7 淋巴细胞的发育与存活

第七章淋巴细胞的发育与存活

正如第三章和第四章讲到的，B和T淋巴细胞携带的抗原受体其结合抗原的特异性是极其庞大的，这就赋予个体在其一生中对接触到的广泛的病原体产生免疫应答。这种B细胞受体（B-cell receptors）和T细胞受体（T-cell receptors）的多样性谱是B细胞和T细胞在发育过程中形成的，分别来自于它们各自未分化的前体。新淋巴细胞的产生或称淋巴细胞增殖（lymphopoiesis），发生在特定的淋巴组织即中枢淋巴组织（central lymphoid tissue），对B细胞而言是在骨髓，而对T细胞来说则在胸腺。像所有造血细胞一样，淋巴细胞前体起源于骨髓，但B细胞在骨髓中完成其大部分的发育过程，而T细胞则由骨髓迁移入胸腺，并从前体细胞发育而成。

单个淋巴细胞的抗原特异性是在其早期分化阶段决定的，在B细胞，编码其免疫球蛋白可变区的DNA序列，以及在T细胞编码其T细胞受体可变区的DNA序列都是由基因片段组装起来的，在第四章中我们已经介绍过了。由于基因片段都需要进行重排，因此B细胞和T细胞的早期发育所经历的过程是非常相似的。在B细胞和T细胞中，这方面的发育调控方式都是相似的，以确保淋巴细胞谱作为整体的多样性以及单个淋巴细胞具有独特的抗原特异性。

淋巴细胞表面抗原受体的表达代表着细胞发育的一个分水岭，因为此时淋巴细胞就可以检测与其受体结合的配体了。在淋巴细胞发育的下一阶段，就需要检测这些受体对附近环境中出现分子的抗原识别特性。受体与这些配体的特异性和亲和力决定了这些未成熟淋巴细胞的命运，这就是说决定细胞是被选择存活下去并进一步发育，还是在未成熟时就死亡。

一般来说，发育中的淋巴细胞其受体与自身抗原发生微弱的相互作用，或以一种特殊的方式与自身抗原结合的，则会收到使之存活的信号，这种选择称为阳性选择（positive selection）。阳性选择在α︰β T细胞的发育中尤其重要，因为这类细胞识别的抗原是与MHC 分子结合的肽段（见第三章）。由于不能识别机体自身MHC分子的T细胞将不能启动针对任何抗原的免疫应答，因此阳性选择的过程确保了个体将拥有一个T细胞库，当外来抗原肽与他或她自身的MHC分子结合时，能对外来抗原肽作出应答的。

另一方面，淋巴细胞其受体与自身抗原强烈结合的话，则会收到导致其死亡的信号，这一过程称为阴性选择（negative selection）。因此，强烈自身反应性的淋巴细胞在它们完全成熟并可能引发破坏性自身免疫应答之前，就从淋巴细胞库中被清除了。通过这种方式对普遍存在的自身抗原建立了免疫耐受（tolerance）。发育中的淋巴细胞当其受体没有收到任何信号时其命运就是死亡，正如我们将会看到的，绝大多数发育中的淋巴细胞在离开中枢淋巴器之前或在外周淋巴器官中成熟之前就会发生死亡。

因此，存活下来并组成成熟淋巴细胞种群的淋巴细胞仅占骨髓或胸腺中产生的淋巴细胞的一小部分。然而，这些细胞表达了一个大的受体谱，能对几乎无限多样化的异己结构作出应答。这个受体谱为获得性免疫应答中发生的克隆选择提供了原材料。

本章中我们将描述小鼠和人B细胞及T细胞发育的不同阶段，从无分化的干细胞到成熟、具有特定功能、携带有独特抗原受体并随时能对外来抗原作出应答的淋巴细胞。在本章的前两个部分，我们会定义淋巴细胞在中枢淋巴器官中发育的各个阶段，以及未经过选择的初级受体库是如何产生的。随后我们将讨论通过什么样的机制进行阳性选择，又是什么机制一旦细胞表面表达了抗原受体，就会出现自身耐受。

本章的最后部分，我们将跟踪关注新生淋巴细胞当其离开中枢淋巴器官迁移至外周淋巴

组织时的命运，某些进一步的成熟就在外周淋巴组织中发生。成熟淋巴细胞连续不断地在血液和外周淋巴组织中再循环。没有发生感染时，尽管不断有新的细胞产生，但淋巴细胞的总数是保持相对恒定的。我们还将探讨控制外周淋巴器官中初始淋巴细胞存活的因素，从而使淋巴细胞保持动态平衡的。

图7.1显示了B细胞生活史的主要阶段，T细胞则见图7.2。本章中我们将介绍这些细胞要在外周成熟淋巴细胞种群中占有一席之地的发育阶段。我们将在8-10中讨论成熟淋巴细胞生活史的最后一个阶段，在这个阶段中淋巴细胞接触外来抗原，并活化成为效应细胞或记忆细胞。本章的最后一部分内容包括对淋巴瘤的探讨，淋巴瘤代表了在细胞增殖中逃脱了正常控制的细胞，另外也因为它们具有T细胞和B细胞不同发育阶段的特征而引人关注。

图7.1 B细胞来源于骨髓中的淋巴祖细胞

在发育的第一阶段，骨髓中的祖B细胞重排了它们的免疫球蛋白基因。这个阶段是不依赖于抗原的，但依赖于它们与骨髓基质细胞的相互作用（第一张图）。这个阶段终止于未成熟B细胞，它们携带有以细胞表面IgM形式存在的抗原受体，并且能与所处环境中的抗原发生相互作用。这个阶段的未成熟B细胞受到抗原强烈刺激后会死亡或在阴性选择过程失活，因此许多自身反应性B细胞会清除出细胞库中（第二张图）。发育的第三阶段，存活下来的未成熟B细胞迁移到外周，成熟并表达IgD和IgM。当它们在二级淋巴器官中遇到相特异性外来抗原时，就可以活化（第三张图）。活化的B细胞增殖并分化为分泌抗体的浆细胞和长寿的记忆细胞（第四张图）。

图7.2 T细胞的发育

T细胞发育的过程大部分与B细胞相似。T细胞前体从骨髓迁移至胸腺，并重排T细胞受体基因（第一张图），与自身MHC分子相配的α∶βT 细胞受体在与胸腺上皮的相互作用中，给细胞传递了一个生存信号，使该细胞通过阳性选择。自身反应性受体转导了导致细胞死亡的信号，因而在阴性选择过程中这些细胞就从谱中被清除（第二张图）。通过选择存活下来的T细胞则成熟并离开胸腺至外周循环，它们不断地离开血液迁移通过外周淋巴器官，并

在此它们可能会接触到其特异性外来抗原并活化（第三张图）。活化后引起细胞增殖并分化为效应T细胞。这些细胞被吸引至感染位点，杀伤感染细胞或者激活巨噬细胞（第四张图）；其他T细胞则被吸引至B细胞聚居区，可以辅助并激活抗体应答（未显示）。

哺乳动物中淋巴细胞的发育很大部分是在中枢淋巴器官的特定环境中发生的——B细胞在骨髓（胎儿在肝脏）而T细胞在胸腺。对于胎儿和青少年来说，这些组织是大量新生淋巴细胞的来源地，然后这些新生淋巴细胞会迁移并居留于外周淋巴组织中。在成熟个体中，胸腺中新生T细胞的发育减缓，T细胞的数量是通过中枢淋巴器官以外的成熟T细胞分裂而维持的。另一方面，新生的B细胞却能不断地从骨髓产生，成年个体也不例外。

本章这一部分介绍的是这些主要淋巴细胞增殖环境的本质以及淋巴细胞经过的发育阶段。这些阶段的确定主要是根据功能性抗原受体基因在组装和表达过程中的不同步骤，以及区分不同功能类型T和B细胞的形态特征。在淋巴细胞发育过程中的每一步，基因重排的进程都受到监控。一次成功的基因重排导致蛋白链的合成就是细胞进入下一步发育的一个信号。

T细胞的发育比B细胞的发育复杂得多，因为T细胞具有两种不同的细胞类群，分别携带两种不同类型的T细胞受体即α︰β和γ︰δ，因此这两种类型细胞的产生必需协调（见4-13）。α︰β和γ︰δT细胞在T细胞的发育早期就分开了。α︰βT细胞进一步分化为CD4和CD8 T细胞（见3-12），这发生在T细胞受体基因已重组并表达后的未成熟T细胞中。

