Specificity of CD8 cells in islets from type 1 diabetes patients

描述（由申请人提供）：人1型糖尿病（T1 D）的特征在于特异性免疫介导的胰岛素产生胰腺β细胞的破坏。重要的是，早期的研究表明，对β细胞抗原的免疫记忆在其破坏中起着重要作用，这构成了胰岛细胞移植物长期接受的主要障碍（1-3）。目前的共识是CD 8 T细胞构成了近期发病患者胰岛炎的主要T细胞类型（4）。这样的CD 8 T细胞是潜在地非常有害的，因为它们已经显示出在体内容易杀死人β细胞，如果足够的MHC I类在它们的表面上被上调（5）。然而，对胰岛中存在的总体CD 8特异性和频率以及它们进入和激活的原因知之甚少。可能的靶标是已知的来源于β细胞的自身抗原，例如胰岛素、HRP、IA-2和GAD（缩写见表1）和细胞基质蛋白，当β细胞被破坏时，细胞基质蛋白可以被呈递，但也有病毒蛋白，例如肠道病毒决定簇。因此，该提案的总体目标是揭示在人类胰岛中发现的CD 8 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。[In通过平行研究，我们的目的是证实我们的初步数据，这些数据表明胰岛浸润可能持续很长时间超过临床诊断，并且在这种情况下，在定量和定性方面都遵循连续的过程。后一项发现将对长期患者的实验性耐受化疗法的可行性产生广泛影响。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CD 8 T细胞，并将其数量和激活状态与胰岛的局部组织病理学相关联。将使用目前可通过独特的nPOD器官库获得的新鲜冷冻人胰腺和胰腺淋巴结的原位四聚体染色。此外，表达人源化HLA A2的转基因小鼠将用于绘制对新的细胞基质（自身）表位的应答，然后在人组织上验证。我们的第二个目标是在胰岛内寻找CVB特异性T细胞，并解决重要的问题，即病毒感染是否使胰岛更容易获得自身反应性T细胞，反之亦然。为此，将用[CVB特异性]四聚体探测糖尿病患者胰腺的人类切片，并与Heikki Hyoty和Stephen Tracy合作检测病毒核酸。此外，糖尿病易感的HLA-A2人源化小鼠和荧光标记的科萨基B病毒株将用于解决基本的“鸡-蛋”问题，即病毒感染是否对调节胰岛的自身反应性淋巴细胞进入和破坏更重要，或者相反，自身反应性攻击是否使胰岛更容易受到肠道病毒感染。

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.