Signals Regulating T Cell Development

我们的研究集中在三个方面。 第一个涉及T细胞抗原受体（TCR）信号的作用的表征，并且特别是单个TCR信号转导亚基和信号转导基序在T细胞发育中的作用。 我们已经将我们的研究扩展到包括分析在TCR下游起作用或抑制TCR信号传导的信号转导分子。这些研究的目的是了解这些分子如何参与TCR介导的信号传导，并确定它们和它们调节的信号通路在T细胞成熟和T细胞活化中发挥的作用。第二，我们继续研究在发育中的T细胞上表达的趋化因子受体的功能。 这些细胞表面蛋白介导对胸腺离散区域表达的特异性配体的趋化性。趋化因子受体是调节胸腺祖细胞归巢和调节胸腺细胞胸腺内迁移的候选者。最后，该实验室已经启动了一个新的研究领域，即控制造血干细胞生成和维持的基因。 T细胞抗原受体（TCR）信号在胸腺细胞发育中的作用。 信号转导序列（称为基于免疫受体酪氨酸的活化基序; ITAM）包含在多聚体TCR复合物的四个不同亚基（ζ、CD 3-γ、-δ、-δ）内。ITAM内的二酪氨酸残基在TCR接合时被磷酸化，并起到将信号传导分子如蛋白酪氨酸激酶募集到TCR复合物的作用，从而启动T细胞活化级联反应。为了确定TCR信号转导亚基是否在发育中执行不同或类似的功能，我们先前通过基因靶向产生了zeta缺陷和CD 3-κ B缺陷小鼠，用编码野生型或信号缺陷（ITAM突变体）形式的zeta和CD 3-κ B的转基因对这些小鼠进行遗传重建，并表征了这些改变对TCR信号传导的发育和功能后果。这些研究的结果表明，TCR-ITAM在功能上是等同的，但在放大TCR信号方面一致起作用。发现TCR信号放大对于胸腺细胞选择是关键的，通过该过程，潜在有用的未成熟T细胞被指示存活并进一步分化（正选择），并且可能导致自身免疫疾病的潜在自身反应性细胞在胸腺中被删除（负选择）。 因此，TCR的多亚基结构可能已经进化为使复杂的生物体能够发展广泛的、自限制的但自身耐受的T细胞库。在目前的研究中，我们正在使用条件基因表达系统来分析TCR信号在特定发育阶段的重要性。此外，我们正在使用微阵列和消减克隆来鉴定参与T细胞信号传导和T细胞发育的基因。 趋化因子受体CCR 9在T细胞发育中的作用 胸腺细胞通过不同发育阶段的有序进展也与它们暴露于不同生长因子和信号的不同胸腺微环境中和之间的迁移有关。趋化因子是一组结构相关的小分子，其通过与七跨膜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CCL 25在胸腺和小肠中高度表达，这是T淋巴细胞生成的两个已知位点。CCL 25的受体CCR 9在大多数胸腺细胞上表达，这增加了CCR 9及其配体可能在胸腺细胞发育中发挥重要作用的可能性。为了研究CCR 9在淋巴细胞发育过程中的作用，我们产生了CCR 9缺陷型（CCR 9-/-）和CCR 9转基因小鼠。这些研究表明，当被迫与来自CCR 9 +/+小鼠的祖细胞竞争时，来自CCR 9-/-小鼠的淋巴细胞祖细胞重建胸腺的能力显著降低。 在其他实验中，CCR 9在转基因小鼠中的过表达抑制了早期胸腺细胞的发育，并阻断了未成熟胸腺细胞在胸腺内的正常迁移。这些结果表明，CCR 9参与调节祖细胞向胸腺的迁移和胸腺内发育中的胸腺细胞的迁移。 控制造血干细胞特化和维持的基因。 造血系统由一组功能多样的细胞组成，这些细胞来源于能够长期自我更新和多谱系分化的常见造血干细胞（HSC）。自我更新确保HSC池在整个生命中持续存在，而分化导致所有循环血细胞（包括淋巴细胞、髓细胞、红细胞和血小板）的连续产生。我们最近启动了旨在鉴定对HSC生成和维持重要的基因的实验。我们的初步研究集中在LIM结构域结合蛋白-1（Ldb 1）在造血中的作用。这些实验的结果揭示了Ldb 1在调节造血干细胞中的自我更新/分化细胞命运决定中的关键功能，并进一步表明Ldb 1有核转录复合物可以控制谱系特异性干细胞的维持。

Our studies are focused in three areas. The first involves characterization of the role of T cell antigen receptor (TCR) signals, and in particular, individual TCR signal transducing subunits and signal transducing motifs in T cell development. We have extended our studies to include analysis of signal transducing molecules that function downstream of the TCR or that inhibit TCR signaling. The aim of these studies is to understand how these molecules participate in TCR mediated signaling and to determine what roles they and the signaling pathways they regulate play in T cell maturation and T cell activation. Second, we have continued studies characterizing the function of chemokine receptors that are expressed on developing T cells. These cell surface proteins mediate chemotaxis in response to specific ligands that are expressed in discreet regions of the thymus. Chemokine receptors are candidates for regulating homing of progenitor cells to the thymus and for regulating intrathymic migration of thymocytes. Finally, the lab has initiated a new area of investigation of the genes controlling the generation and maintenance of Hematopoietic Stem Cells. Role of T cell antigen receptor (TCR) signaling 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. To determine if TCR signal transducing subunits perform distinct or analogous functions in development, we previously 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. In current studies we are using conditional gene expression systems to analyze the importance of TCR signaling at specific stages of development. In addition, we are using microarray and subtractive cloning to identify genes involved in T cell signaling and T cell development. Role of the chemokine receptor CCR9 in T cell development 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 forcedforced to compete with progenitor cells from CCR9+/+ mice. In other experiments, overexpression of CCR9 in transgenic mice inhibited early thymocyte development and blocked the normal migration of immature thymocytes within the thymus. These results indicate that CCR9 participates in regulating both the migration of progenitor cells to the thymus and the migration of developing thymocytes within the thymus. Genes controlling Hematopoietic Stem cell specification and maintenance. The hematopoietic system is composed of a functionally diverse group of cells that originate from a common hematopoietic stem cell (HSC) capable of long-term self-renewal and multi-lineage differentiation. Self-renewal ensures that a pool of HSCs persists throughout life, whereas differentiation leads to the continuous generation of all circulating blood cells including lymphocytes, myeloid cells, erythrocytes and platelets. We have recently initiated experiments aimed at identifying genes important for HSC generation and maintenance. Our initial studies focused on the role of LIM domain binding protein-1 (Ldb1) in hematopoiesis. The results of these experiments revealed a critical function for Ldb1 in regulating the self-renewal/differentiation cell fate decision in hematopoietic stem cells and suggest further that Ldb1 nucleated transcription complexes may control maintenance of lineage specific stem cells.