Genetic Analysis Of Thymocyte Development

胸腺细胞发育的遗传分析

• 批准号: 6541226
• 负责人: PAUL E LOVE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6541226
• 关键词: CD3 molecule; CD5 molecule; T cell receptor; T lymphocyte; biological signal transduction; developmental genetics; gene expression; gene targeting; genetic regulation; immunogenetics; laboratory mouse; leukocyte activation /transformation; leukopoiesis; molecular cloning; polymerase chain reaction; protein structure function; receptor expression

Research is directed at understanding the cellular and genetic events that control T cell development. Current studies center on the signal transduction molecules and pathways that regulate thymocyte maturation and mature T cell function. The T cell antigen receptor (TCR). A major focus of research in the lab has been investigating TCR signal transduction in thymocyte development. TCR signal transduction sequences (termed Immunoreceptor Tyrosine-based Activation Motifs; ITAMs) are contained within four distinct subunits of the multimeric TCR complex (zeta, CD3-gamma, -delta, -epsilon). Di-tyrosine residues within ITAMs are phosphorylated upon TCR engagement and function to recruit signaling molecules, such as protein tyrosine kinases, to the TCR complex, thereby initiating the T cell activation cascade. Though conserved, ITAM sequences are nonidentical, raising the possibility that the diverse developmental and functional responses controlled by the TCR may be regulated, in part, by distinct ITAMs. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we generated zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR signaling. The results of these studies demonstrate that TCR-ITAMs are functionally equivalent but act in concert to amplify TCR signals. TCR signal amplification was found to be critical for thymocyte selection (the process by which auto-reactive cells are deleted in the thymus). CD5. Other cell surface structures can influence the TCR signaling response. The analysis of one such molecule, CD5, which has been shown to negatively regulate TCR signaling and to participate in thymocyte selection, constitutes another area of investigation in the laboratory. Examination of CD5 expression during T cell development revealed that surface levels of CD5 are regulated by TCR signal intensity and by the affinity of the TCR for selecting ligands. To determine if the ability to regulate CD5 expression is important for thymocyte selection, we generated transgenic mice that constitutively express high levels of CD5 throughout development. Over-expression of CD5 significantly impaired positive selection of some thymocytes (those that would normally express low levels of CD5) but not others (those that would normally express high levels of CD5). These findings support a role for CD5 in modulating TCR signal transduction and thereby influencing the outcome of thymocyte selection. The ability of individual thymocytes to regulate CD5 expression represents a mechanism for "fine tuning" of the TCR signaling response during development. The current results indicate that this potential for signal modulation may be particularly useful for generating the maximum possible TCR diversity in the mature T cell repertoire. Since a probable mechanism for CD5 function is via the activation-induced binding of regulatory molecule(s) to sequences within the CD5 cytoplasmic domain, transgenic mice that express a tail-less form of CD5 (mCD5) were generated. Both the intact and mCD5 transgenes were then used to reconstitute CD5 surface expression in CD5-/- mice. These experiments revealed a critical function for the cytoplasmic domain in CD5 signaling. The laboratory is currently attempting to identify molecules that interact with CD5 and that may be involved in regulating signal transduction by the TCR. LAT. Linker for Activation of T cells (LAT) is an integral membrane protein that functions as a critical adaptor linking the T cell antigen receptor (TCR) to multiple downstream signaling pathways required for T cell activation. LAT-deficient cell lines exhibit defects in activation of the two major signaling patways in T cells: the PLC-gamma mediated calcium pathway and the Ras/MAP Kinase (MAPK) pathway. The distal four tyrosines in LAT (tyr136, tyr175, tyr195, tyr235) are necessary and sufficient for LAT activity in T cells and function by recruiting downstream effectors. The calcium and MAPK signaling pathways are also activated by a large number of other receptors and are required for the development and function of many different cell types. Because these signaling pathways function to regulate cellular events unrelated to TCR signaling, their inactivation would likely result in embryonic lethality or pleiotropy. Significantly, the four LAT tyrosines exhibit preferential binding to specific effector molecules, and mutation of different residues in cell lines results in distinct biochemical and signaling consequences. These observations suggested that by mutating specific LAT tyrosines it may be possible to uncouple the TCR from downstream signaling pathways in T cells without effecting the ability of other receptors to utilize these pathways. To explore the role of LAT-coupled signaling pathways in T cell development, we generated in collaboration with Dr. L. Samelson's group (NCI)"knock-in" mutant mice that express LAT proteins containing single or multiple tyrosine-phenylalanine mutations of the four critical tyrosine residues. Knock-in mice that express the wild-type version of the protein exhibit normal T cell development thereby validating the targeting strategy. Conversely, inactivation of all four distal LAT tyrosines yielded a null phenotype, demonstrating the critical role of these residues for T cell development. Surprisingly, knock-in mutation of the first tyr residue (tyr136) resulted in a profound fatal lymphoproliferative disorder characterized by expansion and multi-tissue infiltration of CD4+ T cells. Consistent with previous data demonstrating that tyr136 preferentially binds PLC-gamma, examination of the signaling response of T cells from these mice revealed a severe defect in TCR induced calcium flux. However, MAPK signaling was intact in these cells, indicating that the TCR was specifically uncoupled from the calcium pathway. These results reveal an inhibitory role for calcium signaling in T cell homeostasis.

