【作者】 许涛；

【导师】 沈传来；

【作者基本信息】 东南大学 ， 免疫学， 2017， 博士

【摘要】 当今医学检验试剂领域,病原体特异性基因、抗原和抗体的检测试剂盒很多,竞争性也很强,但是却没有厂家生产针对病原体特异性T细胞的检测试剂盒。抗原特异性T细胞(Antigen-specific Tcell,AST)是介导适应性免疫应答的核心细胞,在清除与控制病原体感染、监视和杀伤肿瘤细胞及自身免疫性疾病的免疫损伤中发挥着至关重要的作用。众所周知,在抗病毒感染和抗肿瘤中,细胞免疫比体液免疫更加重要,即T细胞的作用比B细胞产生的抗体更重要。抗原特异性T细胞数量的多少和功能的强弱直接反映了人体特异性细胞免疫的功能状态,在监测疾病进程、指导治疗方案、评价临床疗效、预测疾病慢性转归以及疫苗效果评估等方面都有着不同于抗体的重要参考价值。尽管抗原特异性T细胞的检测技术从最初的51Cr释放法、有限稀释法、细胞内细胞因子染色法到现在的ELISPOT法、pMHC多聚体流式分析法、蛋白芯片法等已经有了快速发展。尽管这些技术方法各有优点,但是各自的缺点也非常明显。目前临床上依然极少对患者进行抗原特异性T细胞数量和功能的监测。因此,进一步探索操作简便、无需特殊仪器、无需HLA基因分型、能同步检测抗原特异性T细胞数量和功能的新方法和新试剂非常重要和急迫,为精准医疗提供重要的诊疗手段。研究目的:将磁珠式人工抗原提呈细胞技术、ELISPOT技术、磁性细胞分选技术以及微孔反应板有机整合一体,建立特异性细胞免疫功能状态的检测平台:即磁珠式人工抗原提呈细胞微孔反应板(aAPC-microplate)。首先,自制H-2Kb/OVA257-264单链三聚体(SCT),利用aAPC-microplate同步检测OT-1转基因鼠中OVA257-264抗原特异性CD8+ T细胞的数量和功能,进行方法学评估;然后用同样技术制备HLA-A2/HBV peptide多聚体,利用aAPC-microplate技术对慢性乙肝患者和健康体检者的外周血中HBV特异性CD8+ T细胞的数量与功能进行同步检测,对该方法进行初步临床验证。研究方法:1)利用重叠延伸 PCR 技术将 OVA257-264、(GS4)3、β2m、(GS4)4和 H-2Kb (α1/α2/α3)依次串联为H-2Kb/OVA257-264单链三聚体基因(SCT),联合同源重组克隆技术将其插入pET28a原核表达载体,制备pMHC单体和四聚体,验证其空间构象和与TCR的结合能力;然后包被磁珠,制备成人工抗原提呈细胞微孔反应板,同步检测OT-1转基因鼠中OVA257-264抗原表位特异性CD8+T细胞的频率和反应活性,进行特异性、准确性、灵敏度、重复性或精密度等方法学指标的评估,并与进口商品化H-2Kb-Ig/OVA257-264二聚体荧光流式检测法进行配对t检验和Pearson相关性分析,同时与传统ELISPOT法比较反应活性的检测;2)用金标准的PCR-Sequencing-Based Typing技术对86例健康体检人员血液样本和118例江苏地区汉族2型糖尿病血液标本分别进行HLA-A和HLA-B等位基因分型;利用基因重组克隆技术构建和表达所有频率大于1%的13种HLA-A等位基因重链重组质粒和8种常见HBV抗原表位与β2m轻链的重组质粒;利用6种在线表位预测数据库和软件对 HLA-A*01:01、A*03:01、A*30:01、A*31:01、A*32:01、A*33:03和A*6801等7种HLA-A等位基因分子限制性的HBV相关抗原表位肽进行预测;3)利用重叠延伸PCR和同源重组克隆技术构建和表达HLA-A*0201/HBc18-27、HLA-A*0201/HBs183-191、 HLA-A*0203/HBc18-27、 HLA-A *0203/HBs183-191、HLA-A*0206/HBc18-27等五种HLA-A2/HBVpeptide的单体和四聚体,包被磁珠,制备成制备成人工抗原提呈细胞微孔反应板;对临床慢性乙肝患者、健康体检者外周血中HBc18-27和HBs183-191抗原表位特异性CD8+ T细胞的数量与功能进行同步检测;并与进口商品HLA-A2-Ig二聚体的荧光流式分析法和传统ELISPOT法进行比较。研究结果:1)成功构建和表达H-2Kb/OVA257-264 SCT融合基因,制备成H-2Kb/OVA257-264 SCT单体,包被磁珠后用H-2Kb构象特异性荧光单抗染色和流式分析,证实能强烈结合,提示构象正确;制备成荧光标记的H-2Kb/OVA257-264 SCT四聚体,流式法检测4只OT-1鼠脾淋巴细胞群中OVA257-264抗原表位特异性CD8+T细胞频率。结果与进口商品化H-2Kb-Ig/OVA257-264二聚体的流式检测结果无统计学差异,证实H-2Kb/OVA257-264 SCT与TCR的结合能力;2) 7只OT-1鼠脾淋巴细胞群同时经aAPC-microplate法和H-2Kb-Ig/OVA257-264二聚体流式染色法检测OVA257-264表位特异性CD8+T细胞的频率,结果经配对t检验显示p=0.2464,表明无统计学差异,Pearson回归分析显示,相关系数r=0.946,P<0.01,相关方程为:Y=0.936X + 3.262,显示两种检测方法的吻合度很高;用H-2Kb-Ig/OVA257-264二聚体流式染色法检测aAPC-microplate孔内剩余细胞中OVA257-264特异性CD8+ T细胞的纯度,三次实验分别为92.35%、91.22%和92.10%,表明aAPC-microplate法的特异性达到90%以上;aAPC-microplate法检测7只OT-1鼠时,分析内 CV 值分别为 6.44%、4.01%、6.31%、9.08%、6.63%、4.24%和 3.11%,对其中1只进行三次独立重复检测,结果分别为25.47%、25.59%和24.49%,平均分析内CV值分别为3.11%、4.41%和10.62%,三次检测结果的分析间CV值为2.40%,平均分析内CV值均低于15%,表明该检测方法有良好的重复性或精密度,符合我国2000版生物制品鉴定标准;灵敏度分析显示,在5个不同稀释频率(10%, 1%,0.1%,0.05%,0.01%)的样品检测中 aAPC-microplate 法与 H-2Kb-Ig/OVA257-264 二聚体流式染色分析法的配对t检验无统计学差异,p=0.3973, Pearson分析的相关系数r=0.998,P<0.01,相关性方程为:Y=0.943X +0.21,aAPC-microplate方法可检测的频率下限低至0.01% - 0.05%,与传统的MHC多聚体染色加流式分析法相似,该灵敏度能满足临床标本检测的需要;3) 3只OT-1鼠脾淋巴细胞经aAPC-microplate检测频率后,分选后的OVA257-264特异性CD8+T细胞群经ConA刺激后分泌IFN-γ的反应活性比分别为43.83%、41.87%和48.99%;同时对3只OT-1鼠脾淋巴细胞用传统ELISPOT方法进行检测,在OVA257-264抗原肽刺激后脾淋巴细胞群中分泌IFN-γ的细胞比例经直线回归方程校正后分别为4.44%、4.01%和4.98%。