The overall objective of this research program is to understand how cytokines of the tumor necrosis factor (TNF) ligand and receptor families orchestrate T cell mediated defenses against malignant and virus-infected cells. We have recently cloned a novel cytokine, termed LT-beta, a type II transmembrane protein, which shares structural and genetic homology with TNF and lymphotoxin (LT-alpha). LT-beta complexes with LT-alpha to provide a membrane anchor for this cytokine on activated T cells. LT-beta and LT- alpha assemble as trimeric molecules that differ in their subunit composition forming ligands that bind distinct sets of receptors. The LT- alpha 3 homotrimer and lT-alpha2beta1 bind to TNFR 60 and TNFR80, whereas LT-alpha1beta2 and LT-beta3 bind to a novel receptor, termed lT-betaR. LT- betaR shares structural homology with both TNFR60 and TNFR80 including a Cys-rich binding domain characteristic of this receptor family and an apoptosis signalling domain. However, the molecular mechanisms regulating tahe formation of these heteromeric ligands, the basis of receptor specificity and the cellular responses initiated via the lT-betaR are not well defined and are the focus of this project. Our approach will be to utilize advanced protein modeling programs to develop testable hypothesis concerning the formation and receptor interactions of surface LT. Computer aided models of LT-beta and the LT-betaR will be built upon the crystal structure of lT-alpha and the TNFR60 to predict regions important in the specific interactions leading to cellular responses. The validity of the models will be tested using site-directed mutations to generate muteins which will be assessed for complex assembly, receptor specificity and biologic functions. Soluble receptors formed as dimeric Fc fusion proteins will be used to purify soluble forms of the LTR-alpha beta complexes and LT-beta 3 produced in a baculovirus expression system. Cell lines transduced with the LT-betaR or TNFR will be used investigate the binding properties and cellular responses of the purified ligands. Antibodies produced against LT-beta, LT-betaR, and mAb recognizing epitopes specific to LT-alphabeta complex, in conjunction with TNFR;Fc and lT-betaR:Fc ligand antagonists, will be used as modulators in defined tissue culture models of immune responses. These tissue culture models will include assays of antigen recognition, lymphocyte proliferation, differentiation and effector function. The development of these reagents and tissue culture systems will allow us to define the likely roles for this cytokine family in immune and inflammatory responses. It is anticipated that this work will spawn novel reagents and approaches in designing cytokine modulators for immunotherapy.
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