7-1 淋巴细胞在特定的环境中发育并受到抗原受体基因体细胞重排的调节

在前体细胞发育成表达抗原特异性受体——分别是免疫球蛋白和T细胞受体——功能性B或T细胞的过程中，都应用了某些基本原则。在小鼠和人中，淋巴细胞的分化过程依次发生在有序的各个阶段，这些阶段包括抗原受体基因连续的重排步骤及其蛋白质产物的表达，以及其他细胞表面和胞内蛋白质的表达变化。这一内在发育程序的顺利完成需要淋巴细胞发育时所处特定微环境的信号——B细胞是骨髓和胎儿肝脏，而T细胞则是胸腺。这些组织提供了一个特定非淋巴基质细胞（stromal cells）组成的网络，它们与发育中的淋巴细胞密切地相互作用，并通过分泌生长因子以及表达细胞表面分子通过与淋巴细胞前体的受体结合而提供信号。

每个淋巴细胞的抗原特异性是由抗原受体可变区（V）基因决定的，是通过V、D和J 基因片段组装产生的重排V基因编码的（见4-2和4-11）。表达一个完整的抗原受体需要两个不同基因位点的成功重排，这样才能产生抗原受体的蛋白链，即免疫球蛋白的重链和轻链或者T细胞受体的α和β链（或γ和δ链）。然而并不是所有的基因重排都是成功的。由于重排的过程并不严密，因此并不非所有的重排都能产生完整的、可以翻译成蛋白质的DNA 阅读框内序列。V区的成功组装需要对每个基因位点进行监控，并由此界定了淋巴细胞发育的不同阶段。一个成功的基因重排称为生产性重排（productive rearrangement），能够导致蛋白质产物的合成；这也是细胞进入下一个发育阶段的信号。

例如，B细胞重链阅读框内VDJ序列的组装导致了重链的表达；而这是细胞作为停止重链位点重排的信号，细胞分裂几次后开始轻链位点的重排。不能产生蛋白质的不成功重排称为非生产性重排（nonproductive rearrangements），如果没有发生进一步的重排挽救这种局面的话，淋巴细胞就会死亡。

因此，B细胞为了通过这些发育的最初阶段而存活，必须在免疫球蛋白的重链位点以及两个轻链位点κ或λ中任一个基因位点上进行生产性重排。而T细胞则必须在α和β链位点上进行生产性重排，产生α︰β T细胞，或者在γ和δ链位点上进行生产性重排，产生γ︰δ T细胞。那些未能进行必需的生产性重排的细胞会在原位发生凋亡。

导致B细胞携带有免疫球蛋白B细胞受体的事件以及Ｔ细胞携带有α︰β T细胞受体的事件是紧密相关的，我们在此要讨论这些事件，而把同样在最早Ｔ细胞前体中也发生的γ和δ链基因位点重排等复杂因素搁置一边，本章的后面部分将会回到这些内容上。一次只能在一个基因位点发生重排，并且每个位点都按照固定顺序进行重排。B和T细胞都是首先重排含有D基因片段的位点；对B细胞来说是免疫球蛋白重链位点，对T细胞来说则是在T 细胞受体的β链位点。只有进行了生产性重排，发育中的B细胞才能继续进行轻链位点的重排，而T细胞才能继续重排其α链位点。

每一个抗原受体基因位点的蛋白质产物都倾向于与另一条链配对表达；例如α和β链组成T细胞受体（见图3.12）。因此，发育中的T细胞在其α链位点还没有进行重排时，它是如何检测已发生重排的β链基因是否是生产性重排的呢？解决的办法是在发育的这一阶段，B和T细胞都产生恒定的替代性配对链。这些替代链与重链或β链配对产生能表达于细胞表面的“受体”。这些受体的形成就给细胞发出了信号停止VDJ的重排。紧接着细胞经过几轮分裂后进入下一阶段的发育，即B细胞轻链位点和T细胞α链位点的V与J的重排。如果这个重排是生产性的，那么淋巴细胞就能在细胞表面表达真正的免疫球蛋白或者T细胞受体了。

7-2 骨髓中的B细胞在基质细胞的辅助下发育并在外周淋巴器官达到成熟

B细胞发育依赖于骨髓中的非淋巴基质细胞；从骨髓中分离得到的干细胞在培养基中生长时不能分化成B细胞，除非骨髓基质细胞也存在的情况下才能分化。基质，该名称来源于希腊语，意为床垫，即为B细胞发育提供必要的支持。基质细胞有两方面的作用：第一，基质细胞通过细胞粘附分子及其配体的相互作用，与发育中的B系细胞形成了特定的粘附性接触；第二，基质细胞提供生长因子促进淋巴细胞的分化和增殖（见图7.3）。

图7.3 B细胞发育的早期阶段依赖于骨髓基质细胞

上图表示B细胞前体与基质细胞之间的相互作用是未成熟B细胞阶段发育所必需的。B细胞发育所定义的祖B细胞和前B细胞相应情况见图7.5。淋巴样祖细胞和早期祖B细胞通过整合素VLA-4以及其他细胞粘附分子（CAMs）与基质细胞上的粘附分子VCAM-1结合。这些粘附分子间的相互作用促进了祖B细胞表面的受体酪氨酸激酶Kit与基质细胞上的干细胞因子（SCF）结合，激活了激酶并诱导祖B细胞的增殖。稍后阶段的增殖和进一步发育则需要白介素-7（IL-7）。图a：光学显微镜显示小的圆形细胞是祖B淋巴细胞，与培养的基质细胞紧密接触，基质细胞有一些延展的突起，在其生长时将细胞固定于培养皿上。图b：高倍的电子显微镜照片显示细胞培养中的两个淋巴细胞，可见它们附着于扁平的基质细胞上。照片由A. Rolink（a）；P. Kincade and P.L. Witte（b）提供。

已经确定了许多骨髓生长因子并且阐明了其功能。干细胞因子（SCF）可以促进早期B

系细胞的生长，SCF是基质细胞表面递呈的一种膜结合型细胞因子，可以与B细胞前体细胞表面受体酪氨酸激酶Kit发生相互作用。发育中的B细胞在稍后阶段需要分泌型的细胞因子白介素-7（IL-7）。趋化因子基质细胞来源的因子-1或前B细胞生长刺激因子（SDF-1/PBSF）在B细胞发育的早期阶段发挥着重要作用，该分子基因缺陷小鼠其B细胞不能发育就证实了这一点。SDF-1由骨髓基质细胞组成性生产，它的作用之一可能是将发育中的B细胞前体保留在骨髓的微环境中。由基质细胞产生的其他粘附分子和生长因子已知在B细胞的发育中也发挥作用；这是一个研究的热门领域，目前还不能全面了解调节B细胞分化的因子。

随着B系细胞的成熟，它们在骨髓内迁移并一直保持与基质细胞的接触。最早期的干细胞位于称之为骨内膜下区的部位，这是与骨骼内表面相连的区域。随着B系细胞的成熟进程，它们会朝着骨髓腔的中心轴移动（见图7.4）。成熟过程的后期与基质细胞的接触依赖性逐渐变少，未成熟B细胞发育为成熟B细胞的最后阶段在外周淋巴器官如脾脏中进行。

图7.4 B系细胞在其成熟过程中迁移至骨髓中心轴

大鼠大腿骨横切面的荧光显微镜照片，细胞（绿色）是通过末端脱氧核苷酸转移酶（TdT）染色而鉴定的，TdT是祖B细胞阶段的标志（见图7.5）。表达TdT的早期祖B细胞聚集在骨内膜附近（骨髓内腔面），见图中右上角部分。随着发育进程，B系细胞失去TdT，并向髓腔中心移动（左下角），并停留在髓窦中做好输出的准备。照片由D. Opstelten和M. Hermans 提供。

7-3 B细胞发育的不同阶段可以通过表达的免疫球蛋白链和特定的细胞表面蛋白加以鉴别

主要B细胞发育阶段通过免疫球蛋白重链和轻链基因相继重排及表达来界定（图7.5）。首先将B系细胞划分为不同发育阶段，是根据它们是否表达免疫球蛋白链，或只表达免疫球蛋白重链，或重链和轻链都表达。随后，中期分化阶段的鉴别则根据其他细胞表面蛋白的表达，以及对免疫球蛋白基因位点状态的DNA直接分析结果。

H segment occurs in early pro-B cells, generating late pro-B cells in which V H to DJ H rearrangement occurs. A successful VDJ H rearrangement leads to the expression of a complete immunoglobulin

heavy chain as part of the pre-B-cell receptor, which is found mainly in the cytoplasm and to some degree on the surface of the cell. Once this occurs the cell is stimulated to become a large pre-B cell which actively divides. Large pre-B cells then cease dividing and become small resting pre-B cells, at which point they cease expression of the surrogate light chains and express the heavy chain alone in the cytoplasm. When the cells are again small, they reexpress the RAG proteins and start to rearrange the light-chain (L-chain) genes. Upon successfully assembling a light-chain gene, a cell becomes an immature B cell that expresses a complete IgM molecule at the cell surface. Mature B cells produce a heavy chain as well as a heavy chain, by a mechanism of alternative mRNA splicing, and are marked by the additional appearance of IgD on the cell surface.

