负载HCMV pp65_（341-349）抗原肽的HLA-A*2402四聚体的制备及其应用
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摘要
人巨细胞病毒(human cytomegalovirus，HCMV)是一种广泛存在的β疱疹病毒，其感染普遍存在于所有人群中，在免疫功能低下者可发生严重的HCMV疾病。最近研究表明，HCMV血清阳性和免疫衰老的许多生物标记相关，是预测老年群体死亡率的免疫参数之一。抗原特异性细胞毒T淋巴细胞(cytotoxic T lymphocytes，CTL)是抗细胞内感染和杀伤肿瘤细胞的重要效应细胞，在机体免疫防御中发挥着重要作用。主要组织相容性复合体Ⅰ类分子(MHCⅠ)四聚体技术(tetramer technology)灵敏度高、特异性强，目前已成为研究CTL免疫应答的核心技术。然而四聚体通用性差，每个MHCⅠ-肽四聚体均需针对特定MHCⅠ分子而设计，制备四聚体应首选基因频率相对较高的等位基因。HLA-A*2402是东亚群体中最常见的等位基因之一，尤其在日本(基因频率58.1％)及中国(基因频率32.9％)群体中常见，这说明值得制备HLA-A*2402四聚体。
为研究HLA-A24~+人群中HCMV特异性CD8~+T细胞免疫应答规律，本文制备了负载HLA-A*2402限制性HCMV pp65_(341-349)抗原肽(QYDPVAALF，QYD)的可溶性HLA-A*2402-QYD四聚体，对HLA-A24~+健康供者外周血中HCMV特异性CD8~+T细胞的频率、表型及功能状态进行了深入分析。以RT-PCR方法克隆HLA-A*2402重链基因的cDNA，构建了HLA-A*2402重链胞外域与BSP(BirA substrate peptide)融合蛋白(HLA-A*2402-BSP)的原核表达载体，然而该融合蛋白在E.coli未见任何表达。通过同义突变将氨基端(N端)区域编码区的稀有密码子突变为E.coli偏好的密码子，并有效降低其G／C含量，突变型HLA-A*2402-BSP(mHLA-A*2402-BSP)在E.coli中获得了高水平表达，表明通过密码子优化提高HLA-A*2402在E.coli中的表达策略是成功的。在此基础上复性获得负载HLA-A*2402限制性HCMV pp65_(341-349)抗原肽QYD的可溶性HLA-A*2402-QYD单体分子和具有特异结合活性的HLA-A*2402-QYD四聚体，并对其最佳用量进行了滴定，结果表明0.35μg四聚体用于100μl全血时，特异性CTL染色结果最佳，即染色的CTL荧光强度保持较高水平而非特异性染色明显减少。进一步利用所构建的HLA-A*2402-QYD四聚体结合流式细胞术，对HLA-A24~+健康中国供者外周血中HCMV特异性CD8~+T细胞的频率、多种表型(CD62L、CCR7、CD127、CD28、CD27、PD-1)及穿孔素分泌水平进行了详细分析。结果显示QYD肽特异性CD8~+T细胞占总CD8~+T细胞的频率为0.11％～1.52％，这些细胞的表型及穿孔素分泌水平存在高度的异质性，提示这些细胞是由于处于不同分化阶段和不同功能状态的多种类型的细胞所组成。
总之，我们通过密码子优化策略使mHLA-A*2402-BSP重组蛋白在E.coli获得了高效表达，并成功制备了负载HLA-A*2402限制性HCMV抗原肽QYD的可溶性HLA-A*2402-QYD四聚体；进一步利用该四聚体结合结合流式细胞术，对HLA-A24~+健康中国供者外周血中HCMV特异性CD8~+T细胞的频率、多种表型及穿孔素分泌水平进行了分析，从而为深入研究中国HLA-A24~+人群中HCMV特异性CD8~+T细胞应答提供了有价值的数据。
Human cytomegalovirus(HCMV) is a ubiquitous human persistent virus infecting most of the world populations and HCMV diseases are frequently seen in immunosuppressed individuals. Furthermore, recent studieshave demonstrated that HCMV seropositivity is one of immunological parameters which predicts incipient mortality in an elderly population. Major histocompatibility complex class I(MHC I) tetramer technology has been widely used for quantitation, phenotypic and functional characteristics of antigen-specific cytotoxic T lymphocytes(CTL), which are believed to play an essential role in the immune defense against cancer and infectious diseases. However, the high polymorphism of MHC I molecules-human leukocyte antigen(HLA) in humans—hampers the universal application of tetramer technology, thus the most frequently observed HLA class I alleles are preferable for tetramer construction. HLA-A*2402 is one of the most common HLA class 1 allele in East Asian populations, especially in the Japanese(allelic frequency 58.1％) and Chinese populations(allelic frequency 32.9％).
