HLA-A*0201-BSP融合基因的构建与原核表达

克隆表达可生物素化的HLA-A＊0201的重链胞外区是制备HLA-A＊0201-肽四聚体的先决条件。本研究构建重组HLA-A＊0201-BSP融合基因的表达载体，以制备HLA—A2四聚体。根据已报道的序列设计特异引物，利用逆转录聚合酶链反应（RT-PCR）方法从已鉴定为HLA—A＊0201基因型的健康人白细胞中克隆HLA—A＊0201重链胞外区的基因．拼接上依赖生物素-蛋白连接酶（biotin-protein ligase，BirA）的可生物素化序列（BirA substrate peptide．BSP），构建HLA—A＊0201-BSP的原核表达载体，在大肠杆菌DH50t中进行表达。结果表明：成功扩增出HLA—A＊0201-BSP融合基因并测序证实，构建的pBV220-HLA—A＊0201-BSP可在大肠杆菌DH50t中高效表达HLA—A＊0201-BSP融合蛋白．表达量占菌体总蛋白的28％，以包涵体的形式表达，经洗涤、变性后，以Sepharcyl S-300HR（S-300）柱层析纯化，纯度可达90％以上。结论：获得了高效表达HLA—A＊0201-BSP的大肠杆菌工程菌株，建立了简便有效的包涵体纯化方法，为制备HLA-A＊0201-肽四聚体奠定了基础。     

去甲基化和组蛋白去乙酰化转移酶抑制剂对K562细胞增殖和肿瘤相关基因表达的影响

为观察去甲基化和组蛋白乙酰化对白血病细胞系K562增殖及肿瘤相关基因表达的影响，本研究将处于对数生长期的K562细胞系与5-杂氮脱氧胞嘧啶(DAC)和组蛋白去乙酰化转移酶抑制剂一曲古抑菌素(trichostatin，TSA)共同培养，观察细胞生长指数并利用流式细胞术检测细胞周期变化；利用Atlas7742—1基因芯片筛查给药前后的基因表达差异。结果表明，DAC和TSA的联合应用能够抑制K562细胞的增殖，使大多数细胞阻滞在G0期，并促进多个基因的表达上调及少数基因表达下调。结论：去甲基化药物及组蛋白去乙酰化转移酶抑制剂联合应用具有抗白血病作用，与基因芯片联合应用可以用来筛查白血病抑癌候选基因。     

免疫球蛋白重链框架区九肽诱导抗急性B淋巴细胞白血病的细胞毒T细胞应答

目的 用免疫球蛋白(Ig)重链框架区几肽体外诱导抗急性B淋巴细胞白血病(B-ALL)的细胞毒T细胞应答。方法 合成Ig重链可变区第1家族框架区3～11位置上的九肽QLVQSGAEV(IgHV13-11)，进行T2细胞结合实验。IgHV13-11负荷抗原呈递细胞(APC)，体外刺激HLA-A*0201正常外周血单个核细胞(PBMNC)，每周1次，共3次：HLA-A*0201／IgHV13-11四聚体监测培养细胞中IgHV1-311特异性CD8^ T细胞的增殖。对B-ALL初治患儿的7个IgHV基凶家族进行PCR扩增及PCR产物的测序，选IgHV1和IgHV3家族单等位基冈功能性重排克隆，并鉴定其HLA-A*0201位点。MTT比色法分析IgHV13-11，特异性CD8^ T细胞对HLA-A*0201^ IgHV1和IgHV3家族B-ALL细胞克隆的杀伤活性。结果 IgHV13-11可上调HLA-A*0201分子在T2细胞表面的表达1．63倍、HLA-A*0201*正常PBMNC中IgHV13-11特异性CD8^ T细胞的频率由2轮刺激后1．64％增至3轮刺激后82．57％。IgHV13-11特异性CD8^ T细胞在效靶细胞比(E：T)20：1时对IgHV1家族B-ALL细胞的杀伤率为18．24％，是IgHV3家族B-ALL细胞的1．8倍(P=0．01)。结论 体外诱导的Ig重链框架区九肽特异性CD8*T细胞可杀伤表达该肽的B-ALL细胞。     

Distinct structural TCR repertoires in naturally occurring versus vaccine-induced CD8+ T-cell responses to the tumor-specific antigen NY-ESO-1

