【作者】 陈云；

【导师】 姚堃；

【作者基本信息】 南京医科大学 ， 免疫学， 2007， 博士

【摘要】 EB病毒(Epstein-Barr Virus，EBV)属于疱疹病毒γ亚科嗜淋巴细胞病毒成员，在世界各地广泛分布，为95％以上的成人所携带，是较早认识的与人类肿瘤相关的病毒。自1966年Old在鼻咽癌(nasopharyngeall careinoma，NPC)组织中首先发现存在有EB病毒DNA以来，大量的血清流行病学研究已证明EB病毒与鼻咽癌有关。鼻咽癌是上皮来源的恶性肿瘤，高发于东南亚及我国华南地区。临床上鼻咽癌通常不宜手术治疗，放疗、化疗后也不能完全有效清除肿瘤细胞，部分患者易复发和转移，并且治疗产生的副作用又影响了自身的免疫系统。因此目前，在倡导鼻咽癌综合治疗的同时更加注重免疫治疗。对鼻咽癌的免疫机制研究显示，针对EB病毒特异性细胞毒性T细胞(CTL)细胞在清除肿瘤细胞中起着关键作用。CTL亚群能直接识别并杀伤肿瘤细胞，在杀伤肿瘤细胞中CD4~+和CD8~+T细胞相互协调共同发挥作用。以CTL为基础的免疫治疗需选择合适的靶抗原。鼻咽癌肿瘤细胞中EB病毒蛋白呈潜伏Ⅱ型表达，即仅表达低免疫原性抗原EBNA1、LMP1、LMP2。EBNA1在诱导特异性T细胞的同时，也能诱导CD4~+调节性Treg细胞，抑制机体的免疫应答，且EBNA1记忆性CTL反应主要受HLA B3501，B7等限制。LMP1已被确认为能够使正常细胞发生转化癌基因蛋白，CTL识别的靶位易突变，免疫原性也弱于LMP2。目前认为，LMP2A是免疫治疗理想的靶抗原，序列相对保守，具有潜在性的T细胞表位。在NPC病人血液中能检出针对EB病毒LMP2的CTL前体细胞，但其数目及反应性明显低于健康携带者，导致机体对肿瘤的免疫耐受。所以纠正这种CTL的低应答状态对于机体有效清除肿瘤细胞至关重要，诱导EBV特异性CTL是鼻咽癌免疫治疗的关键。T细胞只能识别由抗原递呈细胞(APC)递呈的抗原肽-MHC分子复合物。树突状细胞(DC)是目前发现功能最强的APC，能激发初始型和记忆型T细胞，产生有效的CTL应答。以DC为基础的免疫治疗已在许多肿瘤的治疗中取得了良好的效果，是当前抗肿瘤免疫治疗研究的热点之一。本课题用DC作为鼻咽癌免疫治疗的APC，选用EB病毒LMP2A作为免疫治疗的靶抗原，以分子生物学和细胞生物学方法构建LMP2A稳定表达细胞株，纯化LMP2A蛋白，并对EBV相关的NPC病人的血清进行了检测；蛋白负载经体外扩增的EB病毒阳性健康携带者的DC；负载的DC分别与自体外周血CD4~+和CD8~+T混合培养，激发出LMP2A特异性CD4~+和CD8~+T，并对其功能进行研究。主要研究内容和结果如下：一、EB病毒LMP2A基因重组逆转录病毒和稳定表达LMP2A细胞株的构建用PCR从质粒pPICZ-α／LMP2A扩增EBV的LMP2A基因片断，经EcoRⅠ、BamHⅠ酶切后定向插入含同样酶切位点的逆转录病毒表达载体pSIV-1的LTR启动子下游，构建重组逆转录病毒载体pSIV-1／LMP2A，重组质粒经PCR、EcoRⅠ和BamHⅠ双酶切及序列分析鉴定。获得的重组质粒与两个辅助病毒载体pHIT456和pHIT60用脂质体共转染包装细胞293T，转化后的细胞用G418筛选，经1周筛选培养后获得抗性细胞克隆，将抗性细胞扩大培养，收集含有重组逆转录病毒颗粒抗性细胞克隆培养上清。PCR结果显示在抗性细胞克隆基因组中存在LMP2A基因。用重组逆转录病毒感染NIH3T3细胞，计算病毒滴度，并于感染后48小时用PCR、间接免疫荧光、Westem blot检测目的基因的整合和表达。结果显示，扩增到的病毒效价为5×10~5CFU／ml，PCR阳性结果提示LMP2A能整合于MH3T3细胞中，间接免疫荧光显示NIH3T3细胞有LMP2A的表达，Western blot显示在54KDa处出现特异的蛋白条带。表明重组逆转录病毒能有效地介导LMP2A基因在真核细胞中表达。用重组逆转录病毒颗粒重复3次感染L929细胞，72小时后置于含G418选择性培养基中，筛选培养2周后，获得抗性细胞克隆。PCR阳性结果提示LMP2A能整合于L929细胞中，RT-PCR结果表明LMP2A基因能转录成相应的mRNA。流式细胞仪显示91.3％的细胞能表达LMP2A，Western blot出现特异的蛋白条带。提取细胞蛋白，经His Bind Ni-NTA亲和层析柱纯化，得到纯度达90％以上的LMP2A蛋白。采用纯化的LMP2A蛋白作为抗原，用ELISA法检测EB病毒相关鼻咽癌患者血清中的抗体，结果显示鼻咽癌患者血清中存在着高滴度的抗LMP2A的抗体，检出率为72.29％。比较NPC病人LMP2A血清学检测结果与临床上VCA、EA法结果，三者检出率相当，大部分病例呈正相关，但一些病例中VCA、EA检出率低的LMP2A检出率高，而一些病例VCA、EA检出率高的LMP2A检出率低。因此，如将三者结合有助于提高鼻咽癌患者的检出率，LMP2A对鼻咽癌的检测具有辅助诊断意义。二、人外周血树突状细胞的培养鉴定及EB病毒LMP2A蛋白的负载分离EBV阳性的健康人外周血单核细胞，在含重组hGM-CSF和hIL-4的培养液中培养，用hTNF-a诱导DC成熟。共聚焦显微镜观察其形态，流式细胞仪检测细胞的表面分子。结果显示，从健康人外周血分离得到的单核细胞，体外经重组hGM-CSF、hIL-4的培养可获得未成熟的DC，流式细胞仪检测DC相对特异性标记CD1a、CD83及共刺激分子CD80、HLA-DR等表达较低，而单核细胞特异性标志CD14相对较高；当加入hTNF-α继续培养2天后，得到大量成熟DC，DC特异性标记CD1a、CD83及共刺激分子CD80、HLA-DR表达显著升高，CD14表达下调。光学显微镜下观察诱导成熟的DC具有典型树突状特征。用不同浓度的LMP2A蛋白负载未成熟的DC，诱导成熟后激光共聚焦检测DC俘获抗原的能力，FACS选择最佳蛋白负载浓度；用最佳LMP2A蛋白浓度负载未成熟的DC，FACS检测负载前后诱导成熟DC表面分子CD1a、CD83、CD14、CD80、HLA-DR变化及LMP2A表达情况；并用~3H-TdR掺入法检测负载前后诱导成熟DC刺激同种淋巴细胞增殖能力。结果显示，40μg／ml为LMP2A蛋白转染DC的最佳浓度，共聚焦和FACS结果显示约80％的DC表达LMP2A蛋白；LMP2A负载后诱导的成熟DC对细胞表面共刺激分子及特征性表面标志无影响；负载后诱导成熟DC仍具有较强的刺激同种淋巴细胞增殖能力。研究结果为蛋白负载DC免疫治疗NPC提供可能。三、EB病毒LMP2A蛋白负载树突状细胞激发特异性CTL及体外杀伤活性的研究免疫微磁珠分离获得自体CD4~+和CD8~+T细胞，FACS鉴定分选细胞纯度均大于95％。3H_TdR掺入法细胞增殖实验检测CD4~和CD8~T细胞经LMP2A蛋白负载自体DC或未负载DC刺激后的增殖水平。ELISA法检测CD4~T细胞经抗原负载后的DC刺激分泌IFN-γ和IL-10的水平。