Specificity of CD8 cells in islets from type 1 diabetes patients

描述（由申请人提供）：人类1型糖尿病（T1D）的特征是产生胰岛素的胰腺β细胞的特异性免疫介导破坏。重要的是，早期的研究表明，对β细胞抗原的免疫记忆在它们的破坏中起着重要作用，这构成了胰岛细胞移植长期接受的主要障碍（1-3）。目前的共识是，CD8 T细胞是新发患者胰岛素炎的主要T细胞类型(4)。这样的CD8 T细胞是潜在的非常有害的，因为它们已经被证明很容易杀死体内的人类β细胞，如果它们表面有足够多的MHC I类上调(5)。然而，对胰岛中CD8的总体特异性和频率以及它们进入和激活的原因知之甚少。可能的靶标是来自β细胞的已知自身抗原，如胰岛素、IGRP、IA-2和GAD（缩写见表1）和细胞基质蛋白，这些蛋白可能在β细胞被破坏时呈现，也可能是病毒蛋白，如肠病毒决定因子。因此，本提案的总体目标是揭示在人类胰岛中发现的CD8 T淋巴细胞的特异性，并解决病毒感染是否可能在这种情况下发挥作用。[在平行研究中，我们的目标是证实我们的初步数据，表明胰岛浸润可能在临床诊断后长期存在，并且在这种情况下，在数量和质量上都遵循连续的过程。后一项发现将对长期患者的实验性耐受性治疗的可行性产生广泛的影响。我们的第一个目标是系统地检测人类胰岛内的自身反应性CD8 T细胞，并将它们的数量和激活状态与胰岛的局部组织病理学相关联。将使用通过独特的nPOD器官库可获得的新鲜冷冻人类胰腺和胰腺淋巴结的原位四聚体染色。此外，表达HLA A2的人源化转基因小鼠将被用于绘制对新细胞基质（自身）表位的反应，然后在人体组织上进行验证。我们的第二个目标是在胰岛中寻找[CVB]特异性T细胞，并解决一个重要的问题，即病毒感染是否使自身反应性T细胞更容易进入胰岛，反之亦然。为此，将与Heikki Hyoty和Stephen Tracy合作，用[cvb特异性]四聚体探测糖尿病患者胰腺的人体切片，并检测病毒核酸。此外，易患糖尿病的HLA-A2人源化小鼠和荧光标记的柯萨奇B病毒株将用于解决基本的“先有蛋还是先有蛋”的问题，即病毒感染是否对胰岛自身反应性淋巴细胞进入和破坏更为重要，或者相反，自身反应性攻击是否使胰岛更容易受到肠病毒感染。

DESCRIPTION (provided by applicant): Human type 1 diabetes (T1D) is characterized by the specific immune-mediated destruction of insulin- producing pancreatic beta cells. Importantly, earlier studies demonstrated that immune memory to beta cell antigens plays a significant role in their destruction, which constitutes a major obstacle for long-term acceptance of islet cell grafts (1-3). There is now consensus that CD8 T cells constitute the principal T cell type in insulitis in recent-onset patients (4). Such CD8 T cells are potentially very harmful, since they have been shown to readily kill human beta cells in vivo, if enough MHC class I is upregulated on their surface (5). However, not much is known about the overall CD8 specificities and frequencies present in islets and the cause for their entry and activation. Possible targets are known autoantigens derived from beta cells such as insulin, IGRP, IA-2 and GAD (see Table 1 for abbreviations) and cellular matrix proteins, which could become presented when beta cells are destroyed but also viral proteins for example enteroviral determinants. The overall objective of this proposal is therefore to reveal the specificity of CD8 T lymphocytes that are found in human islets and address whether viral infections may play a role in this scenario. [In parallel studies, we aim to confirm our preliminary data showing that islet infiltration may persist long beyond clinical diagnosis and in such cases follows a continuous course both quantitatively and qualitatively. The latter finding would have broad implications on the feasibility of experimental tolerization therapies in longstanding patients.] Our first goal is to systematically detect autoreactive CD8 T cells within human islets and correlate their numbers and activation status with the local histopathology of the islet. In situ tetramer staining of freshly frozen human pancreata and pancreatic lymph nodes now available through the unique nPOD organ repository will be used. In addition, humanized HLA A2 expressing transgenic mice will be utilized to map responses to novel cellular matrix (self) epitopes which will then be validated on human tissues. Our second goal is to search for [CVB]-specific T cells within islets and address the important question, whether viral infection makes islets more accessible for autoreactive T cell or vice versa. To this purpose human sections from pancreata of diabetic patients will be probed with [CVB-specific] tetramers and viral nucleic acids will be detected in collaboration with Heikki Hyoty and Stephen Tracy. In addition, diabetes- prone HLA-A2 humanized mice and fluorescently labeled Coxsackie B virus strains will be used to address the fundamental 'chicken-egg' question, whether viral infections are more important to condition islets for autoreactive lymphocyte entry and destruction, or, conversely, whether autoreactive attacks make islets more accessible for enteroviral infections. PUBLIC HEALTH RELEVANCE: We wish to systematically investigate the specificity of the immune cells that destroy insulin-producing beta cells in pancreas tissue obtained from patients with type 1 diabetes. Additionally, we will evaluate the potential role of enteroviruses in type 1 diabetes development in both patients and mouse models. This research will expand our knowledge on how the disease is triggered in susceptible individuals and on how we may therapeutically target these cells in order to restore tolerance.