Spontaneous immune responses to the cancer testis antigen NY-ESO-1 are frequently found in cancer patients bearing antigen-expressing tumors. In HLA-A2-expressing patients, naturally elicited NY-ESO-1-specific, tumor-reactive cytotoxic T lymphocytes (CTLs) are mostly directed against an immunodominant epitope corresponding to peptide NY-ESO-1 157-165. NY-ESO-1-specific CTLs can also be induced by synthetic peptide vaccines, but they are heterogeneous in terms of functional avidity and tumor reactivity. The authors investigated the structural bases of this phenomenon by analyzing the TCR features of natural and vaccine-induced NY-ESO-1-specific CTLs. The results indicate that CTLs from the two groups exhibit highly structurally conserved but distinct TCR features, suggesting that the synthetic peptides used for vaccination may fail to faithfully mimic the naturally processed antigen. Together, the results of this study underline the strength of TCR molecular monitoring and will be instrumental for the development and monitoring of vaccines aimed at eliciting CTLs with high tumor reactivity.     

肝癌患者外周血中针对NY-ESO-1/LAGE-1表位的自发性CTL的定量检测

目的检测原发性肝细胞癌（HCC）患者外周血中针对HLA-A2限制性NY-ESO-1b表位的特异性细胞毒性T细胞（cytotoxic Tlymphocyte,CTL）的水平,评价该表位多肽诱导HCC患者自发性细胞免疫应答的能力。方法采用流式细胞技术分析HCC患者的HLA表型,并通过RT-PCR技术检测其癌组织中NY-ESO-1/LAGE-1基因的表达情况;采用HLA-A2/多肽五聚体复合物即pentramer对HCC患者外周血中NY-ESO-1b表位特异性CTL进行标记并用流式细胞仪进行定量检测。结果在HLA-A2阳性、且其癌组织中NY-ESO-1/LAGE-1mRNA为阳性29例HCC患者中,13例（41.4%）患者外周血中可检测到针对HLA-A2限制性NY-ESO-1b表位（p157-165,SLLMWITQC）的特异性CTL应答,而在19例HLA-A2阳性的健康志愿者和12例HLA-A2阳性的肝炎后肝硬化患者外周血中均未检测到相应的自发性CTL应答（P值分别为0.004和0.023）。结论NY-ESO-1b表位多肽可诱发HCC患者体内产生较高频率的自发性CTL应答,提示该多肽可作为备选疫苗用于HCC的主动免疫治疗。     

Lack of strong immune selection pressure by the immunodominant, HLA-A*0201-restricted cytotoxic T lymphocyte response in chronic human immunodeficiency virus-1 infection.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.     

可溶性HLA-A*0201-PR1复合物的构建与鉴定

为制备HLA-A＊0201-PR1四聚体,本课题构建了可溶性的HLA-A＊0201-PR1复合物。以原核表达的HLA-A＊0201-BSP融合蛋白为重链,β2-微球蛋白（β2m）为轻链,与人工合成的HLA-A2限制性抗原肽PR1（蛋白酶3的第169-177氨基酸,VLQELNVTV）进行共折叠复性,以生成可溶性HLA-A＊0201-PR1复合物。应用BirA酶对复性混和物进行生物素化,再经阴离子交换柱纯化,得到生物素化的可溶性HLA-A＊0201-PR1复合物。应用凝胶过滤高效液相色谱分析可溶性HLA-A＊0201-PR1复合物的生成率。利用HLA-A2构象特异性抗体（BB7.2）及链霉亲和素进行Western blot和ELISA分析,对生物素化的可溶性HLA-A＊0201-PR1复合物进行鉴定。结果表明：在折叠体系中,主要含有HLA-A＊0201-BSP聚合体、HLA-A＊0201-PR1复合物及β2m,其中可溶性复合物生成率约18%。获得的HLA-A＊0201-PR1复合物可在BirA酶作用下被有效生物素化。结论：成功地获得了生物素化的可溶性HLA-A＊0201-PR1复合物,为进一步制备HLA-A＊0201-PR1四聚体创立了基础。     

Cancer-testis antigens: promising targets for antigen directed antineoplastic immunotherapy

