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6.用CD4+CD25+抗原特异性调节性T细胞作为治疗性疫苗
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| 克服肿瘤固有的抵抗免疫效应功能 | |||
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王明永 贵阳医学院微生物学教研室
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| 人类和鼠T淋巴细胞中MHC-II类分子的功能与调节 | |||
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石 瑛 译 新乡医学院 医学检验系
简介
在动物王国中表达MHC II类分子的T细胞 II类转录活化子（CIITA）是MHC II类分子表达的主要调节子。与MHC II类分子相一致，仅APCs可以组成性表达CIITA，但是其它各种细胞可以诱导表达。CIITA基因至少被3个独立的启动子单元（CIITA-PI,CIITA-PIII和CIITA-PIV）控制，每一个启动子控制转录唯一一个外显子。最近我们和其他的实验室已经建立了单独用CIITA-PIII可以在正常人体内活化T细胞；然而未受刺激的T细胞缺少CIITA的表达。尽管在活化的人类T细胞中，已经确定了涉及CIITA诱导表达的几种转录因子，但是精确的遗传和后天调节机制和T细胞中各种因子如何组装到CIITA-PIII上的问题还需要解决。
活化的T细胞能递呈抗原吗？
共刺激分子的表达
T细胞的APC功能（T-APC）
MHC II类分子配体介导的细胞内信号
MHC II类分子配体诱导的凋亡
在胸腺中MHC II类分子介导的T-T相互作用
总结 ---------
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| 调节性T细胞研究进展 |
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吴祖群 许以平 上海第二医科大学附属仁济医院呼吸科
提要：调节性T细胞是不同于Th1和Th2的具有调节功能的T细胞群体，因其具有免疫抑制作用，近来受到人们的广泛关注，本文综述调节性T细胞近来的研究进展。 关键词：调节性T细胞；细胞因子；免疫
调节性T细胞是不同于Th1和Th2的具有调节功能的T细胞群体，具有免疫抑制功能，在多种免疫性疾病中起重要的调节作用，成为近年来免疫学领域研究的重要内容。
1 调节性T细胞分类 调节性T细胞（Tr）在体内外具有调节功能，根据其表面标记、产生的细胞因子和作用机制的不同，Tr可分为CD4+CD25+Tr细胞、Tr1和Th3等多种亚型。 1.1 CD4+CD25+Tr 目前研究得较为清楚的亚型为CD4+CD25+Tr。研究表明，在正常人和小鼠的外周血及脾脏组织的CD4+T细胞中约有5%～10%的细胞持续表达CD25分子（IL-2受者α链），同时这一亚群是CD45RB分子低表达的。CD4+CD25+Tr具有免疫无能性和免疫抑制性两大功能特性。其免疫无能性表现在对高浓度IL-2的单独刺激，固相包被或可溶性抗CD3单抗，以及抗CD3单抗、抗CD28单抗的联合作用呈无应答状态，也不分泌IL-2。当经TCR介导信号刺激并有高浓度外源IL-2存在的情况下，CD4+CD25+Tr可活化并增殖，但其增殖程度较CD4+CD25-T细胞弱很多。 CD4+CD25+Tr的免疫抑制性表现在经TCR介导的信号刺激活化以后能够抑制CD4+和CD8+T细胞的活化和增殖。 1.2 Tr1 Tr1是另一类亚型的CD4+Tr。在IL-10存在的情况下，可通过异源抗原活化的人类T细胞经克隆产生，IFN-α可增强IL-10诱导产生Tr1的能力。另外 ，抗原与CD2共刺激可通过IL-10非依赖途径诱导产生抗原特异性Tr，产生大量IL-10，并具有调节功能。最近Kemper等研究发现，在IL-2存在的情况下，人CD4+T细胞与CD3和补体调节剂CD46结合诱导生成Tr1特异性细胞因子表型的T细胞。这种细胞增殖能力强，并具有旁观者抑制效应和免疫记忆力。Tr1与 Th1 和Th2之间的区别于Tr1可产生高水平的IL-10，中等量的TGF-β、IFN-γ和IL-5，少量的IL-2，不产生IL-4。