【作者】 欧阳礼辰；

【导师】 吴雄文； 翁秀芳；

【作者基本信息】 华中科技大学 ， 免疫学， 2013， 博士

【摘要】 乙型肝炎病毒（HBV）是一种嗜肝性，非细胞病变的DNA病毒，感染后很容易导致慢性化。在HBV慢性感染的病人中，由于特异性的抗病毒T细胞免疫缺乏或损坏，常常导致病毒持续性感染并发展为严重的肝脏疾病。虽然有关T细胞对HBV抗原低反应或耐受的机制目前还不完全清楚，大量的研究提示有调节功能的CD4⁺和CD8⁺T细胞可能在抑制HBV特异性的T细胞应答中发挥重要作用。CD3⁺CD8^lowT细胞是细胞表面CD8分子下调的一群CD8⁺T细胞，在很多慢性持续性抗原刺激的情况下增多，如HIV感染病人，器官移植病人以及慢性寄生虫感染的小鼠。研究发现这群细胞属于2型极化T细胞，具有较低的细胞毒性，在某些情况下可通过其分泌的IL-10或细胞表面表达的膜型TGF-β1发挥抑制作用。在本研究中，考虑到慢性HBV感染也是一种持续性的抗原刺激，我们的目的是探索CD8^lowT细胞在慢性HBV感染病人受损的CD8⁺T细胞应答中是否发挥作用以及何种作用。一．慢性HBV感染病人外周血中CD8^lowT细胞的频率高于健康人本研究中我们选择了47个慢性HBV感染病人和19个健康人对照，分离这些个体的外周血单个核细胞并通过流式细胞术检测其中CD8^lowT细胞的频率。我们发现慢性HBV感染病人外周血中CD8^lowT细胞的频率高于健康人对照，同时发现感染HBV时间长于5年的病人较低于5年的病人含有更高的CD8^lowT细胞。二．病人外周血中CD8^lowT细胞的频率与血浆中HLAI类分子的浓度正相关我们通过ELISA检测了病人和健康人血浆HLAI类分子的浓度，发现病人血浆HLAI类分子的浓度显著高于健康人。同时发现，病人外周血中CD8^lowT细胞的频率与血浆中HLAI类分子的浓度成正相关，而健康人中并未发现相关。三．病人外周血中CD8^lowT细胞的表型通过流式细胞术检测CD8^lowT细胞一些与效应和抑制有关的标记的表达，发现相比健康人，病人中的CD8^lowT细胞在表型上更接近2型极化T细胞（较多的IL-4表达和较少的IFN-γ表达），并显示出调节或抑制性的特征（抑制性标志上调，特别是膜型TGF-β1）。四．病人中CD8^lowT细胞的频率与HBc18-27特异性CD8⁺T细胞应答负相关通过IFN-γELISPOT检测病人和健康人HBc18-27特异性CD8⁺T细胞应答。发现相比正常人，病人的HBc18-27特异性CD8⁺T细胞应答显著降低，并且与CD8^lowT细胞的频率成负相关，提示病人中的CD8^lowT细胞可能具有抑制功能。五．结论慢性HBV感染病人外周血中CD8^lowT细胞群显著增多，其在表型上趋向2型极化T细胞，且一些抑制性标志表达升高，这可能是导致慢性HBV感染病人CD8⁺T细胞应答低下的其中一个原因。T细胞通过其细胞膜上的T细胞受体（TCR）与抗原提呈细胞上的肽-主要组织相容复合物（pMHC）结合发挥其细胞免疫及免疫调节功能。通过对TCR的研究既可明确T细胞抗原识别的分子机制，又可发展基于TCR的免疫治疗手段。近期的研究提示TCR基因转导后的T细胞具备相应的特异性免疫调节或细胞毒特性，在治疗恶性肿瘤，自身免疫病，慢性病毒感染疾病等方面有巨大发展潜能。获得目的表位特异性单克隆TCR是研究TCR免疫治疗的关键。TCR通过数目繁多的编码基因不同的排列组合使其多样性达到1018之多，从如此庞大的T细胞库中钓取任意表位特异性的TCR犹如大海捞针。现有单克隆TCR的获得主要是通过特异性T细胞体外扩增及单克隆化技术，然而却存在T细胞难以长期在体外单克隆化增殖以及培养周期长等问题。本研究以HLA-A2限制性黑色素瘤相关抗原表位gp100为例，通过体外单一表位同种特异性T细胞扩增体系及pMHC四聚体分选技术获得高亲和力表位特异性T细胞，并建立TCR单细胞扩增技术，结合快速表达和鉴定体系，为利用基于TCR的免疫干预提供有效可行的方法。一．设计扩增TTCCRRα与β链的引物群并验证各条引物的有效性根据IMGT/Gene-DB （http://imgt.cines.fr/）提供的所有有编码功能的TCR α与β链可变区（V）和不变区（C）基因片段序列，经过基因扫描、比对，利用primerpremier5软件共设计出59条上游引物和4条下游引物，分别可与TCR α与β链的V基因和C基因配对。应用所设计的引物群对健康人外周血T细胞进行PCR扩增，发现几乎每对TCR α链引物均可扩增获得与TCR的α链编码基因长度一致的片段，而且每对TCRβ链引物均可扩增获得与TCR的β链编码基因长度一致的片段，说明我们设计的TCR α与β链的引物群可用于扩增TCR的编码序列。二．TCR单细胞RT-PCR的建立1.从单个Jurkat细胞中扩增TTCCRRα及β链选择表达TCR的人T细胞瘤细胞系Jurkat细胞，对其进行单克隆化，摸索单细胞RT-PCR的条件。将Jurkat细胞有限稀释成理论上的单细胞并裂解细胞得到单个细胞的RNA，用特异性引物即α及β链C区外侧引物（共两条）逆转录得到单个Jurkat细胞的cDNA，利用35对α链混合引物与24对β链混合引物扩增出单个Jurkat细胞的TCRα及β链基因，并将PCR扩增获得的基因克隆入T-easy载体中，对克隆基因测序并在NCBI中比对测序结果。结果显示，扩增序列与NCBI中显示一致，α及β链使用的V区分别为TRAV8-6与TRBV12-3。2.从健康人外周血单个CD8+T细胞中扩增TTCCRRα及β链分离健康人外周血单个核细胞（PBMC），并用CD8磁珠阴性分选出CD8+T细胞。将CD8+T细胞有限稀释成理论上的单细胞，调整单个Jurkat细胞TCR扩增方法，对单个CD8+T细胞进行RT-PCR扩增TCRα及β链基因，在12个单细胞中可成功的从其中的5个单细胞中获得TCR的α链基因，从其中8个单细胞中成功获得TCR的β链基因。将扩增获得的TCRα及β链基因经过测序获得编码序列。三．体外混合淋巴细胞培养扩增单一表位（pMHC）特异性T细胞将荷载抗原肽gp100后的T2细胞（TAP缺陷,仅表达HLA-A2，T2/gp100）作为刺激细胞，与HLA-A2阴性个体（HLA-A2-ve）的PBLs长期共培养14天后，诱导产生出T2/gp100表位特异性的细胞毒性T细胞（CTL-T2/gp100）。利用制备的gp100/HLA-A2四聚体和无关HBc/HLA-A2四聚体对诱导产生的CTL-T2/gp100染色发现gp100/HLA-A2四聚体对CTL-T2/gp100染色阳性率显著高于无关HBc/HLA-A2四聚体的同型对照染色，说明诱导出的CTL是T2/gp100表位特异性的。四．利用TCR单细胞扩增技术对分选获得的gp100/HLA-A2特异性T细胞进行TCR扩增通过流式细胞仪分选出gp100/HLA-A2四聚体染色阳性的CTL-T2/gp100，将分选得到的细胞单细胞化，用建立的TCR单细胞RT-PCR方法扩增表位特异性TCR的α及β链基因，经测序比对确定从10个单细胞中得到5个α链及5个β链基因（其中4对配对）。五．gp100/HLA-A2特异性TCR的表达及鉴定选择扩增成功的gp100/HLA-A2特异性TCR的其中一对α及β链基因,将其序列进行密码子优化后转入昆虫杆状病毒pfast-dual双表达载体中，表达可溶性gp100/HLA-A2特异性TCR。用western-blot检测蛋白具有正确的构象和分子量，用ELISA检测蛋白的特异性结果不太理想，分析可能是我们选取的TCR亲和力不足以检测到或是可溶性TCR分子的浓度较低所致。六．结论我们建立了一种从人T细胞库中扩增表位特异性单克隆TCR的方法，该方法不受表位的限制，为进一步利用基于TCR的免疫干预提供了基础。更多还原