图7.5 B系细胞发育经过的几个阶段是以免疫球蛋白基因的重排和表达为标志的

干细胞还没有开始重排免疫球蛋白（Ig）基因片段；这些基因片段还是胚系结构，与所有非淋巴细胞中发现的相同。重链（H链）位点首先发生重排。在早期祖B细胞中，出现D基因片段与J H基因片段的重排，这一连接产生了晚期原B细胞，V H和DJ H的连接就发生在晚期原B细胞中发生。成功的VDJ H链的重排导致了一条完整的免疫球蛋白重链的表达，它是前B细胞受体的一部分，主要分布在细胞质中，少部分会位于细胞表面。以上事件一旦发生，细胞就会被刺激成一个大的前B细胞，它能够活跃地分裂。随后大前B细胞停止分裂成为静息状的小前B细胞，此时它们停止表达代理轻链而只在胞质中表达重链 。当细胞又一次变小时，它们会重新表达RAG蛋白并且开始重排轻链（L-链）基因。成功组装了一个轻链基因后，细胞就变成了未成熟B细胞，在细胞表面表达完整的IgM分子。通过独特的mRNA拼接机制，成熟B细胞除了生产重链μ外还生产重链δ，且以细胞表面附加的IgD 的出现为标志。

The earliest B-lineage cells are known as pro-B cells, as they are progenitor cells with limited self-renewal capacity. They are derived from pluripotent hematopoietic stem cells and are identified by the appearance of cell-surface proteins characteristic of early B-lineage cells. Rearrangement of the immunoglobulin heavy-chain locus takes place in pro-B cells; D H to J H joining at the early pro-B cell stage is followed by V H to DJ H joining at the late pro-B cell stage.

最早的B系细胞称为祖B细胞，因为它们是自我更新能力有限的祖先细胞。它们起源于多功能造血干细胞，并根据早期B系细胞表面特异性蛋白的出现加以区分。免疫球蛋白重链基因位点的重排在祖B细胞中发生；在早期祖B细胞阶段，重链的D H连接到J H；在随后的晚期祖B细胞阶段，重链的V H连接到DJ H。

Productive VDJ H joining leads to the expression of an intact heavy chain, which is the hallmark of the next main stage of development, the pre-B cell stage. The chain in l arge pre-B cells is expressed intracellularly and possibly in small amounts at the cell surface, in combination with a surrogate light chain, to form the pre-B-cell receptor. Expression of the pre-B-cell receptor signals the cell to halt heavy-chain locus rearrangement and production of the surrogate light chain, and to divide several times before giving rise to small pre-B cells, in which light-chain rearrangements begin. Once a light-chain gene is assembled and a complete IgM molecule is expressed on the cell surface, the cell is defined as an immature B cell. The expression of the immunoglobulin heavy and light chains are key milestones in this differentiation pathway. These events do more than simply delineate stages of the pathway; the expression of an intact heavy chain, and later of a complete immunoglobulin molecule, actively regulates progression from one stage to the next.

生产性的VDJ H连接导致了一条完整的μ重链的表达，μ重链是下一个主要的发育阶段即前B细胞阶段的特征。巨型前B细胞的μ链在细胞内表达，也可能会有少数μ重链与代替性轻链结合并在细胞表面表达，形成前B细胞受体。前B细胞的受体的表达给了细胞一个信号，使细胞停止重链位点的重排和替代性轻链的生产，并进行数次分裂随后产生小前B 细胞，轻链的重排就是在小前B细胞阶段开始的。一旦轻链基因完成组装并且细胞表面表达了完整的IgM分子，那么这个细胞就被定义为未成熟B细胞。Ig的重链和轻链的表达是这种分化途径上非常重要的里程碑；完整的重链和随后的完整的免疫球蛋白分子的表达有效地调节了从一个阶段到下一个阶段的进程。

All development up to this point has taken place in the bone marrow and is independent of antigen. Immature B cells now undergo selection for self-tolerance and subsequently for the ability to survive in the peripheral lymphoid tissues. B cells that survive in the periphery undergo further differentiation to become mature B cells that express IgD in addition to IgM. These cells, also called naive B cells until they encounter their specific antigen, recirculate through peripheral lymphoid tissues, where they may encounter and be activated by the appropriate foreign antigen.

在这个关键点之前的发育都发生在骨髓中，并且不依赖于抗原。接着未成熟的B细胞会经历对自身耐受及在外周淋巴组织存活能力的选择。在外周存活下来的B细胞进一步分化成为不仅表达IgM也表达IgD的成熟B细胞。这些细胞在与相应的特异性抗原相遇之前也被称为幼稚细胞，它们会在外周淋巴组织中重复循环，也可能会在其中遇到合适的外来抗原并被激活。

As B cells develop from pro-B cells to mature B cells, they express proteins other than immunoglobulin that are characteristic of each stage. Many of these proteins are expressed on the cell surface and are useful markers for B-lineage cells at different developmental stages. Fig. 7.6 summarizes their expression patterns. The functions of some of these proteins are understood, whereas others serve at present simply as useful signposts for the study of B-cell development. One of the first identifiable proteins expressed on the surface of B-lineage cells is CD45R (known in mice as B220). This is a B-cell-specific form of the CD45 protein originally known as the common leukocyte antigen; T cells, monocytes, and neutrophils express other variants of this protein. CD45R, which is expressed throughout B-cell development from pro-B cells right up to the antibody-secreting plasma cells, is a protein tyrosine phosphatase that functions in B-cell receptor signaling (see Section 6-7). Another protein that is expressed throughout B-cell development is CD19, which also participates in B-cell receptor signaling. Since signaling through the pre-B and B-cell receptors guides B-cell development, the earliest B-cell precursors begin to assemble the signaling components of the receptor complex including CD45R and CD19. The earliest B-cell precursors also express the receptor for IL-7, which is an essential growth factor for both developing B cells and T cells. Blocking signaling by infusion of an anti-IL-7 antibody will halt B-cell development, as will mutations that inactivate either IL-7 or its receptor.

随着B细胞从祖B细胞发育到成熟B细胞，它们除表达Ig之外还表达每一阶段的标志性蛋白。很多标志性蛋白表达在细胞表面并能作为B系细胞不同发育阶段的有用标记。图7.6总结了它们的表达模式。我们已了解了其中某些蛋白质的功能，而其他的蛋白质在目前仅被当成研究B细胞发育时有用的路标。在B系细胞表面表达的首次被鉴定的蛋白之一是CD45R（鼠中是B220）。这是CD45的B细胞特异性形式，最初被认为是普遍白细胞抗原；T细胞、单核细胞和中心粒细胞则表达这一蛋白的其他变体。从祖B细胞到分泌抗体的浆细胞的发育全过程中都表达的CD45R是一种在B细胞受体信号转导中起作用的酪氨酸磷酸酶（见6-7）。另一种在B细胞发育的全过程都表达的是CD19，它也参与了B细胞受体的信

号转导。前B和B细胞受体的信号转导支配着B细胞的发育，因此最早期的B细胞前体就开始组装受体复合物的信号转导组分包括CD45R和CD19。最早期的B细胞前体也会表达IL-7的受体，IL-7是发育中的B细胞和T细胞必需的生长因子。通过注入抗IL-7的抗体传达的阻断信号可以阻止B细胞的发育，而导致IL-7或其受体失活的突变也会产生同样的结果。

segment rearrangement and expression of cell-surface proteins. Stages of B-cell development are defined by which gene segments are undergoing rearrangement as well as by cell-surface proteins. The earliest B-lineage surface markers are CD19 and CD45R (B220 in the mouse). The expression of these persists throughout B-cell development. A pro-B cell is also distinguished by expression of CD43, c-Kit, and the IL-7 receptor. An early pro-B cell is rearranging D to J H and a late pro-B cell V H to DJ H. A late pro-B cell starts to express CD24 and CD25. When a late pro-B cell makes a successful VDJ join, it can then express an immunoglobulin heavy chain, which defines it as a pre-B cell. A pre-B cell is phenotypically distinguished by expression of BP-1, whereas c-Kit and the IL-7 receptor are no longer expressed. Before going on to rearrange a light-chain locus, a pre-B cell enlarges and undergoes several cycles of cell division. It then becomes a small pre-B cell, which can rearrange light-chain gene segments, first at the locus and, if not successful, then at the locus. A cell that has made a productive light chain becomes an immature B cell expressing surface IgM, and has completed the antigen-independent phase of B-cell development.