由于原理和结果表现形式不同,两种方法的活性检测结果不能直接比较;4)在对人群血液标本进行HLA-A/B位点基因分型中,共获得所有频率大于1%的13种HLA-A等位基因型和32种HLA-B等位基因;成功构建和表达13种HLA-A等位基因的重链重组质粒和8种常见HBV抗原表位与β2m轻链的重组质粒;利用在线表位预测数据库预测获得 HLA-A*01:01、A*03:01、A*30:01、A*31:01、A*32:01、A*33:03和A*6801等7种等位基因型限制性的HBV抗原表位肽序列,每种等位基因均获得多个高亲和力的候选表位肽,分别来自HBsAg(P03138)、HBcAg(P03146)、HBpol(P03156)和HBx(P03165)等4种HBV抗原蛋白分子;5) 成功构建和表达HLA-A*0201/HBc18-27、HLA-A*0201/HBs183-191、HLA-A*0203/HBc18-27、HLA-A*0203/HBs183-191、HLA-A*0206/HBc18-27 等五种 HLA-A2/HBVpeptide的SCT分子,分别包被磁珠,制备成5种aAPC-beads,按照1:1的比例混合,制备成 HLA-A2 限制性、HBc18-27和 HBs183-191 表位特异性的 aAPC-microplate;6)用 aAPC-microplate 法和进口 HLA-A2-Ig/HBc18-27/HBs183-19 二聚体流式染色法对 10例HLA-A2阳性慢性乙肝患者、15例HLA-A2阴性慢性乙肝患者和15例HLA-A2阳性健康体检者的PBMC进行检测。配对t检验显示,两种方法对三组临床标本的检测结果均无统计学差异,p值分别为0.0821、0.0529和0.0594; Pearson相关性分析显示,相关系数r=0.980, P<0.01,相关性方程为:Y=0.984X-0.05,两者的吻合度很高;HLA-A2阳性慢乙肝患者外周血中HBc18-27和HBs183-191表位特异性的CD8+T细胞的频率明显高于两个对照组(0.1957% ± 0.0672% vs. 0.0176% ± 0.0068%,0.1957% ± 0.0672% vs. 0.0249% ± 0.0077%);7) 10名HLA-A2阳性慢性乙肝患者的PBMC经aAPC-microplate法检测频率后,分选后的HBc18-27和HBs183-191表位特异性的CD8+T细胞群经PHA刺激后分泌IFN-γ的反应活性比为22.79%± 3.40%;而同时患者PBMC中整体T细胞群经PHA刺激后的反应活性比为36.35% ± 3.58%,明显高于HBc18-27和HBs183-191表位特异性的CD8+ T细胞群的反应活性。这些结果提示:慢性乙肝患者体内针对乙肝病毒的特异性CD8+T细胞尽管频率高于健康者人群,但是其针对HBV抗原刺激后的活化、增值和分化能力低下。这可能是导致患者感染HBV后慢性转化的重要原因。研究结论:利用自制MHC多聚体建立的aAPC-microplate技术能对OVA257-264抗原特异性CD8+T细胞的数量和功能进行同步检测,具有很好的准确性、特异性、灵敏度、重复性或精密度,可检测的比例下限低至0.01% - 0.05%;利用自制HLA-A2/HBc18-27/HBs183-191 SCT多聚体建立的aAPC-microplate技术能对慢性乙肝患者临床血液标本中的HBc18-27和HBs183-191抗原特异性CD8+ T细胞数量和功能进行同步检测,与传统的MHC多聚体流式染色法检测结果无显著性差异,两者吻合度极高。更多还原

【Abstract】 In the current market of medical diagnostic reagents, the kits detecting pathogen-specific genes, antigens and antibodies are very popular, thus face to strong competition. But no manufacturer focuses on the pathogen-specific T cells detection kit.Antigen-specific T lymphocytes (AST) are the core cells that mediate the adaptive immune response. They play a crucial role in controlling pathogen infections, monitoring tumor cells,and pathogenesis of autoimmune diseases, and so on. It is well known that cellular immune responses are more important than humoral immunity in antiviral and anti-tumor effectes, thus T cells are more important than B cells secreting antibody in infection or tumor cases. The enumeration and functional evaluation of diverse T cell populations in an individual’s T cell repertoire may provide a detailed picture of the physiological status, pathological course, and dynamic immune response to certain antigens associated with pathogens, allergens, cancer cells, self-proteins, allograft, as well as vaccines, and can be used to characterize individual-specific cellular immune response patterns that might help guide the design of immunological therapies.The detection techniques for AST have been developed rapidly from the traditional 51Cr