＜0.05 was considered statistically significant.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was inversely correlated with Th1（r=-0.798,p＜0.05）,and Tc1（r=-0.746,P＜0.05） in CA patients. Conclusion:1.From the set of possible combinations of nanomer or decamer peptides in the HPV-11E7 proteins,five peptides including HPV11E7 7-15（TLKDIVLDL）, 15-23（LQPPDPVGL）,47-55（PLTQHYQIL）,81-89（DLLLGTLNI）and 82-90 （LLLGTLNIV）were predicted as having a high binding ability to HLA-A2 molecules based on favorable amino acid combinations.2.We have successfully established an in vitro culture system for the derivation of dendritic cells.3.We have successfully established 293 cell lines expressing HPV11E7 by transfection with pcDNA3.1-HPV11E7 plasmid,to serve as target cell models in subsequent experiments.4.IL-12 concentration in DCs cultured with the selected peptides was higher than that of DCs cultured in medium alone.DCs cultured with E7 15-23 peptides was higher than in DCs cultured with E7 7-15 peptides,and IL-12 concentration in these two groups was remarkably higher than in DCs stimulated with the other three peptides and controls.5.Secretion of IFN-γwas higher in T cells cocultured with peptide-loaded DCs.T cells cocultured with E7 7-15 peptide-loaded DCs showed the highest level of IFN-γsecretion.6.Autologous T cells stimulated with E7 7-15（TLKDIVLDL）peptide-loaded DCs showed the highest frequency of specific tetramer⁺ CD8⁺ cells as compared to other peptides.T cells cocultured with E7 7-15 peptide-loaded DCs had a stronger cytotoxicity than others When T lymphocytes acted as effector cells adhering to HPV11E7-expressing 293 cells. 7.E7 7-15 is a HLA-A^*0201-restricted CTL epitope CTL of HPV type 11 E7.This was confirmed by measuring the IFN-γsecretion of T cells and the frequency of specific tetramer⁺ CD8⁺ T cells following stimulation of E7 7-15 peptide-loaded DCs.8.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells within the total CD4⁺ population was significantly increased in the blood of total CA patients and relapsed patients compared to health controls.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was significantly increased in the blood of relapsed CA patients compared to first onset patients.The results may indicate that Tregs be involved in the mechanisms allowing CA to occur and the relative prevalence of Tregs may be a determinant for predicting the prognosis of CA patients.9.CA patients showed a decreased proportion of Th1 and Tc1 cells,and a decreased ratio of Th1/Th2 and Tc1/Tc2.Especially remarkable decreased ratios of Th1/Th2 was found in relapsed CA patients.10.The frequency of Foxp3⁺CD4⁺CD25⁺ T cells was inversely correlated with Th1 and Tc1 in CA patients.Tregs appear to downregulate cytokine expression in both Tc1 and Th1 subsets of effector T cells,which may be responsible for antivirus responses.更多还原