Immunological Mechanisms Underlying T1dm Pathogenesis

T1dm 发病机制的免疫学机制

基本信息

批准号：
7734282
负责人：
David Harlan
金额：
$ 26.39万
依托单位：
NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734282
关键词：
Adoptive Transfer Antigen-Presenting Cells Antigens Autoantigens Autoimmune Diabetes Autoimmune Diseases Autoimmune Process Autoimmune Responses Automobile Driving Beta Cell Blood Glucose Blood Vessels C-terminal CD80 gene CD8B1 gene Catalytic Domain Cell Line Cell surface Cells Cessation of life Characteristics Chronic Clinical Cytotoxic T-Lymphocytes Defect Dendritic Cells Development Diabetes Mellitus Diabetic mouse Discipline Disease Dose Epitopes Exercise Experimental Genetics Fibroblasts Gene Expression Genes Genetic Predisposition to Disease Glycoproteins Goals HLA-A2 Antigen Health Homing Hormonal Human Immune Immune response Immune system Immunization Inbred NOD Mice Incidence Individual Inflammatory Injection of therapeutic agent Insulin Insulin-Dependent Diabetes Mellitus Integrins Lead Left Life Ligands Link Lymphocyte Function Lymphoid Mediating Membrane Memory Modeling Mus Myotonic Dystrophy Nerve Non obese Organ Pancreas Pathogenesis Patients Peptides Peripheral Phenotype Phosphotransferases Population Process Proliferating Protein-Serine-Threonine Kinases Proteins Publishing Rattus Relative (related person)Reporting Research Signal Transduction Structure of beta Cell of islet Surface System T-Cell Activation T-Lymphocyte Testing Thinking Tissues To autoantigen Transgenes Transgenic Mice Transgenic Organisms Viral Virus Work autoreactive T cell cell killing chemokine cytokine diabetic glucose monitor glucose-6-phosphatase insulin secretion islet killings lymph nodes man migration mouse model prevent promoter protective effect receptor response trait

