Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Immunology

First Advisor

Gary A. Koretzky

Second Advisor

Paula Oliver

Abstract

Signals transduced through the T cell receptor (TCR) lead to T cell differentiation, proliferation, and elaboration of cytokines, all of which are required for optimal immunity. Phosphoinositide (PI) mediated signaling plays a particularly prominent role in this process. TCR signaling is amplified by the activation of phospholipase C γ1 (PLCγ1), which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2) to form the second messengers diacylglycerol (DAG) and inositol triphosphate (IP3). Regulation of PI and products such as DAG are therefore essential for normal TCR signaling. DAG levels are reduced by diacylglycerol kinases (DGKs), which metabolize DAG and diminish DAG-mediated signaling. In T cells, the predominant DGK isoforms expressed are DGKα and DGKζ. Deletion of either isoform enhances DAG-mediated signaling, yet the relative importance of these enzymes is unknown. Here, we describe that DGKζ but not DGKα suppresses natural regulatory T cell development and predominantly controls Ras and AKT signaling. The differential functions of DGKα and DGKζ are not attributable to differences in expression levels or localization to the T cell-APC contact site. However, RasGRP1, a key activator of Ras signaling, associated to a greater extent with DGKζ than DGKα. In addition, DGKζ displayed greater metabolism of DAG to PA after TCR stimulation than DGKα. In silico modeling of TCR-stimulated Ras activation in DGKα- versus DGKζ-deficient T cells suggested that a greater catalytic rate for DGKζ than DGKα could lead to increased suppression of Ras-mediated signals by DGKζ. DGKζ dominant functions over DGKα, therefore, are in part due to DGKζ's greater effective enzymatic activity and association with RasGRP1. Future experiments will establish how DGK isoform function is regulated by TCR signaling. To examine how the DAG precursor PI is regulated, we performed a preliminary analysis of mice with a T cell specific deficiency of phosphatidylinositol transfer protein (PITP) α and β, which regulate PI-mediated signaling in vitro. Our initial in vivo studies suggest that deletion of PITPα and PITPβ at the double positive stage of T cell development results in loss of mature T cells in the thymus and periphery. Further experiments will establish why PITPs are required for normal T cell development.

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