Adaptor proteins play a crucial role in immunoreceptor signaling. Even though they lack enzymatic activity, these proteins have modular domains that are able to interact with other proteins and form signaling complexes, leading to the activation of signaling cascades. While some adaptors, such as LAT and SLP-76, have a positive and essential role in T cell receptor-mediated signaling and thymocyte development, other adaptor proteins negatively regulate T cell activation. One such adaptor is LAB (Linker of Activation of B cells), a LAT-like molecule that is expressed in B cells, myeloid cells, and activated T cells. It is tyrosine phosphorylated and interacts with Grb2 upon the engagement of immunoreceptors. Our data show that T cells in LAB-/- mice are hyperactivated due to enhanced TCR-mediated signaling, causing the development of a lupus-like syndrome. In addition, LAB-/- mice have an enhanced T-independent secondary antibody response. The mechanism by which LAB regulates T cell activation and autoimmunity is not clear. Based on our data, we propose the following hypothesis: after T cell activation, LAB is upregulated to inhibit TCR- mediated signaling by antagonizing LAT function. In addition to its negative role, LAB can also function positively in T cells.
Three specific aims are designed to test this hypothesis.
In specific aim 1, we will investigate the mechanism by which LAB negatively regulates TCR-mediated signaling and cellular activation. We will examine whether LAB expression impairs LAT recruitment into the immune synapse by live imaging. We will map the inhibitory region in LAB and identify the important tyrosine residues required for its negative function. We will further use LAB knock-in mice to study whether LAB phosphorylation is important in the control of T cell activation and autoimmunity in vivo.
In specific aim 2, we will investigate the function of LAT and LAB in CTL-mediated cytotoxicity. We will examine the ability of CTLs deficient in LAB, LAT, or both, to eliminate target cells. Biochemical analyses of TCR-mediated signaling pathways will be performed to determine which signaling events are required for CTL-mediated killing. Furthermore, we will examine the immune synapse formation, MTOC reorientation, and movement and delivery of lytic granules in these CTLs by live imaging.
In specific aim 3, we will investigate the role of LAB in the regulation of T cell function during immune responses. We plan to identify which T cell subset is hyperresponsive in vivo and in vitro. The OT-1 TCR transgenic system will be used to study LAB function in CD8+ T cell expansion, contract, and memory response.
T cells are the central components of our immune system, and transmembrane adaptor proteins are critical in the regulation of T cell activation. Understanding the function of adaptor proteins could facilitate the design of a rational approach to augment or inhibit T cell proliferation in autoimmunity, allergy, transplantation, and host defense.
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