Genetic Analysis Of Thymocyte Development

胸腺细胞发育的遗传分析

• 批准号: 6813782
• 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/6813782
• 关键词: T cell receptor; T lymphocyte; biological signal transduction; cell migration; chemokine; cytokine receptors; developmental genetics; functional /structural genomics; gene expression; gene targeting; genetic regulation; genetically modified animals; immunogenetics; laboratory mouse; leukopoiesis; membrane proteins; protein structure function

Our research is directed at understanding the cellular and genetic events that regulate T lymphocyte development. Current studies focus on the role of T lymphocyte signal transducing molecules in immature T lymphocyte (thymocyte) selection, a process essential for formation of the mature T cell repertoire. To analyze the function of specific signal transducing proteins in T lymphocyte development, transgenic and gene targeting methods are used to create overexpression, dominant-negative, and loss-of-function mutants in mice. In addition, molecular genetic techniques are being employed to identify and characterize the function of novel genes that are expressed in T lymphocytes. Role of T cell antigen receptor (TCR) signaling in thymocyte development. A major theme of our research has been investigating the role of TCR signal transduction in thymocyte development. 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 non-identical, 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 demonstrated 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 potentially useful immature T cells are instructed to survive and differentiate further-(positive selection), and potentially auto-reactive cells that may cause auto-immune disease are deleted in the thymus (negative selection). Thus, the multi-subunit structure of the TCR may have evolved to enable complex organisms to develop a broad, self-restricted yet auto-tolerant T cell repertoire. Role of LAT in T cell development. 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. The distal four tyrosines in LAT (tyr136, tyr175, tyr195, tyr235) are necessary and sufficient for LAT activity in T cells, which includes activation of the calcium and MAP Kinase (MAPK) downstream signaling pathways. These 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. Thus, their inactivation in all cells would likely result in embryonic lethality. However, by mutating specific LAT tyrosines we have been able to uncouple the TCR from downstream signaling pathways in T cells without affecting the ability of other receptors or cells to utilize these pathways. We generated ?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 exhibited normal T cell development, thereby validating the targeting strategy. 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 fatal lymphoproliferative disorder characterized by expansion and multi-tissue infiltration of CD4+ T cells. Consistent with previous data demonstrating that tyr136 preferentially binds phospholipase C-gamma, examination of the signaling response of T cells from these mice revealed a severe defect in TCR induced/phospholipase C-gamma-mediated calcium flux. However, MAP Kinase signaling was intact in these cells, indicating that the TCR was selectively uncoupled from the calcium but not the MAPK pathway. These results reveal a critical role for LAT in coordinating downstream signals initiated by TCR engagement and demonstrate that this function is essential for normal T cell homeostasis. Structure and signaling potential of the gamma/delta TCR complex. Most vertebrate species contain two separate lineages of T cells that are distinguished by the antigen binding clonotype-specific chains contained within their TCRs: alpha/beta-T cells and gamma/delta-T cells. Although the more abundant alpha/beta TCR has been extensively characterized, much less is known about the structure or function of the gamma/delta TCR which is expressed on the smaller subset of gamma-delta T cells. We found that the subunit composition of the gamma/delta TCR differs from that of the alpha/beta TCR in that a component of the alpha/beta TCR, the CD3delta chain, is not present in gamma/delta TCRs. These results revealed a major difference in the subunit structure of the alpha/beta and gamma/deltaTCRs. Interestingly, signal transduction by the gamma/delta TCR was found to be superior to the alpha/betaTCR as assessed by several criteria. Our data suggest that the structural difference between alpha/beta and gamma/delta TCRs may influence the signaling potential of the TCR complex and that this may have important functional consequences on T cell activation. Current studies involve further analysis of the effect of TCR subunit structure on signaling responses and determining if TCR subunit composition influences T development and T cell lineage commitment. Role of the chemokine receptor CCR9 in T cell development 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 thymus 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. These studies demonstrated that lymphocyte progenitors from CCR9-/- mice had a markedly reduced capacity to repopulate the thymus when forced to compete with progenitor cells from CCR9+/+ mice indicating that CCR9 participates in regulating both the migration of progenitor cells to the thymus and the migration of developing thymocytes within the thymus

