T细胞耐受的诱导及其机理研究

目的　阐明抗原特异性 Ｔ 细胞无能的诱导条件、无能细胞的特性及其耐受的机理。方法　抗 Ｂ７１ 单抗与 Ｃｓ Ａ 联用诱导抗原特异性 Ｔ 细胞无能, 通过３ Ｈ Ｔｄ Ｒ 掺入法测定 Ｔ 细胞增殖和 Ｍ Ｌ Ｒ, 利用 Ｒ Ｔ Ｐ Ｃ Ｒ 检测细胞因子基因表达。结果　耐受 Ｔ 细胞与异体淋巴细胞比例为０ ．０１∶１时, 可显著抑制 Ｍ Ｌ Ｒ, 转染 Ｂ７１ 分子的 Ｍ Ｄ Ａ４５３ 和３ Ａ Ｏ 能协同刺激 Ｃ Ｄ３ 诱导的 Ｔ 细胞增殖,不表达 Ｂ７ 分子的 Ｍ Ｄ Ａ４５３ 和３ Ａ Ｏ 无此作用。 Ｐ Ｈ Ａ、 Ｃ Ｄ３ 单抗、 Ｐ Ｍ Ａ＋ Ａ２３１８７ 可以逆转本试验所用诱导方法所致的 Ｔ 细胞的耐受状态。无能 Ｔ 细胞 Ｉ Ｌ２ 和 Ｉ Ｆ Ｎγｍ Ｒ Ｎ Ａ 不表达, 而 Ｉ Ｌ４ 和 Ｉ Ｌ１０ ｍ Ｒ Ｎ Ａ可表达。无能 Ｔ 细胞活化后, Ｉ Ｌ２ 和 Ｉ Ｆ Ｎγｍ Ｒ Ｎ Ａ 能够表达。结论　抗原特异性 Ｔ 细胞耐受是可以人为诱导的, 无能 Ｔ 细胞细胞因子基因格局向 Ｔ Ｈ２ 细胞偏离。     

表达B7分子的小鼠肺癌细胞的构建

目的 改善肿瘤细胞Ｂ７分子表达,方法 将小鼠ＡＬＡ９７０２肺癌细胞与活化的Ｂ淋巴细胞用ＰＥＧ进行细胞融合,经ＥＬＩＳＡ及免疫细胞化学筛选后,观察了融合细胞的体外生长曲线,并用Ｓ－Ｐ免疫细胞化学染色观察了融合细胞的肺癌抗原及Ｂ７分子的表达。结果 经筛选获得１株晤细胞ＦＬＢ２Ｃ,既表达肺癌抗原又表达分子,该细胞在体外培养中贴壁性减弱,生长变慢。结论 通过细胞融合方法可以有铲改善肿瘤细胞的Ｂ７分子表达,     

McAb共激活T细胞介导肝癌细胞前后的膜分子与TCRVβ基因的表达

Ｔ淋巴细胞活化是一个涉及多种膜表面分子和受体以及一系列相关多肽的复杂过程，为了使Ｔ细胞发挥更好的识别和杀伤癌细胞的功能，采用抗ＣＤ３、ＣＤ２８、ＣＤ８０（Ｂ７１）、ＣＤ２、ＣＤ５８ＭｃＡｂ分别刺激健康人ＰＢＬｓ后作用肝癌细胞，对作用前后ＰＢＬｓ用ＦＡＣＳ进行表型分析，结果发现：作用后ＣＤ３和ＣＤ８分子表达比作用前明显增高，而ＣＤ４分子无显著变化，同时基因家族采用ＲＴＰＣＲＳｏｕｔｈｅｒｎ印迹分析ＴＣＲＶβ基因１～２０亚家族表达水平与特征，健康人ＰＢＬｓ分别加入ＩＬ２、ＰＨＡ、抗ＣＤ３和ＣＤ３＋ＣＤ２８、ＣＤ２８＋ＣＤ８０、ＣＤ２＋ＣＤ５８作用肝癌细胞（ＢＥＬ７４０２）前表达水平平均约为５％，作用ＢＥＬ７４０２后表达水平约为１３％～２５％，其特征为Ｖβ７增高，这提示在癌抗原的参与下ＭｃＡｂ共刺激的Ｔ细胞活化，ＴＣＲ接受ＡＰＣ相应抗原的刺激，具有该ＴＣＲ的淋巴细胞迅速增殖而成为针对抗原的Ｔ细胞克隆，发挥其识别和杀伤癌细胞的作用，这将为肿瘤生物治疗的研究提供分子免疫学依据。     

