<p>The homeostasis of naïve T lymphocytes is maintained by several mechanisms involving basal TCR and cytokine signaling, and nutrient factors. One of the common net results of these input signals is the production and stabilization of anti-apoptotic Bcl-2 family members. A second result of these processes is the induction of autophagy, an intracellular, catabolic, lysosomal targeting pathway. Autophagy induction in most systems involves the class III phosphatidylinositol-3 kinase (PI3K), Vps34, to produce phosphatidylinositol-3-phosphate (PI(3)P). To test this in T lymphocytes, I generated mice specifically lacking Vps34 in T cells (Vps34f/fLck-cre mice). However, Vps34-deficient T lymphocytes have normal levels of basal autophagy, and upregulate autophagy normally in response to cytokine or nutrient withdrawal, or TCR stimulation. Therefore I conclude that Vps34 activity is not required for autophagy induction in T lymphocytes. T lymphocytes lacking Vps34 do have enhanced rates of apoptosis, but this is due to defects in intracellular trafficking, specifically of the Interleukin-7 receptor alpha subunit (IL-7Rα). Additionally, multivesicular body (MVB) maturation is impaired in T cells lacking Vps34 such that extracellular ligands are not efficiently targeted to the lysosome. </p><p>Autophagy induction in Vps34-deficient T lymphocytes is still sensitive to pan-PI3K inhibitors, such as wortmannin and 3-methyladenine (3MA). Therefore, I hypothesized that other classes of PI3K are necessary to induce autophagy in T lymphocytes through the production of PI(3)P. Autophagy induction is sensitive to specific class I PI3K (PI3KI) inhibitors, such as PIK75. Additionally, T cells lacking the p85 regulatory subunit of PI3KI also have severe defects in T cell receptor (TCR) mediated autophagy induction. PI3KI activity results in the production of PI(3,4,5)P3, though, and not PI(3)P. Because of this specificity, I hypothesize that additional inositol polyphosphatases (Inpp) are required for autophagy induction downstream of PI3KI activity. Indeed, utilizing both inhibitors of pharmacological inhibition and siRNA-mediated knockdown of two classes of phosphatidylinositol phosphatases, inositol polyphosphate-4-phosphatase (Inpp4) and SH2 containing inositol phosphatase (SHIP), had dramatic impacts on autophagy induction. Furthermore, exogenous addition of PI(3,4)P2, a hypothesized intermediate in this pathway, positively regulates autophagy induction and leads to enhanced progression of autophagy. These observations indicate that PI3KI activity, linked to Inpp activity, are necessary and positive regulators of autophagy through the production of PI(3)P.</p> / Dissertation
| Identifer | oai:union.ndltd.org:DUKE/oai:dukespace.lib.duke.edu:10161/7138 |
| Date | January 2013 |
| Creators | McLeod, Ian Alexander |
| Contributors | He, You-Wen, Zhuang, *Yuan |
| Source Sets | Duke University |
| Detected Language | English |
| Type | Dissertation |
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