Regulated diacylgylcerol metabolism during antigen receptor stimulation of T cells and mast cells

Diacylglycerol (DAG) is a critical lipid second messenger (LSM) generated following stimulation of a number of different cell surface receptor including G protein-coupled and tyrosine kinase associated receptors. Analyses of the importance of this LSM have been difficult to perform by classical gain- or loss-of-function approaches. The experiments described in this dissertation have tested the importance of DAG metabolism to immune receptor signaling by manipulating the function of key enzymes involved in DAG metabolism, Diacylglycerol Kinases (DGKs). DGKs phosphorylate DAG to generate phosphatidic acid (PA), another important LSM. We hypothesized that DGKs function as negative regulators of immune receptor signaling by removing DAG and attenuating DAG-dependent signals. To test the functional consequences of altered DAG metabolism in T cells and mast cells, we have overexpressed DGK isoforms, studied DGK-deficient cells, and inhibited DGKs pharmacologically. We demonstrate that overexpression of DGKζ or DGKα in T cells results in a T cell receptor (TCR) signaling defect resembling that seen in anergic T cells. Conversely, loss of DGKα and DGKζ function impairs T cell anergy. We provide evidence that one function of CD28 co-stimulation is to increase DAG production, and we demonstrate that impaired DAG metabolism during TCR-alone stimulation is sufficient to abort anergy differentiation. Our studies of DGKζ-/- mast cells have revealed an unexpected function of this enzyme in linking antigen stimulation to optimal activity of PLCγ and PLCβII. We demonstrate that in the absence of DGKζ, FcϵRI-induced PIP2 hydrolysis and IP3 generation are impaired, while PIP3 production and Akt phosphorylation are enhanced. In addition, we demonstrate impaired PKCβII membrane localization in DGKζ-/- BMMC following stimulation with antigen or phorbol esters. These studies suggest that DGKζ regulates phosphatidylinositol metabolism and effector molecule activation through mechanisms in addition to termination of DAG signaling