~3H-TdR掺入法结果显示，CD4~+和CD8~+T细胞经LMP2A蛋白负载自体DC刺激后的增殖水平明显高于未负载组(P＜0.001，P＜0.001)，表明DC能够俘获抗原并将抗原提呈给自体的T淋巴细胞，同时LMP2A蛋白含有MHC-Ⅰ和Ⅱ类分子限制性的靶位。ELISA法检测结果显示，CD4~+T细胞经负载后的DC刺激分泌IFN-γ明显高于未负载DC组(P=0.034)，而IL-10在各组中的分泌水平均很低。构建含有绿色荧光蛋白报告基因(GFP)荧光标记的重组逆转录病毒载体pPGEz-Term／LMP2A，与辅助病毒载体PHT 456和PHI 60共转染包装细胞293T，收集含有Zeocine的抗性细胞培养上清。用此重组逆转录病毒感染第1天诱导的未成熟DC，感染后继续在含hGM-CSF、hIL-4的培养液中培养，重复感染3次，用hTNF-α诱导DC成熟。荧光显微镜观察重组逆转录病毒转染293T细胞情况及FACS检测LMP2A在DC上的表达，并以此作为靶细胞。结果显示，荧光显微镜下观察293细胞可见绿色荧光，FACS检测出56.4％的DC能表达LMP2A。采用重复刺激技术获得LMP2A特异性CD8~+T细胞，分别以转染重组逆转录病毒pPGEZ-Term／LMP2A或空载体的自体成熟DC、LCL、K562细胞作为靶细胞，用LDH释放法检测CTL的杀伤活性。FACS检测CTL杀伤靶细胞(重组逆转录病毒pPGEZ-Term／LMP2A转染的自体成熟DC)后CD4~+和CD8~+T细胞胞内分泌细胞因子IFN-γ水平。LDH检测结果显示，LMP2A蛋白负载DC可以诱导出针对EBV-LMP2A特异的CTL，能够特异性的杀伤重组逆转录病毒pPGEZ-Term／LMP2A转染自体的成熟DC(P=0.023)。FACS结果显示，经LMP2A负载的DC诱导的特异性CD4~+和CD8~+T与靶细胞孵育后，胞内分泌细胞因子IFN-γ水平明显高于对照组(P＜0.01)。我们研究结果表明：EB病毒潜伏膜蛋白2A在L929细胞上稳定的表达，鼻咽癌患者血清中存在着高滴度的抗LMP2A的抗体；LMP2A蛋白负载的DC在体外能激发针对LMP2A特异性CTL，对重组逆转录病毒pPGEZ-Term／LMP2A转染自体的成熟DC具有杀伤效应。本研究为进一步开展EB病毒相关鼻咽癌的临床免疫治疗奠定基础，也为其它病毒相关性肿瘤的免疫治疗研究提供实验和理论依据。更多还原

【Abstract】 Epstein-Barr virus (EBV), infecting over 95 percent of humans andpersisting for the lifetime of the person, belongs to the gama-herpesvirinaesubfamily and was known its relationship with human tumors early. SinceEBV DNA was detected in tissues from patients with nasopharyngealcarcinoma (NPC) in 1966, serological studies have confirmed EBVinfections are the crucial factors in the pathogenesis of NPC. NPC is amalignant tumor derived from the epithelial cells and endemic in theSoutheast Asia and South of China. NPC usually are not suitable forsurgery and tumor cells are not eliminated effectively and completely byradio therapeutic and chemotherapeutic methods. Relapse and metastasismay occur in a portion of NPC patients and side effect arising fromconventional therapeutic methods will affect self immune system greatly.Therefore, it is critically important to develop an immunotherapeuticmethod for NPC except for conventional therapy.Studies on immune system of NPC show that cytotoxicT lymphocytes(CTLs) specific for EBV antigens are crucial for eliminating tumor cells.CTL subtype can recognize and kill tumor cells directly and CD4+ andCDS+ T cells co-contribute it. However, suitable target antigen should beselect in immunotherapy based on CTL.Within NPC tumor cells, the viruses establishes a predominantly latentinfection, with laminated expression of three viral proteins, nuclearantigens 1 (EBNA 1) and three latent membrane proteins (LMPs 1, 2A and2B). EBNA1 can induce CTL specific for it and also produce CD4+regulatory Treg cells, which suppress immune response. CDS+ CTLresponses specific for EBNA1 epitopes are mainly restricted by HLA-B3501、B7. LMP1 has been confirmed to be consistence with itsability to transform rodent fibroblasts, and its target epitopes recognized byvirus-specific cytotoxic T cells (CTL) in NPC patients are easy to mutate.Responses detected in healthy virus carriers indicate that LMP1 is poorlyimmunogenic, thus, the