Individual and Collaborative Roles in T Cell Activation

描述（由申请方提供）：T细胞的发育是一个有序的过程，由T细胞受体（TCR）或前TCR发出的信号严格控制。CD 4-CD 8-双阴性（DN）胸腺细胞向CD 4-CD 8+双阳性（DP）胸腺细胞的转变由前TCR控制。在DP阶段，已经成功重排其TCR的胸腺细胞经历选择过程，该过程允许自身MHC限制的和功能上有能力但不具有自身反应性的T细胞存活和成熟。目前接受的假说提出，TCR参与的亲和力决定了选择的结果。据推测，TCR/MHC相互作用的亲和力程度决定了TCR信号强度的水平，从而导致胸腺选择。然而，尚不清楚哪些信号分子参与调节与细胞应答相关的远端TCR信号通路。T细胞活化的早期事件之一是磷脂特异性磷脂酶C（PLC）-γ的刺激，其介导钙动员、PKC活化和Ras途径的活化，所有这些对于TCR信号传导都是重要的。PLC γ有两种亚型，PLC-γ 1和PLC-γ 2。据信PLC-γ 1解释了T细胞中所有PLC-γ活性。然而，我们的初步数据表明，PLC-γ 2也参与TCR信号转导，但其作用仅在PLC-γ 1表达降低时才显示。尽管先前的细胞系研究表明PLC-γ 1在TCR信号传导中的重要作用，但PLC-γ 1在T细胞发育中的功能尚未在体内直接检测。在这里，我们提出，PLC-γ活性是至关重要的T细胞的发展，在前TCR和TCR信号阶段。我们相信PLC-γ活性对于T细胞阳性和阴性选择是重要的，并且PLC-γ的定量刺激导致与胸腺选择相关的下游信号传导途径的差异活化。PLC-γ 1和PLC-γ 2都参与TCR信号传导的发现为我们提供了一个模型来分析PLC-γ的逐渐减少对T细胞发育和TCR活化的影响。我们建议研究PLC-γ在目标1中T细胞阳性和阴性选择中的作用。将在目的2中检查定量PLC-γ活化对与T细胞选择相关的远端TCR信号传导事件的影响。在目标3中，我们将通过检查T细胞谱系中PLC-γ 1单独或PLC-γ 1和PLC-γ 2缺失的小鼠中的T细胞发育来检查PLC-γ 1和PLC-γ 2在前TCR和TCR信号传导中的作用。总体而言，这些研究应该确定PLC-γ 1以及PLC-γ 2在TCR信号传导和T细胞发育中的作用，并可能为设计能够选择性抑制PLC-γ下游通路从而操纵T细胞活化的药物提供基础。

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.