【Abstract】 Hepatitis B virus (HBV) is known as a hepatotropic, noncytopathic DNA virus, infecting more than300million people worldwide and readily establishing chronicity. In patients chronically infected with HBV, specific antiviral T-cell response is weak or undetected, resulting in persistence of infection and development of severe liver diseases. Although the mechanisms responsible for T cell hyporesponsiveness or tolerance to HBV antigens are not fully elucidated, substantial studies suggest both CD4+and CD8+T cells of regulatory property would play an important role in inhibiting HBV-specific T-cell responses.CD3+CD8low T cells are recognized as a subset of CD8+T cells with down-regulated CD8expression, whose increase is observed in chronic and persistent antigen exposure, e.g. patients infected with HIV, patients underwent transplantation and mouse chronically infected with parasite. The CD8low T cells are reported to be type-2polarized and poorly cytolytic, and even exhibit suppressive function via IL-10 or membrane-bound TGF-β1(mTGF-β1).Given that chronic HBV infection is also a persistent antigen exposure, in this study, we explored the role of CD8low T cells in the impaired CD8+T cell response in patients with chronic HBV infection.1. Frequency of CD8low T cells is increased in peripheral blood of chronic HBV patients19healthy individuals and47chronic HBV patients were selected. PBMC were isolated and flow cytometry was performed to determine the circulating CD8low T-cell frequency. There was a significantly higher frequency of circulating CD8low T cells in chronic HBV patients than that in healthy controls (the CD8low cells in CD8+T cell:15.20%±1.04%vs.5.66%±0.57%[mean±SEM];p<0.001). we also found the patients with the disease course longer than5years (n=21) showed a markedly higher frequency of CD8low T cells compared to those shorter than5years (n=10)(the CD8low cells in CD8+T cell:18.64%±1.70%vs.10.37%±1.84%[mean±SEM]; p<0.01).2. Soluble HLA class I plasma levels show a positive relationship with frequency of CD8low T cells in chronic HBV patientsThe concentrations of plasma HLA class I molecules from47chronic HBV patients and19healthy donors were measured. In comparison to healthy donors, soluble HLA class I plasma levels were significantly higher in chronic HBV patients (ng/mL,850.1±79.77vs.263.1±29.67[mean±SEM];/p<0.001). There was a positive correlation between soluble HLA class I plasma levels and the percentage of CD8low cells in CD8+T cells (R2=0.2155;p=0.001). In contrast, no correlation was found in healthy donors.3. Phenotype of CD8low T cells in chronic HBV patientsThe CD8low T cells in chronic HBV patients and healthy donors were typed for the commonly used effector and suppressive markers. We found CD8low T cells in the patients, compared to that in healthy donors, seemed phenotypically to be type-2polarized (more IL-4and less IFN-y expression) and of regulatory/suppressive properties (elevated expression of suppressive markers, especially mTGF-β1).4. CD8low T-cell frequency is negatively correlated with HBc18-27-specific CD8+T-cell responses in chronic HBV patientsCD8+T-cell responses to the HBc18-27peptide were evaluated with HLA-A2+ve samples by IFN-y Elispot assay. The