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）是一种由工作场所铍暴露引起的肉芽肿性肺部疾病，其特征是肺中铍特异性CD4+ T细胞的积累。疾病易感性与HLA-DPB1等位基因有关，包括DPB1*0201，它在HLA-DP2 2-链的第69位具有谷氨酸残基（2Glu69）。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背景下识别铍。重要的是，呈现铍的DP分子与那些与疾病易感性相关的分子相匹配，证实HLA对疾病的贡献是基于这些分子结合并将铍呈现给T细胞的能力。我们最近结晶了HLA-DP2，并确定了一个潜在的铍结合位点，涉及到在¿26和¿68位的Glu69和另外两个不变谷氨酸。功能研究证实铍的识别依赖于这一簇负电荷。到目前为止，还没有铍诱发疾病的动物模型。以前试图建立的小鼠模型未能证明铍特异性适应性免疫反应。重要的是，小鼠缺乏与HLA-DP2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，产生含有HLA-DP2特有的负电荷簇的人源化转基因小鼠将产生疾病特异性小鼠模型，其病理反映人类疾病。NIOSH的研究人员在FVB/N背景下产生了HLA-DP2和-DP4转基因小鼠。然而，这些小鼠表达小鼠MHC，缺乏人类CD4，这是最佳T细胞激活的重要辅助受体。没有研究表明这些小鼠对吸入铍的反应。初步数据显示，氧化铍（BeO）暴露后，仅在暴露于HLA-DP2转基因小鼠中发生支气管血管周围单核细胞浸润，强烈提示炎症反应是由于HLA-DP2转基因的存在，并支持我们的假设，即含有gl69的HLA-DP分子是产生铍诱导疾病小鼠模型所必需的。在第一个特定目标中，我们将在缺乏小鼠MHCII分子和表达人类CD4的C57BL/6小鼠中重新获得HLA-DP2转基因。在第二个目标中，我们将直接比较铍在这些动物和表达dp2的FVB/N小鼠中诱导的适应性免疫反应，验证稳定HLA-DP2和T细胞受体与人CD4辅助受体之间的相互作用以及消除小鼠MHCII的混淆效应将优化铍特异性免疫反应的假设。总之，这些研究将导致一种疾病特异性的小鼠模型，针对人类疾病发展所需的确切分子。

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.