Document Type

Dissertation

Date of Degree

Spring 2015

Degree Name

PhD (Doctor of Philosophy)

Degree In

Immunology

First Advisor

Jon C. Houtman

Abstract

CD4+ T cells are critical in the fight against parasitic, bacterial, and viral infections, but are also involved in many autoimmune and pathological disorders. Ligation of the T Cell Receptor (TCR) is the primary signal required for T cell activation proliferation, differentiation and cytokine release. Upon TCR activation, several kinases and adaptor proteins are assembled at the TCR/linker for activation of T cells (LAT) signaling complexes, a process indispensable for optimal signal transduction. One important group of proteins recruited to the TCR/LAT complexes is the GRB2 family of adaptors.

Due to their role in mediating signaling complexes, the GRB2 family of adaptors are critical for development, proliferation, and survival of diverse cell types. These proteins have been linked to the initiation and progression of numerous pathological conditions including diabetes, asthma/allergy, and solid and hematopoietic malignancies. Therefore, it is essential to characterize and understand the complete functions of these proteins for the generation of safe and efficient targeting treatments for diseases mediated by these proteins. In T cells, GRB2 and its homologs, GADS and GRAP, are crucial for the propagation of signaling pathways through the TCR and adaptor protein LAT. These proteins recruit distinct sets of proline-rich ligands to LAT thereby inducing multiple signaling pathways such as MAP kinase activation, calcium influx and cellular adhesion. However, the role of GRB2 family members in controlling TCR and LAT mediated signaling in mature human T cells is not completely understood. Moreover, the relative role of GRB2 family members in the extent and timing of the recruitment of SH3 domain ligands to the LAT complex is unknown. Our hypothesis is that these proteins recruit distinct sets of ligands to the LAT complex that can drive differential downstream signaling events.

As presented in CHAPTER III, we developed microRNA and shRNA targeting viral vectors to effectively inhibit the expression of GRB2 and GADS in human CD4+ T cells to examine the role of these adaptors in mature human T cells. We also established optimized protocols for high efficacy retro or lentiviral transduction of human T cell lines, activated and "hard-to-transduce" non-activated primary human CD4+ T cells. In CHAPTER IV, we demonstrate the requirement for GRB2 in TCR-induced IL-2 and IFN-γ release. The defects in cytokine release in the absence of GRB2 were attributed to diminished formation of LAT signaling microclusters, which resulted in reduced MAP kinase activation, calcium flux and PLC-γ1 recruitment to LAT signaling clusters. Overall, the data presented in this chapter demonstrate that the ability of GRB2 to facilitate protein clustering is as important in regulating TCR-mediated functions as its capacity to recruit effector proteins. This highlights that GRB2 regulates signaling downstream of adaptors and receptors by both recruiting effector proteins and regulating the formation of signaling complexes. In CHAPTER V, we describe the role for GADS in mediating TCR-induced IL-2 and IFN-γ production. GADS was critical for the recruitment of SLP-76 and PLC-γ1 to the LAT complex and subsequent calcium influx. We also show, in contrast to the current paradigm, that recruitment of GADS/SLP-76 complexes to LAT is not required for TCR-mediated adhesion and cytoskeletal arrangement.

Overall, our studies reveal novel mechanisms for the role of GRB2 family members in TCR-mediated signaling. They also provide insight into the mechanisms that regulate growth factor, cytokine and insulin receptors. Importantly, studies presented in this thesis will help us understand the mechanisms of T cell activation and highlight potential new therapies for T cell-mediated diseases, including leukemia, lymphomas, autoimmune disorders and cardiovascular disease.

Public Abstract

CD4+ T cells are a type of white blood cell important in the fight against infections, but they also can cause many human diseases when not controlled correctly. The main way for T cells to fight infections is through the activation of the T cell antigen receptor (TCR). Upon TCR activation, several proteins that are needed to transmit the TCR signal inside of the cell are brought together at the T cell plasma membrane, including a family of proteins known as the GRB2 family of adaptors. The GRB2 family members are important for fighting infections but also can be involved in autoimmune disorders and cancer. Therefore, it is important to understand how GRB2 family members work in T cells to better develop the next generation of safe and efficient treatments for diseases caused by these proteins. In this thesis, we first describe techniques that allow us to reduce the levels of two members of the GRB2 family of proteins, GRB2 and GADS. We next show that, through different means, both GRB2 and GADS control signals caused by the activation of the TCR that are needed for the immune response to fight infections. These studies will help us understand how T cells fight infections, while also potentially finding new therapies for diseases caused by inappropriate T cell activation, including leukemia, lymphomas, autoimmune disorders and cardiovascular disease.

Keywords

publicabstract, Cell signaling, GADS, GRB2, LAT microclusters, shRNA/microRNA, T cell receptor

Pages

xviii, 167

Bibliography

152-167

Copyright

Copyright 2015 Mahmood Yousif Bilal

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