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

描述（由申请人提供）：慢性铍病（CBD）是一种肉芽肿性肺部疾病，由工作场所的铍暴露引起，其特征是铍特异性CD 4 + T细胞在肺中蓄积。疾病易感性与HLA-DPB 1等位基因（包括DPB 1 *0201）有关，该等位基因在HLA-DP 2 2-链的69位具有谷氨酸残基（2Glu 69）。我们已经表明铍直接结合HLA-DP 2分子，并且大多数铍特异性CD 4 + T细胞在HLA-DP的背景下识别铍。重要的是，呈现铍的DP分子与涉及疾病易感性的DP分子相匹配，证实了HLA对疾病的贡献是基于这些分子结合铍并将铍呈递给T细胞的能力。我们最近结晶HLA-DP 2，并确定了一个潜在的铍结合位点，涉及<$Glu 69和其他两个不变的谷氨酸在位置<$26和<$68。功能研究证实，铍识别依赖于这簇负电荷。迄今为止，没有铍诱发疾病的动物模型存在。以前尝试产生小鼠模型未能记录铍特异性适应性免疫应答。重要的是，小鼠缺乏与HLA-DP 2序列和结构相似的主要组织相容性复合体II类（MHCII）分子。因此，我们假设含有HLA-DP 2特有的负电荷簇的人源化转基因小鼠的产生将产生具有反映人类疾病的病理学的疾病特异性小鼠模型。NIOSH的研究人员已经在FVB/N背景下产生了HLA-DP 2和-DP 4转基因小鼠。然而，这些小鼠表达鼠MHC并且缺乏人CD 4，这是最佳T细胞活化的重要辅助受体。没有研究表明这些小鼠对吸入铍的反应。初步数据显示，支气管血管周围单核细胞浸润只发生在铍暴露的HLA-DP 2转基因小鼠氧化铍（BeO）暴露后，强烈表明，炎症反应是由于存在的HLA-DP 2转基因，并支持我们的假设，即含Glu 69的HLA-DP分子是必要的铍诱导的疾病的小鼠模型的产生。在第一个具体目标中，我们将在缺乏鼠MHCII分子并表达人CD 4的C57 BL/6小鼠中重新获得HLA-DP 2转基因。在第二个目标中，我们将直接比较铍诱导的适应性免疫应答在这些动物和DP 2表达的FVB/N小鼠，测试的假设，HLA-DP 2和T细胞受体与人CD 4辅助受体之间的相互作用的稳定和小鼠MHCII的混杂效应的消除将优化铍特异性免疫应答。总之，这些研究将导致疾病特异性小鼠模型，靶向人类疾病发展所需的确切分子。 公共卫生相关性：铍诱导疾病的HLA-DP 2转基因小鼠模型的发展将提高我们的能力，以确定精确的环境抗原引起的人类肉芽肿性肺病，并确定金属抗原如何触发免疫反应。

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. PUBLIC HEALTH RELEVANCE: The development of an HLA-DP2 transgenic mouse model of beryllium-induced disease will enhance our ability to define the precise environmental antigen causing a human granulomatous lung disease and to determine how metal antigens trigger an immune response.