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).

项目摘要 人1型糖尿病（T1 D）的特征在于免疫介导的对胰岛素产生细胞的破坏。 胰腺β细胞CD 8 T细胞是胰岛炎病变中最常见的[免疫]细胞， 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[和新的]发现，包括：1）首次鉴定出自体反应性CD 8 T细胞在 使用特别[设计的]四聚体染色的T1 D患者的胰岛和2）[第一描述 与非糖尿病患者相比，T1 D患者中浸润胰腺外分泌的CD 8 T细胞数量较高 个体基于这些发现，我们预计只有一部分浸润胰腺的细胞 是自动反应的因此，识别其他，例如病毒， 抗原可能对器官的炎症环境有显著贡献。目前整体 胰腺中CD 8 T细胞的特异性和频率以及它们进入和激活的原因并不清楚。 完全理解可能的靶点是已知的来源于β细胞的自身抗原，如胰岛素、HRP、IA-2 和GAD（缩写见表2）和细胞基质蛋白，这些蛋白可以被呈递[和 当β细胞被破坏时，病毒蛋白，例如肠道病毒决定簇也被破坏。 可能的候选人。因此，本次更新申请的总体目标是比较 人胰岛CD 8 T淋巴细胞的特异性、表型和功能与 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）。我们将研究近期发作和长期 T1 D，以建立具体性路线图，并进一步了解 CD 8 T细胞浸润和疾病过程可能随着时间的推移而演变[定量和定性。一 独特的优势不仅在于能够获得非常独特的器官储存库，而且还在于新建立的 与Alessandro Sette博士和Bjoern Peters博士的实验室合作， 表位数据库（IEDB）在拉霍亚研究所，是一个表位发现计划的一部分，由诺 诺德。使用IEDB和体外测定中包含的信息，修饰的新表位可以在体外测定。 自身抗原和新的病毒CD 8 T细胞表位限制到HLA-A2将被映射和信息 与我们的实验室分享。总的来说，我们的研究结果应该让我们更好地了解T1 D如何以及为什么 发展，从而最终有助于新的治疗选择的发展。 我们的第一个目标是系统地和定量地检测人类胰岛内的自身反应性CD 8 T细胞， 外分泌胰腺，并将这些细胞的数量和激活状态与局部 器官的组织病理学。新鲜冷冻人胰腺的原位四聚体染色，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 将在奥斯陆大学医院进行生物样本库检查。[The发现新的后修饰的 表位使用IEDB中包含的信息和拉霍亚研究所的专业知识， 我们的原位实验将首次提供关于抗原的精确组织分布的信息- 胰腺中的特异性CD 8 T细胞。此外，我们将进一步表征其表型和功能。这 将允许鉴定对胰岛具有显著极化的关键抗原， 可能参与疾病的发病机制。这也将帮助我们理解为什么胰腺外分泌 炎症通常存在于T1 D中。 我们的第二个目标是在胰岛和外分泌组织中寻找病毒特异性T细胞。[This将有助于 确定是否一种病毒可能引发疾病（肇事逃逸事件）或是否多次病毒攻击 需要（多次命中）或相同属的几种病毒株或甚至不同的病毒， 共同途径可能在疾病的发病机制中起作用（多种病毒）]。胰腺组织 因此，将用病毒特异性探针探测来自最近发作且持续时间较长的T1 D供体的切片。 四聚体，我们将检测这些样品中的任何病毒蛋白质和核酸， nPOD-病毒学小组，以研究病毒感染的方式（打了就跑;多次击中或多次击中） 病毒）。我们首先将注意力集中在肠道病毒（EV）上，因为只有与B组的关联 柯萨奇病毒迄今已有充分的文献记载。[For为此，我们将致力于确定新的EV 表位通过我们的合作与博士塞特和博士彼得斯的实验室和使用IEDB以及 由于他们在发现和验证HLA I类呈递的细胞毒性和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， 巨细胞病毒（CMV）和EB病毒（EBV）。我们还将调查可能存在的 疱疹病毒-6（HHV 6）和HHV 6特异性CD 8 T细胞，因为最近的证据表明，这种病毒可能 存在于T1 D捐赠者的胰腺中。此外，我们将把我们的发现与当地的 器官的组织病理学（MHC-I表达、干扰素特征、CD 8 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）。