小鼠B7-1基因转染的黑色素瘤细胞免疫原性探讨

目的 探讨Ｂ７－１分子对肿瘤细胞免疫原性的影响。方法 将小鼠Ｂ７－１基因转染的黑色素瘤细胞（Ｂ１６－ｍＢ７．１）与野 生型及空载体转染细胞（Ｂ１６－ｗｔ和Ｂ１６－ｎｅｏ）的免疫原性在体内外进行了比较。同源淋巴细胞肿瘤细胞混合培养（ＭＴＬＣＳ）后测定淋巴增殖指数和ＣＴＬＳ活性,将Ｂ１６－ｎｅｏｐ和Ｂ１６－ｍＢ７．１细胞接种于小鼠皮下,观察肿瘤生长速度。结果 Ｂ１６ｍＢ７．１在体外刺激淋巴细胞增殖和诱     

CTLA-4及其在T细胞活化中的作用

细胞毒性Ｔ淋巴细胞相关分子－４（ＣＴＬＡ－４）和ＣＤ２８的结构相似，在转录水平和蛋白的亚细胞运输水平对其表达进行调控，和Ｂ７－１、Ｂ７－２的亲和力极高，主要与Ｂ７－１分子形成受体／配体对，相互作用可以抑制Ｔ细胞的增殖、活化，是机体维持淋巴细胞稳态平衡的关键环节。被磷酸化的ＣＴＬＡ－４和Ｓｙｐ磷酸酯酶的ＳＨ２功能区结合，可导致Ｔ细胞活化的Ｒａｓ途径关闭，此可能为ＣＴＬＡ－４发挥抑制作用的信号传导机制。ＣＴＬＡ－４也为肿瘤和自身免疫性疾病的免疫治疗研究开辟了新的路径。     

CD28与B7.1共刺激PBLs受肝癌细胞诱导后的TCR Vβ基因表达

目的 研究Ｔ细胞受体（ＴＣＲ）Ｖβ在外周血淋巴细胞（ＰＢＬｓ）中的表达。方法 用单克隆抗体ＣＤ２８＋Ｂ７．１（ＣＤ８０）共刺激正常人外周血淋巴细胞后作用于肝癌细胞（ＢＥＬ－７４０２）。结果 发现ＰＢＬＳＴＣＲＶβ７基因在体外选择性扩增,表达水平为１５￣２５％,对照组为４￣８％。结论Ｖβ７基因对识别肿瘤抗原具有特异性,为肿瘤基因治疗和研究提供新的线索和实验依据。     

HBVx基因在肝癌细胞中的表达及其对肝癌细胞凋亡的影响

目的：研究乙型肝炎病毒 Ｘ 基因在肝癌细胞中的表达及其对肝癌细胞凋亡的影响。方法：用分子生物学的方法构建 Ｈ Ｂ Ｖｘ 基因真核表达载体ｐ Ｃ Ｄ Ｎ Ａ３１ Ｈ Ｂ Ｘ，用脂质体将真核表达载体ｐ Ｃ Ｄ Ｎ Ａ３１ Ｈ Ｂ Ｘ转染入肝癌细胞 Ｈ Ｃ Ｃ９２０４ ， Ｇ４１８ ５００ ｍｇ· Ｌ－ １ 筛选，获得稳定表达 Ｈ Ｂｘ 蛋白的细胞，阿霉素２０ μｍｏｌ· Ｌ－ １ 诱导细胞凋亡。电镜、琼脂糖凝胶电泳和流式细胞仪检测凋亡细胞。结果：２０ μｍｏｌ· Ｌ－ １ 阿霉素可诱导肝癌细胞凋亡，稳定表达 Ｈ Ｂｘ 蛋白的肝癌细胞凋亡率比未进行基因转染的肝癌细胞凋亡率低。结论： Ｈ Ｂｘ 蛋白可抑制阿霉素诱导的肝癌细胞调亡。     