项目摘要

Type 1 diabetes mellitus (T1DM) is an autoimmune disease resulting from the T cell mediated destruction of insulin-producing beta cells located in the pancreas. Current treatment, which includes insulin replacement by injection, frequent blood glucose monitoring, and dietary/exercise discipline, can prevent death from hormonal insufficiency, but is not curative and does not completely prevent the long-term complications including nerve damage, and vascular damage to both large and small blood vessels. In previous few years, we developed a transgenic mouse to express the T cell costimulatory receptor CD80 on its pancreatic insulin producing beta cells (under the control of the rat insulin promoter abbreviated RIP) and demonstrated the mouse's extraordinary sensitivity to autoantigen induced immune mediated beta cell destruction, and thus to diabetes. We refer to the diabetes induced in these RIP-CD80 mice as experimental autoimmune diabetes (EAD). Using this EAD model, we've found that relatively weak anti-beta cell autoimmune responses can cause chronic progressive and eventually complete beta cell destruction resulting in symptomatic and irreversible disease. The slow but inexorable process is highly reminiscent of the beta cell destruction leading to clinical T1DM in man, typically months to years of anti-beta cell immune activity precedes sufficient beta cell killing for the blood sugars to rise. Most other autoimmune diabetes studies involve the non-obese diabetic (NOD) mouse, which develops spontaneous diabetes, or virus-induced diabetes models. Only the RIP-CD80 transgenic mouse diabetes model is characterized by an experimental genetic susceptibility trait (in the EAD model the trait is the CD80-transgene) rendering the mouse susceptible to autoantigen-specific T lymphocyte sensitization as is thought to be present in T1DM patients. We have reported that immunizing with either experimentally-introduced autoantigen (e.g. pancreatic beta cell-expressed viral glycoprotein) or endogenous beta cell autoantigen (e.g. insulin) could lead to diabetes in RIP-CD80 mice. Further, most studies now support that the normal individual's T cell repertoire contains potentially autoreactive but quiescent T cells. We concluded that pancreatic beta cell likely contains many autoantigens and that effective control mechanisms must exist to prevent autoimmune responses in healthy individuals. Current immunological dogma suggests that naove CD8 cytotoxic T cell (CTL) precursors respond to strong antigen stimulation in a characteristic fashion by: (i) proliferating and releasing inflammatory cytokines, (ii) differentiating into CTLs and, (iii) down modulating certain surface interaction molecules to allow the CTLs to leave secondary lymphoid organs while increasing other receptors, such as integrins, to promote entry into peripheral tissues, and (iv) changing chemokine signals necessary to facilitate the relocation of activated CTL from lymph nodes (LN) to inflamed peripheral tissues. The EAD model has allowed us to study the response of beta cell-specific CD8 T cells to cognate antigen presented by either professional antigen-presenting cells (APC) like mature dendritic cells DC), or by non-professional (np) APCs like fibroblast-like cell lines (FCL). While DC-stimulated T cells produced the expected effector CTL phenotype described above, FCL-activated T cells were quite different. Relative to DC activated CTLs, the FCL-activated CTLs proliferated less, released equivalent proinflammatory cytokines, and surprisingly displayed increased cytolytic function. Moreover, FCL-stimulated T cells largely failed to switch their homing receptors predicting poor migration from the lymph nodes to the periphery. Most strikingly, however, FCL-stimulated but not DC-activated CTL expressed many of the features associated with memory CTLs; both multiple memory cell surface marker expressioon and predominant homing into secondary lymphoid organs upon adoptive transfer into naive mice. Importantly, while every CTL response gives rise to a small population of long-lived memory CTL, the mechanism that decides the fate of an individual CTL to become a memory cell remains largely unknown. Finally, the FCL-stimulated CTL induced diabetes by a slow, chronic process, suggesting that these central memory CTL might also be involved in driving human T1DM. Research goals pursued in FY 2008: 1. We developed a potent cell immunization strategy using mature dendritic cells loaded with antigenic peptides (derived from beta cell-made proteins), and established a clear antigen-dose diabetes-response relationship for two experimental autoantigen determinants (peptides), encoded by the transgenic LCMV-GP specifically expressed by beta cells. We hypothesize (and we are testing) that antigenic peptides representing genuine beta cell target epitopes will similarly mediate beta cell killing and diabetes development in this test system. 2. We have identified two beta-cell derived peptides (one from a serine/threonine kinase called dystrophia myotonica kinase (DMK) aa143-151, and the other from the islet-specific glucose-6-phosphatase catalytic subunit-related gene (IGRP) aa225-233) capable of sensitizing immune cells to destroy beta cells. These antigenic peptides have previously been reported to be able to mediate diabetes in another mouse diabetes model, the non-obese diabetic (NOD) mouse. Other H2b-restricted antigenic peptides, several published as autoantigenic peptide candidates, did not activate a genuine anti-beta cell response in our test system. 3. We tested comparable approaches in mice "humanized" to express a chimeric human HLA-A2 linked to the murine H2K(b) C-terminal sequences. This membrane molecule has been shown to function as a MHC classs I molecule in mice yet present human (HLA-A2-restricted) epitopes to mouse T cells. Autoantigenic peptides identified using these mice could be used to test autoreactive T cells in HLA-A2 positive subjects with T1DM (about 50 percent of the population). We found a surprising protective effect conferred by the HLA-A2 gene expression. 4. Immune cells in draining lymph nodes are continuously exposed to autoantigen from peripheral organ cells. We have observed that beta cell specific T cell activation depends on the cognate antigen but also on the context of T cell activation. Sub-optimally activated T cells (using either altered peptide ligands (APL) or non-professional (immature) APC) display both bridled effector lymphocyte functions (killing, cytokine secretion) and migration. These defects resulted in markedly reduced diabetes incidence in our EAD model. Current efforts are focused on strategies to induce migration-defective T cells that would be maintained in the draining lymph nodes by presence of autoantigen and there work to down-modulate the anti-beta cell immune response.

