REGULATION OF EAE WITH RECOMBINANT TCR LIGANDS

描述（由申请人提供）：该项目的总体目标是严格检验髓磷脂抗原反应性T细胞和MS风险相关的HLA-DR2等位基因与多发性硬化症（MS）发病机制有关的一般假设。为此，我们开发了一种重组TCR配体（RTL），这是一种与免疫优势MOG-35-55表位共价连接的单链双结构域人HLA-DR2 II类分子，可在转基因（Tg） dr2表达小鼠中诱导长期耐受性并逆转mog肽诱导的EAE的既定临床症状。我们生产的最初的DR2/MOG-35-55构建物（VG312）具有自粘附表面，并且倾向于形成稳定的低聚物，平均组成为14个亚基。低聚化对结构体抑制功能的影响尚不清楚，但低聚物可能具有较低的功能摩尔浓度，因此在体内的抑制活性低于单体。另一方面，低聚物可能比单体更有效地交联TCR，从而通过TCR产生更多或不同的信号传导，从而影响抑制活性。我们现在通过修饰自接触表面的氨基酸残基生产了DR2/MOG-35-55单体（VG342），这将允许与14-mer VG312结构进行功能比较。此外，我们已经生产了含有小鼠(m)MOG和人类(h)MOG肽的构建物，它们对EAE小鼠的tcr具有不同的亲和力，并且一种新的单体形式（VG342-T）缺乏凝血酶(T)切割位点，该位点被设计到原始构建物的肽连接区域。我们的目的是比较这五种形式的DR2/MOG-35-55结构的治疗效果、诱导耐受性以及对小鼠和人类MOG-35-55特异性T细胞系、克隆和我们最近从DR2小鼠中培育的杂交瘤的影响。具体来说，在本应用中，我们将解决以下特定假设：耐受性的程度和机制受以下因素的支配或影响：1)RTL对TCR的亲和力差异；2)RTL与TCR结合的功能亲切度差异；3)RTL的裂解和释放游离肽。最后，我们将评估rtl诱导的旁观者抑制程度，并跟踪rtl处理的T细胞的命运。这是首次在体内评估TCR单体与低聚体阻断对耐受性影响的研究，将为这些重组TCR配体（RTLs）在MS患者中的临床应用提供必要的基础。

DESCRIPTION (provided by applicant): The overall goal of this program is to test critically the general hypothesis that myelin antigen reactive T cells and the MS risk-associated HLA-DR2 allele contribute to the pathogenesis of multiple sclerosis (MS). To this end we developed a recombinant TCR ligand (RTL), a single chain two domain human HLA-DR2 class II molecule covalently linked to the immunodominant MOG-35-55 epitope that induced long-term tolerance and reversed established clinical signs of MOG-peptide-induced EAE in transgenic (Tg) DR2-expressing mice. The initial DR2/MOG-35-55 construct (VG312) that we produced had self-adherent surfaces and tended to form stable oligomers with an average composition of 14-subunits. The effect of oligomerization on the inhibitory function of the constructs is unknown, but potentially the oligomers might have a lower functional molarity and thus less inhibitory activity in vivo than the monomers. On the other hand, the oligomers might be able to cross-link the TCRs more efficiently than monomers, resulting in more or different signaling through the TCR that might affect inhibitory activity. We have now produced a DR2/MOG-35-55 monomer (VG342) by modifying amino acid residues in the self-contact surface that will allow a functional comparison with the 14-mer VG312 construct. Additionally, we have produced constructs that contain mouse (m)MOG versus human (h)MOG peptides with different affinities for TCRs from mice with EAE, and a new monomeric form (VG342-T) that lacks the thrombin (T) cleavage site that was engineered into the peptide-joining region of the original construct. Our goal in this application is to compare these five forms of the DR2/MOG-35-55 construct for therapeutic efficacy, induction of tolerance, and effects on mouse and human MOG-35-55 specific T cell lines, clones, and a hybridoma that we recently developed from the DR2 mice. Specifically, in this application we will address the specific hypothesis that the degree and mechanism of tolerance is governed or influenced by 1) differences in the affinity of the RTL for the TCR, 2) differences in functional avidity of RTL binding to the TCR, and 3) cleavage and release of free peptide from the RTL. Finally, we will evaluate the degree of bystander suppression induced by RTLs and follow the fate of RTL-treated T cells. These are the first studies ever to evaluate the effect on tolerance of monomeric versus oligomeric TCR blockage in vivo, and will provide the necessary foundation for clinical application of these recombinant TCR ligands (RTLs) in patients with MS.