Specificity, Phenotype and Function of Pancreatic CD8 T Cells in Human Type 1 Diabetes

人类 1 型糖尿病中胰腺 CD8 T 细胞的特异性、表型和功能

• 批准号: 9238399
• 负责人: Matthias G. Von Herrath
• 金额: $ 45万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-02 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9238399
• 关键词: Abbreviations; Antigens; Antiviral Agents; Area; Attention; Autoantigens; Autoimmune Diseases; Beta Cell; CD8-Positive T-Lymphocytes; Cell physiology; Cells; Chronic; Collaborations; Collection; Coxsackie B Viruses; Cytomegalovirus; Cytoskeleton; Cytotoxic T-Lymphocytes; Data; Databases; Development; Diabetes Mellitus; Disease; Disease Progression; Drug or chemical Tissue Distribution; Endocrine; Enterovirus; Epitopes; Event; Exocrine pancreas; Freezing; Frequencies; G6PC2 gene; Goals; HLA-A2 Antigen; Herpesviridae; Histocompatibility Antigens Class I; Histopathology; Human; Human Herpesvirus 4; Human Herpesvirus 6; Immune; In Situ; In Vitro; Individual; Infiltration; Inflammation; Institutes; Insulin; Insulin-Dependent Diabetes Mellitus; Interferons; Islets of Langerhans; Knowledge; Laboratories; Lead; Lesion; Maps; Mediating; Methods; Nucleic Acids; Organ; Organ Donor; Pancreas; Pathogenesis; Pathologic; Pathology; Pathway interactions; Patients; Peptides; Phenotype; Play; Population; Production; Proteins; RNA; Research; Role; Running; Sampling; Specificity; Staining method; Stains; Structure of beta Cell of islet; T-Lymphocyte; Time; TimeLine; Tissues; University Hospitals; Up-Regulation; Validation; Viral; Viral Antigens; Viral Genome; Viral Proteins; Virus; Virus Diseases; Work; autoreactivity; base; biobank; cell injury; cell type; cytokine; design; diabetic; experimental study; illness length; in vitro Assay; inflammatory milieu; interest; islet; non-diabetic; novel; novel therapeutics; phenotypic biomarker; repository; therapeutic target; viral detection; virology

PROJECT SUMMARY Human type 1 diabetes (T1D) is characterized by the immune-mediated destruction of insulin-producing pancreatic beta cells. CD8 T cells are the most common [immune] cell found in insulitic lesions and are the principal T cell type implicated in beta cell destruction. Studies performed within this project have already revealed significant [and novel] findings including: 1) the first identification of auto-reactive CD8 T cells in the islets of patients with T1D using a specially [designed method of] tetramer staining and 2) [the first description of high] numbers of CD8 T cells infiltrating the exocrine pancreas in T1D patients compared with non-diabetic individuals. Based on these findings, we anticipate that only a proportion of the cells that infiltrate the pancreas are indeed auto-reactive. Thus, the pool of `bystander' CD8 T cells that recognize other, for example viral, antigens might significantly contribute to the inflammatory environment of the organ. At present, the overall specificities and frequencies of CD8 T cells in the pancreas and the cause for their entry and activation are not fully understood. Possible targets are known autoantigens derived from beta cells such as insulin, IGRP, IA-2 and GAD (see Table 2 for abbreviations) and cellular matrix proteins, which could become presented [and modified] when beta cells are destroyed, but viral proteins, for example enteroviral determinants are also possible candidates. The overall objective of this renewal application is therefore to compare the specificity, phenotype and function of CD8 T lymphocytes from human islets with those found in exocrine tissue, and to assess whether their presence correlates with islet-specific pathology (e.g. viral infections and their detectable footprints). We will study patients with recent-onset and longstanding T1D in order to build a road map of specificities