Immunoregulation of Myelin-Specific T Lymphocytes

多发性硬化症（MS）及其鼠模型，实验性自身免疫性脑脊髓炎（EAE），是慢性的， 中枢神经系统（CNS）的衰弱性自身免疫性疾病，其特征在于广泛的 脱髓鞘和轴突损伤。一种被认为是驱动早期炎症阶段的关键细胞因子， 巨噬细胞移动抑制因子（MIF-1）是最早被描述的巨噬细胞移动抑制因子， 细胞因子/趋化因子。MIF-1水平在MS中增加，并且该关键因子已被认为是MS的标志物。 MS的临床恶化和EAE疾病进展的要求。我们的实验室设计了一个 一种名为RTL 1000的有效生物构建体和第二代衍生物DRα1-MOG-35-55， 与MIF-1受体CD 74紧密结合，竞争性抑制MIF-1结合和下游信号传导。 最近，作为MIF-1的祖先功能同源物的CD 74的第二个配体，称为D-多巴色素， tautamerase（DDT或MIF-2）。MIF-2蛋白与MIF-1只有35%的同源性， 在大多数组织中以相同的水平表达。MIF-2的生物活性仍然需要与CD 74结合， 与MIF-1的结果高度重叠，在评价MIF时需要同时评估MIF-1和MIF-2 致病活性我们以前已经证明，部分MHC II类结构，包括RTL 1000 和DRα1-MOG-35-55在EAE中具有有效的治疗活性，预测两者都选择性地结合于 与MIF-1相同的CD 74三聚化结构域残基。然而，MIF-2是否结合到 RTL 1000和DRα1-MOG-35-55可竞争性抑制CD 74或MIF-2的这些相同区域。 基于我们的建模数据，我们假设CD 74的一个共同区域与CD 74的不同区域结合。 MIF-1与MIF-2同源三聚体，RTL 1000和DRα1-MOG-35-55将竞争性阻断结合， MIF-1和MIF-2两者的下游信令。因此，本提案将针对以下目标：目标1： 使用突变的CD 74构建体测定对MIF-1和MIF-2结合和信号传导的影响， 基于预测的结合相互作用的干扰肽。目的2：比较MIF-1与 在WT、MIF-1 K、MIF-2KO和MIF双KO C57 BL/6中，MIF-2对EAE的发作、严重程度和进展的影响 小鼠目的3：测试RTL 1000和DRα1-MOG-35-55构建体抑制MIF-1与MIF-2结合的能力 和下游信号传导，并治疗MIF-1 KO、MIF-2KO和MIF双KO小鼠的急性和慢性EAE vs. WT小鼠。这些研究将确定RTL 1000和DRα1-MOG-35-55是否也可竞争性抑制 MIF-2和MIF-1通过阻断CD 74介导的信号传导在EAE中发挥疾病促进作用。 这些新型MS治疗药物的开发可能对MS的治疗产生重大影响。 MS的治疗，这仍然是VA的重要临床问题。 版本：2-14-16

Multiple sclerosis (MS) and its murine model, experimental autoimmune encephalomyelitis (EAE), are chronic, debilitating autoimmune diseases of the central nervous system (CNS) characterized by extensive demyelination and axonal damage. One of the key cytokines thought to drive the early inflammatory stage of MS to a chonic progressive phase is Macrophage Migration Inhibitory Factor (MIF-1), the first described cytokine/chemokine. MIF-1 levels are increased in MS and this key factor has been implicated as a marker of clinical worsening in MS and as a requirement for disease progression in EAE. Our laboratory designed a potent biological construct called RTL1000 and a second generation derivative, DRα1-MOG-35-55, that bind tightly to the MIF-1 receptor, CD74, and competitively inhibit MIF-1 binding and downstream signaling. Recently, a second ligand for CD74 that is an ancestral functional homolog of MIF-1, called D-dopachrome tautamerase (DDT or MIF-2) was reported. The MIF-2 protein has only 35% homology with MIF-1 and is expressed at equivalent levels in most tissues. MIF-2 biological activities still require binding to CD74 and strongly overlap with those of MIF-1, requiring assessment of both MIF-1 and MIF-2 when evaluating MIF pathogenic activity. We have demonstrated previously that partial MHC class II constructs, including RTL1000 and DRα1-MOG-35-55 have potent therapeutic activity in EAE both are predicted to bind selectively to the same CD74 trimerization domain residues as MIF-1. However, it remains unstudied whether MIF-2 binds to these same regions of CD74 or if MIF-2 can be competitively inhibited by RTL1000 and DRα1-MOG-35-55. Based on our modeling data, we hypothesize that a common region of CD74 binds to distinct regions of the MIF-1 vs. MIF-2 homotrimers and that RTL1000 and DRα1-MOG-35-55 will competitively block binding and downstream signaling of both MIF-1 and MIF-2. This proposal will thus address the following Aims: Aim 1: Determine the effects on MIF-1 and MIF-2 binding and signaling using mutated CD74 constructs and interfering peptides based on predicted binding interactions. Aim 2: Compare the contributions of MIF-1 vs. MIF-2 on the onset, severity and progression of EAE in WT, MIF-1K, MIF-2KO and MIF double KO C57BL/6 mice. Aim 3: Test the ability of RTL1000 and DRα1-MOG-35-55 constructs to inhibit MIF-1 vs. MIF-2 binding and downstream signaling and to treat acute and chronic EAE in MIF-1KO, MIF-2KO and MIF double KO mice vs. WT mice. These studies will establish if RTL1000 and DRα1-MOG-35-55 can also competitively inhibit MIF-2 as well as MIF-1 disease-promoting effects in EAE through blockade of CD74-mediated signaling. Development of these novel therapeutic agents for MS could have a significant impact on the treatment of MS, which remains an important clinical problem for the VA. Version: 2-14-16