刺激TCR不能使Tr1有效增殖，有研究显示IL-15对于刺激Tr1在体外增殖至关重要。Tr1抑制免疫反应的主要机制依赖于产生具有免疫调节功能的细胞因子如IL-10和TGF-β。 1.3 Th3 Th3型CD4+Tr是在研究口服耐受机制的过程中发现的，Th3主要分泌TGF-β，对Th1和Th2都具有抑制作用。Th3可从IL-4缺陷小鼠产生，显示Th3是与Th2不同的一种独特类型。TGF-β、IL-4、IL-10可促进TCR转基因小鼠Th前体分化为Th3。低剂量抗原口服诱导产生Th3，同时口服IL-4可促进Th3的产生。 由于不成熟树突状细胞（DC）在体外诱导产生的CD4+Tr和体内诱导产生的CD8+Tr与Tr1具有相同的特性，即都产生高水平的IL-10，但不产生IL-4或IL-2。然而，与Trl比较，不成熟DC诱导产生的CD4+Tr在体外的抑制活性不依赖于IL-10。并且，由不成熟DC在体外诱导产生的CD4+Tr通过抗原非依赖机制直接抑制成熟Th1的增殖反应，该过程需要细胞与细胞之间的接触，并可被外源性的IL-2所抑制。因此，从功能上讲，这些Tr更类似于上述的CD4+CD25+Tr。 目前还不清楚CD4+CD25+Tr与Trl之间的关系，有人认为它们可能是处于不同分化阶段的同一Tr亚型，CD4+CD25+Tr可能来源于胸腺部分分化的T细胞，并在外周遇到抗原时最终分化为产生IL-10和TGF-β的Trl。但较多倾向认为CD4+CD25+Tr与Tr1是两种不同的细胞亚型，有研究观察到小鼠CD4+CD25+Tr属于CD45RBlow群体，而Tr1在体外由CD45RA+初始细胞分化而来。
2 调节性T细胞的分离、纯化及克隆扩增方法 获得大量的调节性T细胞是对其进行研究的前提，正常情况下淋巴结、脾脏及外周血存在一定数量的CD4+CD25+Tr，可通过CD标记，免疫磁珠分选法获得大量CD4+CD25+Tr。Trl主要通过克隆扩增的方法获得，较为复杂。 2.1 CD4+CD25+Tr的分离、纯化 制备单个细胞悬液，以T细胞纯化柱分离小鼠淋巴结或脾脏T淋巴细胞，以抗-CD8-FITC抗体和抗FITC标记磁珠相结合的阴性选择法剔除CD8+T细胞，再以抗-CD25-FITC抗体和抗FITC标记磁珠相结合的阳性选择法选出CD25+T淋巴细胞。也可采用CD4+T细胞亚型纯化柱分离得到CD4+T细胞，再结合抗-CD25-FITC抗体，以流式细胞仪分选出纯化的CD4+CD25+T细胞。 2.2 Trl的克隆、扩增 Groux等较早克隆扩增成功人和小鼠Tr1，人Trl是在外源性IL-10存在情况下，以外源性单核细胞刺激人外周血CD4+T细胞，10 d后，以流式细胞仪筛选出单个CD4+T细胞克隆并接种于培养孔，加入经照射的饲养细胞（JY，105/ml,PBMC,106/ml）,以10U/ml的rIL-2和高剂量交叉结合的抗-CD3单抗（100μg/ml）反复刺激扩增。小鼠Trl克隆通过有限稀释法获得，培养基中加入OVA（2 μmo/L）和经照射的脾APC（107/ml），以IL-2（20 U/ml）和IL-4（20 U/ml）刺激进行扩增。
3 调节性T细胞作用机制 活化的CD4+CD25+Tr主要通过接触抑制的方式抑制T细胞的活化和增殖。另外，CD4+CD25+Tr还可分泌细胞因子IL-10和TGF-β抑制免疫反应。CD4+CD25+Tr接触抑制的机制在于该细胞表达CTLA-4，CTLA-4跨膜分子胞内段携带免疫受者酷氨酸抑制基序（ITIM），与B7配接后传递抑制信号，抑制T细胞的增殖和活化，用抗CTLA-4单抗可阻断CD4+CD25+Tr的抑制作用。 前已提及，Trl发挥免疫调节作用主要是通过分泌IL-10和TGF-β来实现的，IL-10和TGF-β都是具有免疫抑制作用的细胞因子，二者抑制效应广泛。IL-10可通过直接和间接机制明显降低抗原特异性T细胞增殖。IL-10对T细胞的直接作用包括抑制IL-2的产生以及延长细胞增殖周期。间接机制包括下调MHC Ⅱ类分子的表达、下调单核细胞CD80和CD86的表达以及T细胞共同刺激分子CD28的配体。并且，通过抑制APC，IL-10有效抑制IL-12的产生，而IL-12是Thl细胞分化的关键因子。IL-10还可阻止T细胞受体介导的CD4+T细胞活化。以IL-10处理T细胞导致持久的抗原特异性T细胞无反应，以抗CD3和CD28抗体重新刺激细胞不能逆转IL-10诱导的T细胞无能，加入外源性的IL-2也不能逆转IL-10诱导的T细胞无能。