T CELL ANTIGEN RECOGNITION

Dana-Farber Cancer Inst, Boston MA

Investigators

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Abstract

The T cell receptor (TCR) is a multi-subunit complex that mediates[unreadable] recognition of peptide antigens in the context of MHC molecules and is[unreadable] central for initiating signal transduction events including those[unreadable] initiated through accessory structures. During the last funding period,[unreadable] we utilized Xenopus oocyte reconstitution as well as lymphoid cell line[unreadable] transfection studies to show that the dimeric TCR component CD3zelu is[unreadable] structurally and functionally similar to FcepsilonRIgamma, a signaling[unreadable] subunit of the high affinity IgE receptor. We also found that[unreadable] FcepsilonRIgamma substitutes for CD3zelu to form the TCR in naturally[unreadable] occurring LGL. In addition, we isolated the 23KD murine CD3eta protein,[unreadable] microsequenced CNBr fragments and cloned CD3eta at both cDNA and genomic[unreadable] levels. CD3eta was shown to be an alternatively spliced product of the[unreadable] same genetic locus encoding CD3zelu on murine chromosome 1 where the[unreadable] FcepsilonRIgamma gene is also found. The different functional roles of[unreadable] CD3eta vs. CD3zelu are not yet apparent, although CD3eta is thought to[unreadable] be involved in thymic development. During the next granting period, we[unreadable] will definitively characterize the role of CD3eta in thymic selection.[unreadable] B6D2 transgenic (tg) mice overexpressing CD3eta protein by >100 fold have[unreadable] already been created and will be backcrossed onto C57BL/6 (H-2b) and[unreadable] DBA/2 (H-2d) mice. Differential Vbeta usage and susceptibility to anti-[unreadable] CD3eta mAb induced DNA fragmentation in vivo will be assessed in[unreadable] comparison to non-tg littermates. Disruption of the CDeta locus by[unreadable] targeted recombination of ES cells will also be exploited. Second, the[unreadable] constitutive or induced association of novel intracellular proteins with[unreadable] the TCR complex will be systematically investigated using a battery of[unreadable] detergents and ionic conditions with and without chemical crosslinkers.[unreadable] 31-13 TCR-Jurkat or other variant cells transfected with[unreadable] CD8alpha/CD3zelu/eta chimeric constructs, or as control, CD8alpha will[unreadable] serve as incisive tools to detect TCR associations since such a chimera[unreadable] mediates all the signals of the native TCR. Microsequencing and cDNA[unreadable] cloning of candidate proteins will be performed and their CD3zelu/eta[unreadable] association verified by immunoprecipitation and western blotting analysis[unreadable] with specific antibodies. In parallel, direct screening of lambdagt11[unreadable] T cell cDNA expression libraries will be performed with DC3zelu/eta[unreadable] derived peptides or fusion proteins containing the HMK phosphorylation[unreadable] site for 32P labeling. Finally, reconstitution studies in Xenopus[unreadable] oocytes will allow us to determine the minimal components of the TCR[unreadable] complex required for signaling phosphoinositide hydrolysis and.or protein[unreadable] tyrosine kinase activity. Oocyte microinjections will be performed with[unreadable] synthetic RNAs encoding CD8alpha/CD3zelu+/-p56lck+/-CD45 and signaling[unreadable] after anti-CD8alpha mAb crosslinking assessed. Any new cDNAs derived[unreadable] from aim 2 will be tested in this system as will others obtained by[unreadable] complementation methodologies.[unreadable]