图7.6 B细胞发育各阶段与基因片段重排及细胞表面蛋白质表达之间的相互关系

B细胞发育阶段是由什么基因正在重排以及细胞表面的蛋白质来确定的。最早期的B系细胞的表面标志是CD19和CD45R（在老鼠中是B220）。这些蛋白质的表达贯穿了B细胞的发育过程。原B细胞也可以根据CD43，c-Kit，和IL-7受体的表达来鉴别。一个早期的原B

细胞正在进行D和J H的连接，而晚期原B细胞则将V H连接到DJ H上。晚期原B细胞开始表达CD24和CD25。当晚期原B细胞成功地将VDJ连接到一起时，它就会表达免疫球蛋白重链，标志着它成为了一个前B细胞。前B细胞在表型上以BP-1的表达来区别，而c-Kit 和IL-7受体则不再表达。在重排轻链基因位点之前，前B细胞会膨胀并且经历几轮的细胞分裂。随后它们就变成了小前B细胞，能对轻链基因片段进行重排，首先是在κ基因位点，如果没有成功，就会在λ基因位点继续重排。完成了轻链生产性重排的细胞就成为了未成熟B细胞，会表达表面IgM，且它已经完成了抗原依赖性的B细胞发育阶段。

Another cell-surface protein first detected at the pro-B cell stage is CD43 (the mucin leukosialin), but this is lost as cells progress to become immature B cells. CD43 functions both as an adhesion molecule that may guide cell-cell inter-actions for example those of B-cell precursors with stromal cells and also as a signaling molecule, although not as part of the B-cell receptor complex. Exactly why CD43 is expressed at these early stages of B-cell development, but not later, is not yet known. Other cell-surface molecules expressed during early stages of B-cell development include the heat-stable antigen (HSA, CD24) and the aminopeptidase BP-1. The functions of these molecules in B-cell development are unknown, although B cells apparently develop normally in mice lacking BP-1. At the late pro-B cell stage, Kit, the receptor for SCF-1 mutates. Kit is a factor that stimulates both lymphoid and myeloid development in the bone marrow. After Kit turns off, at the large pre-B cell stage, the low affinity IL-2 receptor, CD25, is expressed. Signaling through Kit, in concert with IL-7 and other stromal-derived signals, promotes pro- and pre-B cell proliferation (see Fig. 7.3).

在祖B细胞阶段首先检测到的另外一个细胞表面蛋白质是CD43（白细胞唾液酸蛋白粘液素），但它会随着细胞逐步发展成未成熟B细胞而被丢失。CD43的功能既作为一个引导细胞与细胞间相互作用——如B细胞前体与基质细胞的相互作用——的粘附分子，也作为B 细胞受体复合物的一部分。目前我们还不确知为什么CD43要在B细胞发育的这个阶段而非稍后的阶段表达。其他在B细胞发育早期阶段表达的细胞表面分子包括热稳定抗原（HAS，CD24）和氨基肽酶BP-1。这些分子在B细胞发育过程中的功能我们还不了解，尽管缺失BP-1的老鼠中B细胞明显能够正常地发育。在晚期祖B细胞阶段，SCF-1的Kit受体发生变异。Kit是一个能刺激淋巴系和髓系细胞在骨髓中发育的因素。在Kit关闭之后的巨型前B细胞阶段，低亲合力的IL-2受体CD25开始表达。通过Kit发生的信号转导与IL-2及其他来源于基质的信号一起刺激祖B细胞和前B细胞增殖（见图7.3）。

7-4 T细胞也起源于骨髓，但在其发育过程中所有的重要事件都发生于胸腺

T lymphocytes develop from a common lymphoid progenitor in the bone marrow that also gives rise to B lymphocytes, but those progeny destined to give rise to T cells leave the bone marrow and migrate to the thymus (see Fig. 7.2). This is the reason they are called thymus-dependent (T) lymphocytes or T cells. The thymus is situated in the upper anterior thorax, just above the heart. It consists of numerous lobules, each clearly differentiated into an outer cortical region the thymic cortex and an inner medulla (Fig. 7.7). In young individuals, the thymus contains large numbers of developing T-cell precursors embedded in a network of epithelia known as the thymic stroma, which provides a unique microenvironment for T-cell development analogous to that provided by the stromal cells of the bone marrow.

T淋巴细胞是由一个普通的淋巴细胞前体在骨髓中发育而来，这种淋巴细胞前体也能产生B淋巴细胞，但它们的将要发育成T细胞的子代细胞离开骨髓并迁移入胸腺中（见图7.2）。这就是为什么它们被称为胸腺依赖的T淋巴细胞或T细胞的原因。胸腺位于胸腔的前上方，也是紧挨着心脏的正上方。它具有许多小叶，每一叶分化成一个外部的皮质区——胸腺皮质区——和一内部的髓质区（见图7.7）。年轻个体的胸腺中含有大量正在发育的T细胞前体，一个被称为胸腺基质的上皮细胞组成的网络将它们包围其中并为T细胞发育提供了一个独特的微环境，与骨髓中基质细胞提供的微环境相类似。

midline of the body, above the heart, is made up of several lobules, each of which contains discrete cortical (outer) and medullary (central) regions. As shown in the diagram on the left, the cortex consists of immature thymocytes (dark blue), branched cortical epithelial cells (pale blue), with which the immature cortical thymocytes are closely associated, and scattered macrophages (yellow), which are involved in clearing apoptotic thymocytes. The medulla consists of mature thymocytes (dark blue), and medullary epithelial cells (orange), along with macrophages (yellow) and dendritic cells (yellow) of bone marrow origin. Hassall’s corpuscles are probably also sites of cell destruction. The thymocytes in the outer cortical cell layer are proliferating immature cells, whereas the deeper cortical thymocytes are mainly immature T cells undergoing thymic selection. The photograph shows the equivalent section of a human thymus, stained with hematoxylin and eosin. The cortex is darkly staining; the medulla is lightly stained. The large body in the medulla is a Hassall’s corpuscle. Photograph courtesy of C.J. Howe.

图7.7 人胸腺细胞的细胞组成

胸腺位于身体的中线上，心脏的正上方，由几个小叶组成，每片小叶包括离散的皮质区（外层）和髓质区（中心区）。如上图左半部分所示，皮质区罕有未成熟的胸腺细胞（深蓝色），带分枝的皮层细胞（浅蓝色），未成熟的皮质胸腺细胞与带分枝的皮层细胞之间有密切的联系，还有分散的巨噬细胞（黄色），巨噬细胞参与了凋亡胸腺细胞的清除。髓质中则包括成熟的胸腺细胞（深蓝色），髓质皮层细胞（桔黄色），巨噬细胞（黄色）和来源于骨髓的树突状细胞（黄色）。哈氏小体也很有可能是清除细胞的场所。外皮质层中的胸腺细胞是正在增殖的未成熟细胞，而更深处的皮质胸腺细胞则主要是正在经历胸腺选择的未成熟T细胞。图中显示的是人胸腺的横截面，用的染料是苏木精和曙红。皮质区染色很深，而髓质区则染色较浅。髓质中很大的小体是哈氏小体。照片源于C.J. Howe。

The thymic stroma arises early in embryonic development from the endo dermal and ectodermal layers of embryonic structures known as the third pharyngeal pouch and third

branchial cleft. Together these epithelial tissues form a rudimentary thymus, or thymic anlage. The thymic anlage then attracts cells of hematopoietic origin, which colonize it; these give rise to large numbers of thymocytes, which are committed to the T-cell lineage, and the intrathymic dendritic cells. The thymocytes are not simply passengers within the thymus; they influence the arrangement of the thymic epithelial cells on which they depend for survival, inducing the formation of a reticular epithelial structure that surrounds the developing thymocytes (Fig. 7.8). The thymus is independently colonized by numerous macrophages, also of bone marrow origin.