1型糖尿病（T1DM）是一种自身免疫性疾病，是由于T细胞介导的胰腺中产生胰岛素β细胞的破坏而引起的。当前的治疗包括通过注射胰岛素替代，频繁的血糖监测以及饮食/运动学科，可以防止激素不足，但不能治愈，并且不能完全防止长期并发症，包括神经损害，以及对大型和小血管的血管损害。在过去的几年中，我们开发了一种转基因小鼠，以表达T细胞的胰腺胰岛素产生β细胞（在大鼠胰岛素启动子缩写的RIP的控制下）上表达T细胞共刺激受体CD80，并证明了该小鼠对自身抗体介导的免疫介导的β细胞细胞的非凡敏感性。我们将这些RIP-CD80小鼠诱导的糖尿病称为实验性自身免疫性糖尿病（EAD）。使用这种EAD模型，我们发现相对较弱的抗抗抗抗毒细胞自身免疫反应会导致慢性进行性，并最终完成β细胞破坏，从而导致有症状和不可逆性疾病。缓慢但不可侵蚀的过程高度让人联想到β细胞破坏导致人类的临床T1DM，通常数月至数年的抗β细胞免疫活性在足够的β细胞杀死之前，使血糖升高。大多数其他自身免疫性糖尿病研究涉及非肥胖糖尿病（NOD）小鼠，该小鼠会发展为自发性糖尿病或病毒诱导的糖尿病模型。仅RIP-CD80转基因小鼠糖尿病模型的特征在于实验性遗传易感性特征（在EAD模型中，性状是CD80-转基因），使小鼠对自身抗原特异性T淋巴细胞敏感的小鼠易感为T1DM患者。我们报道说，用实验引入的自身抗原（例如胰腺β细胞表达的病毒糖蛋白）或内源性β细胞自身抗原（例如胰岛素）可以导致RIP-CD80小鼠的糖尿病。此外，现在大多数研究都支持正常人的T细胞库中包含潜在的自身反应性但静止的T细胞。我们得出的结论是，胰腺β细胞可能包含许多自身抗原，并且必须存在有效的控制机制，以防止健康个体的自身免疫反应。当前的免疫学教条表明，NAOVE CD8细胞毒性T细胞（CTL）前体以以下方式以特征性的方式对强抗原刺激反应：整联蛋白，促进进入周围组织，以及（iv）改变趋化CTL从淋巴结（LN）迁移到发炎的外围组织所需的趋化因子信号。 EAD模型使我们能够研究Beta细胞特异性CD8 T细胞对由专业抗原呈递细胞（APC）（如成熟的树突状细胞DC）或非专业（NP）APC（如成纤维细胞样细胞系（FCL））所呈现的响应。尽管DC刺激的T细胞产生了上述预期效应的CTL表型，但FCL激活的T细胞却完全不同。相对于DC激活的CTL，FCL激活的CTL较少增殖，释放等效促炎细胞因子，并且出人意料地显示出细胞溶解功能的增加。此外，FCL刺激的T细胞在很大程度上未能切换其归因受体，预测从淋巴结迁移到周围的迁移不佳。然而，最引人注目的是，FCL刺激但没有DC激活的CTL表达了许多与内存CTL相关的功能。在收养到幼稚的小鼠中，多个记忆细胞表面标记表达和主要归位为二级淋巴机器人。重要的是，尽管每个CTL响应都会产生少量长寿命的记忆CTL，但决定单个CTL成为记忆细胞的命运的机制仍然在很大程度上未知。最后，通过缓慢而慢性的过程，FCL刺激的CTL诱导的糖尿病，表明这些中央记忆CTL也可能参与驱动人T1DM。 2008财年实现的研究目标： 1。我们使用装有抗原肽（源自β细胞制成的蛋白）的成熟树突状细胞制定了有效的细胞免疫策略，并建立了两个实验性自动抗原剂（肽）（肽）的明确抗原剂量糖尿病 - 糖尿病 - 反应关系，并由Transgenic LCMV-GP特定于BETA的细胞编码。我们假设（并且正在测试）表明代表真正的β细胞靶位鉴定的抗原性肽将同样介导该测试系统中的β细胞杀伤和糖尿病发育。 2。我们已经鉴定了两种β细胞衍生的肽（一种来自丝氨酸/苏氨酸激酶，称为肌酸肌张型激酶（DMK）AA143-151，另一种来自胰岛特异性葡萄糖-6-磷酸酶催化酶催化亚基相关基因（IGRP）供应量（IGRP）AAA AAA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA AA BETA。以前据报道，这些抗原性肽能够介导另一种小鼠糖尿病模型，即非肥胖糖尿病（NOD）小鼠中的糖尿病。其他H2B限制的抗原肽，其中几种以自身抗原肽的候选物发表，在我们的测试系统中没有激活真正的抗β细胞反应。 3。我们测试了“人性化”小鼠中的可比较方法，以表达与鼠H2K（B）C末端序列相关的嵌合人HLA-A2。该膜分子已被证明是小鼠中的MHC I类I分子的作用，但呈现到小鼠T细胞的人（HLA-A2限制性限制）表位。使用这些小鼠鉴定的自身抗原肽可用于测试具有T1DM（约占人群的50％）的HLA-A2阳性受试者中的自身反应性T细胞。我们发现HLA-A2基因表达赋予了令人惊讶的保护作用。 4。排水淋巴结中的免疫细胞不断暴露于外周器官细胞的自身抗原。我们已经观察到β细胞特异性T细胞激活取决于同源抗原，也取决于T细胞激活的背景。亚次优化的T细胞（使用改变的肽配体（APL）或非专业（未成熟）APC）同时表现出Bridled效应的淋巴细胞功能（杀伤，细胞因子分泌）和迁移。这些缺陷导致在我们的EAD模型中显着降低了糖尿病的发病率。当前的努力集中在诱导迁移缺陷的T细胞的策略上，这些策略通过自动抗原的存在将保持在排水淋巴结中，并在那里工作以减少抗β细胞免疫反应。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cytotoxic T cell-mediated diabetes in RIP-CD80 transgenic mice: autoantigen peptide sensitivity and fine specificity.

