Activation of naive CD4 T helper cells results in the development of at least two distinct effector populations, Th1 and Th2. Th1 cells produce pro-inflammatory cytokines and are commonly associated with cell mediated immunity and clearance of intracellular pathogens. Th2 cells on the other hand produce anti-inflammatory cytokines that are crucial for control of extracellular pathogens and promote atopic and allergic reactions. Th1 cells have been implicated in the induction of many organ specific autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), a model for the human disease multiple sclerosis (MS). Although much is known about the functions of these two helper subsets, there are few surface molecules that differentiate between them. We have recently cloned and characterized a cell surface protein, Tim-3 (T cell, Immunoglobulin and Mucin containing molecule), which is expressed only on differentiated Th1 cells. Administration of monoclonal antibodies to Tim-3 in-vivo results in exacerbation of EAE, with extensive demyelination and macrophage activation and expansion. Based on these data, we hypothesize that a cognate interaction between Tim-3 and its ligand regulates T cell and macrophage activation and expansion. A genetic interval (locus) containing the Tim-3 molecule has shown linkage with susceptibility in murine asthma models and also in a number of autoimmune diseases, including EAE. In a preliminary molecular analysis, we have identified an allelic polymorphism in the Tim-3 gene in EAE-susceptible (SJL) and EAE-resistant (B 10.S) mice. In this grant application, we propose to: 1) characterize the phenotype and function of T cells and macrophages following in-vitro and in-vivo crosslinking of Tim-3 and its ligand. This will be accomplished by further characterizing the alternately spliced, secreted form of Tim-3 and testing the in-vivo effects of the soluble Tim-3 (administered as an Ig-fusion protein) on T cell activation and Th1/Th2 differentiation; 2) determine whether the interaction of Tim-3 and its ligand is an activating or inhibitory signal for T cell and macrophage activation and function by using Fab fragments of the anti-Tim-3 antibody and by using T cells and non-T cells from Tim-3 -/- deficient mice; 3) study the role of Tim-3 in changing disease susceptibility and resistance to EAE by utilizing the variant forms of soluble Tim-3 (as soluble Ig-fusion proteins) derived from susceptible (SJL) and resistant (B 10.S) mice. By characterizing the interaction of Tim-3 with its ligand, we will be able to identify the of role of Th1 specific molecule Tim-3 in macrophage activation and in the induction of autoimmune disease.
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