During the last decade, the aberrant expression of normal testicular proteins in neoplastically transformed cells became common knowledge. Cancer-testis antigens (CTAs) represent a novel family of immunogenic proteins. The genes MAGE, BAGE, GAGE, LAGE and NY-ESO-1 code for antigens that are recognised on various neoplastically transformed cells by autologous, cytolytic CD8 ( + ) T lymphocytes. The MAGE genes were initially analysed from melanomas and turned out to have an almost exclusively neoplasm specific expression pattern. In normal adult tissues, most 23 human MAGE genes are expressed only in the testis, with expression patterns suggesting that this gene family is involved in germ cell development. The SSX (synovial sarcoma on X chromosome) gene family, located on the X chromosome, encode a family of highly homologous nuclear proteins. A number of observations confirmed that all five SSX genes were expressed in normal testis. The newly detected CTA, NY-ESO-1, is regarded as one of the most immunogenic antigens ever isolated, inducing spontaneous host immune responses in 50% of patients with NY-ESO-1-expressing neoplasms. The identification of neoplasm-associated markers recognised by cellular or humoral effectors of the immune system has opened new perspectives for antigen directed, individualised antineoplastic immunotherapy. In preparation for this new era of targeted immunotherapy, a number of neoplasm-associated antigen families have been identified as targets for CD8+, cytolytic T lymphocytes in vitro and in vivo : (1) CTAs expressed in various neoplasms and in normal testis, restricted to male germ cells; (2) melanocyte differentiation antigens; (3) point mutations of normal genes; (4) antigens overexpressed in neoplastic tissues; and (5) viral antigens. Immunotherapeutic protocols directed against the CTAs have already been initiated to analyse the induction of antigen-specific cellular and humoral immune responses in vivo.     

Induction of tumor-reactive cytotoxic T-lymphocytes using a peptide from NY-ESO-1 modified at the carboxy-terminus to enhance HLA-A2.1 binding affinity and stability in solution

NY-ESO-1 is an attractive candidate tumor antigen for the development of immunotherapy for a wide variety of cancers. It is expressed in multiple types of tumors, but its normal tissue distribution is predominantly limited to the testes and ovaries; furthermore, both humoral and cellular immune responses can be mounted against this protein. Three overlapping HLA-A2.1-restricted T-cell epitopes have been identified within NY-ESO-1. In this investigation, the authors evaluated the in vitro immunogenicity of these peptides. From 2 of 12 HLA-A2.1+ patients with metastatic melanoma, peptide-reactive cytotoxic T-lymphocytes were generated using either NY-ESO-1:157-167 or NY-ESO-1:157-165 but not NY-ESO-1:155-163. Because NY-ESO-1:157-165 is a 9 amino acid peptide completely contained within NY-ESO-1:157-167, it seemed likely that this peptide was the minimal determinant, and thus it was selected for continued study. An amino acid substitution of C to V was introduced into NY-ESO-1:157-165 at P9 to attempt to improve its immunogenicity by enhancing its binding affinity to HLA-A2.1 and increasing its stability in solution, because the C residue is readily oxidized, leading to dimerization of the peptide. From 5 of 20 HLA-A2.1+ patients with metastatic melanoma, NY-ESO-1:157-165(165V) stimulated cytotoxic T-lymphocytes in vitro, which recognized peptide-pulsed target cells and HLA-A2.1+ NY-ESO-1+ tumor cells, suggesting that this peptide may be clinically valuable for the treatment of patients with NY-ESO-1+ tumors.     

A histone deacetylase inhibitor enhances recombinant adeno-associated virus-mediated gene expression in tumor cells.

The transduction of cancer cells using recombinant adeno-associated virus (rAAV) occurs with low efficiency, which limits its utility in cancer gene therapy. We have previously sought to enhance rAAV-mediated transduction of cancer cells by applying DNA-damaging stresses. In this study, we examined the effects of the histone deacetylase inhibitor FR901228 on tumor transduction mediated by rAAV types 2 and 5. FR901228 treatment significantly improved the expression of the transgene in four cancer cell lines. The cell surface levels of alpha v integrin, FGF-R1, and PDGF-R were modestly enhanced by the presence of FR901228. These results suggest that the superior transduction induced by the HDAC inhibitor was due to an enhancement of transgene expression rather than increased viral entry. Furthermore, we characterized the association of the acetylated histone H3 in the episomal AAV vector genome by using the chromatin immunoprecipitation assay. The results suggest that the superior transduction may be related to the proposed histone-associated chromatin form of the rAAV concatemer in transduced cells. In the analysis with subcutaneous tumor models, strong enhancement of the transgene expression as well as therapeutic effect was confirmed in vivo. The use of this HDAC inhibitor may enhance the utility of rAAV-mediated transduction strategies for cancer gene therapy.     