鼠抗人B7-1分子功能性单克隆抗体的制备及生物学特性

目的：制备鼠抗人Ｂ７－１分子的功能性单克隆抗体,研究其对高表达相应配基分子细胞的生物学效应。方法：用两株转入Ｂ７－１基因细胞株ＸＧ７－Ｂ７和Ｌ－Ｂ７分别作为免疫原及检测细胞株,利用Ｂ淋巴细胞杂交瘤技术制备单克隆抗体。以快速定性试纸分析法鉴定单抗所属的小鼠ＩｇＧ亚类,采用竞争抑制及间接免疫荧光法分析单抗的特异性和亲和力。以高表达Ｂ７－１分子的恶性淋巴瘤细胞Ｒａｊｉ和Ｄａｕｄｉ为靶细胞,分析单抗对其生     

共刺激分子B7-1与IL-6协同诱导T细胞活化的研究

目的：探讨Ｂ７－１单独或者与ＩＬ－６联合体外诱导Ｔ细胞活化的能力。方法：在混合淋巴细胞肿瘤细胞培养（ＭＬＴＣＳ）后,测定淋巴细胞增殖指数和ＣＴＬ杀伤活性。结果：Ｂ７－１＾＋的Ｂ１６细胞能够较有效地刺激淋巴细胞增殖,诱导特导ＣＴＬ杀伤活性,当与ＩＬ－６结合时效更加显著。结论：Ｂ７－１基因在肿瘤细胞中表达可增强其免疫原性在体外有效诱导Ｔ细胞活化;在此过程中ＩＬ－６与Ｂ７－１分子存在协同效应。     

B7-1分子诱导体外抗肝癌免疫反应

目的：了解Ｂ７分子在体外抗肝癌免疫中的作用。方法：健康人外周血单个核细胞（ＰＢＭＣ）与ＨｅｐＧ２／ｈＢ７－１，ＨｅｐＧ２／ｎｅｏ及亲代ＨｅｐＧ２瘤苗混合培养（ＭＬＴＣ），检测淋巴细胞活化增殖能力，淋巴细胞ＨＬＡ－Ｉ类抗原的表达，培养上清ＩＦＮ－γ水平，ＴＮＦ活性及ＬＡＫ，ＣＴＬ细胞活性。结果：ＨｅｐＧ２／ｈＢ７－１瘤苗促淋巴细胞增殖最高达１４．６倍，明显高于ＨｅｐＧ２／ｎｅｏ和ＨｅｐＧ２瘤苗的作用     

Priming of immune responses against transporter associated with antigen processing (TAP)-deficient tumours: tumour direct priming

We previously showed that introduction of transporter associated with antigen processing (TAP) 1 into TAP-negative CMT.64, a major histocompatibility complex class I (MHC-I) down-regulated mouse lung carcinoma cell line, enhanced T-cell immunity against TAP-deficient tumour cells. Here, we have addressed two questions: (1) whether such immunity can be further augmented by co-expression of TAP1 with B7.1 or H-2K^b genes, and (2) which T-cell priming mechanism (tumour direct priming or dendritic cell cross-priming) plays the major role in inducing an immune response against TAP-deficient tumours. We introduced the B7.1 or H-2K^b gene into TAP1-expressing CMT.64 cells and determined which gene co-expressed with TAP1 was able to provide greater protective immunity against TAP-deficient tumour cells. Our results show that immunization of mice with B7.1 and TAP1 co-expressing but not H-2K^b and TAP1 co-expressing CMT.64 cells dramatically augments T-cell-mediated immunity, as shown by an increase in survival of mice inoculated with live CMT.64 cells. In addition, our results suggest that induction of T-cell-mediated immunity against TAP-deficient tumour cells could be mainly through tumour direct priming rather than dendritic cell cross-priming as they show that T cells generated by tumour cell-lysate-loaded dendritic cells recognized TAP-deficient tumour cells much less than TAP-proficient tumour cells. These data suggest that direct priming by TAP1 and B7.1 co-expressing tumour cells is potentially a major mechanism to facilitate immune responses against TAP-deficient tumour cells.     

