086 抗原提呈的研究进展

抗原提呈过程涉及从抗原被提呈细胞摄取并消化成免疫原性肽,以ＭＨＣ肽复合物的形式呈现于细胞表面,通过ＴＣＲ激活Ｔ细胞,直至触发免疫应答反应的复杂过程。本文综述了抗原提呈的总过程,ＭＨＣ、肽、ＴＣＲ三者组装及其结构,免疫原性肽的胞内运输以及抗原交叉提呈等同最新进展。     

抗原提呈的研究进展

抗原提呈过程涉及从抗原被提呈细胞摄取并消化成免疫原性肽 ,以MHC 肽复合物的形式呈现于细胞表面 ,通过TCR激活T细胞 ,直至触发免疫应答反应的复杂过程。本文综述了抗原提呈的总过程 ,MHC、肽、TCR三者组装及其结构 ,免疫原性肽的胞内运输以及抗原交叉提呈等方面的最新进展。     

四聚体技术检测人外周血巨细胞病毒抗原特异性CTL

为深入了解抗病毒免疫机制,探索人在健康状态及巨细胞病毒(CMV)急性感染时抗原特异性细胞毒T淋巴细胞(CTL)数量的动态变化。以CMV抗原肽、HLA-A*0201重链和轻链制备CMV四聚体;分离并检测12名健康被检者外周血单个核细胞(PBMC)中CMV抗原特异CTL数量;PBMC体外培养建立CTL细胞系,于末次刺激的不同时相进行四聚体染色,流式细胞仪(FACS)检测抗原特异CTL数量。结果发现9名被检者PBMC中均检出抗原特异CTL;细胞系中CMV特异性CTL数量急剧增多,细胞系与PBMC相比,差异有显著的统计学意义(P<0.001);研究结果提示,9名被检者感染过CMV,血液中存在少量免疫记忆性T细胞,当再次遭遇同一抗原后发生克隆扩增。     

脂多糖对抗原提呈细胞作用的影响

脂多糖的免疫调节效应能辅助机体的细胞免疫和体液免疫，促进机体的抗感染能力，但是大多数细菌脂多糖同时还有强的毒性作用，引起机体病理损伤。因此，了解脂多糖在免疫应答的重要环节——抗原提呈中的作用及其机制，对开发LPS的临床应用是非常重要的。     

脂多糖对抗原提呈细胞作用的影响

脂多糖的免疫调节效应能辅助机体的细胞免疫和体液免疫,促进机体的抗感染能力,但是大多数细菌脂多糖同时还有强的毒性作用,引起机体病理损伤。因此,了解脂多糖在免疫应答的重要环节——抗原提呈中的作用及其机制,对开发LPS的临床应用是非常重要的。     

胸腺基质细胞的抗原提呈作用

目的 研究胸腺基质细胞的抗原提呈能力。方法 应用ＯＶＡ－特异的、受Ｉ－Ａ＾ｄ分子识别限制的辅助Ｔ细胞杂交瘤（３ＤＯ．１８．３）识别提呈的ＯＶＡ的ＣＮＢｒ水解片段而被活化后产生ＩＬ－２,测定ＩＬ－２活性来分析胸腺基质细胞的抗原提呈作用。结果ＩＦＮ－γ能促进ＭＴＥＣＩ和ＭＴＳＣ４表达Ｉ－Ａ＾ｄ分子,并促进ＭＴＳＣ４表达Ｂ７－１分子。经ＩＦＮ－γ作用后,ＭＴＥＣ１和ＭＴＳＣ４均有抗原提呈能力,ＭＴＳＣ     

巨噬细胞型骨髓基质细胞对肿瘤抗原提呈的体外实验研究

目的　探讨骨髓基质细胞对肿瘤抗原的提呈功能。方法　小鼠骨髓贴壁细胞经 Ｇ Ｍ Ｃ Ｓ Ｆ 诱导,形成以成熟巨噬细胞为主的基质细胞,用小鼠红白血病细胞 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激,然后再与 Ｆ Ｂ Ｌ３ 肿瘤抗原致敏的 Ｔ 淋巴细胞混合培养。结果　骨髓基质细胞经 Ｆ Ｂ Ｌ３ 肿瘤抗原刺激后, Ｔ Ｎ Ｆα和 Ｉ Ｌ１β的分泌水平明显升高,经抗原预激的骨髓基质细胞能特异性地刺激同种抗原致敏的 Ｔ 淋巴细胞增殖和分泌高水平的 Ｉ Ｌ２ 。单抗阻断试验发现, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子的联合阻断能有效地抑制致敏 Ｔ 淋巴细胞分泌 Ｉ Ｌ２ 。结论　本实验证实骨髓基质细胞具有抗原提呈功能, Ｍ Ｈ ＣⅡ类分子和 Ｂ７２ 分子在其抗原提呈中发挥了重要作用。     