Cancer-testis antigens: promising targets for antigen directed antineoplastic immunotherapy

During the last decade, the aberrant expression of normal testicular proteins in neoplastically transformed cells became common knowledge. Cancer-testis antigens (CTAs) represent a novel family of immunogenic proteins. The genes MAGE, BAGE, GAGE, LAGE and NY-ESO-1 code for antigens that are recognised on various neoplastically transformed cells by autologous, cytolytic CD8⁺ T lymphocytes. The MAGE genes were initially analysed from melanomas and turned out to have an almost exclusively neoplasm specific expression pattern. In normal adult tissues, most 23 human MAGE genes are expressed only in the testis, with expression patterns suggesting that this gene family is involved in germ cell development. The SSX (synovial sarcoma on X chromosome) gene family, located on the X chromosome, encode a family of highly homologous nuclear proteins. A number of observations confirmed that all five SSX genes were expressed in normal testis. The newly detected CTA, NY-ESO-1, is regarded as one of the most immunogenic antigens ever isolated, inducing spontaneous host immune responses in 50% of patients with NY-ESO-1-expressing neoplasms. The identification of neoplasm-associated markers recognised by cellular or humoral effectors of the immune system has opened new perspectives for antigen directed, individualised antineoplastic immunotherapy. In preparation for this new era of targeted immunotherapy, a number of neoplasm-associated antigen families have been identified as targets for CD8⁺, cytolytic T lymphocytes in vitro and in vivo: (1) CTAs expressed in various neoplasms and in normal testis, restricted to male germ cells; (2) melanocyte differentiation antigens; (3) point mutations of normal genes; (4) antigens overexpressed in neoplastic tissues; and (5) viral antigens. Immunotherapeutic protocols directed against the CTAs have already been initiated to analyse the induction of antigen-specific cellular and humoral immune responses in vivo.     

结核杆菌抗原Rv0173的HLA-A*0201限制性CTL表位的预测及鉴定

目的 鉴定结核杆菌(Mycobacterium tuberculosis,Mtb)抗原Rv0173中的HLA-A*0201限制性CTL表位.方法 应用数据库SYFPETTHI预测结核杆菌Rv0173中可能存在的HLA-A*0201限制性CTL表位,经流式细胞术分析各抗原肽与HLA-A*0201的亲合力,经时间分辨荧光法检测外周血单个核细胞(PBMCs)对各抗原肽产生的增殖反应,经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性,逐步鉴定Rv0173的HLA-A*0201限制性CTL表位.结果 位于Rv0173氨基酸序列肽3(21-30aa)和抗原肽7(161-170aa)与HLA-A*0201分子具有较高的亲合力,并都能刺激HLA-A*0201阳性个体PBMCs增殖,并诱导产生特异性杀伤活性.结论 肽3(RLSQSADQYL)(21-30aa)和肽7(LLRGGGLVNL)(161-170aa)是抗原Rv0173上HLA-A*0201限制性CTL的优势表位,可作为结核疫苗设计的候选表位.     