CD8+T cells of chronic HBV patients showed significantly lower IFN-y production in response to the HBc peptide (15.39±1.59vs.36.78±10.01[mean±SEM];p<0.01). Notably, a significantly negative correlation was observed between frequency of CD8low T cells and HBc-specific CD8+T-cell responses in chronic HBV patients (r2=0.376,p=0.003). In contrast, no correlation was found in healthy donors (r2=0.195,p=0.234).5. ConclusionCD8low T-cell subpopulation is significantly increased in chronic HBV patients, which is type-2polarized and expresses elevated levels of suppressive markers. The prevalence of CD8low T cells in patients with chronic HBV infection may be one of the factors that resulting in impaired CD8+T-cell response. T cells execute cellular immune response and immuno-regulatory functionthrough interaction of their membrane TCRs with the antigenic peptides presented bythe major histocompatibility complex molecule on APCs. Investigation of TCRmolecules could not only dissect the molecular mechanism of antigen recognition, butalso develop immunotherapeutic approaches based on TCRs. Recent studies haveindicated T cells transduced with specific TCR gene display specificimmuno-regulatory or cytotoxic characteristics, suggesting their great potential intreatment of malignant tumors, autoimmune diseases, chronic viral infections, etc.Obtaining epitope-specific monoclonal TCR is cruial for investigation of TCR genetherapy.The diversity of TCR repertoire is amazingly enormous (reaches1018) because ofvarious and complex gene combination rearrangement process. To select randomepitope-specific TCR from this huge T cell repertoire is like looking for a needle in ahaystack. At present, single-cloning following in vitro expansion of specific T cells isthe main method to obtain monoclonal TCRs. However, the difficulty of long-termsingle-cloning expansion in vitro and time-consuming culture limited the widespreaduse of this method. In this study, we chose HLA-A2restricted melanoma-relatedantigenic peptide gp100as an example. A combination of in vitro epitope-specificalloreactive T cell expansion, pMHC tetramer sorting and single cell RT-PCRtechnique was employed to clone single epitope-specific TCR with high-affinity. TheTCR gene was then expressed in a vector and identified. The method proposed hereprovides an efficient and practical way to employ the TCR-based immune intervention.1. Design of primers for TCR α and β chain amplification and validation of thedesigned primersSequences corresponding to the functional variable and constant region genes forTCR alpha, beta chains were obtained from IMGT/Gene-DB (http://imgt.cines.fr/).After analysis and alignment of all the sequences,59forward primers and4reverseprimers were designed with the aid of the Primer Primer5.0software, which wereannealed to the variable region and the constant region, respectively. To furthervalidate all the primers we designed, RT-PCRwere performed. For each reaction,cDNA derived from PBLs of healthy donors was used as template. Nearly each pair ofprimer of TCR α chain produced bands of expected size. Similarly, each pair ofprimer of TCRβ chain obtained the positive results. It suggested that the primers wedesigned were suitable for cloning TCR genes.2. Establishment