The CD2-CD58 counter-receptor pair facilities activation and effector function of T lymphocytes, regulates their IL-12 responsiveness and restores immune reactivity from an anergic state. The activities are mediated by mechanisms which include conformational changes in the CD2 ectodomain. During the last funding interval, we produced the hCD2 adhesion domain and defined its structure using electrospray ionization mass spectrometry and NMR. The CD58 binding site was mapped, the structure of the glycan determined and its role in stabilizing the V-set Ig domain ascertained. Studies of CD2-based signal transduction were initiated and the CD3 zeta requirement in association with either the TCR or CD16 complex shown. The importance of the CD2-CD58 interaction for enhancing APC-dependent IL-12 stimulated T cell proliferation and IFN-gamma was uncovered. The present proposal will provide further molecular detail on the CD2 extracellular and cytoplasmic segments. First, we will complete mutagenesis studies to precisely map the conformational CD2R site within the interdomain (DlD2) linker region and in conjunction with NMR and electron paramagnetic resonance, investigate the mobility of Dl relative to D2 in unligated CD2 NMR and electron paramagnetic resonance and investigate the mobility of Dl relative to D2 in unligated CD2 and CD2-CD58 complexes. Cellular activation stimuli which induce CD2R expression investigated. CD2-CD58 complexes will be made for structure determinations using NMR as well as x-ray crystallography. Second, the role of p59fyn in CD2-mediated signaling will be explored by analyzing the ability of concatamerized fyn SH3 binding sites to stimulate T cell activation or alternatively, dominant negative fyn variants to inhibit activation. The functional significance of these proteins will be ascertained in vitro through transfection analysis using anti-sense or mutational strategies and in vivo by creating transgenic and/or knockout mice. Third, studies employing CD58 vs hCD48 CHO transfectants will ascertain whether or not both CD2 ligands can augment IL-12 production. CD2 regulation of IL-12 responsiveness will be examined in a parallel mouse T cell system assaying TH2 vs. TH1 directed immune responsiveness to soluble antigens in CD2-/- vs. CD2-/+ littermates.
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