Individual and Collaborative Roles in T Cell Activation

描述(由申请人提供)：T细胞的发育是一个由T细胞受体(TCR)或前TCR发出的信号严格控制的有序过程。胸腺细胞由CD4-CD8-双阴性(DN)向CD4-CD8+双阳性(DP)的转变受TCR前状态的控制。在DP阶段，成功重排TCR的胸腺细胞经历了一个选择过程，允许自身MHC限制性和功能正常但不是自身反应性T细胞存活和成熟。目前公认的假说认为，TCR参与的亲和力决定了选择的结果。推测TCR/MHC相互作用的亲和力程度决定了TCR信号强度的水平，从而导致胸腺的选择。然而，目前还不清楚哪些信号分子参与调节与细胞反应有关的远端TCR信号通路。T细胞活化的早期事件之一是刺激磷脂特异性磷脂酶C(PLC)-γ，它介导钙动员、PKC激活和RAS通路的激活，所有这些都对TCR信号转导起重要作用。PLC-Gamma有两种亚型，PLC-Gamma1和PLC-Gamma2。据认为，PLC-Gamma1解释了T细胞中PLC-Gamma的全部活性。然而，我们的初步数据表明，PLC-Gamma2也参与了TCR信号转导，但只有当PLC-Gamma1的表达减少时，才能揭示其作用。尽管以前的细胞系研究表明PLC-Gamma1在TCR信号转导中起重要作用，但PLC-Gamma1在T细胞发育中的作用尚未在体内直接检测。在此，我们认为PLC-γ活性在TCR前和TCR信号转导阶段对T细胞的发育都是至关重要的。我们认为PLC-γ活性对T细胞的阳性和阴性选择很重要，定量刺激PLC-γ导致与胸腺选择相关的下游信号通路的差异激活。PLC-Gamma1和PLC-Gamma2均参与TCR信号转导，为分析PLC-Gamma1和PLC-Gamma2的进行性降低对T细胞发育和TCR激活的影响提供了一个模型。我们建议在目标1中研究PLC-Gamma在T细胞阳性和阴性选择中的作用。在目标2中，我们将研究定量PLC-Gamma激活对与T细胞选择相关的远端TCR信号事件的影响。在目标3中，我们将通过检测T细胞谱系中PLC-Gamma1缺失或PLC-Gamma1和PLC-Gamma2共同缺失的小鼠T细胞发育，来研究PLC-Gamma1和PLC-Gamma2在TCR前和TCR信号转导中的作用。总之，这些研究应该确定PLC-Gamma1和PLC-Gamma2在TCR信号和T细胞发育中的作用，并可能为选择性抑制PLC-Gamma下游通路从而操纵T细胞激活的药物的设计提供基础。

DESCRIPTION (provided by the applicant): Development of T cells is an ordered process tightly controlled by signals emanating from the T cell receptor (TCR) or pre-TCR. The transition of CD4-CD8- double negative (DN) to CD4-CD8+ double positive (DP) thymocytes is governed by the pre-TCR. At the DP stage, thymocytes that have successfully rearranged their TCR undergo a selection process that allows self MHC-restricted and functionally competent but not autoreactive T cells to survive and mature. The current accepted hypothesis proposes that the avidity of TCR engagement determines the outcome of the selection. It is presumed that the degrees of affinity of TCR/MHC interaction determine the levels of TCR signaling strength, which consequently result in thymic selection. However, it is not clear what signaling molecules are involved in regulating the distal TCR signaling pathways linked to cellular responses. One of the early events in T cell activation is stimulation of phospholipid-specific phospholipase C (PLC)-gamma, which mediates calcium mobilization, PKC activation and activation of the Ras pathway, all of which are important for TCR signaling. PLCgamma has two isoforms, PLC-gamma1 and PLC-gamma2. It is believed that PLC-gamma1 accounts for all the PLC-gamma activity in T cells. However, our preliminary data suggest that PLC-gamma2 is also involved in TCR signaling, but its role is only revealed when PLC-gamma1 expression is reduced. Although previous cell line studies indicated an important role of PLC-gamma1 in TCR signaling, the function of PLC-gamma1 in T cell development has not been directly examined in vivo. Here, we propose that PLC-gamma activity is crucial for T cell development at both the pre-TCR and TCR signaling stages. We believe PLC-gamma activity is important for T cell positive and negative selection, and quantitative stimulation of PLC-gamma results in differential activation of the downstream signaling pathways relevant to thymic selection. The finding that both PLC-gamma1 and PLC-gamma2 are involved in TCR signaling provides us a model to analyze the influence of progressive reduction of PLC-gamma on T cell development and TCR activation. We propose to study the role of PLC-gamma in T cell positive and negative selection in Aim 1. The influence of quantitative PLC-gamma activation on the distal TCR signaling events related to T cell selection will be examined in Aim 2. In Aim 3 we will examine the role of PLC-gamma1 and PLCgamma2 in pre-TCR and TCR signaling by examining T cell development in mice null for PLC-gamma1 alone or for both PLC-gamma1 and PLC-gamma2 in the T cell lineage. Overall, these studies should define the role of PLC-gamma1 as well as PLC-gamma2 in TCR signaling and T cell development, and may provide the basis for the design of drugs that can selectively inhibit PLC-gamma downstream pathways and thus manipulate T cell activation.