Structural, functional, and mechanistic anlaysis of autoreactive CD8 T cells

摘要 CD8 T细胞是许多自身免疫性疾病的重要致病因素， 包括1型糖尿病。尽管自身反应性CD8 T细胞很重要，但许多基础知识 在几个主要方面缺乏，包括：1)I类MHC等位基因的特性，这些等位基因赋予易感性或 对疾病的抵抗力；ii)构成混杂抗原识别的决定因素通常表现为 自体反应性T细胞；以及iii)CD8CD8αβ共受体在促进自身免疫过程中的作用。 这些是我们寻求填补的关键知识空白，我们将使用多学科方法来做到这一点 以及一系列创新的策略、试剂和动物模型。在目标1中，我们将定义 人类I类MHC等位基因的生化、结构特征及体内多肽递呈特性 与1型糖尿病的易感性或抵抗力有关。这些研究将集中在人类白细胞抗原-B*39：06， B*39：01和B*38：01，这是一组密切相关的I类MHC等位基因，与 疾病。我们将检验这三个等位基因对疾病的区分能力是 某些与疾病相关的β细胞肽的不同结合和呈现的结果。我们的新品 开发的表达人胰岛素或人IGRP的NOD小鼠模型将是实现这一目标的核心。在目标2中， 我们将使用来自一组混杂和自身反应性CD8T细胞克隆的T细胞受体在结构和 突变研究以确定T细胞受体交叉反应的基础机制。除其他外， 方法，我们将利用我们开发的新的高通量分析方法来识别T细胞 细胞表位和MAP蛋白结合界面。在目标3中，我们将使用生化和生物物理 更好地理解CD8MHC和多肽-αβ相互作用的方法和体内模型。 尽管CD8αβ需要CD8T细胞的选择和外周细胞的激活，但它作为一种 治疗靶点，其与pMHC相互作用的结晶学分析仅限于 H-2DD诱导的小鼠CD8αβ。我们最近对小鼠模型的研究结果表明， 人CD8MHC可能以单一可用结构未揭示的方式与I类αβ相互作用。 虽然在这里研究的自体反应性CD8T细胞可能表现出一些独特的特征，因为它们 逃避容忍机制的必要性，将学到的大部分将是基础知识，即 普遍适用于CD8T细胞生物学，这将有助于指导今后更有效的发展 在健康和疾病中操纵和利用免疫系统的方法。

ABSTRACT CD8 T cells are important contributors to the pathology observed in many autoimmune diseases, including type 1 diabetes. Despite the importance of autoreactive CD8 T cells, much fundamental knowledge is lacking in several major areas, including i) the properties of class I MHC alleles that confer susceptibility or resistance to disease; ii) the determinants that underlie the promiscuous antigen recognition often displayed by autoreactive T cells; and iii) the contribution of the CD8αβ co-receptor in facilitating autoimmune processes. These are the critical knowledge gaps that we seek to fill, and we will do so using a multidisciplinary approach and a collection of innovative strategies, reagents, and animal models. In Aim 1, we will define the biochemical and structural features and in vivo peptide presentation properties of human class I MHC alleles associated with susceptibility or resistance to type 1 diabetes. These studies will focus on HLA-B*39:06, B*39:01, and B*38:01, a closely related group of class I MHC alleles that are differentially associated with disease. We will test the hypothesis that the differential ability of these three alleles to mediate disease is the consequence of differential binding and presentation of certain disease-relevant beta cell peptides. Our newly developed NOD mouse models expressing human insulin or human IGRP will be central to this aim. In Aim 2, we will use the T cell receptors from a set of promiscuous and autoreactive CD8 T cell clones in structural and mutagenesis studies to define the mechanisms that underlie T cell receptor cross-reactivity. Among other approaches, we will take advantage of novel high-throughput assays that we have developed both to identify T cell epitopes and map protein binding interfaces. In Aim 3, we will use biochemical and biophysical approaches and in vivo models to better understand the interaction between CD8αβ and peptide-MHC. Despite the requirement of CD8αβ for CD8 T cell selection and peripheral activation, and its potential as a therapeutic target, crystallographic analysis of its interaction with pMHC is limited to a single structure of murine CD8αβ with H-2Dd. Our own findings from mouse models we have recently characterized suggest that human CD8αβ may be interacting with class I MHC in a manner not revealed by the single available structure. While autoreactive CD8 T cells under study here are likely to exhibit some unique features due to their necessity to escape tolerance mechanisms, much of what will be learned will be fundamental knowledge that is generally applicable to CD8 T cell biology and that will help guide the future development of more effective approaches to manipulate and harness the immune system in health and disease.