除了主动抑制T细胞增殖，在特定条件下，IL-10可诱导Trl的分化，人和小鼠CD4+T细胞在IL-10存在时经慢性活化可产生Trl，增强Trl的抑制作用。IL-10对其他炎症细胞也具有较强的抑制作用，可抑制单核细胞、巨噬细胞、中性粒细胞及嗜酸粒细胞产生前炎症因子和趋化因子，并可抑制单核细胞产生IL-10，提高IL-1α和IL-1β的自然拮抗剂IL-1RA的产生。IL-10还可抑制APC HLA-DR及多种共同刺激分子如CD54、CD80和CD86的表达。IL-10还可通过抑制嗜酸粒细胞表达CD40发挥抗过敏效应，导致嗜酸粒细胞加速凋亡。在体内，IL-10表现出较强的免疫抑制和抗炎效应，在内毒素血症、自身免疫性甲状腺炎以及过敏原诱导的气道炎症小鼠模型中，IL-10都表现出保护作用。 TGF-β从三个方面对免疫功能起抑制作用：一是抑制免疫效应细胞的增殖；二是抑制免疫效应细胞的分化和活性；三是抑制细胞因子的产生及其免疫调节作用。将TGF-β加入到非纯化入T淋巴细胞培养基抑制IL-2依赖性ConA诱导的增殖，同时T细胞表达的IL-2Rα和CD71下降。TGF-β可使葡萄球菌毒素刺激的鼠CD8+T细胞毒性减低90%。混合淋巴细胞反应开始加入TGF-β可抑制细胞溶解效应细胞的产生，加入IFN-α可逆转该效应。TGF-β抑制新鲜分离的T细胞IL-4及IFN-γ的产生。
4 调节性T细胞的应用前景 Tr所具有的免疫抑制特性使得其在自身免疫性疾病的调节中具有广泛的应用前景，具有许多研究显示调节性T细胞在Th1或Th2介导的疾病中具有良好效果。 4.1 炎症性肠病 在炎症性肠病（IBD）小鼠模型，同时输入Trl细胞和致病性CD4+CD45RBhiT细胞可抑制严重免疫缺陷小鼠IBD的形成。该研究同时证实Trl必须在体内被活化才能发挥调节作用，因为只有在接受的抗原可被Trl识别的小鼠中才能阻止IBD的形成。Trl克隆抑制免疫反应的特性与CD45RBlowCD4+T细胞相似，后者也可通过IL-10和TGF-β依赖机制抑制IBD。 4.2 移植免疫 诱导免疫耐受是移植成功的关键，Tr在移植耐受中起重要调节作用。Taylor等研究显示，CD4+CD25+Tr在移植物抗宿主病（GVHD）的发生过程中起重要作用，在数个动物品系的组合中，不论全身放射剂量如何，去除供者T细胞接种物中CD4+CD25+Tr或移植前去除受者CD25+T细胞将导致GVHD的升高。输注新鲜纯化的供者CD4+CD25+Tr和等量的CD4+T细胞可适度抑制GVHD的发生。而体外培养的活化CD4+CD25+Tr与等量的CD4+T细胞或去除CD25+T细胞输注明显抑制快速致死性GVHD。 4.3 支气管哮喘 支气管哮喘是由Th2细胞介导的气道慢性炎症性疾病，结合在肥大细胞、嗜碱粒细胞表面的抗原特异性IgE通过桥联抗原促使上述细胞释放炎症介质，在速发相及迟发相哮喘反应中起重要作用。Cottrez等研究发现，将Trl克隆转移入OVA诱导的速发型超敏反应小鼠模型中，使抗原特异性IgE减少90%，抗IL-10抗体可逆转Trl抑制抗原特异性IgE的作用，说明IL-10在Trl的调节功能中起重要作用。给予Trl的小鼠淋巴结T细胞经OVA刺激后分泌的IL-10较对照组细胞明显升高，仅分泌少量的IL-5，无IL-4。将抗原特异性Tr转移入致敏小鼠也可阻止气道高反应的形成。Zuany-Amorim等研究发现，经灭活分析杆菌悬液SRP299处理的小鼠产生CD4+CD45RBloqTr，可通过产生IL-10和TGF-β抑制气道炎症的形成。 4.4 口服耐受及实验性自身免疫性脑脊髓炎 口服耐受是指通过预先口服抗原特异性抑制细胞和（或）体液免疫，阻止机体对食物蛋白和细菌抗原的高反应性。口服低剂量抗原可诱导产生分泌免疫抑制性细胞因子TGF-β的调节性细胞，抑制实验性自身免疫性疾病的发生。低剂量抗原经Peyer's patch肠相关APC递呈后，优先诱导产生抗原特异性调节性细胞，这种调节性细胞在体内外遇到抗原后可分泌TGF-β。而高剂量抗原口服不仅诱导克隆无能和消除，也可诱导产生TGF-β的调节性细胞，OVA TCR转基因小鼠口服抗原也可诱导产生CD4+CD25+Tr，其抑制效应部分由TGF-β介导。Chen等研究发现，小鼠口服给予髓磷脂碱蛋白可诱导周围耐受，其肠系膜淋巴结可分离出产生TGF-β、IL-10及IL-4的T细胞克隆，抑制实验性自身免疫性脑脊髓炎（experimental autoimmune encephalomyelitis,EAE）的形成，此病是由Th1介导的多发生硬化动物模型。