肿瘤特异性TCRαβ基因转染诱导记忆性T细胞分化及其免疫功能的研究

目的研究特异性TCR基因转染T细胞被肿瘤抗原激活后,记忆性T细胞的分化情况,并明确其表型特征和免疫功能。方法密度梯度离心法分离PBMC,重组TCR腺病毒感染T细胞,流式细胞术检测外源TCR表达效率。AFP表位肽刺激T细胞,流式细胞术检测TCR基因转染T细胞经抗原刺激后,记忆性T细胞标志分子表达。MTT法检测T细胞对不同肿瘤细胞株的杀伤活性。Annexin V-PI双染法检测靶细胞凋亡比例。ELISA法检测T细胞作用于靶细胞后IFN-γ与IL-2分泌水平。结果重组腺病毒载体感染3 d后,外源TCR表达比例接近30%。特异性TCR基因转染可有效促进T细胞识别肿瘤抗原后活化,CD45RO+细胞比例逐渐上升至接近50%。CD45RO+细胞以CD62L–CD44+表型为主。此后CD62L+细胞比例逐渐上升。最终出现分群明显的CD62L+CD44+TCM表型细胞。特异性TCR基因转染能够促进T细胞杀伤AFP+靶细胞,诱导靶细胞凋亡,并促进IFN-γ分泌。抗原预先刺激能够进一步增强TCR基因转染T细胞抗肿瘤免疫效应。结论 TCR基因转染能够有效促进T细胞识别肿瘤抗原后活化。经肿瘤抗原预先刺激的TCR基因转染T细胞将启动记忆性T细胞分化,在再次遭遇表达相同抗原的肿瘤细胞时发挥更为强烈的免疫效应。     

TCRαβ分子识别抗原肽-MHC复合物的争议问题探讨

T细胞抗原受体(TCR)是T细胞表面的特征性标志分子,其与CD3分子组成的TCR-CD3复合体,是进行抗原识别,发挥细胞免疫重要功能的基础.一般认为,TCR必须识别抗原肽-MHC分子复合物才能活化T细胞,其中CD4+T细胞识别MHCⅡ类抗原,CD8+T细胞识别MHC Ⅰ类抗原.TCR分子是由两条不同肽链构成的跨膜蛋白,肽链的种类有α、β、γ、δ四种,根据组合的差异,可分为TCRαβ+T细胞和TCRγδ+T细胞,其中TCRαβ+T细胞在发挥特异性细胞免疫过程中发挥重要作用.有关TCRαβ分子识别抗原肽-MHC分子复合物的基本分子机制已形成了经典的免疫学理论,但其中有关MHC分子限制性、CD4及CD8分子的辅助性以及TCR分子α链与β链在识别过程中的地位及作用等问题还存在不少存有争议的地方,本文将介绍相关领域的研究进展,并结合自己的综合分析,对TCRαβ分子识别抗原肽-MHC复合物过程中的一些争议问题进行探讨.     

自身免疫模型小鼠T细胞受体可变区β链(TCR Vβ)偏向取用研究进展

T细胞是机体的主要免疫细胞,且在适应性免疫中起到主要作用。而T细胞受体(TCR)是T细胞表面特异性受体,是识别抗原的主要部位;TCR具有多样性和特异性,其中,最具多样性的是T细胞受体β链可变区(TCR Vβ),TCR Vβ的研究可为临床疾病的诊断和治疗提供新的思路和方向。TCR Vβ家族在各种疾病的动物模型的研究中具有重要的意义和应用价值,本文将对Vβ亚家族偏向取用在小鼠模型中的研究进展进行综述。     

T细胞等位基因排斥研究进展

T淋巴细胞通过其表面受体(T cell receptor,TCR)与抗原提呈细胞(antigen presenting cell,APC)表面的MHC-肽复合物结合识别外来抗原.TCR由异源二聚体α、β或γ、δ组成.其中参与特异性免疫应答的T细胞是α/βT细胞,占外周T细胞库的90%～95%[1].     