在胚胎发育早期胸腺基质开始出现，它起源于胚胎内胚层和外胚层中被称为第三咽囊和第三鳃裂的结构。同时，这些上皮组织也会形成一个未发育的胸腺，或称为胸腺原基（thymic anlage）。随后胸腺原基诱导产生造血细胞的祖先，它们会定位于胸腺原基中；这些祖先细胞能产生大量的胸腺细胞（thymocytes）——之后将发育为T系细胞——和胸腺内树突细胞（intrathymic dendritic cells）。胸腺细胞不仅仅作为胸腺的组成部分，它们还影响胸腺上皮细胞的排列，同时诱导发育中胸腺细胞周围网状上皮细胞结构的形成（图7.8）。胸腺被大量的巨噬细胞占据，它们也来源于骨髓。

图7.8 胸腺上皮细胞形成了包围发育中胸腺细胞的网络

在这张胸腺的扫描电子照片中，发育中的胸腺细胞（球形细胞）占据在上皮细胞网络中的空隙中。照片由W. van Ewijk提供。

The cellular architecture of the human thymus is illustrated in Fig. 7.7. Bone marrow derived cells are differentially distributed between the thymic cortex and medulla; the cortex contains only immature thymocytes and scattered macrophages, whereas more mature thymocytes, along with dendritic cells and macrophages, are found in the medulla. This reflects the different developmental events that occur within these two compartments, as we will discuss further in Section 7-7.

人类胸腺的细胞组成结构见图7.7。源于骨髓的细胞在胸腺皮质和髓质中被分割开来；皮质区仅包含未成熟胸腺细胞和散布着的巨噬细胞，而髓质中则包含更多的成熟胸腺细胞、树突细胞和巨噬细胞。这能反映出在这两个不同的部位发生的不同的发育事件，我们将在7-7中进一步讨论。

The importance of the thymus in immunity was first discovered through experiments on mice, and indeed, most of our knowledge of T-cell development within the thymus comes from the mouse. It was found that surgical removal of the thymus (thymectomy) at birth resulted in immunodeficient mice, focusing interest on this organ at a time when the difference between T and B lymphocytes in mammals had not yet been defined. Much evidence has accumulated since to establish the importance of the thymus in T-cell development, including observations of immunodeficient children. Thus, for example, in DiGeorge's syndrome in humans, and in mice with the nude mutation (which also causes hairlessness), the thymus fails to form and the affected individual produces B lymphocytes but few T lymphocytes.

胸腺在免疫中的重要性首先在小鼠实验中被发现，其实我们所知的大部分关于T细胞在胸腺中发育的知识都是在小鼠身上发现的。如果在刚出生时胸腺就被通过外科手术去除（胸腺切除术，thymectomy），那么小鼠就会变成免疫缺陷鼠。这使得人们在对哺乳动物的T和B淋巴细胞之间的区别未有具体定义时的研究焦点都聚集到这一器官。自从确立了胸腺在T细胞发育中的重要作用以来，我们已搜集了许多证据，包括对患有免疫缺陷的儿童的

观察。因此，例如在人的乔治症候群（DiGeorge's syndrome）以及小鼠的裸突变（还会造成没有毛发）中，胸腺都不能形成，个体能产生B淋巴细胞但却几乎没有T淋巴细胞。

The crucial role of the thymic stroma in inducing the differentiation of bone marrow-derived precursor cells can be demonstrated by tissue grafts between two mutant mice, each lacking mature T cells for a different reason. In nude mice the thymic epithelium fails to differentiate, whereas in scid mice B and T lymphocytes fail to develop because of a defect in T-cell receptor gene rearrangement (see Section 4-5). Reciprocal grafts of thymus and bone marrow between these immunodeficient strains show that nude bone marrow precursors develop normally in a scid thymus (Fig. 7.9). Thus, the defect in nude mice is in the thymic stromal cells. Transplanting a scid thymus into nude mice leads to T-cell development. However, scid bone marrow cannot develop T cells even in a wild-type recipient.

胸腺基质在诱导骨髓来源的前体细胞分化时起的关键作用可以通过在两个突变小鼠之间进行器官移植而得到验证，每只突变小鼠因为不同的原因缺乏成熟T细胞。在裸鼠中胸腺上皮不能分化，而在重症免疫缺陷老鼠（scid mice）中的B和T淋巴细胞都不能发育，因为T细胞受体基因的重排有缺陷（见4-5）。在这些免疫缺陷品系中进行胸腺和骨髓的相互移植，结果表明裸鼠的骨髓前体细胞能在scid老鼠的胸腺中正常地发育（图7.9）。因此，裸鼠的缺陷是在胸腺基质细胞。把一个scid老鼠的胸腺移植入裸鼠中T细胞发育就能进行。然而，即使将scid的骨髓移植入野生型的接受者中不能使T细胞发育。

Figure 7.9. The thymus is critical for the maturation of bone marrow-derived cells into T cells. Mice with the scid mutation (upper left photograph) have a defect that prevents lymphocyte maturation, whereas mice with the nude mutation (upper right photograph) have a defect that affects the development of the cortical epithelium of the thymus. T cells do not develop in either strain of mouse: this can be demonstrated by staining spleen cells with antibodies specific for mature T cells and analyzing them in a flow cytometer (see Appendix I, Section A-22), as represented by the blue line in the graphs in the bottom panels. Bone marrow cells from nude mice can restore T cells to scid mice (red line in graph on left), showing that, in the right environment, the nude bone marrow cells are intrinsically normal, and capable of producing T cells. Thymic epithelial cells from scid mice can induce the maturation of T cells in nude mice (red line in graph on right), demonstrating that the thymus provides the essential microenvironment for T-cell development.

图7.9 胸腺对起源于骨髓的细胞成熟为T细胞至关重要

scid突变的小鼠（图左上角）有缺陷，能够阻止淋巴细胞的成熟，然而nude突变的老鼠（图右上角）的缺陷则能影响胸腺细胞皮质上皮细胞的发育。T细胞在两个品系中的小鼠中都不会发育：用成熟T细胞特异性抗体对脾脏细胞染色并用流式细胞仪进行分析（见附录I，A-22），图中底部用蓝线表示的就是。nude小鼠骨髓细胞可以使scid小鼠中重新出现T细胞

（图中左边部分的红线），这表明在适当的环境中，nude骨髓细胞实际上是正常的，能够产生T细胞。scid小鼠中的胸腺上皮细胞能够诱导nude小鼠中的T细胞成熟（图中右边部分的红线），这表明胸腺给T细胞发育提供了一个不可或缺的微环境。

In mice, the thymus continues to develop for 3 to 4 weeks after birth, whereas in humans it is fully developed at birth. The rate of T-cell production by the thymus is greatest before puberty. After puberty, the thymus begins to shrink and the production of new T cells in adults is lower, although it does continue throughout life. In both mice and humans, removal of the thymus after puberty is not accompanied by any notable loss of T-cell function. Thus, it seems that once the T-cell repertoire is established, immunity can be sustained without the production of significant numbers of new T cells; the pool of peripheral T cells is instead maintained by the division of mature T cells.

老鼠的胸腺在出生后会继续发育3到4个星期，然而人类的胸腺则在出生前已经完成发育。青春期之前胸腺产生T细胞的速度最快。青春期后，胸腺开始萎缩，虽然在人的一生中新生T细胞一直在生成，但成年人中新生T细胞的产量较低。在小鼠和人中，青春期以后切除胸腺并不会伴随着T细胞功能的显著缺失。因此，似乎一旦T细胞库被建立，即使不能产生大量的新生T细胞免疫力也能得到维持；外周的T细胞库能够通过成熟T细胞的分裂稳定地维持。

7-5 大部分正在发育的T细胞在胸腺中死亡

T-cell precursors arriving in the thymus from the bone marrow spend up to a week differentiating there before they enter a phase of intense proliferation. In a young adult mouse the thymus contains around 108 to 2 × 108 thymocytes. About 5 × 107 new cells are generated each day; however, only about 106 to 2 × 106 (roughly 24%) of these will leave the thymus each day as mature T cells. Despite the disparity between the numbers of T cells generated daily in the thymus and the number leaving, the thymus does not continue to grow in size or cell number. This is because approximately 98% of the thymocytes that develop in the thymus also die within the thymus. No widespread damage is seen, indicating that death is occurring by apoptosis rather than by necrosis (see Section 1-11).