most likely target antigen for a CD8 CTL-basetherapy is LMP2. LMP2 amino acid is more conserved and containsHLA-restrict CTL target epitopes which can elicit strong specific CTLresponse. Pre-CTL cells specific for EBV-LMP2 can be detected in the seraof NPC patients, but its number and response are obviously lower thanhealthy carders, which results in immune tolerance. Therefore, it’s veryimportant to correct this low CTL-response status and elicit EBV-specificCTL in NPC patient is one of the key immunotherapeutic strategies.T cells can only recognize antigen peptide-MHC monocular complexpresented by antigen presenting cells (APCs). Dendritic cells (DC) areknown as the most powerful and the only APC which are capable ofactivating naive T cells and initiating primary immune response. Resultsfrom DC-based immunotherapy have revealed a promising approach forcancer treatment and become one of the hot points in immunotherapyresearch at present.In this study, DC was utilized as APC, and EBV-LMP2A was chosenas the target antigen of NPC immunotherapy. LMP2A stable expressioncell line was constructed by monocular and cellular biological methods,and was purified. We then loaded DC induced from monocytes of EBVhealthy carriers with the purified LMP2A protein. The loaded DC wereco-cultured with autologous CD4~+ and CD8~+ T cells separately to elicitEBV-specific CD4~+ and CD8~+ T cells which were further analyzed itsfunctions. The main methods and results are as following:PartⅠ. Establishment of EBV LMP2A gene recombinant retrovirusvector and L929 cell line stable expressing LMP2AThe full-length EBV LMP2A gene was generated by PCRamplification from pPICZ-α/LMP2A which contain complementnucleotide sequence of EBV LMP2A gene with a pair of primers. Afterdigestion with EcoRⅠ、BamHⅠ, PCR products were inserted downstream of LTR of retroviral vector pSIV-1 to obtain recombinant retroviralexpression vector pSIV-1/LMP2A. Recombinant retroviral expressionvector pSIV-1/LMP2A was detected positive by PCR, enzyme digestionwith EcoRⅠand BarnHⅠas well as sequence analysis. To produceretroviral virus, packing cells, 293T cells were co-transfected withrecombinant retroviral expression vector pSIV-1/LMP2A and two auxiliaryviral vectors pHIT456 and pHIT60 by lipofectAMINE2000. Colonies wereisolated by neomycin (G418) selection and obtained after 1 week toexpand. Supematants of cloned packing cells were harvested, whichcontain recombinant retrovirus. The PCR results showed that EBV LMP2Agene had been integrated into the genome of resistant 293T cells. Viraltitration was performed on NIH3T3 according to the instructions of themanufacturer yielding a titer of 5×10~5 infectious particles/ml. The PCRpositive results indicated that NIH3T3 genome had contained LMP2A gene.Indirect immunofluorescence showed that LMP2A gene had beenexpressed in the murine flbroblast, NIH3T3. Transduction rate was 91.3%determined by fluorescence activated cell sorter (FACS). Western blotresults showed a specific protein lane at 54KDa position. These resultssuggested that transfer of LMP2A could be efficiently mediated byrecombinant retrovirus. Western blot results also confirmed it. LMP2Aprotein was extracted and purified by His Bind Ni-NTA affinitychromatography column, the purity of over 90% LMP2A protein wasobtained.The purified recombinant protein LMP2A was used as an antigen todetect specific antibodies in the sera from NPC patients. 72.29% patientswere positive for anti-LMP2A IgG. These studies demonstrate the potentialsignificance of LMP2A-specific Abs for the diagnosis and prognosis ofEBV-associated malignancies, especially of NPC.PartⅡ. Induction and identification of dendritic cells from humanperipheral blood monoeytes and load of DC with EBV-LMP2Apurified proteinThe monocytes were isolated from PBMC of a healthy EBV carrier and cultured with the cytokines (rhGM-CSF, rhIL-4), followed by additional 2days with TNF-α, yielding mature DCs. The morphological changes wereobserved by confocal analysis. The surface molecule of DCsIncluding CD80 and MHC classⅡwere measured by FACS. The resultsshowed that immature could be obtained from PBMC cultured in themedium containing GM-CSF and IL-4. DCs relative specific markerCDla, CD83 and co-stimulational monocular CDS0, HLA-DR wereexpressed low, while monocyte specific marker CD14 was relative high.When the immature DCs were cultured for additional two days in thepresence of TNF-α, mature DCs were harvested and express high level ofCD1a、CD83 and CD80、HLA-DR, while CD14 express low. DCs typicalcharacteristics were observed by morphological microscope. In addition,mature DC had potent ablility to stimulate the proliferation of allogeneic Tcells.Immature DCs were loaded with EBV-LMP2A purified protein atdifferent concentrations. The ability of DCs capture antigen was observedby confocal analysis and a suitable protein concentration was selected byFACS. The alteration of surface markers on mature DC including CDla,CD83, CD80 and HLA-DR was detected by means of FACS before andafter loaded with suitable LMP2A protein. The stimulatory effect of DCson proliferation of T lymphocytes was examined by ~3H-TdR incorporation.The results showed that above 80% of mature DC were expressed LMP2Aprotein after loaded with LMP2A protein at the concentration of 40μg/ml.No significant changes in the surface markers and the cytomorpholosis ofmature DC were examined during loading protein. It showed that DCsloaded antigen had no influence to the differentiation and maturation. DCsstill have strong potential to stimulate the proliferation of allogeneic T cells.The study could be the foundation of the immunotherapy with DC.PartⅢ. Generation and cytotoxieity assay of EBV LMP2A-specificCTL elicited by DC loaded with LMP2A protein in vitroImmuno-magnetic beads were used to prepare CD4~+ and CD8~+T cells.Median purity of the cell preparations was determined by flow cytometryanalysis