T细胞受体与白血病

<正> T、B淋巴细胞是人体特异性免疫反应中,两类主要的效应细胞,B细胞通过其表面的免疫球蛋白(Ig)对抗原的识别,发挥体液免疫作用,而T细胞则通过T细胞受体(TCR)对呈现在细胞表面的外来抗原进行识别,介导细胞免疫反应。本文着重介绍和分析白血病细胞中TCR基因的重排与表达的情况。     

T淋巴细胞活化及其代谢变化

近年来,国外关于T淋巴细胞活化机理的研究非常活跃。T和B淋巴细胞是人体特异性免疫反应中两类主要的效应细胞,B 细胞通过其表面的免疫球蛋白(SIg)对抗原的识别发挥体液免疫作用。而T 细胞则通过T 细胞受体(TCR)对呈现在细胞表面的外来抗原进行识别,介导细胞免疫反应。本文着重从T 细胞受体的结构、膜E 受体的结构及T 细胞内的代谢变化等方面,综述了T 细胞活化免疫机制的研究进展,同时讨论了活化T 细胞内的代谢变化。     

小鼠γδT细胞抗原呈递功能的实验研究

目的:证实小鼠γδT细胞抗原也具有呈递功能.方法:观察了高度纯化的小鼠γδT细胞体外激活后MHC Ⅱ分子及其他共刺激分子的表达与其表面特征性TCR的关系.用自身反应抗原IRBP/MOG特异性T细胞株作为反应细胞,以增殖反应及细胞因子表达作为效应指标,观察γδT细胞的抗原呈递功能.结果:小鼠γδT细胞在激活后可表面表达MHC Ⅱ分子,并能有效呈递抗原给T细胞,使之发生抗原特异性免疫应答.结论:小鼠γδT细胞具有抗原呈递功能,在启动特异性免疫应答的过程中起重要作用.另一方面,不象人γδT细胞,表达MHC Ⅱ分子限于新激活的小鼠γδT细胞,后者失去了表面γδTCR.当γδT细胞重新获得表面γδTCR时,其MHC Ⅱ抗原表达则逐渐减少.     

T细胞受体免疫组库研究技术概述

T淋巴细胞能够同时对外环境中的病原体及其毒素和内环境中因基因突变产生的肿瘤抗原产生免疫应答.然而环境中的抗原千变万化,它们何以识别自然界中天文数量的抗原?经典的克隆选择理论(clonal selection theory)认为,机体内存在众多的淋巴细胞克隆(1012个以上),每一个克隆携带一种抗原受体,特异性识别一种抗原,从而组成一个多样性极其丰富的抗原受体库(repertoire)[1].抗原受体的多样性由T细胞发育过程中,决定其表面受体(T cell receptor,TCR)结构的基因发生重排(rearrangement)而产生[2].TCR由异源二聚体α/β或γ/δ组成.其中参与特异性免疫应答的T细胞主要是α/β T细胞,占外周T细胞库的90％ ～95％[3].     

阻断病毒抗原提呈途径在免疫逃逸机制中的研究

抗原的加工和提呈在适应免疫应答过程中发挥着中枢作用,是淋巴细胞活化、增生、发挥效应的始动环节,也是启动特异性免疫的关键步骤。病毒抗原肽提呈的过程包括病毒蛋白经胞质中的蛋白酶体降解成抗原多肽,由抗原加工相关转运子( transporter associated with antigen presentation, TAP)转运至内质网( endoplasmic reticulum,ER)腔内,与糖基修饰的主要组织相容性复合体( major histocompatibility complex,MHC)分子共同组成MHC-抗原肽分子复合体,经高尔基复合体（Golgi complex,GC）提呈至细胞表面,被T细胞受体(T cell receptor,TCR)识别,激活特异性细胞毒性T淋巴细胞（cytotoxic T lymphocytes,CTL)等一系列反应。     

CDR3谱型分析在肿瘤研究中的应用进展

肿瘤特异性T细胞通过其表面T细胞受体(Tcell receptor,TCR)识别肿瘤,进而杀伤肿瘤细胞,其所介导的细胞免疫在机体抗肿瘤免疫应答中发挥极其重要的作用.寻找肿瘤特异性T细胞克隆是当前肿瘤研究领域的热点,而应用最广泛、最灵敏地寻找肿瘤特异性T细胞的方法是通过基因扫描(Ge-neScan)对TCR互补决定区3(complementary deter-mining region 3,CDR3)谱型进行分析.     

