Determinants of Natural Killer Cell Ly49 Receptor Specificity

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Natural killer (NK) cells are lymphocytes that act as a first line of defense against viruses and tumours, destroying pathologically altered cells. NK cells express inhibitory receptors specific for class I (CI) major histocompatiblity complex molecules ubiquitously expressed on healthy cells. Lack of engagement of NK inhibitory receptors, due to loss of CI expression that typically occurs with virus-infected cells or tumour cells, releases inhibition of NK cells, allowing them to destroy such altered cells. Ly49 are large families of major class of CI-specific receptors expressed by mouse and rat NK cells, but not humans. CI associate with beta-2 microglobulin (b2m) and bind and present peptide fragments from intracellular proteolytic events. We, and others, have found that the identity of the CI-bound peptide can determine Ly49 recognition of CI. What is unclear, is the nature of endogenous peptides in healthy cells that regulate NK cells, and the in vivo significance of peptide selectivity by Ly49. Our long-term goal is to understand the molecular basis of peptide selectivity of Ly49 recognition. The significance and uniqueness of this undertaking is that it directly addresses the very fundamental biological question of the molecular basis of healthy cell recognition by NK cells. Furthermore, understanding Ly49 recognition, may provide novel insights into host resistance to infections such as hanta viruses in mice, and possibly diseases in horses, a species which also has an expanded Ly49 gene family. We hypothesize that certain and specific endogenous peptides support CI recognition by Ly49, identification of which will provide a window into the purpose of Ly49 surveillance. We also hypothesize that peptide selectivity determines Ly49 affinity thresholds, and has in vivo significance. To test these hypotheses, we propose the following objectives:***1. Assess diversity and identity of endogenous peptides controlling Ly49C recognition. Advances in mass spectrometry have identified endogenous peptides bound to CI. We will examine individual endogenous peptides bound to CI (Kb) in supporting Ly49C recognition, including their molecular properties and proteins of origin (from peptide sequence database matching), in contributing to the control of Ly49C recognition.****2. Define the influence of CI bound peptide identity in determining Ly49 affinity. We will determine the affinities of Ly49C for different CI (Kb)-peptide complexes by surface plasmon resonance. This should reveal whether there are threshold affinities for Ly49C interaction controlled by identities of bound peptides.***3. Determine the influence of Ly49 peptide selectivity on NK cell activity in vivo. By transgenesis on a b2m-/- background we will express genetically engineered single chain peptide-CI complexes to determine the ability of distinct individual peptides to support licensing and function of NK cells in vivo.**