PTEN基因对人肝癌细胞系HHCC作用的研究

目的：研究PTEN基因对人肝癌细胞系HHCC细胞恶性表型及凋亡的影响。方法：将PTEN真核表达载体pBabe-PuroPTEN及空质粒pBabe-Puro，通过脂质体介导的基因转染方法，转入该基因表达缺失的HHCC细胞系中，嘌呤霉素筛选获得稳定表达PTEN基因的转染细胞。通过平板克隆形成试验、DNA凝胶电泳、相差显微镜和电镜，观察PTEN基因对人肝癌细胞系HHCC恶性表型及凋亡的影响。结果：转染后的细胞，经原位杂交、免疫组织化学检测证实，有PTEN mRNA及其蛋白的表达；平板克隆形成试验证实，转染PTEN基因后，细胞形成克隆的能力降低；转染PTEN的细胞在相差显微镜和电镜下可出现典型的凋亡形态学改变；DNA琼脂糖凝胶电泳呈现出明显的梯状条带。结论：外源性PTEN基因导入HHCC细胞后，可降低HHCC的细胞的恶性表型，并促进凋亡发生。     

Interleukin-12 and B7.1 co-stimulation cooperate in the induction of effective antitumor immunity and therapy of established tumors

Interleukin-12 (IL-12) promotes specific and long-lasting anti-tumor immunity mediated by T cells in a variety of murine tumor models. IL-12 also synergizes with B7.1 (CD80) co-stimulation to induce proliferation and cytokine production by both human and murine T cells in vitro. We evaluated the combined antitumor efficacy of IL-12 and B7.1 gene delivery in two apparently poorly immunogenic tumor models (TS/A and MCA207). In both of these models, expression of B7.1 and production of IL-12 in the inoculum led to improved anti-tumor immunity, with up to 80% long-term tumor-free animals (vs 0– 20% of mice remaining tumor free when inoculated with either B7.1- or IL-12-transfected tumors alone). Tumor-free mice were capable of rejecting a subsequent rechallenge with the wild-type tumor in 66% of the cases. Cooperativity was dependent upon the level of IL-12 secreted by engineered cells. IL-12 delivery required B7 expression of therapeutic effects to be observed in these models. Vaccines provided at a site distal to a control, non-transfected tumor slowed (TS/A) or abrogated (MCA207) the progression of wild-type tumors. The synergistic anti-tumor effects associated with combined application of B7.1- and IL-12-transfected tumors were partially negated by systemic administration of the CD28-B7.1/B7.2 antagonist CTLA4-Ig or by inoculation with neutralizing antibodies directed against murine interferon-γ or tumor necrosis factor-α, two cytokines elicited in response to IL-12 stimulation. These data support the potential clinical utility of combined gene therapy using IL-12- and B7.1-engineered autologous cells (tumor or fibroblasts) as a vaccine to elicit specific anti-tumor immunity.     

人肝癌细胞系中肿瘤相关基因MAGE的克隆

目的 克隆人肝癌细胞系HHCC中的肿瘤相关基因MAGE－1的全长cDNA。方法 根据GenBank中MAGE－1基因编码区，设计PCR引物，用RT-PCR手稿,人人肝癌细胞系HHCC中获得MAGE－1全长cDNA，双酶切后连接入质粒pUC19中，转化入大肠杆菌DH5α。挑选阳性克隆提取质粒，进行DNA序列分析，并将测得的核苷酸序列与GenBank中的DNA序列进行BLAST同源性分析。结果 获得MAGE－1全长cDNA,MAGE-6基因463bp片段，以及1个与MAGE－6基因编码区有93％同源性的片段，可能为MAGE家族中的新成员。结论 人肝癌细胞系HHCC可表达多种MAGE基因，可能有效的MAGE基因出现，为研究MAGE基因在HCC中的表达模式及其在HCC免疫治疗中的靶点提供了新的资料。     