另外，Kohm等研究发现，在体外，CD4+CD25+Tr可有效抑制MOG（35～55）特异性Th1的增殖和细胞因子的产生。在体内，过继转移CD4+CD25+Tr明显抑制自身反应性Th1介导的EAE的形成，同时MOG（35～55）特异性Th2出现的频率增加，中枢神经系统浸润降低。 5 Tr研究展望 Tr是近年来发现的一种具有免疫调节功能的细胞群体，深入研究其发生、生长及功能将有助于进一步阐明机体免疫调节机制。另外，已有多项研究将Tr用于自身免疫性病及超敏反应性疾病的试验性调节，并取得良好的结果。随着研究的不断深入，将有可能在治疗此类疾病方面取得新的突破。
----------- 摘自:《现代免疫学》2004年第1期
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基因疫苗和免疫治疗 |
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Margaret Liu , Bruce Acres, Jean-Marc Balloul, et al
DNA vaccines, comprised of plasmid DNA encoding proteins from pathogens,
allergens, and tumors, are being evaluated as prophylactic vaccines
and therapeutic treatments for infectious diseases, allergies, and cancer;
plasmids encoding normal human proteins are likewise being tested as
vaccines and treatments for autoimmune diseases. Examples of in vivo
prophylaxis and immunotherapy, based on different types of immune responses
(humoral and cellular), in a variety of disease models and under evaluation
in early phase human clinical trials are presented. Viral vectors continue
to show better levels of expression than those achieved by DNA plasmid
vectors. We have focused our clinical efforts, at this time, on the
use of recombinant viral vectors for both vaccine as well as cytokine
gene transfer studies. We currently have four clinical programs in cancer
immunotherapy. Two nonspecific immunotherapy programs are underway that
apply adenoviral vectors for the transfer of cytokine genes into tumors
in situ. An adenovirus-IFN construct (TG1042) is currently being tested
in phase II clinical trials in cutaneous lymphoma. A similar construct,
adenovirus-IL2 (TG1024), also injected directly into solid tumors, is
currently being tested in patients with solid tumors (about one-half
of which are melanoma). Encouraging results are seen in both programs.