and to further understand how the relationship between the CD8 T cell infiltrate and the disease course might evolve [both quantitatively and qualitatively over time. A unique strength is not only the access to very unique organ repositories but also the newly established collaboration with Dr. Alessandro Sette and Dr. Bjoern Peters' laboratories, which maintain the Immune Epitope Database (IEDB) at La Jolla institute and are part of an epitope discovery initiative led by Novo Nordisk. Using the information contained in the IEDB and in vitro assays, neo-epitopes to modified autoantigens and new viral CD8 T cell epitopes restricted to HLA-A2 will be mapped and the information shared with our laboratory. Overall, our findings should give us a better understanding of how and why T1D develops and thus help ultimately with the development of new therapeutic options.] Our first goal is to systematically and quantitatively detect autoreactive CD8 T cells within human islets and exocrine pancreas and to correlate the number and activation status of these cells with the local histopathology of the organ. In situ tetramer staining of freshly frozen human pancreata available from the Network for Pancreatic Organ Donors with Diabetes (nPOD), the Diabetes Biobank Brussels (DBB) and the Biobank at Oslo University Hospital will be performed. [The discovery of new post-translationally-modified epitopes using the information contained in the IEDB and the expertise at La Jolla Institute in conjunction with our experiments in situ will provide, for the first time,] information on the precise tissue distribution of antigen- specific CD8 T cells in the pancreas. In addition, we will further characterize their phenotype and function. This will allow for the identification of key antigens with significant polarization towards the islets, which are more likely to be involved in the pathogenesis of disease. It will also help us to understand why exocrine pancreas inflammation is often present in T1D. Our second goal is to search for virus-specific T cells within islets and exocrine tissue. [This will help to ascertain whether one virus might trigger the disease (hit-and-run event) or whether numerous viral attacks are required (multiple hits) or several viral strains of the same genus or even different viruses that trigger common pathways might play a role in the pathogenesis of the disease (multiple viruses)]. Pancreas tissue sections from donors with recent-onset and long duration of T1D will therefore be probed with virus-specific tetramers, and we will detect any viral proteins and nucleic acids in these samples in collaboration with the nPOD-Virology group in order to investigate the manner of viral infection (hit-and-run; multiple hits or multiple viruses). We will initially focus our attention on enteroviruses (EV), as only the association with group B coxsackieviruses has thus far been sufficiently documented. [For this purpose, we will aim to identify new EV epitopes through our collaboration with Dr. Sette and Dr. Peters' laboratories and the use of the IEDB as well as their expertise on the discovery and validation of cytotoxic and T cell epitopes presented by HLA Class I molecules]. The frequency of EV-specific cells will be compared to that of other common viruses such as cytomegalovirus (CMV) and Epstein-Barr virus (EBV). We will also investigate the possible presence of herpesvirus-6 (HHV6) and HHV6-specific CD8 T cells, because recent evidence suggests that this virus might be present in the pancreas of T1D donors. In addition, we will correlate our findings with the local histopathology of the organ (MHC-I expression, interferon signature, CD8 T cell phenotype, cytokine production and presence/absence of viral protein or genome).