Predominant rearrangements of the T cell receptor β-chain gene in blood lymphocyte populations stimulated with autologous tumor cells suggest clonal T cell expansion in two of fourteen cases

T cell responses against autologous tumors with samples from patients with a variety of tumors were examined. The abilities of T lymphocytes to lyse the autologous tumor cells were analyzed after short-term mixed lymphocyte/tumor cell cultures (MLTC). Southern blot analysis was used to evaluate whether particular rearrangements of the TCR β-chain gene predominate in these cultures. Tumor specific lysis could be induced in a proportion of the mixed cultures. In two cases enrichment of T lymphocytes with similar TCR β-chain gene rearrangements was detected after repeated stimulations with autologous tumor cells.     

Prospects and limitations of T cell receptor gene therapy

Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer, as it can circumvent the demanding process of in vitro generation of large numbers of specific immune cells and it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments and mouse model systems. Furthermore, we discuss the prospects of clinical application of this gene therapy approach, and the possible barriers on the route towards clinical use.     

Prospects and limitations of T cell receptor gene therapy

Adoptive transfer of antigen-specific T cells is an attractive means to provide cancer patients with immune cells of a desired specificity and the efficacy of such adoptive transfers has been demonstrated in several clinical trials. Because the T cell receptor is the single specificity-determining molecule in T cell function, adoptive transfer of TCR genes into patient T cells may be used as an alternative approach for the transfer of tumor-specific T cell immunity. On theoretical grounds, TCR gene therapy has two substantial advantages over conventional cellular transfer. First, it circumvents the demanding process of in vitro generation of large numbers of specific immune cells. Second, it allows the use of a set of particularly effective TCR genes in large patient groups. Conversely, TCR gene therapy may be associated with a number of specific problems that are not confronted during classical cellular therapy. Here we review our current understanding of the potential and possible problems of TCR gene therapy, as based on in vitro experiments, mouse model systems and phase I clinical trials. Furthermore, we discuss the prospects of widespread clinical application of this gene therapy approach for the treatment of human cancer.     

Dual T cell receptor T cells with two defined specificities mediate tumor suppression via both receptors

Grafting T cells with new antigen specificity by T cell receptor (TCR) gene transfer could greatly facilitate adoptive T cell immunotherapy. Little is known about how two TCR on one T cell influence each other. Among other reasons, this is often due to the fact that only one TCR specificity is known. We have genetically generated murine dual TCR T cells (OT-I/P14), specific for ovalbumin(ova257) and lymphocyte choriomeningitis virus glycoprotein (gp33). These cells retain both specificities and can be stimulated by either antigenic peptide to proliferate and produce IFN-gamma. Even though one TCR (P14) is expressed at reduced levels on dual TCR T cells, the peptide sensitivity of these cells is similar to that of single TCR T cells of the same specificity. TCR down-modulation on dual TCR T cells depends primarily on binding of the specific ligand. Adoptively transferred dual TCR T cells suppress the growth of both B16-ova and B16-gp33 melanoma cells, regardless of the peptide used for in vitro activation. Taken together, despite a certain dominance of expression between two TCR on the same T cell, this need not necessarily have functional consequences.     

Control of natural killer cell-mediated innate resistance against the intracellular pathogen Listeria monocytogenes by gamma/delta T lymphocytes.

Listeria monocytogenes is an intracellular bacterium which causes an acute infectious disease in mice. Initial host resistance depends on innate immunity mediated primarily by natural killer (NK) cells followed by specific alpha/beta T cells, which are central to acquired specific immunity. Gamma/delta T lymphocytes seem to provide a link between the innate and the specific immune response. All these lymphocyte populations produce gamma interferon (IFN-gamma), which, because of its macrophage-activating potential, is central to antibacterial protection. IFN-gamma from NK cells not only contributes to early host resistance but also promotes development of protective T-cell responses of helper T type 1 (Th1) type. Here, we show that innate resistance and early IFN-gamma production in listeriosis are markedly impaired in T-cell receptor (TCR)-delta-/- but not TCR-beta-/- gene disruption mutant mice. By two-color cytofluorimetry, we demonstrate that NK cells rather than gamma/delta T lymphocytes are the major cellular source of IFN-gamma in immunocompetent mice and that IFN-gamma production by NK cells is impaired in the TCR-delta-/- mutants. Probably, reduced tumor necrosis factor production in listeria-infected TCR-delta-/- mutants contributed to impaired NK cell activation. Our data reveal a novel function of gamma/delta T cells as regulators of innate resistance against sublethal infection with an intracellular pathogen.     