In order to study the CD8~+ T cell responses in Chinese populations, we described here the generation and application of HLA-A*2402 tetramer loaded with HCMV pp65_(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A*2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A*2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide(BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type HLA-A*2402-BSP was not expressed in Escherichia coli(E. coli), while mutant HLA-A*2402-BSP with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A*2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence ofβ_2-microglobulin and OYD peptide. The tetramer was formed by mixing HLA-A*2402-QYD monomers with streptavidin-PE at a molar ratio of 4: 1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24~+ donors. The titration of HLA-A*2402-QYD tetramer dosage was performed and the optimal dosage for 100μL whole blood was determined to be 0.35μg, under which the mean fluorescence intensity(MFI) of specific staining was higher while unspecific staining of CD8~+ T cells was quiet low. Furthermore, the HLA-A*2402-QYD tetramer was utilized and the the quantitation, phenotypic and functional characteristics(CD62L、CCR7、CD127、CD28、CD27、PD-1 and Perforin) of HCMV specific CTL from healthy Chinese HLA-A24~+ donors was investigated. The results showed the frequencies of tetramer~+ CTL were 0.11％-1.52％(mean 0.42％) within total CD8~+ T cells, while phenotypic and functional characteristics of tetramer~+ CD8~+ T cells indicated that they are highly heterogeneous, suggesting they may be composed of CD8~+ T cells at various differentiated stages.
In conclusion, the cDNA of HLA-A*2402 heavy chain was successfully cloned and HLA-A*2402-BSP was overexpressed in E. coli by codon optimization. Furthermore, HLA-A*2402-QYD tetramer was prepared from this fusion protein and it was biologically functional. This tetramer was utilized and the quantitation, phenotypic and functional characteristics of HCMV-specific CTL from healthy Chinese HLA-A24~+ donors was investigated, which provides valuable data for further characterization of antigen-specific CD8~+ T cells from HLA-A24~+ subjects.
为研究HLA-A24~+人群中HCMV特异性CD8~+T细胞免疫应答规律，本文制备了负载HLA-A*2402限制性HCMV pp65_(341-349)抗原肽(QYDPVAALF，QYD)的可溶性HLA-A*2402-QYD四聚体，对HLA-A24~+健康供者外周血中HCMV特异性CD8~+T细胞的频率、表型及功能状态进行了深入分析。以RT-PCR方法克隆HLA-A*2402重链基因的cDNA，构建了HLA-A*2402重链胞外域与BSP(BirA substrate peptide)融合蛋白(HLA-A*2402-BSP)的原核表达载体，然而该融合蛋白在E.coli未见任何表达。通过同义突变将氨基端(N端)区域编码区的稀有密码子突变为E.coli偏好的密码子，并有效降低其G／C含量，突变型HLA-A*2402-BSP(mHLA-A*2402-BSP)在E.coli中获得了高水平表达，表明通过密码子优化提高HLA-A*2402在E.coli中的表达策略是成功的。在此基础上复性获得负载HLA-A*2402限制性HCMV pp65_(341-349)抗原肽QYD的可溶性HLA-A*2402-QYD单体分子和具有特异结合活性的HLA-A*2402-QYD四聚体，并对其最佳用量进行了滴定，结果表明0.35μg四聚体用于100μl全血时，特异性CTL染色结果最佳，即染色的CTL荧光强度保持较高水平而非特异性染色明显减少。进一步利用所构建的HLA-A*2402-QYD四聚体结合流式细胞术，对HLA-A24~+健康中国供者外周血中HCMV特异性CD8~+T细胞的频率、多种表型(CD62L、CCR7、CD127、CD28、CD27、PD-1)及穿孔素分泌水平进行了详细分析。结果显示QYD肽特异性CD8~+T细胞占总CD8~+T细胞的频率为0.11％～1.52％，这些细胞的表型及穿孔素分泌水平存在高度的异质性，提示这些细胞是由于处于不同分化阶段和不同功能状态的多种类型的细胞所组成。
总之，我们通过密码子优化策略使mHLA-A*2402-BSP重组蛋白在E.coli获得了高效表达，并成功制备了负载HLA-A*2402限制性HCMV抗原肽QYD的可溶性HLA-A*2402-QYD四聚体；进一步利用该四聚体结合结合流式细胞术，对HLA-A24~+健康中国供者外周血中HCMV特异性CD8~+T细胞的频率、多种表型及穿孔素分泌水平进行了分析，从而为深入研究中国HLA-A24~+人群中HCMV特异性CD8~+T细胞应答提供了有价值的数据。
