The role of Notch in T cell activation and development
Notch is crucial for multiple stages of T cell development, including the CD4+CD8+ double positive (DP) to CD8 + single positive (SP) transition, but regulation of Notch activation is not well understood. In this thesis, I explored the potential of p53, endocytosis, and Cbl-b to regulate Notch activation. p53 regulates Presenilin1 (PS1) expression, and PS1 cleaves Notch, releasing its intracellular domain (NIC), leading to the expression of downstream targets, e.g. the HES1 gene. One aim of this thesis was to determine if p53 regulates Notch activity during T cell development. I found that Notch1 expression and activation were negatively regulated by p53 in several thymoma lines. Additionally, NIC was elevated in Trp53 −/− thymocytes as compared to Trp53 +/+ thymocytes. To determine if elevated Notch1 activation in Trp53−/− thymocytes had an effect on T cell development, CD4 and CD8 expression were analyzed. The CD4+ SP:CD8+ SP T cell ratio was decreased in Trp53 −/− splenocytes and thymocytes. This alteration in T cell development correlated with the increased Notch1 activation observed in the absence of p53. These data indicate that p53 negatively regulates Notch1 activation during T cell development. Skewing of T cell development toward CD8 + SP T cells in Trp53−/− mice is reminiscent of the phenotype of NIC-overexpressing mice. Thus, I suggest that p53 plays a role in T cell development, in part by regulating Notch1 activation. In the second aim of my thesis I present preliminary data showing that endocytosis does not appear to be involved in mammalian Notch activation although there is evidence in Drosophila for a positive endocytic role in Notch activation. The ability of Cbl-b to regulate Notch activation was the final aim of this thesis. Cbl-b, like Notch, has been shown to play a role in regulating the T cell signaling threshold. With this aim, I wanted to address the possibility of Cbl-b regulating T cell signaling via regulating Notch activation. Due to technical difficulties I was only able to obtain preliminary data suggesting that Cbl-b does positively regulate Notch activation in peripheral T cells. In this dissertation I have shown that Notch activation is regulated by controlling the expression of cellular components needed for cleavage, not just by encountering ligand on neighboring cells.
0307: Molecular biology