of single cell RRTT--PPCCRRfor cloning TCR2.11Amplification of TCR α and β chain from single jurkat cellsTo explore the optimum condition of single cell RT-PCR, we chose a humanlymphoblastic T jurkat cell line which expresses TCR and single cloned it. Singlejurkat cells were isolated using serial dilution method and lyzed to obtain RNA. ThenRNA was reverse transcribed to cDNA using constant-specific primers (2in total) forα and β chain. TCR α and β chain of single jurkat cells were amplified using35pairsof mix primers for α chain and24pairs of mix primers for β chain. The amplifiedPCR products were cloned to T-easyvector for sequencing and aligned to NCBI. Asexpected, sequence we amplified was right. Variableregion of TCR α and β chain ofjurkat cells were TRAV8-6and TRBV12-3, respectively.2.2Amplification of TCR α and β chain from single CD8+T cells frompheripheral blood of healthy donorsPheripheral blood mononuclear cells (PBMCs) were isolated from healthy donors. And then CD8+T cells were enriched by negative selection using a naive CD8+T CellIsolation Kit and diluted at one cell per PCR tube. TCR α and β chain of each cell wasamplified using modified single cell RT-PCR for jurkat cells. We successfullyamplified5TCR α chains and8TCR β chains from12single T cells. Sequences ofamplified TCR α and β chain were obtained after sequencing.3. Expansion of single epitope-specific T cells through mixed lymphocytecoculture in vitroT2cells (TAP deficient, only express HLA-A2molecule) pulsed with gp100peptide were used as stimulators and named as T2/gp100. Cytoxic T cells specific toT2/gp100epitope (CTL-T2/gp100) were induced by coculturing HLA-A2-ve PBLswith T2/gp100for14days. The bulk cells were tested for their binding ability to thegp100/HLA-A2tetramer or irrelevant HBc/HLA-A2tetramer. The frequency ofT2/gp100tetramer-stained CD8+T cells was significantly higher compared with thatof irrelevant HBc/HLA-A2tetramer-stained CD8+T cells. It suggested CTL specificto T2/gp100were successfully induced.4. Cloning TCR from sorted single gp100/HLA-A2specific T cells using singlecell RT-PCRGp100/HLA-A2tetramer-stained CTL-T2/gp100were sorted by flow cytometryand diluted to single cell per tube. Then α and β chain of epitope-specific TCR wereamplified by single cell RT-PCR decribed above. We determined5α and5β chainsequences from10single cells after sequencing and alignment (4pairs).5. Expression and identification of soluble gp100/HLA-A2specific TCROne pair of α and β chain gene of gp100/HLA-A2specific TCR was selected andtransduced to pfast-dual expression vector after codon optimization for expression ofsoluble gp100/HLA-A2specific TCR molecule. Western-blot detection indicated theconformation and molecular size of the protein were right. Unfortuately, specificitydetection of the protein using ELISA with gp100/HLA-A2tetramer was not desirable. It is supposed that the TCR we chose may have an intrincially affinity not highenough for detection, or maybe the concentration of produced TCR molecule was toolow for detection.6. ConclusionWe establish an approach to amplify epitope-specific monoclonal TCR fromhuman T cell repertoire which is not restricted to epitope, providing basis for furtheremployment of TCR-based immunotherapy.更多还原