release assay, limiting dilution assay (LDA), intracellular cytokine staining (ICS) to enzyme-linked immunospot assay (ELISPOT), pMHC multimer plus flow analysis, and pMHC protein array or chip. Although each technique has its advantages, but AST detection still sufferes from the obvious disadvantages of these motheds. Few patients have been monitored for AST in clinics in China by now. It is worthy to develop a convenient method for the concurrent emueration and functional evaluation of AST cells without the requirement of expensive instruments and HLA genotyping, which will facilitate the routine analysis of patient-specific immune response pattern to certain antigen in translational studies in the anticipation of precision medicine.Objective:In the present study, pMHC multimers, artificial antigen-presenting cells (aAPCs),magnetic-activated cell sorting (MACS), and ELISPOT techniques have been integrated into a micro well by coupling pMHC multimers onto cell-sized magnetic beads to characterize AST cell populations in a magnetic field in a 96-well microplate pre-immobilized with cytokine-capture antibodies. This method, termed artificial antigen-presenting cell microplate(aAPC-microplate), allows enumeration and local cytokine production of multiple AST cells in a single assay without using flow cytometry or fluorescence intensity scanning. First, we used the in-house H-2Kb/OVA257-264 single-chain trimer (SCT) molecules to prepare aAPC-beads, and detect the frequency and reactivity of OVA257-264 antigen-specific CD8+ T cell in the OT-1 trangenic mice by aAPC-microplate followed by methodological evaluation.Then,we generated HLA-A2/HBVpeptide mutilmers in a same manner, and fabricated aAPC-microplate to enumerate and functionally evaluate HBc18-27/HBs183-191antigen-specific CD8+ T cells in the peripheral blood mononuclear cells (PBMCs) from chronic hepatisis B patients, in a preliminary clinical validation for this new method.Methods:1) H-2Kb-Ig/OVA257-264 SCT fusion gene which contains OVA257-264, (GS4)3, β2m,(GS4)4,and H-2Kb (α1/α2/β3) fragments was synthesized using the overlap extension PCR, and inserted into plasmid pET28a by homologous recombination technology followed by expression in BL21 (DE3). Then,H-2Kb/OVA257-264 SCT molecues and tetramers were prepared and the structural conformation and binding ability to antiden-specific TCR were further confirmed. Finally, we used the in-house H-2Kb/OVA257-264 SCT aAPC-microplate to detect OVA257-264 antigen-specific CD8+ T cell from OT-1 transgenic mice, with the methodological evaluation for specificity, accuracy, sensitivity,reproducibility,and the comparison with commercial H-2Kb-Ig/OVA257-264 dimer staining plus flow cytometry by the two-tailed, paired Student’s test and Pearson correlation analysis. Meanwhile, aAPC-microplate method was also compared with traditional ELISPOT assay.2) Eighty-six healthy donors and 118 patients with type 2 diabetes