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型或胰岛素依赖型糖尿病）是一种自身免疫性疾病，由胰岛中产生胰岛素的β细胞破坏引起。胰岛素注射使100万患有1型糖尿病的美国人得以延续生命;然而，它们既不能治愈疾病，也不能预防微血管病、肾病和神经病的破坏性并发症，这些并发症降低了生活质量，并使糖尿病患者的预期寿命平均缩短15年。非肥胖糖尿病（NOD）小鼠提供了一个相关的模型系统，1型糖尿病，共享许多人类疾病的特征。重要的是，患者和NOD小鼠都发生胰岛的淋巴细胞浸润（胰岛炎）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，β细胞自身反应性CD 8 + T细胞限制于I类主要组织相容性复合体（MHC）分子是疾病发展绝对需要的。这些效应物也被怀疑是新发和移植复发的1型糖尿病患者中β细胞消除的贡献者。尽管它们的重要性，很少有人知道的抗原特异性的CD 8 + T细胞参与的初始阶段的细胞破坏，最终导致疾病。本提案的总体目标是定义这些限制于鼠或人I类MHC分子的T细胞的起始特异性。从标准NOD小鼠或转基因表达HLA-A2的NOD小鼠的早期胰岛炎病变分离的CD 8 + T细胞将用于此目的。将采用生物化学和表达克隆方法的组合来鉴定这些T细胞识别的肽和它们所来源的蛋白质。本研究提出了三个具体的目的：（1）从NOD小鼠早期胰岛病变的CD 8 + T细胞克隆中分离并测序可被CD 8 + T细胞识别的I类MHC结合的α细胞肽;（2）利用表达克隆方法直接鉴定标准NOD小鼠早期胰岛病变的CD 8 + T细胞所靶向的自身抗原，以补充目的1中提出的肽分离研究;（3）用NOD.HLA-A2.1小鼠鉴定糖尿病前期早期人类I类MHC分子HLA-A2识别的抗原。完成所提出的目标将导致对1型糖尿病的发病机制和分子基础的更好理解，并且可以想象地提出新的基于抗原的干预策略和检测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.