应用免疫球蛋白重链可变区上框架区来源的抗原九肽获得的细胞毒T细胞功能分析

目的明确存B淋巴细胞肿瘤表面的免疫球蛋白重链可变区(IgHV)的框架区(FR)上是否存在可以激发细胞毒T淋巴细胞(CTL)的抗原表位，这些来源于FR的抗原肽是否能够激发家族特异性的免疫反应，探讨按照B淋巴细胞肿瘤的IgHV基因分型进行家族性的免疫治疗的可能性，方法利用生物信息学系统预测了40例B淋巴细胞肿瘤表面的免疫球蛋白序列的抗原表位，人工合成不同基因家族的抗原几肽，利用1、2细胞结合实验检测预测的抗原肽和HLA分子的亲合力利用体外CTL刺激扩增体系。诱导特异性的CTL细胞增殖，用肽／HLA四聚体检测特异性CTL的数目，应用LDH释放实验检测CTL的细胞毒活性并验证其家族特异性。结果在B淋巴细胞肿瘤的7个IgHV基因家族中找到了12个高亲和力的抗原九肽，其中10个(83％)位于FR，以HLA-A*0201正常供的外周血单个核细胞(PBMNC)负荷该九肽作为抗原呈递细胞去刺激自身PBMNC，经过4轮刺激，CD8和肽／HLA四聚体双阳性细胞从0．38％上升到49．38％LDH释放实验证明对HLA—A*0201( )、IgHV1( )靶细胞有明显的杀伤作用，并且被IgHV1肽刺激出的CTL不能识别负荷IgHV3肽的靶细胞：结论IgHV基因FR抗原儿肽可以刺激产生具有HLA限制性的并且肽特异性的CTL，同时这样的杀伤作用具有家族特异性，以此为靶位有望对B淋巴细胞肿瘤进行家族特异性的免疫治疗。     

可溶性HLA-A2-IgGFc融合蛋白在昆虫细胞的表达及鉴定

目的利用杆状病毒-昆虫细胞表达体系制备可溶性HLA-A2与免疫球蛋白IgGFc段的融合蛋白。方法首先将可溶性HLA-A*0201和IgG3分子Fc段的cDNA基因插入杆状病毒表达载体pFastHTa,形成重组载体pFHT-sHLA-A*0201-IgGFc。将该重组载体转化DH10Bac大肠杆菌,sHLA-A*0201-IgGFc基因同源重组入菌体内杆粒(Bacmid)中。抽提阳性杆粒,转染昆虫细胞Sf9,72 h收集病毒上清。再将病毒上清感染Sf9细胞,96 h收集细胞并裂解,SDS-PAGE观察其表达。细胞裂解液经Ni+-NTA树脂纯化Western-blot法鉴定其蛋白质序列,间接ELISA法鉴定是否形成正确构象。结果融合蛋白不仅与HLAⅠ类分子的构象特异性抗体(W6/32)结合,还与羊抗人IgG抗体结合。结论所制备可溶性HLA-A2-IgGFc融合蛋白序列正确,并在体外形成正确空间构象。     

Human papillomavirus 16 E6-specific CD45RA+ CCR7+ high avidity CD8+ T cells fail to control tumor growth despite interferon-gamma production in patients with cervical cancer

We defined the nature of the cellular immune response in 5 women with human papillomavirus (HPV) 16+cervical carcinoma at a single time point when surgery was performed for treatment. To monitor the differences in T-cell recognition, 2 approaches of tetramer-guided technology were employed: (i) the in situ localization of major histocompatibility complex class I peptide complexes in the tumor lesions and (ii) the ex vivo sorting of HLA-A*0201-restricted and HPV16 E6-reactive T cells. CD8 T cells from the periphery (peripheral blood lymphocytes), the tumor (tumor-infiltrating lymphocytes), and T cells harvested from draining lymph nodes (T-LN) were analyzed. HPV16 E6 tetramer-sorted lymphocytes from the different anatomic sites recognized an HLA-A*0201-restricted E6 peptide irrespective of the type of antigen-presenting cells used for stimulation as determined by interferon-gamma production: autologous tumor cells, HLA-A*0201 surrogate antigen-presenting cells pulsed with the nominal peptide, and an HLA-A*0201-matched human dendritic cell line transgenic for HPV16 E6. Further analysis showed that the HPV16 E6-reactive CD8 T cells were of high avidity defined by blocking with an anti-CD8-alpha specific monoclonal antibody. We found that HPV16 E6-reactive T cells reside preferentially within the CD45RA+ CCR7+ T-cell subpopulation of tumor-infiltrating lymphocyte, peripheral blood lymphocyte, and T-LN in cervical cancer patients, suggesting that successful immune surveillance of HPV16+ tumor cells in cervical cancer patients is impaired. The CD45RA+/CCR7+ phenotype of HPV antigen-reactive T cells may serve as an indicator of dysfunctional T cells, despite effective interferon-gamma production in response to HPV antigens.