: 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.
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