Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

David Straus

Abstract

Proper T-cell activation and effector function are essential for effective immunity. T-cell antigen receptor (TCR) signals are known to regulate the outcome of differentiation, but the mechanisms remain unclear. Recent work from our lab demonstrates that the Src family protein tyrosine kinase, p56Lck, is able to specifically link TCR signals to activation of the Mitogen Activated Protein Kinase (MAPK) pathway through the function of its SH3 domain. The MAPK pathway is known to be involved in T-cell activation downstream of TCR ligation and has previously been implicated in T-helper type 2 (Th2) effector function. We have utilized an Lck SH3 mutant knock-in mouse line (Lck W97A) to investigate the potential role of this regulatory signaling mechanism in determining T-lymphocyte activation and effector function. Our results demonstrate that the Lck SH3 domain function is required for normal activation of T-lymphocytes following TCR stimulation as indicated by significantly reduced proliferation, IL-2 production, and CD69 induction in Lck W97A T-cells. Biochemical studies confirm that activation of the MAPK pathway is selectively altered in Lck W97A T-cells as P-ERK1/2 induction is significantly reduced but phospho-PLCg1 induction and calcium mobilization is unaffected. In vivo experiments demonstrate a specific and significantly impaired Th2 immunity in Lck W97A mice, with reduced serum levels of IgG1, IgE and IL-4 following immunization with DNP-KLH, or infection with the helminth Nippostrongylus brasiliensis. Th1 immunity does not appear differentially regulated in Lck W97A mice as serum levels of IgG3 and IgG2b are similar to WT following immunization with DNP-KLH, as well as serum levels of IFN-g1 following immunization with heat-killed Brucella abortus. In vitro differentiation studies confirm that Lck W97A T-lymphocytes are able to be directed to the Th2 phenotype, as indicated by intracellular staining for IL-4, with significantly increased levels of IFN-g under Th2 differentiating conditions compared to WT. These data indicate that the Lck SH3 domain regulates activation of T-lymphocytes by affecting MAPK pathway induction and demonstrate a novel and critical role for Lck in the regulation of Th2-type immunity. The Lck SH3 domain has also been implicated in the pathogenesis of Plasmodium, the causative agent of malaria. The role of the mosquito vector on malaria pathogenesis is not well understood. Initial studies examining the role of vector salivary gland proteins on cells of the innate immune system indicate that Anopheles stephensi saliva is able to enhance macrophage activation and phagocytosis as well as enhance macrophage Ag-presentation to T-lymphocytes in an in vitro model.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2009

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