mellitus (T2DM) were enrolled in HLA-A and B loci genotyping, respectively, using the gold-standard method,termed polymerase chain reaction with sequence-based genotyping (PCR-SBT). Then,recombinant plasmids of HLA-A (α1/α2/α3) for 13 alleles with a frequency of > 1% in Chinese population were constructed, respectively, while the recombinant genes of HBVpeotide-β2m for 8 antigenic peptides of HBV antigens were fused by traditional technology. The CTL epitopes derived from HBV antigens and restricted by HLA-A*01:01, A*03:01,A*30:01,A*31:01,A*32:01,A*33:03, or A*6801 were predicted by using the data bases of SYFEPITHI, BIMAS, SVMHC, IEDB, EpiJen, and NETMHC.3) Overlap extension PCR and homologous recombination technology were utilized to construct the SCT fusion genes and recombinant plasmids of HLA-A*0201/HBc18-27,HLA-A*0201/HBs183-191, HLA-A*0203/HBc18-27, HLA-A*0203/HBs183-191, and HLA-A*0201/HBc18-27. The resulting HLA-A2/HBc18-27/HBs183-191 complexes,tetramers,aAPC-beads,and aAPC-microplate were prepared. Concurrent enumeration and functional evaluation of HBc18-27/HBs183-191antigen-specific CD8+ T cells in PBMCs was carried out for the chronic hepatisis B patients and healthy donors. The results were further compared with that analyzed by traditional HLA-A2-Ig/HBc18-27/HBs183-191 dimer staining plus flow cytometry and ELISPOT assay.Results:1) H-2Kb/OVA257-264 SCT protein was successfully constructed and expressed. Magnetic beads pre-coupled with H-2Kb/OVA257-264 complexes could be strongly stained by the PE-labeled anti-mouse H-2Kb mAb (AF6-88.5), a conformation-specific mAb, as analyzed in flow cytometry, implying the correct conformation and appropriate orientation as well as accessibility of H-2Kb/OVA257-264 SCT molecules onto beads.Furthermore, PE-labeled H-2Kb/OVA257-264 tetramers were generated to detect OVA257-264 antigen-specific CD8+ T cells from four individual OT-1 mice by flow cytometry. No significant difference was found between the in-house H-2Kb/OVA257-264 tetramers staining and commercial H-2Kb-Ig/OVA257-264 dimers staining, suggesting the TCR-binding ability of in-house H-2Kb/OVA257-264 molecules similar to its commercial counterpart.2) The splenic lymphocytes from 7 OT-1 mice were detected by the aAPC-microplate method and conventional H-2Kb-Ig/OVA257-264 dimers staining plus flow cytometry for the frequency of OVA257-264-specific CD8+ T cells. No statistical difference was found between the aAPC-microplate method and the conventional flow cytometry for seven individual OT-1 mice tests (p=0.2464). The correlation coefficient (r value) between two methods was 0.946, P<0.01,Y=0.936X + 3.262, as analyzed by two-tailed Pearson. The result suggested that the methodological accuracy compared well with conventional pMHC multimer flow staining. Cells were collected from each well after aAPC-bead sorting and stained with H-2Kb-Ig/OVA257-264 dimer followed by flow cytometry. The purity of