T-cell receptor- and anti-inflammatory gene-modulated T cells as therapy for autoimmune disease

Antigen-specific immunotherapy is a future therapy that could achieve high efficacy with limited adverse effects. Although T cells are essential components in antigen-specific immunity, we do not have a practical strategy for manipulating antigen-specific T cells. We propose here that T-cell receptor (TCR) gene transfer is an effective approach for antigen-specific immunotherapy. We have confirmed the efficacy of TCR gene therapy in animal models of systemic autoimmune disease and arthritis. In lupus-prone NZB/W F1 mice, nucleosome-specific TCR and CTLA4Ig-transduced cells suppressed autoantibody production and nephritis development. In the therapeutic experiment of collagen-induced arthritis, arthritis-related TCRs were isolated from single T cells accumulating in the arthritis site. With this TCR, we demonstrated two applications of the TCR-transduced T cells. First, this arthritis-related TCR and the TNFRIg-transduced cells suppressed arthritis as a vector for therapeutic molecule. Second, arthritis-associated TCR and Foxp3-transduced cells suppressed arthritis progression and bone destruction as antigen-specific regulatory T cells. Therefore, engineered antigen-specific cells manipulated to express appropriate functional genes could be applied to specific immunotherapy.     

Immunotherapy through TCR gene transfer

The antigen specificity of T lymphocytes is dictated solely by the T cell receptor (TCR) alpha and beta chains. Consequently, genetic transfer of TCR chains may be an appealing strategy with which to impose a desirable virus- or tumor-antigen specificity onto cytotoxic or helper T cell populations. We describe here the genetic introduction of a virus-specific TCR into peripheral T cells in a mouse model system. These experiments showed that T cells redirected by TCR gene transfer expanded upon viral infection of mice and efficiently homed to effector sites. In this setting, TCR gene transfer was not associated with any significant autoimmune pathology. In addition, small numbers of TCR-transduced T cells promoted the rejection of antigen-expressing tumors in vivo. These data suggest that the redirection of T cells by TCR gene transfer is a viable strategy for the rapid induction of virus- or tumor-specific immunity.     

Staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity

T cells equipped with sophisticated TCR and MHC recognition structures, an efficient cytokine communication network and lethal cytotoxic effector functions constitute one of the bulwarks of the mammalian immune system. However, infective agents have developed strategies to undermine T-cell immunity; for example, certain bacterial toxins serve as 'superantigens' by binding to preserved determinants on MHC class-II-encoded proteins and activating T cells expressing particular sequences of TCR V beta gene products. In this paper, Mikael Dohlsten and colleagues present evidence suggesting that these bacterial superantigens direct T cells to eradicate MHC class-II-expressing antigen-presenting cells, thus counteracting specific T-cell functions.     

Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer

We identified a tumor-associated cytotoxic T lymphocyte (CTL) epitope derived from the widely expressed human MDM2 oncoprotein and were able to bypass self-tolerance to this tumor antigen in HLA-A*0201 (A2.1) transgenic mice and by generating A2.1-negative, allo-A2.1-restricted human T lymphocytes. A broad range of malignant, as opposed to nontransformed cells, were killed by high-avidity transgenic mouse and allogeneic human CTLs specific for the A2.1-presented MDM2 epitope. Whereas the self-A2.1-restricted human T cell repertoire gave rise only to low-avidity CTLs unable to recognize the natural MDM2 peptide, human A2.1+ T lymphocytes were turned into efficient MDM2-specific CTLs upon expression of wild-type and partially humanized high-affinity T cell antigen receptor (TCR) genes derived from the transgenic mice. These results demonstrate that TCR gene transfer can be used to circumvent self-tolerance of autologous T lymphocytes to universal tumor antigens and thus provide the basis for a TCR gene transfer-based broad-spectrum immunotherapy of malignant disease.