从骨髓中出来到达胸腺的前体T细胞进入快速扩增期之前需要花不到一星期以上的时间在胸腺中分化。年轻的成熟小鼠胸腺中大约含有108到2 ×108个胸腺细胞。每天大约产生5 × 107个新生细胞；然而，其中大约只有106到2 × 106个（约为24%）新生细胞能够作为成熟T细胞离开胸腺。尽管每天在胸腺中产生的T细胞的数量与离开胸腺的细胞数量不一致，然而胸腺却并未在体积上持续增大，其中细胞数量也并未持续增多。这是因为约有98%的在胸腺中发育的胸腺细胞也会在其中死亡。并未发现有蔓延开来的损害作用，这就表明死亡是由于凋亡而非坏死造成的（见1-11）。

Changes in the plasma membrane of cells undergoing apoptosis lead to their rapid phagocytosis, and apoptotic bodies, which are the residual condensed chromatin of apoptotic cells, are seen inside macrophages throughout the thymic cortex (Fig. 7.10). This apparently profligate waste of thymocytes is a crucial part of T-cell development as it reflects the intensive screening that each new thymocyte undergoes for the ability to recognize self MHC and for self tolerance.

正在经历凋亡的细胞中质膜的变化包括导致它们很快被吞噬，凋亡小体——凋亡细胞残留染色质的浓缩物——可以在遍及胸腺皮质的巨噬细胞内看到（图7.10）。这种对胸腺细胞

的很显然的肆意浪费是T细胞发育中很关键的一部分，因为它反映了对胸腺细胞严格的筛选，即每个新生胸腺细胞都要经历一个对其识别自身MHC和自身免疫耐受能力的选择过程。

Figure 7.10. Developing T cells that undergo apoptosis are ingested by macrophages in the thymic cortex.Panel a shows a section through the thymic cortex and part of the medulla in which cells have been stained for apoptosis with a red dye. Thymic cortex is to the right of the photograph. Apoptotic cells are scattered throughout the cortex but are rare in the medulla. Panel b shows a section of thymic cortex at higher magnification that has been stained red for apoptotic cells and blue for macrophages. The apoptotic cells can be seen within macrophages. Magnifications: panel a, ×45; panel b, ×164. Photographs courtesy of J. Sprent and C. Suhr, reprinted with permission from Nature 372:100-103, ?1994 Macmillan Magazines Limited.

图7.10 进入凋亡的发育中的T细胞能被胸腺皮质中的巨噬细胞消化

a图展示的是胸腺皮质以及部分髓质的横切面，髓质中染上红色的细胞是凋亡细胞。胸腺皮质直延伸到照片的右半边。凋亡细胞遍及皮质区但在髓质中却很少。b图展示的是放大了更高倍数的胸腺皮质，着红色的是凋亡细胞，着蓝色的是巨噬细胞。巨噬细胞内能够找到凋亡细胞。放大倍数：a图×45；b图×164。照片源自J. Sprent和C. Suhr。此次重新印刷征得了《自然》372:100-103, ?1994 Macmillan Magazines Limited的同意。

7-6 胸腺细胞发育过程中的每个阶段可以通过细胞表面分子的变化加以识别

Developing thymocytes pass through a series of distinct phases that are marked by changes in the status of T-cell receptor genes and in the expression of the T-cell receptor, and by changes in expression of cell-surface proteins such as the CD3 complex and the co-receptor proteins CD4 and CD8. These surface changes reflect the state of functional maturation of the cell. Particular combinations of cell-surface proteins can thus be used as markers for T cells at different stages of differentiation. The principal stages are summarized in Fig. 7.11. Two distinct lineages of T cells : and : , which have different types of T-cell receptor are produced early in T-cell development. Later, : T cells develop into two distinct functional subsets, CD4 and CD8 T cells.

发育中的胸腺细胞会经历一系列不同的阶段，它们以T细胞受体基因状态的变化、T细胞受体的表达以及细胞表面蛋白质如CD3复合物和共受体蛋白质CD4和CD8的表达的变化为标志。这些表面的变化反映了细胞在功能上的成熟情况。因此细胞表面蛋白质的特殊组合就用来作为T细胞在不同分化阶段的特征了。主要的阶段总结在图7.11中。两类不同品系的T细胞——α:β和γ:δ，它们含有不同类型的T细胞受体——是在T细胞发育的早期阶段产生的。随后，α:β T细胞发育为两群功能亚类，CD4和CD8 T细胞。

Figure 7.11. at different stages of maturation to be distinguished.The most important cell-surface molecules for identifying thymocyte subpopulations have been CD4, CD8, and T-cell receptor complex molecules (CD3, and the T-cell receptorɑandβchains). The earliest cell population in the thymus does not express any of these. As these cells do not express CD4 or CD8, they are called ‘double-negative’ thymocytes. These precursor cells give rise to two T-cell lineages, the minority population of ɑ:βT cells (which lack CD4 or CD8 even when mature), and the majority ɑ:βT-cell lineage. Development of prospective ɑ:βT cells proceeds through stages where both CD4 and CD8 are expressed by the same cell; these are known as ‘double-positive’ thymocytes. At first, double-positive cells express the pre-T-cell receptor (pTɑ:β). These cells enlarge and divide. Later, they become small resting double-positive cells in which low levels of the T-cell receptor (ɑ:β) itself are expressed. Most thymocytes (~97%) die within the thymus after becoming small double-positive cells. Those cells whose receptors can interact with self MHC molecules lose expression of either CD4 or CD8 and increase the level of expression of the T-cell receptor. The outcome of this process are the ‘single-positive’ thymocytes, which, after maturation, are exported from the thymus as mature single-positive CD4 or CD8 T cells.

图7.11 细胞表面分子的变化使得处在成熟过程中不同阶段的胸腺细胞类群能被区分

区分胸腺细胞亚群的最重要的细胞表面分子是CD4，CD8和T细胞受体复合物（CD3，和T 细胞受体ɑ和β链）。胸腺中最早期的细胞类群不表达上述分子中的任何一种。因为这些细胞不表达CD4和CD8，所以被称为双阴性胸腺细胞。这些前体细胞能产生两个T细胞系，数量较少的类群ɑ:βT细胞（直到成熟时仍然缺乏CD4或CD8），以及数量众多的ɑ:βT 细胞系。预期的ɑ:βT细胞发育会经历CD4和CD8在一个细胞上共同表达的时期；这个阶段的细胞就被成为双阳性胸腺细胞。首先，双阳性细胞表达前T细胞受体（pTɑ:β）。这些细胞会膨胀并且分裂。稍后，它们变成小的静息状双阳性细胞，它们的T细胞受体（ɑ:β）的表达量很少。大部分胸腺细胞（～97％）变成小的双阳性细胞后会在胸腺中死亡。那些受体能与自身MHC分子相互作用的细胞会失去CD4或CD8的表达能力，并增加T细胞受体的表达水平。这个过程的结果是单阳性胸腺细胞，它在成熟后作为成熟的单阳性CD4或CD8 T细胞会从胸腺中被运走。

When progenitor cells first enter the thymus from the bone marrow, they lack most of the surface molecules characteristic of mature T cells and their receptor genes are unrearranged. These cells give rise to the major population of : T cells and the minor population of : T cells. If injected into the peripheral circulation, these lymphoid progenitors can even give rise to B cells and NK cells (see Section 1-1). Interactions with the thymic stroma trigger an initial phase of differentiation along the T-cell lineage pathway followed by cell proliferation, and the expression of the first cell-surface molecules specific for T cells, for example CD2 and (in mice) Thy-1. At the end of this phase, which can last about a week, the thymocytes bear distinctive markers of the T-cell lineage, but they do not express any of the three cell-surface markers that define mature T cells. These are the CD3:T-cell receptor complex and the co-receptors CD4 or CD8. Because of the absence of CD4 and CD8 such cells are called 'double-negative' thymocytes (see Fig. 7.11).