Human cytomegalovirus(HCMV) is a ubiquitous human persistent virus infecting most of the world populations and HCMV diseases are frequently seen in immunosuppressed individuals. Furthermore, recent studieshave demonstrated that HCMV seropositivity is one of immunological parameters which predicts incipient mortality in an elderly population. Major histocompatibility complex class I(MHC I) tetramer technology has been widely used for quantitation, phenotypic and functional characteristics of antigen-specific cytotoxic T lymphocytes(CTL), which are believed to play an essential role in the immune defense against cancer and infectious diseases. However, the high polymorphism of MHC I molecules-human leukocyte antigen(HLA) in humans—hampers the universal application of tetramer technology, thus the most frequently observed HLA class I alleles are preferable for tetramer construction. HLA-A*2402 is one of the most common HLA class 1 allele in East Asian populations, especially in the Japanese(allelic frequency 58.1％) and Chinese populations(allelic frequency 32.9％).
In order to study the CD8~+ T cell responses in Chinese populations, we described here the generation and application of HLA-A*2402 tetramer loaded with HCMV pp65_(341-349) peptide (QYDPVAALF, QYD). The cDNA of HLA-A*2402 heavy chain was cloned by RT-PCR from one of the donors. DNA fragment encoding the ectodomain of HLA-A*2402 heavy chain fused at its carboxyl-terminal a BirA substrate peptide(BSP) was amplified by PCR with the cloned heavy chain cDNA as a template. The wild-type HLA-A*2402-BSP was not expressed in Escherichia coli(E. coli), while mutant HLA-A*2402-BSP with optimized codons was overexpressed as inclusion bodies in E. coli. Furthermore, the soluble HLA-A*2402-QYD monomers were generated by in vitro refolding of washed inclusion bodies in the presence ofβ_2-microglobulin and OYD peptide. The tetramer was formed by mixing HLA-A*2402-QYD monomers with streptavidin-PE at a molar ratio of 4: 1. Flow cytometry analysis indicated that this tetramer possessed binding activity with specific CTL from HLA-A24~+ donors. The titration of HLA-A*2402-QYD tetramer dosage was performed and the optimal dosage for 100μL whole blood was determined to be 0.35μg, under which the mean fluorescence intensity(MFI) of specific staining was higher while unspecific staining of CD8~+ T cells was quiet low. Furthermore, the HLA-A*2402-QYD tetramer was utilized and the the quantitation, phenotypic and functional characteristics(CD62L、CCR7、CD127、CD28、CD27、PD-1 and Perforin) of HCMV specific CTL from healthy Chinese HLA-A24~+ donors was investigated. The results showed the frequencies of tetramer~+ CTL were 0.11％-1.52％(mean 0.42％) within total CD8~+ T cells, while phenotypic and functional characteristics of tetramer~+ CD8~+ T cells indicated that they are highly heterogeneous, suggesting they may be composed of CD8~+ T cells at various differentiated stages.
In conclusion, the cDNA of HLA-A*2402 heavy chain was successfully cloned and HLA-A*2402-BSP was overexpressed in E. coli by codon optimization. Furthermore, HLA-A*2402-QYD tetramer was prepared from this fusion protein and it was biologically functional. This tetramer was utilized and the quantitation, phenotypic and functional characteristics of HCMV-specific CTL from healthy Chinese HLA-A24~+ donors was investigated, which provides valuable data for further characterization of antigen-specific CD8~+ T cells from HLA-A24~+ subjects.
引文
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