and was routinely more than 95%. The proliferation of autologous CD4~+ cells, CD8~+T cells stimulated with Unloaded DCs (UL-DC) andloaded DCs (L-DC) was measured by methods of ~3H-TdR incorporation.The proliferative responses of CD8~+T and CD4~+ T cells to L-DC weresignificantly increased compared to those from UL-DC (P＜0.001,P＜0.001). The results demonstrate that DCs capture LMP2A protein andpresent their Ags to autologous T cells and LMP2A protein containsMHC-ⅠandⅡclass restricted epitopes. We tested the IFN-γand IL-10secretion from UL-DC and L-DC stimulated CD4~+T cells by ELISA andfound that L-DC stimulated CD4~+T secreted higher levels of IFN-γthanthose from UL-DC(P=0.034), but the level of IL-10 was rarely detected ineither group.EBV LMP2A gene recombinant retrovirus vector pGEZ-Term/LMP2A was established which contains GFP report gene. 293T cells wereco-transfected with recombinant retroviral expression vectorpGEZ-Term/LMP2A and two auxiliary viral vectors pHIT456 and pHIT60by lipofectAMINE2000. Colonies were isolated by zeocine. Supernatantsof cloned packing cells were harvested, which contain recombinantretrovirus. The monocytes were isolated from PBMC and incubated withGM-CSF and IL-4 medium. After a 24-h incubation, the cells weretransduced with retrovirus supernatants and polybrene at a finalconcentration of 8μg/ml. The supematant were replaced with GM-CSFand IL-4 medium 2-3h later. The transduction procedure was repeated threetimes. After 6 days later, TNF-αwas added and mature DCs wereharvested after additional 2 days culture. The surface molecule of DCs andthe efficiency of LMP2A expression were measured by FACS. The resultsshowed that 56.4% of DCs can express LMP2A determined by FACS.Immuno-magnetic beads were used to prepare EBV-LMP2A specificCD8~+ T cells, which were then restimulated with autologous DC loadedwith LMP2A protein. Cytotoxicity of LMP2A specific CTL wasdetermined with LDH release assay, the target cells are autologous DCtransduced with recombinant retrovirus pGEZ-Term/LMP2A、autologousLCL and K562 cells separately. We evaluated IFN-γ-producing cells bothin LMP2A-specific CD4~+ T cells and CD8~+ T cells induced by L-DCstimulated with recombinant retrovirus pGEZ-Term/LMP2A by FACS. CD4~+ T cells and CD8~+ T cells induced by L-DC had significantly higherfrequency of circulating LMP2A-specific IFN-γ-producing CD4~+ T cellsand CD8~+ T cells, compared to controls (P＜0.01).In summary, the results above suggested that LMP2A can beexpressed stably in L929 cell line, LMP2A may be of prognostic valuein NPC patients; LMP2A-specific CTL could be induced by DC loadedwith LMP2A protein in healthy EBV carriers in vitro and elicitcytotoxicity to autologous DC tranceduced with recombinantretrovirus pGEZ-Term/LMP2A. Such results could be the foundationfor the further immunotherapy of NPC in clinical trails; moreover,this research provides experimental and theoretical basis for othervirus related tumor immunotherapy.更多还原