Role of HLA Class II Genes in Demyelination

描述（由申请人提供）：多发性硬化症（MS）是一种炎症性和脱髓鞘性自身免疫性疾病，具有遗传和环境易感性。在所有与MS易感性相关的遗传因素中，DR2/DQ6、DR3/DQ2、DR4/DQ8等hlaⅱ类单倍型的相关性最强。虽然HLA-DR等位基因在MS中的直接作用已被证实，但由于强烈的连锁不平衡，HLA-DQ等位基因在疾病发病机制中的作用一直难以理解。群体研究表明，DQ等位基因可能在MS的发展过程中发挥调节作用。为了更好地了解HLA- dr和-DQ基因对MS易感性和抗性的作用机制，我们构建了表达HLA II类基因和缺乏内源性小鼠II类基因的单转基因和双转基因小鼠。之前我们已经证实HLA-DR3转基因小鼠对PLP91-110诱导的EAE易感，而DQ6 （DQB1*0601）和DQ8 （DQB1*0302）转基因小鼠对EAE有抗性。令人惊讶的是，DQ6/DR3双转基因小鼠具有耐药性，而DQ8/DR3小鼠的发病率和严重程度更高。DQ6对DQ6/DR3小鼠的保护作用是通过抗炎IFN3介导的，而DQ8分子的加重作用是通过IL17介导的。基于这些观察结果，我们假设HLA-DR和-DQ基因之间的上位性相互作用在ms易感性中起重要作用。这一建议旨在加深对HLA-DQ和-DR分子之间的上位性相互作用决定疾病易感性和抗性的机制的理解。我们提出了两个目的来了解HLA-DQ分子调节HLA-DR/DQ双转基因小鼠疾病结局的机制。在第一个目标中，我们将通过分析HLA-DQ8分子如何增加DQ8/DR3双转基因小鼠的疾病发病率和严重程度-i) HLA多态性和肽- mhc亲和力/亲和度在DQ8限制性CD4 T细胞产生促炎il - 17中的作用；ii) IL17导致DR3DQ8小鼠脑致原性增加的机制；DQ8产生的GM-CSF限制CD4 T细胞在DR3DQ8双转基因小鼠EAE加重中的作用。在第二个目标中，我们将研究HLA-DQ6限制免疫反应如何导致产生高IFN3产生的调节性CD4+ T细胞，特别是mhc肽亲和力/功能亲和性的作用。接下来，我们将分析DQ6限制CD4+ T细胞抑制HLA-DR3/DQ6双转基因小鼠EAE的机制。我们还将产生一种表达疾病易感HLA-DR3、疾病保护-DQ6和疾病增强-DQ8基因的三重转基因小鼠，以模拟人类杂合状态。本提案中概述的综合研究应该能够深入了解HLA II类分子塑造T细胞库并调节促炎和抗炎细胞因子谱的机制。这将促进治疗多发性硬化症等炎症性疾病的新疗法的转化研究发展。

DESCRIPTION (provided by applicant): Multiple sclerosis (MS), an inflammatory and demyelinating autoimmune disease has both a genetic and an environmental predisposition. Among all the genetic factors associated with MS susceptibility, HLA-class II haplotypes such as DR2/DQ6, DR3/DQ2, DR4/DQ8, show the strongest association. Although a direct role of HLA-DR alleles in MS have been confirmed, it has been difficult to understand the contribution of HLA-DQ alleles in disease pathogenesis, due to strong linkage disequilibrium. Population studies have indicated that DQ alleles may play a modulatory role in progression of MS. To better understand the mechanism by which HLA-DR and -DQ genes contribute to susceptibility and resistance to MS, we generated single and double transgenic mice expressing HLA class II genes and lacking endogenous mouse class II genes. Previously, we have shown that HLA-DR3 transgenic mice were susceptible to PLP91-110 induced EAE, while DQ6 (DQB1*0601) and DQ8 (DQB1*0302) transgenic mice were resistant. Surprisingly DQ6/DR3 double transgenic mice were resistant while DQ8/DR3 mice showed higher disease incidence and severity. Protective effect of DQ6 in DQ6/DR3 mice was mediated by anti-inflammatory IFN3, while disease exacerbating effect of DQ8 molecule was mediated by IL17. Based on these observations, we hypothesize that epistatic interaction between HLA-DR and -DQ genes play an important role in predisposition to MS. This proposal is aimed to enhance understanding of the mechanism by which epistatic interactions between HLA-DQ and -DR molecules determine the susceptibility vs. resistance to disease. We are proposing two aims to understand the mechanism by which HLA-DQ molecule modulate the disease outcome in HLA-DR/DQ double transgenic mice. In the first aim, we will examine how HLA-DQ8 molecule increases disease incidence and severity in DQ8/DR3 double transgenic mice by analyzing- i) role of HLA polymorphism and peptide-MHC affinity/avidity in generation of pro-inflammatory IL17 production from DQ8 restricted CD4 T cells; ii) mechanism by which IL17 cause increased encephalitogenicity in DR3DQ8 mice; and iii) role of GM-CSF produced by DQ8 restricted CD4 T cells in exacerbation of EAE in DR3DQ8 double transgenic mice. In the second aim, we will investigate how HLA-DQ6 restricted immune response leads to generation of high IFN3 producing regulatory CD4+ T cells, especially the role of MHC-peptide affinity/functional avidity. Next we will analyze mechanisms by which DQ6 restricted CD4+ T cells suppress EAE in HLA-DR3/DQ6 double transgenic mice. We will also generate a triple transgenic mice expressing disease susceptible HLA-DR3, disease protective -DQ6, and disease enhancing -DQ8 gene to simulate human heterozygous condition. The comprehensive studies outlined in this proposal should yield an insight into mechanism by which HLA class II molecules shape the T cell repertoire and regulate the pro-inflammatory and anti-inflammatory cytokine profile. This will facilitate the translational research development of novel therapeutic to treat inflammatory disease such as MS. PUBLIC HEALTH RELEVANCE: Although HLA haplotypes are linked to predisposition and onset of multiple sclerosis, it has been difficult in past to define a clear role of HLA molecule(s) in MS. We have used HLA transgenic mice successfully to understand their role in inflammatory and demyelinating disease of CNS such as MS using an experimental model experimental autoimmune encephalomyelitis (EAE). These humanized class II model of EAE can be used to evaluate potential therapeutic protocols and peptide vaccines applicable in the future to MS patients.