H-2Kb-Ig/OVA257-264 dimer+/CD8+ T cells was increased to 92.35%, 91.22% and 92.10% in tree independent assays. These data indicate a specificity of > 90% for the aAPC-microplate method. Seven OT-1 mice were detected independently by the aAPC-microplate method for the enumeration of OVA257-264-specific CD8+ T cells. The within-run coefficient variation (CV) of five cell density groups (3 wells per density) in each independent test was 6.44%, 4.01%, 6.31%, 9.08%, 6.63%, 4.24% and 3.11%,respectively,representing an acceptable well-to-well reproducibility. Moreover,a cell sample from the same individual OT-1 mouse was detected repeatedly in three independent experiments at different time points by the aAPC-microplate method. The proportion of OVA257-264-specific CD8+ T cells was 25.47%,25.59% and 24.49%. The within-run CV was 3.11%, 4.41% and 10.62%, and the between-run CV was 2.40%. The methodological precision meets to the national standard for biological products approvment of China. These data imply a reliable reproducibility and precision of the aAPC-microplate method. Comparison of the aAPC-microplate method with H-2Kb-Ig/OVA257-264 dimers staining assay was further carried out in the five cell samples with different concentrations of OVA257-264-specific CD8+ T cells (10%,1%,0.1%,0.05%,and 0.01%). No statistical difference was found between the two methods (p=0.3973).The correlation coefficient (r value) between two methods was 0.998, P<0.01, Y=0.943X+ 0.21, as analyzed by two-tailed Pearson. The detection limit of the aAPC-microplate method was 0.01% - 0.05%, which meets the requirement for clinical tests and is comparable to that obtained by standard state-of-the-art flow cytometry.3) Three OT-1 mice were detected independently by both the aAPC-microplate method and traditional ELISPOT assay for the functional evaluation of OVA257-264-specific CD8+ T cells. The percentage of IFN-y-secreting cells in the OVA257-264-specific CD8+ T cell population was 43.83%, 41.87%, and 48.99% after stimulation with ConA. As detected by traditional ELISPOT assay, the percentage of IFN-y-secreting cells in the population of lymphocytes isolated from OT-1 mouse spleen was 4.44%, 4.01% and 4.98%,respectively, after stimulation with OVA257-264 antigenic peptide. Due to the distinct methodological principles and presentation of results, we cannot directly compare the results obtained by the aAPC-microplate method and traditional ELISPOT assay.4) We have collected 13 HLA-A alleles and 32 HLA-B alleles with a frequency of > 1% for each allele in Chinese population; constructed and experessed 13 recombinant plasmids of the HLA-A alleles (al/a2/a3) and 8 fusion genes of peptide-(32m with different antigenic peptides of HBV antigens; and obtained several high-affinity antigenic epitopes derived from HBsAg (P03138),HBcAg (P03146),HBpol (P03156),or HBx (P03165),for each of HLA-A*01:01, A*03:01, A*30:01, A*31:01, A*32:01, A*33:03, and