粘膜抗原提呈的研究进展

粘膜持续地暴露于大量种类繁多的抗原环境中 ,许多感染性病原体通过粘膜表面进入宿主 ,因此产生迅速有效的免疫反应对机体来说至关重要。大多数免疫反应产生的一个限制性步骤是抗原需通过辅佐细胞被提呈给T淋巴细胞 ,抗原的加工与提呈是免疫反应的一个中心事件。粘膜抗原可通过专职抗原提呈细胞和非专职抗原提呈细胞提呈。     

粘膜抗原提呈的研究进展

粘膜持续地暴露于大量种类繁多的抗原环境中，许多感染性病原体通过粘膜表面进入宿主，因此产生迅速有效的免疫反应对体来说至关重要。大多数免疫反应产生的一个限制性步骤是抗原需通过辅佐细胞被提呈给Ｔ淋巴细胞，抗原的加工与提呈是免疫反应的一个中心事件。粘膜抗原可通过专职抗原提呈细胞和非专职抗原提呈细胞提呈。     

永生化EB病毒转化B细胞对肝癌细胞抗原的提呈作用

目的 建立EB病毒(Epstein—Barr virus，EBV)转化的永生化B细胞(命名为LEBV)并研究其对肝癌细胞抗原的提呈作用。方法 从人外周血中分离出外周血单个核细胞(Perpheral blood mononuclear cell,PBMC)，用等体积的EBV上清混合培养4周以上建立永生化EBV转化B细胞(LEBV)，用流式细胞计数仪(FACS)检测其细胞表面功能相关分子CD86、CN40、HLA-DR和HLA-ABC的表达情况。LEBV与自体淋巴细胞和肝癌细胞抗原共培养3d，结束培养前12h加入37kBq／孔^3H—标记的胸腺嘧啶(^3H—TdR)，收获细胞，用液体闪烁计数仪测定cpm。结果 培养3周以上即培养出EBV转化的永生化B细胞，可连续培养1年以上。经FACS检测，细胞表面分子CD86、CD40、HAL-DR和HLA—ABC的表达率分别为74％、91％、83％和86．7％。该细胞接触肝癌细胞抗原后刺激自体淋巴细胞增殖的能力增强。结论 EBV转化的永生化B细胞对肝癌细胞抗原具有提呈作用。     

Multispecific and heterogeneous recognition of the gag protein by cytotoxic T lymphocytes (CTL) from HIV-infected patients: factors other than the MHC control the epitopic specificities.

The HIV gag polyprotein is a major target for recognition by CTL in infected humans. Using recombinant vaccinia viruses (rVV) expressing truncations of the p24gag, and the p18gag, p15gag and HIV-2 p56gag proteins, the characterization of epitope regions recognized by in vitro-stimulated peripheral blood mononuclear cells (PBMC) from 18 infected patients has been studied. The gag-specific response of most individuals is polyclonal and multispecific, and interindividual variations between target epitope regions were frequently observed, despite shared MHC alleles. As CTL may play an important role in the control of HIV replication in infected hosts, these results have important implications for designing vaccine strategies.     

Characteristics of cytotoxic T lymphocytes eluted from allogeneic target cells

The increase in the specific cytotoxic effect of immune lymphocytes after their adsorption on the corresponding target cells (TC) and subsequent elution with pronase is due, not to increased cytotoxic activity of individual cells, but to a quantitative increase in the proportion of effector T cells in the population. The eluted and original immune lymphocytes are indistinguishable in the kinetics of their adsorption on TC. The fraction of eluted lymphocytes contains twice as many T cells and four to five times as many cells synthesizing DNA, on account of an increase in the percentage of medium-sized and large lymphocytes.Laboratory of Immunochemistry and Diagnosis of Tumors, N. F. Gamaleya Institute of Epidemiology and Microbiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR G. V. Vygodchikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 83, No. 1, pp. 50–53, January, 1977.     