项目概要 人类 1 型糖尿病 (T1D) 的特点是免疫介导的胰岛素产生破坏 胰腺β细胞。 CD8 T 细胞是胰岛病变中最常见的[免疫]细胞，也是 参与β细胞破坏的主要T细胞类型。在该项目中进行的研究已经 揭示了重要的[且新颖的]发现，包括：1）首次在细胞中鉴定出自身反应性 CD8 T 细胞 使用特殊[设计的]四聚体染色方法对 T1D 患者的胰岛进行检测，并且 2) [第一个描述 与非糖尿病患者相比，T1D 患者的外分泌胰腺中浸润的 CD8 T 细胞数量较多 个人。根据这些发现，我们预计只有一部分细胞浸润胰腺 确实是自动反应的。因此，识别其他病毒（例如病毒）的“旁观者”CD8 T 细胞池 抗原可能对器官的炎症环境有显着影响。目前，总体 胰腺中 CD8 T 细胞的特异性和频率以及它们进入和激活的原因尚不清楚 完全明白了。可能的靶标是源自 β 细胞的已知自身抗原，例如胰岛素、IGRP、IA-2 和 GAD（缩写见表 2）和细胞基质蛋白，它们可能会被呈现[和 修改]当β细胞被破坏时，但病毒蛋白，例如肠道病毒决定簇也会被破坏 可能的候选人。因此，本续签申请的总体目标是比较 人类胰岛 CD8 T 淋巴细胞与胰岛中发现的 CD8 T 淋巴细胞的特异性、表型和功能 外分泌组织，并评估它们的存在是否与胰岛特异性病理学相关（例如， 病毒感染及其可检测的足迹）。我们将研究近期发病和长期患病的患者 T1D 是为了构建一个具体的路线图并进一步了解它们之间的关系 CD8 T 细胞浸润和病程可能会随着时间的推移在数量和质量上发生变化。一个 独特的优势不仅在于可以访问非常独特的器官库，还在于新建立的 与 Alessandro Sette 博士和 Bjoern Peters 博士的实验室合作，维持免疫 拉霍亚研究所的表位数据库 (IEDB) 是 Novo 领导的表位发现计划的一部分 诺德。利用 IEDB 和体外测定中包含的信息，对新表位进行修饰 自身抗原和仅限于 HLA-A2 的新病毒 CD8 T 细胞表位将被绘制出来，并且信息 与我们的实验室共享。总的来说，我们的研究结果应该让我们更好地理解 T1D 的发生方式和原因 发展并最终帮助开发新的治疗选择。] 我们的首要目标是系统地、定量地检测人类胰岛内的自身反应性 CD8 T 细胞和 外分泌胰腺并将这些细胞的数量和激活状态与局部 该器官的组织病理学。对新鲜冷冻的人胰腺进行原位四聚体染色，可从 糖尿病胰腺器官捐献者网络 (nPOD)、布鲁塞尔糖尿病生物库 (DBB) 和 奥斯陆大学医院的生物样本库将进行。 [新的翻译后修饰的发现 表位使用 IEDB 中包含的信息以及拉霍亚研究所的专业知识 我们的原位实验将首次提供]有关抗原精确组织分布的信息 胰腺中的特异性 CD8 T 细胞。此外，我们将进一步表征它们的表型和功能。这 将允许识别对胰岛具有显着极化的关键抗原，这些抗原更 可能参与疾病的发病机制。它还将帮助我们理解为什么外分泌胰腺 炎症常出现在 T1D 中。 我们的第二个目标是在胰岛和外分泌组织内寻找病毒特异性 T 细胞。 [这将有助于 确定一种病毒是否可能引发疾病（肇事逃逸事件）或是否存在大量病毒攻击 需要（多次点击）或同一属的多个病毒株甚至不同的病毒触发 共同途径可能在疾病的发病机制中发挥作用（多种病毒）]。胰腺组织 因此，来自近期发病且持续时间较长的 T1D 捐献者的切片将采用病毒特异性检测 四聚体，我们将与 nPOD-病毒学小组，旨在调查病毒感染的方式（肇事逃逸；多次袭击或多次袭击） 病毒）。我们首先将注意力集中在肠道病毒 (EV)，因为仅与 B 组相关 迄今为止，柯萨奇病毒已得到充分记录。 [为此目的，我们将致力于识别新的电动汽车 通过我们与 Sette 博士和 Peters 博士实验室的合作以及 IEDB 的使用来确定表位 作为他们在发现和验证 HLA I 类细胞毒性和 T 细胞表位方面的专业知识 分子]。 EV 特异性细胞的频率将与其他常见病毒的频率进行比较，例如 巨细胞病毒 (CMV) 和 Epstein-Barr 病毒 (EBV)。我们还将调查可能存在的 疱疹病毒 6 (HHV6) 和 HHV6 特异性 CD8 T 细胞，因为最近的证据表明该病毒可能 存在于 T1D 捐献者的胰腺中。此外，我们还将把我们的发现与当地的情况联系起来。 器官的组织病理学（MHC-I 表达、干扰素特征、CD8 T 细胞表型、细胞因子 病毒蛋白或基因组的产生和存在/不存在）。