Date of Award

Summer 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Immunology

First Advisor

Gary Koretzky

Abstract

The cytosolic adapter protein src homology 2(SH2) domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) lacks enzymatic activity but nucleates a multi-molecular signaling complex that links early T cell receptor (TCR)-induced phosphorylation events into multiple downstream signaling pathways. The N-terminus of SLP-76 contains three tyrosines at residues 112,128 and 145 that are phosphorylated following TCR ligation and, although the mechanisms are not entirely clear, they are required for optimal TCR signal transduction. TCR signals are required for T cell proliferation, cytokine production, and effector and memory differentiation. The experiments described in this dissertation have first tested the biochemical mechanisms by which the SLP-76 tyrosines transmit signals and second tested how alterations in the TCR signals transmitted through SLP-76 tyrosines influence T cell differentiation and effector function. Experiments were performed using two genomic knock-in (KI) mice that express tyrosine to phenylalanine mutations at residue 145 (Y145F) or 112 and 128 together (Y112/128F). Using biochemistry-, flow cytometry- and microscopy-based approaches we show that mutations in the tyrosines of SLP-76 result in graded defects in TCR-induced signals and function depending on the tyrosine(s) affected. Surprisingly, localization of SH2 domain containing effector proteins to mutant SLP-76-nucleated signaling complexes was not lost and therefore could not account for the observed signaling defects. Infection of SLP-76 KI mice with lymphocytic choriomeningitis virus (LCMV) resulted in normal CD8 expansion but graded enhancement of memory differentiation consistent with a model in which weaker TCR signals preferentially promote memory versus effector differentiation. Furthermore CD8+ effector and memory KI T cells failed to produce appropriate cytokine upon antigen restimulation. Similarly, in vitro polarized KI Th17 and Th2 cells failed to produce IL17a and IL4, respectively, following TCR restimulation. Taken together our data show that SLP-76 tyrosines are essential for optimal TCR signal transduction and, moreover, TCR signals sufficient to promote T cell differentiation are different than those required to elicit inflammatory cytokine production.

Files over 3MB may be slow to open. For best results, right-click and select "save as..."

Share

COinS