Development of an HLA-DP2 Transgenic Murine Model of Chronic Beryllium Disease

慢性铍病 HLA-DP2 转基因小鼠模型的建立

基本信息

批准号：
8389617
负责人：
Andrew P. Fontenot
金额：
$ 17.1万
依托单位：
UNIVERSITY OF COLORADO DENVER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-01-01 至 2014-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8389617
关键词：
Alleles Alveolitis Amino Acids Animal Disease Models Animal Model Animals Antigens Beryllium Binding Binding Sites Blood Breathing C3H/HeJ Mouse C57BL/6 Mouse CD4 Positive T Lymphocytes Cells Charge Chronic berylliosis Data Development Disease Disease susceptibility Exposure to FVB/N Mouse Fibrosis Generations Genetic Predisposition to Disease Glutamic Acid Granulomatous HLA-DP Antigens HLA-DP2 HLA-DP4 HLA-DPB1 gene Hilar Histocompatibility Antigens Class II Human Immune System Diseases Immune response Inbred Strains Mice Individual Inflammation Inflammatory Response Knowledge Lead Link Lung Lung diseases Lymphocyte Lysine Mediating Metals Modeling Mononuclear Mus Nature Oxides Pathology Patients Positioning Attribute Public Health Publishing Research Personnel Risk Sampling Site Spleen Structure T-Cell Activation T-Cell Receptor T-Lymphocyte Testing Threonine Time Transgenes Transgenic Mice Transgenic Organisms Workplace animal model development base cytokine human disease human subject interstitial lymph nodes lymphocyte proliferation mouse model response

项目摘要

DESCRIPTION (provided by applicant): Chronic beryllium disease (CBD) is a granulomatous lung disorder caused by beryllium exposure in the workplace and is characterized by the accumulation of beryllium-specific CD4+ T cells in the lung. Disease susceptibility has been linked to HLA-DPB1 alleles, including DPB1*0201, that possess a glutamic acid residue at position 69 of the HLA-DP2 2-chain (2Glu69). We have shown that beryllium directly binds to HLA-DP2 molecules and that the majority of beryllium-specific CD4+ T cells recognize beryllium in the context of HLA- DP. Importantly, the DP molecules that present beryllium match those implicated in disease susceptibility, confirming that the HLA contribution to disease is based on the ability of those molecules to bind and present beryllium to T cells. We have recently crystallized HLA-DP2 and identified a potential beryllium binding site involving ¿Glu69 and two other invariant glutamic acids at positions ¿26 and ¿68. Functional studies confirmed that beryllium recognition was dependent on this cluster of negative charges. To date, no animal model of beryllium-induced disease exists. Previous attempts to generate a murine model failed to document a beryllium-specific adaptive immune response. Importantly, mice lack a major histocompatibility complex class II (MHCII) molecule of similar sequence and structure to HLA-DP2. Thus, we hypothesize that the generation of a humanized transgenic mouse containing the cluster of negative charges unique to HLA-DP2 will result in a disease-specific murine model with pathology reflective of the human disease. HLA-DP2 and -DP4 transgenic mice on an FVB/N background have been generated by researchers at NIOSH. However, these mice express murine MHC and lack human CD4, an important coreceptor for optimal T cell activation. No studies characterizing the response of these mice to inhaled beryllium have been published. Preliminary data show peribronchovascular mononuclear cell infiltrates occurring only in beryllium-exposed HLA-DP2 transgenic mice after beryllium oxide (BeO) exposure, strongly suggesting that the inflammatory response is due to the presence of the HLA-DP2 transgene and supporting our hypothesis that ¿Glu69-containing HLA-DP molecules are required for the generation of a murine model of beryllium-induced disease. In the first specific aim, we will re-derive the HLA-DP2 transgene in C57BL/6 mice lacking murine MHCII molecules and expressing human CD4. In the second aim, we will directly compare the beryllium-induced adaptive immune response in these animals and DP2-expressing FVB/N mice, testing the hypothesis that stabilization of the interaction between HLA-DP2 and T cell receptor with the human CD4 coreceptor and elimination of the confounding effects of mouse MHCII will optimize the beryllium-specific immune response. Together, these studies will lead to a disease-specific murine model, targeting the exact molecule necessary for the development of a human disease.

描述（由申请人提供）：慢性铍病 (CBD) 是一种由工作场所接触铍引起的肉芽肿性肺部疾病，其特征是肺部特异性 CD4+ T 细胞积聚，该疾病的易感性与 HLA- 相关。 DPB1 等位基因，包括 DPB1*0201，在 HLA-DP2 的第 69 位具有谷氨酸残基我们已经证明铍直接与 HLA-DP2 分子结合，并且大多数铍特异性 CD4+ T 细胞在 HLA-DP 的背景下识别铍，重要的是，呈现铍的 DP 分子与这些分子相匹配。与疾病易感性有关，证实 HLA 对疾病的贡献是基于这些分子与 T 细胞结合和呈递铍的能力。结晶 HLA-DP2 并确定了一个潜在的铍结合位点，涉及 ¿ Glu69 和其他两个不变的谷氨酸在位置 ¿ 26 和 ¿ 68. 功能研究证实，铍识别依赖于这组负电荷。迄今为止，尚不存在铍诱发疾病的动物模型。小鼠缺乏与 HLA-DP2 具有相似序列和结构的主要组织相容性复合物 II 类 (MHCII) 分子，因此，我们发现含有 HLA-DP2 所特有的负电荷簇的人源化转基因小鼠的产生。 NIOSH 的研究人员已经在 FVB/N 背景上建立了 HLA-DP2 小鼠模型，其病理学反映了人类疾病。然而，这些小鼠表达小鼠 MHC 和 MHC。缺乏人类 CD4，这是最佳 T 细胞激活的重要辅助受体，尚未发表有关这些小鼠对吸入铍的反应的研究。仅在接触铍的 HLA-DP2 强转基因小鼠中，在氧化铍 (BeO) 暴露后发生浸润，表明炎症反应是由于 HLA-DP2 转基因的存在，并支持我们的假设 ¿含有 Glu69 的 HLA-DP 分子是生成铍诱发疾病的小鼠模型所必需的。在第一个具体目标中，我们将在缺乏小鼠 MHCII 分子并表达人类的 C57BL/6 小鼠中重新衍生 HLA-DP2 转基因。在第二个目标中，我们将直接比较这些动物和表达 DP2 的 FVB/N 小鼠中铍诱导的适应性免疫反应，测试稳定化的假设。 HLA-DP2 和 T 细胞受体与人类 CD4 辅助受体之间的相互作用以及消除小鼠 MHCII 的混杂效应将优化铍特异性免疫反应，这些研究将共同产生一种针对精确疾病的小鼠模型。人类疾病发展所必需的分子。