Tumor cells cotransfected with interleukin-7 and B7.1 genes induce CD25 and CD28 on tumor-infiltrating T lymphocytes and are strong vaccines

Interleukin-7 (IL-7) and the membrane molecule B7 are both able to provide proliferation and activation signals for T cells. However, tumor cells transfected to express either molecule alone are not reliably rejected in syngeneic hosts or are not sufficiently immunogenic to serve as potent tumor vaccines. Since IL-7 and B7 have shown synergistically to induce activation and proliferation of T cells in vitro, we have expressed B7.1 by means of a retrovirus in the mammary adenocarcinoma TS/A which arose spontaneously in a BALB/c mouse and in the plasmacytoma J558L and their IL-7-transfected sublines to improve vaccine efficacy. Expression of IL-7 or B7.1 alone in tumor cells decreased tumorigenicity, but nevertheless tumors grew in a substantial number of mice. In contrast, IL-7/B7.1 cotransfected cells did not grow as tumor in a single case. This inhibition of tumor growth was completely T cell dependent, because TS/A-IL-7/B7.1 cells retained their full tumorigenic potential in T cell-deficient mice. Analysis of tumor-infiltrating T lymphocytes revealed increased numbers of T cells in B7, IL-7 and IL-7/B7 transfected compared to parental tumors. In IL-7/B7 transfected tumors, T cell numbers were not further increased compared to that in singlegene-transfected tumors. However, T cells in B7 and IL-7 transfected tumors differed phenotypically with respect to activation markers. In B7 transfected tumors, T cells were predominantly CD28⁺ and CD25⁺, while in IL-7-transfected tumors, T cells were mainly CD28^? and CD25⁺. In IL-7/B7 cotransfected tumors, the majority of T cells was CD28⁺ and CD25⁺. Thus, IL-7 and B7 induced an anti-tumor immune response by complementary T cell directed pathways in a cooperative fashion. Importantly, immunization of mice with the transfected cells and subsequent contralateral challenge with parental tumor cells showed that IL-7/B7 co-expressing cells induced the most strongly protective immunity, which is superior to that induced by single-gene transfectants and to the adjuvant Corynebacterium parvum. Vaccine efficacy was abrogated when irradiated cells were used for vaccination. Together, our results show that IL-7 and B7.1 transfected tumor cells induce strong T cell activation and tumor immunity.     

B7.1基因的表达对肝癌细胞生物学特性的影响

目的　观察B7 1基因表达对肝癌细胞增殖特性的影响及诱发杀伤性T淋巴细胞 (CTL)细胞毒活性的变化。方法　用细胞计数法和流式细胞仪 (FCM)测定细胞生长曲线、DNA含量及细胞周期的变化。同时测定CTL对转染B7 1基因前后肝癌细胞的细胞毒作用。结果　转染B7 1基因后 ,肝癌细胞的增殖受到一定影响 :生长延缓、增殖幅度降低和倍增时间延长。CTL对转染B7 1基因的肝癌细胞杀伤明显增强。结论　转染B7 1基因的肝癌细胞出现了一定的增殖抑制现象 ,提示B7 1基因有一定的免疫调节作用     

