Antigens Recognized by CD8+ T Cells in Type 1 Diabetes

描述（由申请人提供）：1型糖尿病（以前称为青少年发病或胰岛素依赖型糖尿病）是一种自身免疫性疾病，由朗格汉斯胰岛中产生胰岛素的β细胞破坏引起。胰岛素注射使100万美国1型糖尿病患者的生命得以延续；然而，它们既不能治愈这种疾病，也不能预防其毁灭性的并发症，如微血管病变、肾病和神经病变，这些并发症会降低生活质量，使糖尿病患者的预期寿命平均缩短15年。非肥胖糖尿病（NOD）小鼠为1型糖尿病提供了一个相关的模型系统，它具有许多人类疾病的特征。重要的是，患者和NOD小鼠都出现胰岛淋巴细胞浸润（胰岛素炎）和随后由T淋巴细胞介导的β细胞破坏。利用NOD小鼠的研究表明，仅限于I类主要组织相容性复合体（MHC）分子的β细胞自身反应性CD8 + T细胞在疾病发展中是绝对必需的。这些效应物也被怀疑对新发和移植物复发的1型糖尿病患者的β细胞消除有贡献。尽管CD8+ T细胞很重要，但它们参与最终导致疾病的a细胞破坏初始阶段的抗原特异性知之甚少。本提案的总体目标是确定这些起始特异性的T细胞限制在小鼠或人类I类MHC分子。从标准NOD小鼠或转基因表达HLA-A2的NOD小鼠的早期胰岛素病变中分离的CD8 + T细胞将用于此目的。生化和表达克隆相结合的方法将被用来鉴定这些T细胞识别的肽和它们的蛋白质来源。提出了三个具体目标：(1)从标准NOD小鼠早期胰岛素损伤中提取CD8 + T细胞克隆识别的I类mhc结合a细胞肽，并对其进行生化分离和测序；(2)利用表达克隆方法直接鉴定标准NOD小鼠早期胰岛素CD8 + T细胞靶向的自身抗原，以补充Aim 1中提出的肽分离研究；(3)利用NOD.HLA-A2.1小鼠鉴定糖尿病前期人类I类MHC分子HLA-A2背景下识别的抗原。完成拟议的Aims将导致对1型糖尿病发病机制和分子基础的更好理解，并可能提出新的基于抗原的干预策略和检测方法来监测患者的自身免疫状态。

DESCRIPTION (provided by applicant): Type 1 diabetes (formerly referred to as juvenile-onset or insulin-dependent diabetes) is an autoimmune disease resulting from destruction of the insulin-producing beta cells in the pancreatic islets of Langerhans. Insulin injections allow for continuation of life for the one million Americans afflicted with type 1 diabetes; however, they neither cure the disease nor prevent its devastating complications of microangiopathy, nephropathy, and neuropathy that diminish quality of life and shorten the life expectancy of a person with diabetes by an average of 15 years. The nonobese diabetic (NOD) mouse provides a relevant model system for type 1 diabetes that shares many of the characteristics of the human disease. Importantly, patients and NOD mice both develop lymphocytic infiltration of pancreatic islets (insulitis) and subsequent beta cell destruction mediated by T lymphocytes. Studies utilizing the NOD mouse have shown that beta cell-autoreactive CD8 + T cells restricted to class I major histocompatibility complex (MHC) molecules are absolutely required for disease development. Such effectors are also suspected contributors to beta cell elimination in both new-onset and graft-recurrent type 1 diabetes patients. Despite their importance, very little is known regarding the antigenic specificities of the CD8+ T cells participating in the initial phase of the a cell destruction that ultimately leads to disease. The overall goal of this proposal is to define these initiating specificities for T cells restricted to either murine or human class I MHC molecules. CD8 + T cells isolated from early insulitic lesions of either standard NOD mice or NOD mice transgenically expressing HLA-A2 will be utilized for this purpose. A combination of biochemical and expression cloning methods will be employed to identify the peptides recognized by these T cells and the proteins from which they are derived. Three Specific Aims are proposed: (1) To biochemically isolate and sequence the class I MHC-bound a cell peptides recognized by CD8 + T cell clones derived from early insulitic lesions of standard NOD mice; (2) To directly identify the autoantigens targeted by early insulitic CD8 + T cells in standard NOD mice using an expression cloning approach to complement the peptide isolation studies proposed in Aim 1; (3) To use NOD.HLA-A2.1 mice to identify the antigens recognized in the context of the human class I MHC molecule HLA-A2 during the early prediabetic period. Completion of the proposed Aims will result in an improved understanding of the pathogenesis and molecular basis of type 1 diabetes and could conceivably suggest new antigen-based intervention strategies and assays to monitor the autoimmune status of patients.