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

项目总结 人类1型糖尿病(T1D)的特征是免疫介导的胰岛素产生的破坏 胰岛β细胞。CD8 T细胞是胰岛病变中最常见的[免疫]细胞，是 与β细胞破坏有关的主要T细胞类型。在这个项目中进行的研究已经 发现了重要的[和新的]发现，包括：1)首次发现自体反应性CD8T细胞在 T1D患者的胰岛使用一种特殊的[设计]四聚体染色方法和2)[第一描述 与非糖尿病患者相比，T1D患者外分泌胰腺中CD8 T细胞数量增多 个人。根据这些发现，我们预计只有一部分渗透到胰腺的细胞 确实是自动反应的。因此，识别其他类型的CD8T细胞池，例如病毒， 抗原可能对器官的炎症环境有重要作用。目前，总体上， CD8T细胞在胰腺中的特异性和频率以及它们进入和激活的原因尚不清楚 完全理解。可能的靶点是来自β细胞的已知自身抗原，如胰岛素、IGRP、IA-2 和GAD(缩写见表2)和细胞基质蛋白，可能会出现[和 当β细胞被破坏时，但病毒蛋白，例如肠道病毒决定簇也 可能的候选人。因此，此次续签申请的总体目标是比较 人胰岛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(例如 病毒感染及其可检测的足迹)。我们将研究新近发病和长期发病的患者 T1D为了构建具体的路线图，并进一步了解 CD8 T细胞渗入，病程可能会随着时间的推移[在数量上和质量上]演变。一个 独特的优势不仅是能够访问非常独特的器官储存库，而且还包括新建立的 与亚历山德罗·塞特博士和比约恩·彼得斯博士的实验室合作，他们维持着免疫 拉霍亚研究所的表位数据库(IEDB)，是Novo领导的表位发现倡议的一部分 诺德。利用IEDB和体外检测中包含的信息，对新表位进行修饰 将绘制自身抗原和限制于人类白细胞抗原A2的新的病毒CD8 T细胞表位图，并提供信息 与我们的实验室共享。总体而言，我们的发现应该会让我们更好地理解T1D的方式和原因 开发并最终帮助开发新的治疗方案。] 我们的第一个目标是系统和定量地检测人胰岛中的自身反应性CD8T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 器官的组织病理学。新鲜冷冻的人胰腺的原位四聚体染色可从 糖尿病患者胰腺器官捐赠者网络(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库的表演。[新的翻译后修饰的发现 利用IEDB中包含的信息和拉霍亚研究所的专业知识 我们的现场实验将首次提供有关抗原的精确组织分布的信息- 胰腺中的特异性CD8 T细胞。此外，我们还将进一步鉴定它们的表型和功能。这 将允许识别对胰岛有显著极化的关键抗原，它们更多 可能与疾病的发病机制有关。这也将有助于我们理解为什么胰腺外分泌 炎症通常出现在T1D。 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。[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)，或者是否有多起病毒攻击 是必需的(多次命中)或同一属的几种病毒株，甚至引发不同病毒 共同途径可能在该病的发病机制中发挥作用(多种病毒)]。胰腺组织 因此，来自最近发病和持续时间较长的T1D的捐赠者的部分将被调查病毒特异性 四聚体，我们将在这些样本中检测任何病毒蛋白质和核酸 NPOD-病毒学小组，以调查病毒感染的方式(肇事逃逸；多次命中或多次 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组有关的 到目前为止，柯萨奇病毒已经有了充分的文献记载。[为此，我们将致力于识别新的电动汽车 通过我们与塞特博士和彼得斯博士的实验室的合作以及对IEDB的使用 作为他们在发现和验证由HLAI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，例如 巨细胞病毒(CMV)和EB病毒(EBV)。我们还将调查是否可能存在 疱疹病毒-6(HHV6)和HHV6特异性CD8 T细胞，因为最近的证据表明，这种病毒可能 存在于T1D供者的胰腺中。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、干扰素信号、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)。

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).