Genetic Analysis Of Thymocyte Development

胸腺细胞发育的遗传分析

• 批准号: 6671886
• 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/6671886
• 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 lymphocyte development. Current studies center on the signal transduction molecules and pathways that regulate immature T lymphocyte (thymocyte) maturation and mature T lymphocyte function. 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. 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 pathways 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 a critical role for LAT in T cell homeostasis through its ability to integrate signals downstream of the TCR. CCR9: T cell development continues into adulthood and requires the periodic migration of T-progenitor cells from the bone marrow to the thymus. The ordered progression of thymocytes through distinct stages of development is also associated with migration into and between different thymic microenvironments where they are exposed to different growth factors and signals. Chemokines are a group of small, structurally related molecules that regulate trafficking of leukocytes through interactions with a subset of seven-transmembrane, G protein-coupled receptors. The chemokine CCL25 is highly expressed in the thymus and small intestine, the two known sites of T lymphopoesis. The receptor for CCL25, CCR9, is expressed on the majority of thymocytes raising the possibility that CCR9 and it ligand may play an important role in thymocyte development. To investigate the role of CCR9 during lymphocyte development, we generated CCR9-deficient (CCR9-/-) and CCR9 transgenic mice. Surprisingly, both T cell and B cell development appeared normal in CCR9-/- mice. However, competitive bone marrow transplantation experiments demonstrated that CCR9-/- bone marrow cells had a markedly reduced capacity to repopulate the thymus compared to bone marrow cells from CCR9+/+ mice. Overexpression of CCR9 in transgenic mice inhibited early thymocyte development and blocked the normal migration of immature thymocytes within the thymus. These results demonstrate that CCR9 participates in regulating the migration of progenitor cells to the thymus and in regulating the migration of developing thymocytes within the thymus. Gamma/delta TCR. Most vertebrate species contain two separate lineages of T cells that are distinguished by the clonotype-specific chains contained within their TCRs: alpha/beta-T cells and gamma/delta-T cells. Although the alpha/betaTCR has been extensively characterized, much less is known about the structure or function of the gamma/deltaTCR. We found that the subunit composition of the gamma/deltaTCR differs fundmentally from that of the alpha/betaTCR in that a major subunit of the alpha/beta TCR, the CD3delta chain, is not a component of the gamma/deltaTCR. Interestingly, signal transduction by the gamma/deltaTCR was consistently superior to the alpha/betaTCR as assessed by several criteria. These results demonstrate a major difference in the subunit structure of the alpha/beta and gamma/deltaTCRs. Moreover, our data suggest that this structural difference may influence the signaling potential of the TCR complex and have important functional consequences on T cell activation.

研究旨在了解控制T淋巴细胞发育的细胞和遗传事件。当前的研究中心关于调节未成熟T淋巴细胞（胸腺细胞）成熟和成熟T淋巴细胞功能的信号转导分子和途径。 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。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通路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LAT在T细胞稳态中通过其在TCR下游集成信号的能力的关键作用。 CCR9：T细胞的发育持续到成年，需要T-progogenit细胞从骨髓到胸腺的周期性迁移。胸腺细胞通过不同发育阶段的有序进展也与迁移到不同胸腺微环境之间的迁移以及暴露于不同生长因子和信号的不同。趋化因子是一组与结构相关的小分子，通过与7跨膜G蛋白偶联受体的子集相互作用来调节白细胞的运输。趋化因子CCL25在胸腺和小肠，这是T淋巴发作的两个已知位点。 CCL25 CCR9的受体在大多数胸腺细胞上表达，这会提高CCR9及其配体在胸腺细胞发育中起重要作用。为了研究CCR9在淋巴细胞发育中的作用，我们产生了CCR9缺陷型（CCR9 - / - ）和CCR9转基因小鼠。令人惊讶的是，在CCR9 - / - 小鼠中，T细胞和B细胞发育均正常。然而，竞争性骨髓移植实验表明，与CCR9+/+小鼠的骨髓细胞相比，CCR9 - / - 骨髓细胞重填充胸腺的能力明显降低。 CCR9在转基因小鼠中的过表达抑制了早期胸腺细胞的发育，并阻止了胸腺中未成熟胸腺细胞的正常迁移。这些结果表明，CCR9参与调节祖细胞向胸腺的迁移，并调节胸腺内发育细胞的迁移。 伽玛/三角洲TCR。大多数脊椎动物都包含两个单独的T细胞谱系，这些谱系由其TCR中包含的clonotype特异性链（α/beta-T细胞和伽马/delta-T细胞）区分。尽管alpha/betatcr已被广泛表征，但对伽马/deltatcr的结构或功能的了解少得多。我们发现，γ/deltatcr的亚基组成与alpha/betatcr的基本不同，因为alpha/beta tcr的主要亚基是CD3DELTA链，不是γ/deltatcr的组成部分。有趣的是，γ/deltatcr的信号转导始终优于通过几个标准评估的α/betatcr。这些结果表明，α/beta和γ/deltatcrs的亚基结构有主要差异。此外，我们的数据表明，这种结构差异可能会影响TCR复合物的信号传导潜力，并对T细胞激活产生重要的功能后果。