Two cancer vaccine immunotherapy programs focus on two cancer-associated
antigens: human papilloma virus E6 and E7 proteins and the epithelial
cancer-associated antigen MUC1. Both are encoded by a highly attenuated
vaccinia virus vector [modified vaccinia Ankara (MVA)] and both are
coexpressed with IL-2. Encouraging results seen in both of these programs
are described.
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| 自身免疫中的治疗性疫苗 | |||
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Michael Sela and Edna Mozes
Similarly to prophylactic vaccines whose purpose is to
prevent infectious diseases, therapeutic vaccines against autoimmune
diseases are based on their similarity to the putative causes of the
disease. We shall describe here two such examples: a copolymer of amino
acids related to myelin basic protein, in the case of multiple sclerosis,
and a peptide derived from the nicotinic acetylcholine receptor (AChR),
in the case of myasthenia gravis (MG). Copolymer 1 (Cop 1, glatiramer
acetate, Copaxone) is a synthetic amino acid random copolymer, immunologically
cross-reactive with myelin basic protein and suppresses experimental
allergic encephalomyelitis in several animal species. Cop 1 slows the
progression of disability and reduces relapse rate in exacerbating-remitting
multiple sclerosis patients. It was approved by the Food and Drug Administration
in 1996, and today is used by tens of thousands of patients. Cop 1 is
a potent inducer of T helper 2 (Th2) regulatory cells in mice and humans,
and Th2 cells are found both in the brains and spinal cords of Cop 1-treated
mice. MG and experimental autoimmune MG are T cell-regulated, antibody-mediated
autoimmune diseases. Two peptides, representing sequences of the human
AChR -subunit, p195-212 and p259-271, are immunodominant T cell epitopes
in MG patients and in two strains of mice. Altered peptide ligand, composed
of the tandemly arranged two single amino acid analogs, inhibits in
vitro and in vivo MG-associated autoimmune responses. The active suppression
is mediated by the CD4+CD25+ immunoregulatory cells and is associated
with the down-regulation of Th1-type cytokines and the up-regulation
of the secretion of IL-10 and the immunosuppressive cytokine, transforming
growth factor .
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| 用CD4+CD25+抗原特异性调节性T细胞作为治疗性疫苗 | |||
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Jeffrey A. Bluestone and Qizhi Tang
Autoimmune disease results from the dysregulation of basic tolerogenic processes designed to control self/non-self-discrimination. Approaches to treat autoimmunity have focused historically on potent immunosuppressives that block the activation and expansion of antigen-specific T cells before they differentiate into pathogenic T cell responses. These therapies are very efficient in reducing clonal expansion and altering early signaling pathways. However, once the pathogenic responses are established (i.e., autoimmunity), the interventions are less effective on activated and differentiated T cell subsets (including memory T cells) or acting in the presence of an inflammatory milieu to abort immune responses at the target tissue and systemically. Moreover, the current immunotherapies require continuous use because they do not redirect the immune system to a state of tolerance. The continuous treatment leads to long-term toxicities and can profoundly suppress protective immune responses targeted at viruses, bacteria, and other pathogens. Over the past decade, there have been tremendous advances in our understanding of the basic processes that control immune tolerance. Among the most exciting has been the identification of a professional regulatory T cell subset that has shown enormous potential in suppressing pathologic immune responses in autoimmune diseases, transplantation, and graft vs. host disease. In this review, we summarize current efforts to induce and maintain tolerance in the autoimmune diabetes setting by using therapeutic vaccination with CD4+CD25+ regulatory T cells. Emphasis will be placed on approaches to exploit regulatory T cells either directly or through the use of anti-CD3 immunotherapy. . |
| 通过脂多糖受体、TLR受体4的信号转导 | |||
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Eva M. Polsson-McDermott &
Luke A. J. O'Neill An understanding of lipopolysaccharide (LPS) signal transduction
is a key goal in the effort to provide a molecular basis for the lethal
effect of LPS during septic shock and point the way to novel therapies.