我们的研究旨在了解调节T淋巴细胞发育的细胞和遗传事件。当前的研究重点是T淋巴细胞信号转导分子在未成熟T淋巴细胞（胸腺细胞）选择中的作用，这是成熟T细胞库形成必不可少的过程。为了分析特异性信号转导蛋白在T淋巴细胞发育中的功能，使用转基因和基因靶向方法来产生小鼠的过表达，显性阴性和功能丧失突变体。另外，正在采用分子遗传技术来识别和表征在T淋巴细胞中表达的新型基因的功能。 T细胞抗原受体（TCR）信号传导在胸腺细胞发育中的作用。 我们研究的一个主要主题是研究TCR信号转导在胸腺细胞发育中的作用。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，-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，可以部分地通过不同的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-ITAM在功能上是等效的，但协同作用以扩大TCR信号。发现TCR信号扩增对于胸腺细胞选择至关重要，胸腺细胞的选择至关重要，该过程指示潜在有用的不成熟的T细胞生存和分化进一步的（阳性选择），并且可能导致可能引起自身免疫性疾病的潜在自身反应性细胞在胸膜中被删除（阴性选择）。因此，TCR的多生产结构可能已经发展为使复杂的生物能够发展出宽阔，自限制但自动耐受性的T细胞库。 LAT在T细胞发育中的作用。 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（Tyr136，Tyr175，Tyr195，Tyr235）中的远端四个酪氨酸对于T细胞中的LAT活性是必要的，其中包括钙和MAP激酶（MAPK）下游信号通路的激活。这些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。因此，它们在所有细胞中的失活可能会导致胚胎致死性。但是，通过突变特定的LAT酪氨酸，我们已经能够使TCR与T细胞中下游信号通路脱离，而不会影响其他受体或细胞利用这些途径的能力。我们生成了吗？表达含有四个关键酪氨酸残基的单个或多个酪氨酸 - 苯丙氨酸突变的LAT蛋白的突变小鼠。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。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）导致致命的淋巴增生性疾病，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优先结合磷脂酶C-Gamma，对这些小鼠的T细胞的信号反应的检查显示，TCR诱导的/磷脂酶C-Gamma介导的钙介导的钙磁通均严重缺陷。但是，这些细胞中MAP激酶信号传导完好无损，表明TCR与钙选择性脱在一起，而不是MAPK途径。这些结果揭示了LAT在协调通过TCR参与启动的下游信号的关键作用，并证明该功能对于正常的T细胞稳态至关重要。 γ/Delta TCR复合物的结构和信号传导潜力。 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中包含的抗原结合链型特异性链（alpha/beta-t细胞）和γ/delta-t细胞区分。尽管较丰富的α/βTCR已得到广泛的表征，但对伽马/delta TCR的结构或功能的了解少得多，该结构或功能在γ-戴尔塔T细胞的较小子集上表达。我们发现，γ/delta TCR的亚基组成与alpha/beta tcr的亚基组成不同，因为alpha/beta tcr的CD3DELTA链的成分不存在于γ/delta tcr中。这些结果表明，α/beta和伽马/deltatcrs的亚基结构有主要差异。有趣的是，发现γ/delta TCR的信号转导比通过多个标准评估的α/betatcr优于α/betatcr。我们的数据表明，α/beta和γ/delta TCR之间的结构差异可能会影响TCR复合物的信号传导潜力，并且这可能对T细胞激活产生重要的功能后果。当前的研究涉及对TCR亚基结构对信号反应的影响的进一步分析，并确定TCR亚基组成是否影响T发育和T细胞谱系承诺。 趋化因子受体CCR9在T细胞发育中的作用 T细胞的发育一直持续到成年，需要T型 - 促基质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。这些研究表明，当被迫与CCR9+/+小鼠的祖细胞竞争时，CCR9 - / - 小鼠的淋巴细胞祖细胞显着降低了胸腺的能力，表明CCR9参与调节祖细胞向胸骨迁移到胸骨thymos的迁移，并调节胸腔内的thymocies inymocius of thyymocies of thymos thyymocius of。