A*6801,by prediction based on the data bases.5) HLA-A*0201/HBc18-27, HLA-A*0201/HBs183-191, HLA-A*0203/HBc18-27 HLA-A*0203/HBs183-191, and HLA-A*0206/HBc18-27 SCT proteins were successfully constructed, experessed and refloded, and were coupled onto magnetic beads, respectively,to prepare HLA-A2/HBc18-27/HBs183-191 beads. Each type of HLA-A2/HBc18-27/HBs183-191 aAPC-beads was mixed together at a ratio of 1:1 to fabricate the HLA-A2/HBc18-27/HBs183-191 SCT aAPC-microplate.6) The percentages of HBc18-27/HBs183-191-specific CD8+ T cells in the PBMCs from 10 HLA-A2-positive chronic hepatisis B patients, 15 HLA-A2-negative chronic gepatisis B patients, and 15 HLA-A2-positive healthy donors were detected by the aAPC-microplatemethod and traditional flow cytometry. No statistical difference was found between the two methods for each subject group, with the p values of 0.0821, 0.0529, and 0.0594,respectively. The correlation coefficient (r value) between two methods was 0.980,P<0.01, Y=0.984X-0.05, as analyzed by Pearson test. Notably, HLA-A2-positive patients with chronic hepatisis B displayed obviously higher frequencies of HBc18-27/HBs183-191-specific CD8+ T cells in PMBCs than HLA-A2-negative patients or HLA-A2-positive healthy donors, who only presented background level of the AST cells in PBMCs (0.1957%±0.0672% vs. 0.0176% ± 0.0068%,0.1957% ± 0.0672% vs.0.0249% ±0.0077%).7) Ten HLA-A2-positve chronic hepatisis B patients were detected independently by the aAPC-microplate for the functional elvaluation of HBc18-27/HBs183-191-specific CD8+ T cells after aAPC-bead sorting. The percentage of IFN-y-secreting cells in the sorted HBc18-27/HBs183-i9i-specific CD8+ T cell population was 22.79% ± 3.40% after stimulation with PHA. However, the percentage of IFN-y-secreting cells in the T cell population was 36.35% ± 3.58% after stimulation with PHA. These data indicate that Chronic Hepatisis B patients displayed obviously higher frequencies of HBV-specific CD8+ T cells in PBMCs than healthy donors, but the reactivity of HBV-specific CD8+ T cells was lower than the unrelated T cells. This may be one of the reasons leading to chronic development of HBV infection.Conclusion:The aAPC-microplate fabricated using in-house pMHC multimer was successfully applied to enumerate and functionally evaluate OVA257-264-specific CD8+ T cells in a single assay. The high accuracy,specificity,reproducibility, and sensitivity of the aAPC microplate process compares well with conventional methods. The detection limit was down to 0.01% -0.05%. Concurrent enumeration and functional evaluation of HBc18-27/HBs183-191-specific CD8+ T cells in the PBMCs of chronic hepatisis B patients were carried out successfully by aAPC-microplate prepared using the in-house HLA,A2/4Bc18-27/HBs183-191 SCT multimers.No statistical difference was found between the aAPC-microplate method and traditional flow cytometry with a high goodness of fit.更多还原