Control of Cytotoxic Lymphocytes by Polymorphic KIR3DL3

1摘要 天然细胞毒性淋巴细胞，包括自然杀伤(NK)细胞，是免疫的重要组成部分。 3感染和恶性肿瘤的监测。它们的效应器功能是通过多个 4淋巴细胞与靶细胞免疫突触上的配体-受体对。在这方面，杀手细胞 5免疫球蛋白样受体(KIR)可以抑制任何表达其配体和 6鼓励杀死不表达其配体的不健康细胞。个体和群体都不同， 7存在KIR基因的数量和这些基因的特异性等位基因。KIR的这种极端多态 8基因家族与感染性、过敏性、炎症性和自身免疫性疾病的易感性有关，在 9造血细胞移植和繁殖的成功。KIR3DL3的独特之处在于它是唯一 10在多个灵长类物种中保守，并在每个人类个体中观察到。 11虽然KIR3DL3的普遍存在强调了它在人类生存中的必要性，但我们缺乏对 12其生物学作用，包括信号传递能力、配体相互作用、对下游效应器功能的影响 13和可以表达它的组织。虽然我们的首要目标是建立功能、绑定 14个KIR3DL3的配对和组织分布，我们还将探索其在免疫治疗中的应用。实质上 15为了证明后者是正确的，我们已经确定了KIR3DL3的一个配体，它可以用来帮助特定的NK细胞 16检测和消除感染或癌细胞。该配体与其他具有 17已成功用于免疫检查点抑制治疗。在目标1中，我们将描述KIR3DL3配体 18，并确定这种相互作用在NK细胞生物学中的作用。我们将测量相互作用的动力学特性， 19生成该复合体的晶体结构，并在受体之后进行效应器功能的分析 20结扎。在目标2中，我们将使用生化方法来表征细胞内对KIR3DL3的需求 21信令并定义相关路径。其他KIR包含两个特定的氨基酸基序 22胞质尾巴，介导抑制信号。KIR3DL3只包含其中一个基序，我们的进化 23分析表明，受体聚集是必要的，使它们接近信号。 24在我们的初步分析中，我们发现KIR3DL3在一组组织中表达，这些表达与 25我们确定要表达的配体的组织。来研究组织驻留的假说 26个细胞毒细胞通过KIR3DL3与特定的组织细胞相互作用，在目标3中，我们将确定体内 27受体和配体的表达谱。我们还将设计一种嵌合的KIR3DL3，它可以引导NK细胞 28杀死而不是保留表达该配体的不健康细胞。KIR3DL3高度多态，是世界上 29个人类杂合子基因。因此，在我们的所有目标中，我们将评估多态对 30 KIR3DL3的性质和功能。

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.