Action of lysosomotropic amines on spontaneous and interferon enhanced NK and CTL cytolysis

Cell-mediated cytotoxicities, such as natural killer (NK) and T-dependent cytotoxic (CTL) activities, are inhibited to the same extent by lysosomotropic amines, even when interferon is added to cultures to enhance lysis. It is postulated that the amines block steps common to spontaneous and IFN-enhanced NK and CTL lytic mechanisms.     

Cell death recognition model for the immune system

It is essential for the immune system to recognize markers or understand rules required for discriminating antigens that should be actively responded to from those be tolerated. Although the classic self-nonself theory over the past five decades has been challenged by "danger" model and "infectious nonself" model, etc., no theories could fit for all. Cell death is important not only for its role in homeostasis, but also for its decisive effects on the immune responses. Different ways of cell death, apoptosis or necrosis, transmit fundamentally opposite driving forces for the immune system, inducing tolerance or initiating adaptive immune responses. The progress in understanding phagocytosis and process of apoptotic and necrotic cells leads the author to propose "cell death" recognition model for the immune system. Four principles are important in this model. First, only antigens shedding from apoptotic or necrotic cells rather than those from healthy cells, can be presented to na?ve T cells. Second, either apoptotic cells or necrotic cells, but not healthy cells, can attract phagocytes, namely dendritic cells (DC) or macrophages that are also antigen presenting cells (APC), to scavenge dead cells. Third, macrophages or DC residing in non-lymphoid tissues phagocytose dying/dead cells, migrate to lymphoid tissues and present antigens to na?ve T cells there. Fourth, tolerance or adaptive responses are not dependent on whether the antigens are self or nonself, but on the ways of cell death during antigen presentation. Importantly, tolerance and adaptive immunity are all dominant responses and the impact of cell death on immune responses is a dynamic balance between them. "Cell death" recognition model could more easily explain various immune phenomena, including infection, self tolerance and autoimmunity, tumor immunity as well as transplant rejection. Investigation into the roles and mechanisms of cell death mediated immune responses and finding out key modulators will prompt better understanding the ways of immune recognition and provide novel strategies for the management of autoimmunity, tumors, infections as well as transplantation.     

人脐带血抗巨细胞病毒和EB病毒的特异性细胞毒性T细胞的建立

目的:探讨用人的脐带血单个核细胞体外同时扩增对抗EB病毒(EBV)和巨细胞病毒(CMV)的特异性细胞毒性T淋巴细胞的可行性。方法:利用人的脐带血单个核细胞(CBMC),通过EBV感染转化成B成淋巴细胞细胞株(BLCL),再通过逆转录病毒载体,将CMV蛋白基因pp65导入BLCL,用这种细胞体外刺激同一供者脐带血的CBMC产生细胞毒性T细胞(CTL),经[⁵¹Cr]释放实验(CRA)检测产生的CTL的杀伤功能。结果: 经免疫印迹 (Western Blot)检测,我们获得了同时表达EBV和CMV特异性抗原的抗原递呈细胞BLCLpp65,免疫组化结果表明,BLCLpp65细胞表达CMVpp65抗原的阳性率高达95%。CRA结果证实,用BLCLpp65刺激产生的CTL同时对EBV和CMV都有细胞毒作用。用免疫磁珠法将CD₄⁺和CD₈⁺ T细胞分离后再进行CRA,表明特异性的细胞毒性作用主要是CD₈⁺ CTL介导的。结论:BLCLpp65是很好的抗原递呈细胞,在体外能同时表达EBV和CMV蛋白抗原,用其刺激血清病毒抗体阴性的CBMC,能够扩增出同时针对EBV和CMV的特异性CTL,其中CD₈⁺CTL起主要作用。     

实验性病毒性心肌炎心肌颗粒酶B表达的研究

目的 探讨颗粒酶B(GzmB)在病毒性心肌炎(VMC)心肌损伤过程中的作用及其发病机制.方法 将36只5-6周龄雄性BALB/c小鼠随机分成两组:正常组1O只,模型组26只.于第11天处死小鼠取出心脏观察心肌病变,并以半定量RT-PCR和免疫组化方法检测心肌GzmB的表达.结果 模犁组小鼠心肌组织中均检测到GzmB阳性细胞和GzmB mRNA的表达,且小鼠心肌浸润细胞中GzmB阳性细胞数与心肌病理积分呈显著正相关(r=O.859,P=O.000),而正常组小鼠心肌中未检测到GzmB的表达.结论 心肌浸润细胞中GzmB的表达在病毒性心肌炎心肌损伤中起一定作用,其介导的细胞毒效应是导致病毒性心肌炎心肌损伤的重要机制之一;心肌浸润细胞中GzmB的表达量与心肌损害严重程度成显著正相关.     