研究旨在了解控制T细胞发育的细胞和遗传事件。当前的研究中心关于调节胸腺细胞成熟和成熟T细胞功能的信号转导分子和途径。 T细胞抗原受体（TCR）。实验室中的主要研究重点是研究胸腺细胞发育中的TCR信号转导。 TCR信号转导序列（称为免疫受体酪氨酸的活化基序； ITAMS）包含在多聚体TCR复合物的四个不同亚基中（Zeta，CD3 -Gamma，-delta，-delta，-epsilon）。 ITAM中的Di-tyrosine残基在TCR参与和功能下被磷酸化，以募集TCR复合物等信号分子，例如蛋白酪氨酸激酶，从而启动T细胞激活级联反应。尽管保守了，但ITAM序列是非相同的，这增加了TCR控制的多种发育和功能响应的可能性，部分可以通过不同的ITAM来调节。 To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we generated zeta deficient and CD3-epsilon deficient mice by gene targeting, genetically reconstituted these mice with transgenes encoding wild-type or signaling-deficient (ITAM-mutant) forms of zeta and CD3-epsilon, and characterized the developmental and functional consequences of these alterations on TCR信号。这些研究的结果表明，TCR-ITAMS在功能上是等效的，但协同起来可以放大TCR信号。发现TCR信号扩增对于胸腺细胞选择至关重要（在胸腺中删除自身反应性细胞的过程）。 CD5。其他细胞表面结构会影响TCR信号响应。对一个这样的分子CD5的分析已被证明对TCR信号进行负调控并参与胸腺细胞选择，这构成了实验室的另一个研究领域。 T细胞发育过程中CD5表达的检查表明，CD5的表面水平受TCR信号强度和TCR对选择配体的亲和力的调节。为了确定调节CD5表达的能力是否对于胸腺细胞选择很重要，我们产生了在整个发育过程中组成型表达高水平CD5的转基因小鼠。 CD5的过表达显着损害了某些胸腺细胞的阳性选择（通常表达低水平的CD5），但没有表达其他胸腺细胞（通常会表达高水平的CD5）。这些发现支持CD5在调节TCR信号转导的作用，从而影响胸腺细胞选择的结果。单个胸腺细胞调节CD5表达的能力代表了发育过程中TCR信号反应“微调”的机制。当前的结果表明，这种信号调制的潜力可能对于在成熟的T细胞库中产生最大可能的TCR多样性可能特别有用。由于CD5功能的可能机制是通过激活诱导的调节分子与CD5细胞质域内序列的结合，因此产生了表达无尾巴CD5（MCD5）的转基因小鼠。然后使用完整的和MCD5转基因来重新构建CD5 - / - 小鼠中的CD5表面表达。这些实验揭示了CD5信号传导中细胞质结构域的关键功能。该实验室目前正在尝试鉴定与CD5相互作用的分子，并且可能与TCR调节信号转导有关。 拉特。激活T细胞（LAT）的接头是一种积分的膜蛋白，它是将T细胞抗原受体（TCR）与T细胞激活所需的多个下游信号通路联系起来的关键适配器。 LAT缺陷细胞系在T细胞中两个主要信号PATWay的激活中表现出缺陷：PLC-GAMMA介导的钙途径和RAS/MAP激酶（MAPK）途径。 LAT（Tyr136，Tyr175，Tyr195，Tyr235）中的远端四个酪氨酸是必需的，足以通过募集下游效应子来进行T细胞的LAT活性和功能。钙和MAPK信号通路也被大量其他受体激活，并且需要许多不同细胞类型的开发和功能。由于这些信号通路的功能可以调节与TCR信号无关的细胞事件，因此它们的失活可能会导致胚胎致死性或多效性。值得注意的是，四个LAT酪氨酸表现出与特定效应分子的优先结合，并且细胞系中不同残基的突变导致不同的生化和信号传导后果。这些观察结果表明，通过突变特定的LAT酪氨酸，可以将TCR与T细胞中下游信号通路的脱离，而不会影响其他受体利用这些途径的能力。 为了探索LAT偶联信号通路在T细胞发育中的作用，我们与L. Samelson博士（NCI）的“敲入”突变小鼠合作，表达了四个关键酪氨酸残基的单一或多个酪氨酸 - 苯基丙氨酸突变。表达蛋白质野生型版本的敲入小鼠表现出正常的T细胞发育，从而验证了靶向策略。相反，所有四个远端LAT酪氨酸的灭活都产生了无效的表型，这表明这些残基在T细胞发育中的关键作用。令人惊讶的是，第一个Tyr残基的敲入突变（Tyr136）导致了严重的致命淋巴增生性疾病，其特征是CD4+ T细胞的膨胀和多组织浸润。与以前的数据一致，表明Tyr136优先结合PLC-伽马，对这些小鼠的T细胞的信号传导反应的检查显示，TCR诱导的钙通量存在严重缺陷。但是，在这些细胞中，MAPK信号传导完好无损，表明TCR是与钙途径的特异性脱在一起的。这些结果揭示了T细胞稳态中钙信号传导的抑制作用。