NK cell inhibitory receptor interactions with MHC class I molecules

Abstract: Natural killer (NK) cells represent the third population of lymphocytes in size after B- and T-cells. NK mediated killing of target cells is strongly influenced by MHC class I molecules expressed on target cells. In contrast to T-cells, NK cells can be inhibited by MHC class I molecules through the action of specific inhibitory NK cell receptors. In the mouse, the Ly49 family represents the dominating receptors for recognition of classical MHC class I molecules. The Ly-49 family consists of at least 24 family members, Ly-49A to Ly-49W, with both inhibitory and activating receptors in the family. This thesis is devoted to the study of the inhibitory Ly-49 NK cell receptors and how they interact with MHC class I molecules. The interaction was studied on a molecular level with the aim to pinpoint the key contacts that determine the outcome of the Ly-49 receptor - ligand interaction. This has been accomplished by studies of Ly-49A contact sites in H-2D, and by studies of the Ly-49C receptor in terms of ligand recognition and characterization of residues involved in MHC class I binding. In the first study the role of the a-domains of H-2D in Ly-49A mediated inhibition was investigated. Studies of exon shuffled chimeric H-2D molecules showed that inhibition of Ly-49A expressing NK cells required the presence of the alpha2-domain of H-2D. In contrast, inhibition mediated by several human NK cell inhibitory receptors had been reported to depend on residues in the alpha1-domain of the MHC class I molecule. In the following study, the Ly-49A recognition area on MHC class I molecules was studied further by site directed mutagenesis of H-2D. Mutations introducing a hydrophobic ridge in the floor of the antigen binding groove of H-2D affected the interaction with Ly-49A. This indicated a possible involvement of the MHC bound peptides in the interaction. It had previously been shown that Ly-49A receptor expression on NK cells is low in presence of the MHC class I ligand in the host. It was therefore of interest to investigate whether the same rules applied to the Ly-49C receptor, using H-2K b as its major ligand. Ly-49C expression did not only vary depending of the MHC haplotype of the mice tested, the expression was also strongly influenced by the NK gene complex of the mice. It was hypothesized that this influence was due to an allelic variation in the Ly49C gene itself, with consequences for receptor specificity. An allelic variation was indeed confirmed by RTPCR cloning and sequencing of Ly-49C cDNA from the mouse strains C57BL/6 and A/Sn. This was the first cloning of the C57BL/6 allele of Ly-49C. Later studies by several groups suggest that this allelic variation is not influencing receptor specificity, and other factors explaining the original interpretation are discussed in the thesis. Inhibition via some human inhibitory NK receptors is known to be influenced by the peptides bound to MHC class I molecules, in contrast to the mouse Ly-49A receptor which is peptide dependent but not selective. The influence of peptides in the recognition by the Ly-49C receptor was investigated in this thesis. Ly-49C+ NK cells were inhibited efficiently by some but not all H-2K b binding peptides loaded on the TAP deficient cell line RMA-S. Residue 7 in the peptide was crucial in mediating the inhibition via Ly- 49C. Thus, Ly-49C mediated inhibition was peptide selective in context of H-2K b . The studies described above involved investigations of different contact areas in the MHC ligand. The last study of this thesis focused on potential contact residues in the Ly-49C receptor, selected on the basis of a molecular model of this protein. Site directed mutated Ly-49C cDNA was expressed in reporter cells, and tested for binding to H-2 class I tetramers refolded with different peptides. Three out of nine mutations in Ly-49C led to reduced or complete loss of binding of H-2D b , H-2D d and H-2Kb AMC class I tetramers. The three mutations that affected MHC class I binding were compared to the crystal structure of the Ly-49A - H-2D d co-complex. It was concluded that Ly-49C used partly the same residues as Ly49A to bind MHC class I ligands, suggesting that both receptors may bind MHC class I molecules in a similar fashion. A model based on this notion and the formation of multiple receptor-ligand lattices in the interface between the NK cell and the target cell is discussed.