Identification of potential CTL epitopes of tumor-associated antigen MAGE-1 for five common HLA-A alleles

Identification of CTL epitopes for tumor-specific responses is important for the development of immunotherapies to treat cancer patients. We have developed a strategy to identify potential CTL epitopes based on screening of sequences of target proteins for presence of specific motifs recognized by the most common HLA-A alleles, and identification of high affinity binding peptides using in vitro quantitative assays. A systematic analysis using the sequence of the product of the tumor-associated MAGE-1 gene has been carried out. All possible peptides of nine and ten residues, containing binding motifs for HLA-A1, -A2.1, A-3.2, -A11 and -A24 were synthesized and tested for binding using a quantitative assay. Out of 237 possible peptide/MHC combinations, 47 cases demonstrated good binding affinity (K_d 500 nM). Several peptides were identified as good MHC binders for each one of the five HLA-A alleles studied (five for HLA-A1, 11 for HLA-A2.1, 10 for HLA-A3.2,16 for HLA-A11 and five for HLA-A24. Furthermore, eight of these peptides were found to bind well to more than one HLA-A allele. These results have important implications for the development of immunotherapeutic vaccines to treat malignant melanoma.     

A role for the histone H2A deubiquitinase MYSM1 in maintenance of CD8+ T cells

Several previous studies outlined the importance of the histone H2A deubiquitinase MYSM1 in the regulation of stem cell quiescence and haematopoiesis. In this study we investigated the role of MYSM1 in T‐cell development. Using mouse models that allow conditional Mysm1 ablation at late stages of thymic development, we found that MYSM1 is intricately involved in the maintenance, activation and survival of CD8⁺ T cells. Mysm1 ablation resulted in a twofold reduction in CD8⁺ T‐cell numbers, and also led to a hyperactivated CD8⁺ T‐cell state accompanied by impaired proliferation and increased pro‐inflammatory cytokine production after ex vivo stimulation. These phenotypes coincided with an increased apoptosis and preferential up‐regulation of p53 tumour suppressor protein in CD8⁺ T cells. Lastly, we examined a model of experimental cerebral malaria, in which pathology is critically dependent on CD8⁺ T cells. In the mice conditionally deleted for Mysm1 in the T‐cell compartment, CD8⁺ T‐cell numbers remained reduced following infection, both in the periphery and in the brain, and the mice displayed improved survival after parasite challenge. Collectively, our data identify MYSM1 as a novel factor for CD8⁺ T cells in the immune system, increasing our understanding of the role of histone H2A deubiquitinases in cytotoxic T‐cell biology.     

Isolation of broadly reactive, tumor-specific, HLA Class-I restricted CTL from blood lymphocytes of a breast cancer patient

Blood lymphocytes of a HLA-A2 positive breast cancer patient were stimulated with either MCF-7 or MDA-MB-231, i.e., HLA-A2-matched allogeneic breast carcinoma cell lines. Several CD8⁺ CTL clones with reactivity against the stimulator cells but not against K562 were generated. Reactivity could be blocked with monoclonal antibody (mAb) W6/32, MA2.1, and/or BB7.2, indicating that the clones are HLA-class I and HLA-A2 restricted. The CTL clones generated following stimulation with MCF-7, recognized various other allogeneic HLA-A2⁺ tumor cell lines, including breast carcinoma, renal cell carcinoma, and melanoma cell lines, but not HLA-A2⁻ tumor cell lines. The CTL clones did not recognize normal HLA-A2⁺ cells including breast epithelial cells, renal proximal tubular epithelial cells (PTEC), or EBV-transformed B cells including the autologous EBV cell line. In contrast to the CTL clones induced with MCF-7, the reactivity of the clones stimulated with MDA-MB-231, was limited to the stimulator cell MDA-MB-231. Cytotoxicity assays utilizing T2 cells loaded with peptides as target cells indicated that none of the examined CTL-epitopes derived from HER-2/neu, Muc-1, Ep-CAM-1, and p53 were recognized by the CTL clones generated. Our findings underscore that breast cancer is an immunogenic tumor and that HLA-class I-matched allogeneic tumor cells can be used as stimulator cells to generate tumor-specific CTL from peripheral blood of a breast cancer patient with specificity for an antigenic determinant that is broadly expressed on tumor cells from various origins or with specificity limited to the breast cancer stimulator cell.