当祖先细胞从骨髓中首先进入胸腺时，它们不具有大部分的成熟T细胞表面的特征分子，它们的受体基因也还未发生重排。这些细胞能产生数量较大的α:βT细胞类群和数量较

小的γ:δT细胞类群。如果直接将这些淋巴细胞祖先注射入外周淋巴循环中，它们也能产生B细胞和NK细胞（见1-1）。与胸腺基质的相互作用引发了T系细胞进入分化通路上在细胞增殖之前的一个很重要的阶段，并诱导第一个T细胞特有表面分子的表达，如CD2和（老鼠的）Thy-1。这个阶段的末期可以持续一个星期左右，这时的胸腺细胞携带了T系细胞特征分子，但是它们不表达三类成熟T细胞的标记分子，即CD3:T细胞受体复合物和共受体CD4和CD8。这些细胞因缺失了CD4和CD8而被称为“双阴性”胸腺细胞（见图7.11）。

In the fully developed thymus, these immature double-negative T cells form approximately 60% of a small, highly heterogeneous pool of CD4- CD8- cells (about 5% of total thymocytes), which also includes two populations of more mature T cells that belong to minority lineages. One of these, representing about 20% of all the double-negative cells in the thymus, or 1% of total thymocytes, comprises cells that have rearranged and are expressing the genes encoding the γ:δT-cell receptor; we will return to these cells in Section 7-13. The second, representing another 20% of all double negatives, includes cells bearing α:β T-cell receptors of a very limited diversity; these cells also express the NK1.1 receptor commonly found on NK cells, hence they are known as 'NK1.1+ T cells' (sometimes simply called NK T cells). NK T cells are activated as part of the early response to many infections; they differ from the major lineage of α:β T cells in recognizing CD1 molecules rather than MHC class I or MHC class II molecules (see Section 5-18) and they are not shown in Fig. 7.11. In this and subsequent discussions, we will reserve the term double-negative thymocytes for the immature thymocytes that do not yet express a complete T-cell receptor molecule. These cells give rise to bothγ:δand α:β T cells (see Fig. 7.11). Most of them develop along the α:β pathway.

在发育完全的胸腺中，未成熟的双阴性T细胞约占数量小但具有高度异源性的CD4- CD8- 细胞库的大约60％（约占全体胸腺细胞的5％），这个细胞库中也包括更多的属于少数世系的成熟T细胞。其中的一个占了胸腺内所有双阴性细胞的总数的20%或全体胸腺细胞的1%的世系包括了已经对γ:δT细胞受体基因进行了重排和表达的那些细胞；我们将在7－13中再次提到这些细胞。第二个占双阴性细胞总数20％的世系包括携带多样性非常有限的α:β T细胞受体的细胞；这些细胞也能表达常能在NK细胞表面找到的NK1.1受体，因此它们也被称为“NK1.1+ T细胞”（有时简称为NK T细胞）。NK T细胞在对很多感染的早期反应中积极地发挥作用；它们不同于主要世系的α:β T细胞，α:β T细胞识别的是MHC I 类或MHC II类分子（见5－18），而NK T细胞识别的是CD1分子，并且图7.11中也未将它们表示出来。在接下来的讨论部分中，我们将继续把那些还未表达完整的T细胞受体分子的未成熟胸腺细胞称为双阴性胸腺细胞。这些细胞可以产生γ:δ和α:βT细胞（见图7.11）。它们中的大部分沿着α:β通路发育。

The α:βpathway is shown in more detail in Fig. 7.12. The double-negative stage can be subdivided on the basis of expression of the adhesion molecule CD44, CD25 (the α chain of the IL-2 receptor), and c-Kit, the receptor for the hematopoietic cytokine, stem cell factor. At first, double-negative thymocytes express c-Kit and CD44 but not CD25; in these cells, the genes encoding both chains of the T-cell receptor are in the germline configuration. As the thymocytes mature further, they begin to express CD25 on their surface and, later still, expression of CD44 and c-Kit is reduced. In these latter cells, which are known as CD44low CD25+cells, rearrangement of the T-cell receptor β-chain locus occurs. Cells that fail to make a successful rearrangement of the β locus remain in the CD44low CD25+ stage and soon die, whereas cells that make productive β-chain gene rearrangements and express the β chain lose expression of CD25 once again. The functional significance of the transient expression of CD25 is unclear; T cells

develop normally in mice in which the IL-2 gene has been deleted by gene knockout (see Appendix I, Section A-47); however, another cytokine, IL-15, also binds to the IL-2 receptor and may be able to compensate for the loss of IL-2. By contrast, c-Kit is quite important for the development of the earliest double-negative thymocytes in that mice lacking c-Kit have a markedly reduced number of double-negative T cells. In addition, the IL-7 receptor is also essential for early T-cell development, as T cells do not develop in either humans or mice when this receptor is defective.

α:β通路在图7.12中有更为详细的描述。双阴性阶段可以根据粘附分子CD44,CD25（IL-12受体的α链），c-Kit，生血细胞因子的受体以及干细胞因子的表达而被进一步划分。一开始，双阴性胸腺细胞表达c-Kit和CD44而不会表达CD25；在这些细胞中，编码T细胞受体两条链的基因处于原型状态。随着胸腺细胞的进一步发育，它们开始在表面表达CD25，稍后CD44和c-Kit也被诱导表达。这些后期的细胞也被称为CD44low CD25+ 细胞，它们的T细胞受体β链基因位点的重排开始了。那些未能完成β链基因位点重排的细胞滞留在CD44low CD25+ 阶段很快就会死亡。然而那些进行了生产性β链基因重排并表达了β链的细胞又再一次不会表达CD25了。CD25瞬时表达的功能特征现在还未研究清楚；T细胞在IL-2基因已被敲除的老鼠中能够正常发育（见附录I，A-47）；尽管如此，另一种细胞因子IL-15也能结合IL-2受体，并且它有可能可以补偿IL-2的缺失。另一方面，c-Kit对于最早期的双阴性胸腺细胞的发育非常重要，因为缺失c-Kit的老鼠的双阴性T细胞的数量明显少很多。另外，IL-7受体对早期T细胞发育也很重要，因为T细胞在缺失这种受体的人和老鼠中都不能发育。

Figure 7.12. The correlation of stages of : T-cell development with T-cell receptor gene rearrangement and expression of cell-surface proteins.Lymphoid precursors are triggered to proliferate and become thymocytes committed to the T-cell lineage through interactions with the thymic stroma. These negative cells express CD44 and c-Kit and, at a later stage, the chain of the IL-2 receptor, CD25. After this, the CD44+ CD25+ cells begin to rearrange the -chain locus, becoming CD44low and c-Kit low as this occurs. The cells are arrested in the CD44low CD25+ stage until they productively rearrange the -chain locus; the in-frame chain then pairs with the surrogate pT chain and is expressed on the cell surface, which triggers entry into the cell cycle. Expression of pT : on the cell surface is associated with small amounts of CD3, and c a uses the loss of CD25, cessation of -chain gene rearrangement, cell proliferation, and the expression of

CD4 and CD8. After the cells cease proliferating and revert to small CD4+ CD8+ double-positive cells, they begin rearrangement at the -chain locus. The cells then express low levels of an : T-cell receptor and the associated CD3 complex and are ready for selection. Most cells die by failing to be positively selected or as a consequence of negative selection, but some are selected to mature into CD4 or CD8 single-positive cells and eventually to leave the thymus.

图7.12 α:β T的发育阶段与T细胞受体基因重排和细胞表面蛋白质表达之间的联系淋巴细胞前体被诱导增殖并且变成T细胞系的胸腺细胞，它们与胸腺基质细胞之间有相互作用。这些阴性细胞会表达CD44和c-Kit，以及在稍后的阶段表达的IL-2受体的α链，即CD25。在这之后，CD44+CD25+细胞开始在 链基因位点发生重排，变成CD44 low和c-Kit low。在β链基因位点完成生产性重排之前细胞会滞留在CD44low CD25+阶段，β链随后就会与替代的pT 链配对，并且表达在细胞表面，诱导细胞进入细胞周期。pT : 在细胞表面的表达与少量的CD3相关，并且导致了CD25的丢失，β链基因重排的停止，细胞的增殖，CD4和CD8的表达。细胞停止增殖并回复到CD4+CD8+双阳性细胞时，它们就开始在 链基因位点上发生重排。随后细胞表达低水平的 : T细胞受体和相关的CD3复合物，并为被选择做好准备。大部分的细胞因未能被阳性选择或因阴性选择的发生而死亡，但是有些则被选择成为CD4或CD8单阳性细胞并最终离开胸腺。

The β chains expressed by CD44low CD25+ thymocytes pair with a surrogate chain called pTa (pre-T-cell α), which allows them to assemble a pre-T-cell receptor that is analogous in structure and function to the pre-B-cell receptor. The pre-T-cell receptor is expressed on the cell surface as a complex with the CD3 molecules that provide the signaling components of T-cell receptors (see Section 6-6). The assembly of the CD3:pre-T-cell receptor complex leads to cell proliferation, the arrest of further β-chain gene rearrangements, and the expression of CD8 and CD4. These double-positive thymocytes comprise the vast majority of thymocytes. Once the large double-positive thymocytes cease to proliferate and become small double-positive cells, the -chain locus begins to rearrange. As we will see later in this chapter, the structure of the locus allows multiple successive rearrangement attempts, so that a successful rearrangement at the -chain locus is achieved in most developing thymocytes. Thus most double-positive cells produce an : T-cell receptor.

