Naive T cells lack the protein 4-1BB, which is not only induced upon T cell activation, but also remains on activated T cells. The natural ligand for 4-1BB is also induced and is found on activated antigen-presenting B cells and macrophages. When 4-1BB is stimulated simultaneously with CD3, IL-2 and IFN-gamma are produced. The applicant's hypothesis is that 4-1BB is a costimulatory protein for T cell activation and that the cytoplasmic domain of 4-1BB (4-1BBcy) and its binding protein (4-1BBbp) are involved in signal transduction. Furthermore, it is proposed that 4-1BB signaling pathway is distinct and results in production of a greater spectrum of cytokines than the CD28 signaling pathway. To determine the molecular basis of 4-1BB-mediated signaling and the function of 4-1BB, this proposal contains five specific aims. 1. To determine the cytoplasmic regions of 4-1BB important for signaling. To conveniently assay the 4-1BBcy-mediated signaling and eliminate the endogenous 4-1BB effect, C8.A3 cells expressing 4-1BBcy chimeric proteins will be prepared. The chimeric proteins will be hTNFRII-4-1BBcy (the ectodomain of human tumor necrosis factor receptor-4-1BBcy) and hEpoR-4-1BBcy (the ectodomain of human erythropoietin receptor-4-1BBcy). IL-2 and IFN-gamma will be measured after triggering the chimeric receptors. 2. To isolate and characterize the intracellular molecules involved with 4-1BB signal transduction. The yeast-based two hybrid system has been employed to obtain genes that encode proteins that bind 4-1BBcy. cDNA clones have been isolated which are potentially associated with 4-1BBcy. Characterization of these gene products will be important for understanding 4-1BB-mediated signaling. 3. To compare and contrast the cytokines induced by CD28 and/or by 4-1BB-triggering. Th clones will be produced from 4-1BB or CD28 knockout mice and recombinant CD28 and 4-1BB will be expressed singly and together. The Th clones will be used to examine whether CD28 or 4-1BB can be engaged on the same T cell clone and produce different patterns of cytokine expression. 4. To examine selected B and T responses of 4-1BB KO mice for deficiencies. To determine a potential role that 4-1BB may play in lymphocyte development and/or activation, comparative studies will be carried out to evaluate different parameters of the immune system and the immune responses in 4-1BB+/+, 4-1BB+/-, and 4-1BB-/- mice. 5. To compare immune responses among 4-1BB KO, CD28 KO, and 4-1BB/CD28 KO mice. In this Aim a hypothesis will be tested that there exist additional costimulatory pathways responsible for part of the normal responses, or that alternative pathways take over in the absence of the CD28/B7 interaction.
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