Control of Cytotoxic Lymphocytes by Polymorphic KIR3DL3

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1 Summary 2 Innate cytotoxic lymphocytes, including natural killer (NK) cells, are essential components of immune 3 surveillance for infection and malignancy. Their effector functions are modulated through interaction of multiple 4 ligand-receptor pairs at the immune synapse between lymphocyte and target cell. In this regard, killer cell 5 immunoglobulin-like receptors (KIR) can suppress killing of any healthy cells that express their ligand and 6 encourage killing of unhealthy cells that do not express their ligand. Individuals and populations vary both in the 7 number of KIR genes present and the specific alleles of those genes. This extreme polymorphism of the KIR 8 gene family is implicated in susceptibility to infectious, allergic, inflammatory, and autoimmune diseases, and in 9 the success of hematopoietic cell transplantation and reproduction. KIR3DL3 is unique in being the only KIR that 10 is conserved through multiple primate species and observed in every human individual. 11 Whereas the ubiquity of KIR3DL3 underlines its necessity in human survival, we lack a basic understanding of 12 its biological role, including signalling capability, ligand interactions, influence on downstream effector functions 13 and the tissues where it can be expressed. While our overarching goal is thus to establish the function, binding 14 partner and tissue distribution of KIR3DL3, we will also explore its utility for immunotherapy. Substantially 15 justifying the latter, we have identified a ligand for KIR3DL3 that could be utilized to aid NK cells in specific 16 detection and elimination of infected or cancerous cells. The ligand is closely related to other markers that have 17 been successfully used for immune checkpoint inhibition therapy. In Aim 1, we will describe the KIR3DL3 ligand 18 and define the role of this interaction in NK cell biology. We will measure the kinetic properties of the interaction, 19 generate a crystal structure of the complex and perform assays of the effector functions following receptor 20 ligation. In Aim 2, we will use biochemical approaches to characterize the requirements for KIR3DL3 intracellular 21 signalling and define the associated pathways. Other KIR contain two specific amino acid motifs in the 22 cytoplasmic tail that mediate inhibitory signals. KIR3DL3 contains only one of these motifs, and our evolutionary 23 analysis suggests that receptor aggregation is necessary to bring them into proximity for signalling. 24 In our preliminary analysis we identified KIR3DL3 expression in a subset of tissues, and these are the same as 25 the tissues where we identified the ligand to be expressed. To investigate the hypothesis that tissue resident 26 cytotoxic cells interact with specific tissue cells through KIR3DL3, in Aim 3, we will determine the in vivo 27 expression profile of receptor and ligand. We will also engineer a chimeric KIR3DL3 that can direct NK cells to 28 kill, rather than spare unhealthy cells that express the ligand. KIR3DL3 is highly polymorphic and one of the most 29 heterozygous human genes. Thus, throughout all our aims, we will assess the impact of polymorphism on the 30 properties and functions of KIR3DL3.