Allelic variation and 'chimerism' of Ly49 natural killer cell receptors

Natural killer (NK) cells comprise a CD3- lymphoid population which demonstrates cytolysis of target cells. Recent advances regarding the molecular mechanisms involved in NK cell target recognition have revealed that this NK cell function is mediated by cell surface receptors encoded by several distinct multigene families. Interestingly, a common theme observed between each of these multigene families is the presence of activating and inhibitory forms. For example, Ly49A, the prototypic member of the C-type lectin-like family, has been shown to act as an inhibitory receptor that binds H-2Dd while Ly49D has been shown to act as an activation receptor. Similar activating and inhibitory pairs have been observed for the other NK cell receptor families. Thus, NK cell function is regulated by several structurally distinct inhibitory and activating receptors that are encoded by multigene families.;While the Ly49 family of NK cell receptors is encoded by a polygenic genetic locus, allelic forms have also been described. In order to determine whether differences between Ly49A alleles would have functional consequences, allelic variants of Ly49A were cloned from several inbred mouse strains representative of different restriction fragment length polymorphic groups. Stable transfectants expressing each Ly-49A allelic variant were generated and tested for reactivity with a panel of monoclonal antibodies specific for the C57BL/6 form of Ly49A. Binding to H-2Dd was also assessed using fluorescently labeled H-2Dd tetramers. Furthermore, cytotoxicity assays were performed using anti-Ly49A mAb separated NK cells. We show that despite binding to fluorescently labeled H-2Dd tetramers that the Ly49A+ NK cells from representative mouse strains displayed significantly different degrees of inhibition with H-2Dd targets. Thus, the Ly49 family displays functionally significant allelic polymorphism which affects the repertoire of NK cell receptors.;In contrast to Ly49A, Ly49D is an activation receptor expressed on C57BL/6 derived NK cells. Ly49D is responsible for lysis of Chinese hamster ovary (CHO) and H-2Dd expressing targets. A novel Ly49 molecule was isolated from C57L mice which is recognized by mAb 4E4 (anti-Ly49D). Interestingly, the Ly49C57L shares extensive similarity to Ly49DB6 in its extracellular domain but the cytoplasmic domain of Ly49 C57L is identical to Ly49AB6 including a cytoplasmic immunoreceptor tyrosine-based inhibitory motif. Cytotoxicity experiments revealed that 4E4+ LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class-I tetramer staining revealed that the Ly49C57L bound H-2Dd while Ly49DB6 did not. Furthermore, 4E4 + C57L LAK cells display decreased lysis of H-2Dd expressing targets. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.;Crystallization of the Ly49A-H-2Dd complex revealed that Ly49A has two ligand binding sites for H-2Dd. The structural basis for ligand binding by Ly49A alleles or the "chimeric" Ly49 can be examined in the context of this crystal structure. Taken together, the results presented in this dissertation suggest that even though each individual Ly49 family member appears to be structurally similar that allelic polymorphisms and "chimerism" can alter specificity and function ultimately increasing the complexity of the Ly49 repertoire.