Rapid progress in this field during the last 6 years has resulted in
the discovery of not only the receptor for LPS - Toll-like receptor
4 (TLR4) - but also in a better appreciation of the complexity of the
signalling pathways activated by LPS. Soon after the discovery of TLR4,
the formation of a receptor complex in response to LPS, consisting of
dimerized TLR4 and MD-2, was described. Intracellular events following
the formation of this receptor complex depend on different sets of adapters.
An early response, which is dependent on MyD88 and MyD88-like adapter
(Mal), leads to the activation of nuclear factor-B (NF-B). A later response
to LPS makes use of TIR-domain-containing adapter-inducing interferon-
(TRIF) and TRIF-related adapter molecule (TRAM), and leads to the late
activation of NF-B and IRF3, and to the induction of cytokines, chemokines,
and other transcription factors. As LPS signal transduction is an area
of intense research and rapid progress, this review is intended to sum
up our present understanding of the events following LPS binding to
TLR4, and we also attempt to create a model of the signalling pathways
activated by LPS.
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在调节B细胞功能上TRAFs的多方面作用 |
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Gail A. Bishop
Tumour-necrosis factor receptor (TNFR)-associated factors (TRAFs) are cytoplasmic adaptor proteins that are important in lymphocyte activation and apoptosis. Many studies of TRAFs have used models of exogenous overexpression by non-lymphoid cells. However, the actions of TRAFs present at normal levels in lymphoid cells often differ considerably from those that have been established in non-lymphocyte overexpression models. As I discuss here, information obtained from studying these molecules in physiological settings in B cells reveals that they have several roles, which are both unique and overlapping. These include activation of kinases and transcription factors, and interactions with other signalling proteins, culminating in the induction or inhibition of biological functions.
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| B细胞系在阳性和阴性选择中的受体编辑 | |||
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Efrat Edry and Doron Melamed
In B lymphopoiesis, Ag receptor expression and signaling are critical
to determine developmental progression, survival, and activation. Several
positive and negative selection checkpoints to test this receptor have
been described in B lymphopoiesis, aiming to ensure the generation of
functionally competent, nonautoimmune repertoire. Secondary Ag receptor
gene recombination allows B lymphocytes to replace an inappropriate receptor
with a new receptor, a mechanism called receptor editing. This salvage
mechanism uncouples the Ag receptor fate from that of the cell itself,
suggesting that B cell repertoire is regulated by a process of receptor
selection. Secondary rearrangements are stimulated in different stages
of B cell development, where editing of the receptor is necessary to fulfill
stage-specific requirements. In this study, we discuss the contribution
of receptor editing in B lymphopoiesis and its regulation by positive
and negative selection signals.
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| NF-kB抑制剂在两个时期阻碍B细胞发育 | |||
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Biao Feng , Shuhua Cheng , Warren S Pear and Hsiou-Chi Liou
Members of the NF-kB transcription factor family are differentially expressed
in the B cell lineage. Disruption of individual or two NF-kB subunits
exhibits distinct defects in B lymphocyte development, activation, and
survival. However, the role each NF-kB plays during B cell development
has been obscured by molecular compensation. To address this issue, a
trans-dominant form of IkBα was transduced into bone marrow cells to act
as a pan-inhibitor of NF-kB using a retroviral system. While the development
of T-lymphocytes and myeloid cell lineages was not grossly affected by
the transduced IkBα gene, a significant reduction in the number and percentage
of B lineage cells was apparent in IkBα transduced chimeric mice. IkBα
expression decreased the percentage of pre-B and immature B cell subsets
in the bone marrow and further impaired the development of follicular
mature B cells and marginal zone B cells in the periphery. Introduction
of the Bcl-X transgene completely restored the pre-B and immature B cell
pool in the bone marrow. However, despite a significant improvement of
overall viability of the B cell lineage, Bcl-X expression was insufficient
to overcome the maturation block resulting from NF-kB inhibition. Together,
our study suggests that NF-kB activity is required for two distinct checkpoints
during B cell development: one is for pre-B/immature B cell viability,
the other is to provide both survival and maturation signals to ensure
the proper development of follicular mature B cells. |
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新乡医学院免疫学网络工作室 主办 深圳晶美生物工程有限公司 协办 |