CD44low CD25+ 胸腺细胞表达的β链与一称为pTa（前T细胞α）的替代链配对，这样它们就可以组装成一个功能和结构都与前B细胞受体相似的前T细胞受体。前T细胞受体以与CD3分子形成复合物的形式表达在细胞表面，CD3分子是一个T细胞受体种转导信号的组分（见6-6）。CD3:前T细胞受体复合物的组装导致了细胞增殖、进一步的β链基因重排的开始以及CD8和CD4的表达。双阳性胸腺细胞包括了大部分的胸腺细胞。一旦大双阳性胸腺细胞不再增殖变成小双阳性胸腺细胞，α链基因位点就开始重排。我们还将在这一张的后面部分提到，？基因位点的结构允许多样性的连续重排，所以大多数正在发育的胸腺细胞都能在？位点成功地重排。因此，大部分双阳性细胞会产生α:β T细胞受体。

Small double-positive thymocytes initially express low levels of the T-cell receptor. Most of these cells bear receptors that cannot recognize self MHC molecules; they are destined to fail positive selection and die. On the other hand, those double-positive cells that recognize self MHC and can therefore undergo positive selection, go on to mature and express high levels of the T-cell receptor. Concurrently, they cease to express one or other of the two co-receptor molecules, becoming either CD4 or CD8 single-positive thymocytes. Thymocytes also undergo negative selection during and after the double-positive stage in development, which eliminates those cells

capable of responding to self antigens. Approximately 2% of the double positives survive this dual screening and mature as single-positive T cells that are gradually exported from the thymus to form the peripheral T-cell repertoire. The time between the entry of a T-cell progenitor into the thymus and the export of its mature progeny is estimated to be around 3 weeks in the mouse.

小双阳性胸腺细胞最初表达T细胞受体的水平很低。这些细胞中的大部分携带的是不能识别自身MHC分子的受体，它们注定不能完成阳性选择而会死亡。另一方面，那些能识别自身MHC且能够通过阳性选择阶段的双阳性细胞就会继续成熟并表达高水平的T细胞受体。同时，它们停止表达两种共受体分子中的其中一种，变成CD4或CD8单阳性胸腺细胞。在发育过程中的双阳性阶段之后胸腺细胞也会经历阴性选择，阴性选择清除了那些能够与自身抗原发生反应的细胞。约2％的双阳性细胞能够在这种二重筛选中存活下来并成熟为单阳性T细胞，单阳性细胞逐渐从胸腺中转移到外周形成外周T细胞库。我们估计从T细胞祖先进入胸腺到成熟的成熟T细胞离开胸腺在老鼠中约要花3个星期的时间。

7-7 不同发育阶段的胸腺细胞分布在胸腺中的不同部位

The thymus is divided into two main regions, a peripheral cortex and a central medulla (see Fig. 7.7). Most T-cell development takes place in the cortex; only mature single-positive thymocytes are seen in the medulla. At the outer edge of the cortex, in the subcapsular region of the thymus (Fig. 7.13), large immature double-negative thymocytes proliferate vigorously; these cells are thought to represent the thymic progenitors and their immediate progeny and will give rise to all subsequent thymocyte populations. Deeper in the cortex, most of the thymocytes are small double-positive cells. The cortical stroma is composed of epithelial cells with long branching processes that express both MHC class II and MHC class I molecules on their surface. The thymic cortex is densely packed with thymocytes, and the branching processes of the thymic cortical epithelial cells make contact with almost all cortical thymocytes (see Fig. 7.8). Contact between the MHC molecules on thymic cortical epithelial cells and the receptors of developing T cells has a crucial role in positive selection, as we will see later in this chapter.

胸腺被分成两个主要区域，外周的皮质和中心的髓质（见图7.7）。T细胞的发育大多数发生在皮质；只有成熟的单阳性胸腺细胞能在髓质中看到。皮质的边缘区的胸腺膜下区（图7.13），大的未成熟双阴性胸腺细胞迅速地增殖；这些细胞被认为是胸腺细胞的祖先，它们随后的子代将会产生所有亚类群的胸腺细胞群体。皮质中稍微深入一点处，大部分的胸腺细胞是小双阳性细胞。皮质基质则由上皮细胞组成，这些上皮细胞具有很长的树突，其表面既表达MHC I也表达MHC II类分子。胸腺皮质中紧密地排列着胸腺细胞，具有长树突的胸腺皮质上皮细胞几乎与所有的皮质上皮细胞都有接触（见7.8）。胸腺皮质上皮细胞上MHC 分子与发育中T细胞的受体之间的接触在阳性选择中发挥关键作用，我们将在这一章后面的部分学到。

Figure 7.13. Thymocytes at different developmental stages are found in distinct parts of the thymus. The earliest cells to enter the thymus are found in the subcapsular region of the cortex. As these cells proliferate and mature into double-positive thymocytes, they migrate deeper into the thymic cortex. Finally, the medulla contains only mature single-positive T cells, which eventually leave the thymus and enter the bloodstream.

图7.13 位于不同发育阶段的胸腺细胞可以在胸腺的不同部位找到最早进入胸腺的细胞位于皮质的被膜下区域。随着这群细胞增殖并成熟为双阳性胸腺细胞，它们会进入胸腺皮质区更深处的部位。最后，髓质中只包含了成熟的单阳性T细胞，它们最终会离开胸腺进入到血液循环中。

The function of the medulla of the thymus is less well understood. It contains relatively few thymocytes, and those that are present are single-positive cells resembling mature T cells. These cells probably include newly mature T cells that are leaving the thymus through the medulla. In addition, they may also include other populations of mature T cells that remain within the medulla or return to it from the periphery to perform some specialized function, such as the elimination of infectious agents within the thymus. Before they mature, the developing thymocytes must undergo negative selection to remove self-reactive cells. We will see that this selective process is carried out mainly by the dendritic cells, which are particularly numerous at the cortico-medullary junction, and by the macrophages that are scattered in the cortex but are also abundant in the thymic medulla.

胸腺髓质的作用我们了解得比较少。它含有的胸腺细胞相对较少，其中出现的胸腺细胞则是类似于成熟T细胞的单阳性细胞。这些细胞很可能包括了新生的正在通过髓质离开胸腺的成熟T细胞。另外，它们可能也会包括另外一些滞留在髓质中或是从外周回到髓质以发挥某些特定功能——如清除胸腺内的感染物质——的成熟T细胞类群。我们将会学到这个选择过程主要是靠树突细胞和巨噬细胞来进行，树突细胞在皮质与髓质相连的部位数量特别多，巨噬细胞则在皮质中分散得很开但在胸腺髓质非常富集。

小结

B cells are generated and develop in the specialized microenvironment of the bone marrow, while the thymus provides a specialized and architecturally organized microenvironment for the development of T cells. As B cells differentiate from primitive stem cells, they proceed through stages that are marked by the sequential rearrangement of immunoglobulin gene segments to generate a diverse repertoire of antigen receptors. This developmental program also involves changes in the expression of other cellular proteins. Precursors of T cells migrate from the bone marrow and mature in the thymus. This process is similar to that for B cells, including the sequential rearrangement of antigen receptor gene segments. Developing T cells pass through a series of stages that can be distinguished by the differential expression of CD44 and CD25, the CD3:T-cell receptor complex proteins, and the co-receptor proteins CD4 and CD8. The development of both T and B cells is guided by the environment, particularly by stromal cells that provide contact-dependent signals and growth factors for developing lymphocytes. In the case of T cells, development is compartmentalized, with different types of stromal cells in the thymic cortex and medulla. Most steps in T-cell differentiation occur in the cortex of the thymus. The