RIP-CD80 转基因小鼠中细胞毒性 T 细胞介导的糖尿病：自身抗原肽敏感性和精细特异性。

DOI：
10.1196/annals.1394.008
发表时间：
2007
期刊：
Annals of the New York Academy of Sciences
影响因子：
5.2
作者：
Pechhold,Klaus;Chakrabarty,Sagarika;Harlan,DavidM
通讯作者：
Harlan,DavidM

Rescue of hematopoietic stem cells following high-dose radiation injury using ex vivo culture on endothelial monolayers.

使用内皮单层离体培养来拯救高剂量辐射损伤后的造血干细胞。

DOI：
发表时间：
2002
期刊：
Military medicine
影响因子：
1.2
作者：
Chute,JohnP;Clark,William;Saini,Abha;Wells,Mark;Harlan,David
通讯作者：
Harlan,David

Islet neogenesis associated protein transgenic mice are resistant to hyperglycemia induced by streptozotocin.

胰岛新生相关蛋白转基因小鼠对链脲佐菌素诱导的高血糖有抵抗力。

DOI：
10.1677/joe.1.06698
发表时间：
2006
期刊：
The Journal of endocrinology
影响因子：
0
作者：
Taylor-Fishwick,DavidA;Bowman,Angela;Hamblet,Natasha;Bernard,Paul;Harlan,DavidM;Vinik,AaronI
通讯作者：
Vinik,AaronI

Beta cell-specific CD80 (B7-1) expression disrupts tissue protection from autoantigen-specific CTL-mediated diabetes.

Beta 细胞特异性 CD80 (B7-1) 表达会破坏组织对自身抗原特异性 CTL 介导的糖尿病的保护。