一个新的MAGE—1表位为肝癌细胞表面HLA—B7分子递呈

目的：肿瘤抗原的存在是机体识别肿瘤并激活免疫系统的物质基础。机体抗肿瘤免疫以细胞免疫为主,故寻找被T细胞识别的肝癌细胞抗原肽,为肝癌免疫治疗奠定基础。方法：对于肝癌细胞系HHCC（HLA-A2,A29,B7,B51）,应用细胞膜酸洗技术使抗原肽从细胞膜表面脱落,经过凝胶层析,反相高效液相色谱层板得到不同组份多肽,高效液相色谱=质谱仪联用技术获得抗原肽的一级结构。通过氨基酸锚定位点分析,预测其HLA配体类型;互联网上氨基酸同源性分析确定其同源性用技术获得抗原肽的一级结构,通过氨基酸锚定位点分析,预测其HLA配体类型,互联网上氨基酸同源性分析确定其同源性序列;人工合成抗原肽,HLA同型树突状细胞递呈合成抗原肽刺激特异性CTL反应,^51Cr杀伤实验检测CTL对肿瘤细胞系的杀伤作用。结果：经过反相高效液相色谱层析后可以获得10多个组份,其中一个组份经过液质联用鉴定和氨基酸同源性分别确定其序列为EPVTKAEML,是黑色素瘤抗原-1,MAGE-1（121-129）表位。由HLA-B7分子递呈,体外诱导实验表明其可以诱导出较强的CTL反应。结论：液质联用技术是寻找抗原肽的有效方法,MAGE-1抗原肽EPVTKAEML地于HLA-B7阳性及阳性的肝癌患者具有潜在的免疫治疗作用。     

LAK-cell-mediated cytotoxicity against tumor cell targets used to monitor the stimulatory effect of interleukin-2: cytotoxicity, target recognition and phenotype of effector cells lysing the Daudi, T24 and K562 tumor cell lines.

The T24 transitional bladder carcinoma cell line, the Daudi Burkitt lymphoma cell line and the K562 erythroleukemia cell line have all been used as target cells in 51Cr release assays to measure the in vivo induced lymphokine-activated killer (LAK) cell cytotoxicity during interleukin-2 (IL-2) therapy of cancer patients. However, different relationships between the clinical response to IL-2 treatment and the LAK cytotoxicity have been reported using these three different target cells. The purpose of the present study was to evaluate whether the LAK cytotoxicities measured against these target cells represent similar effector-to-target-cell interactions, so similar conclusions may be drawn of 51Cr release assay results in which the cell lines are used as target cells. The cytotoxicity of peripheral blood mononuclear cells (PBMC) and PBMC depleted of different natural killer and T cell subsets was measured against the three targets. LAK cell recognition of targets was evaluated by cold target inhibition experiments, and the development of LAK-cell-mediated lysis with time was evaluated in 51Cr release assays of varying duration. This study shows that LAK-mediated lysis of T24 and Daudi cells was closely related and LAK cytotoxicity measured in 51Cr release assays against these two target cells may be measurement of similar effector-to-target cell interactions.     

T cell activation by antigens on human melanoma cells co-stimulation by B7-1 is neither sufficient nor necessary to stimulate IL-2 secretion by melanoma-specific T cell clones in vitro

B7-1 expression, induced by transfection in poorly Immunogenicmurine tumours, was shown to elicit a T cell-mediated rejectionof these tumours and further active immunity against the nontransfectedtumour. We therefore asked to what level similarly induced expressionof B7 on human melanoma cells would affect the antigen-dependentresponses of tumour-specific T cell clones in vitro. Data presentedshow that B7-1 expression by melanoma lines: (i) significantlyinduced, or improved, an IL-2-dependent proliferative responseof such clones to the antigen; (ii) increased the amount ofIL-2 produced by two clones in response to the parental non-transfectedtumour cells;and (iii) increased the TNF responses of all theCD4⁺ clones. However, despite these clear co-stimulatory effectson antigen-induced responses of all T cell clones, which demonstratedaneffective interaction of the B7-1 transfected molecule withone or the other of its counter-receptors expressed on T cellclones, B7 co-stimulation did not correct the defect of IL-2secretion exhibited by many of these clones in response to invitro antigen presentation by melanoma cells. We further showthat defective IL-2 secretion in response to melanoma antigenswas not due to a T cell clone refractoriness induced by theculture, since one of these clones could be induced to secreteIL-2 by an antigen-expressing melanoma line, upon increasedlymphocyte function associated antigen-3expression induced bygene transfectlon. Together these data suggest that defectiveIL-2 secretion by many tumour-infiltrating lymphocytes clonesin response to antigen presentation by melanoma cells in vitrois not exclusively due to the inability of these cells to providean appropriate co-stimulation through the B7-1 molecule.