Functional impact of dendritic cell phenotype in a mouse model of myositis

描述(申请人提供)：在特发性炎症性肌病中，疾病的发病机制在很大程度上仍未明确。然而，我们最近发表的工作表明，用小鼠组氨酰-tRNA合成酶(Jo-1)免疫不同的NOD和C57BL/6同源菌株会产生类似于抗合成酶综合征的肌肉和肺部炎症。本申请中概述的实验方法通过对树突状细胞(DC)表型的功能操纵来扩展这些早期研究，以确定导致疾病表达的Jo-1特异性免疫反应的特征。特异性目的1使用不同的体外细胞因子制剂和成熟刺激来产生Jo-1负载的DC，能够偏向Th1、Th2或Th17免疫应答。与Jo-1/CFA免疫相比，在特定条件下培养的Jo-1冲击的DC随后免疫NOD和C57BL/6同基因小鼠将突出抗原特异性T细胞极化对组织炎症的影响。将DC免疫的器官浸润性淋巴细胞过继转移到幼小鼠身上，将牢固地建立Jo-1特异性自身免疫反应与抗合成酶综合征之间的关系。同时测量CFSE标记的T细胞增殖反应和来自DC免疫小鼠脾和器官引流淋巴结的抗原攻击淋巴细胞的细胞内细胞因子染色，将进一步将观察到的组织表型与Th17和/或Th1极化的Jo-1特异性T细胞反应联系起来。相反，在特定目的2中使用替代细胞因子调节将产生Jo-1负载的“耐受”树突状细胞，能够阻断疾病诱导，更重要的是，在现有疾病中重新建立耐受。补充Jo-1脉冲致耐受树突状细胞免疫在疾病预防中的体内评估不适用。改善，体外转井混合和抗体阻断实验将确定无能、旁观者抑制和接触介导的抑制对抗原特异性调节T细胞功能的相对贡献。这些研究提供的机制洞察力将有助于确定适用于未来人类临床试验的免疫学靶点和治疗方法。公共卫生相关性：该项目使用抗原冲击的树突状细胞的功能操作来确定Jo-1特异性T细胞在炎症性肌病小鼠模型中的作用。这一方法将阐明疾病启动/持续的机制，并建立抗原特异性的、树突状细胞介导的耐受诱导的可行性。后一个目标与多种自身免疫性疾病的治疗有关，全球免疫抑制是这些疾病的唯一治疗选择。

DESCRIPTION (provided by applicant): In the idiopathic inflammatory myopathies, disease pathogenesis remains largely undefined. However, we have recently published work demonstrating that immunization of different NOD and C57BL/6 congenic strains with murine histidyl-tRNA synthetase (Jo-1) generates muscle and lung inflammation resembling features of the anti-synthetase syndrome. The experimental approach outlined in this application extends these earlier studies through functional manipulation of dendritic cell (DC) phenotype to define characteristics of the Jo-1-specific immune response contributing to disease expression. Specific Aim 1 employs different in vitro cytokine preparations and maturation stimuli to generate Jo-1-loaded DCs capable of skewing toward Th1, Th2, or Th17 immune responses targeting this antigen. Subsequent immunization of NOD and C57BL/6 congenic mice with Jo- 1-pulsed DCs cultivated under defined conditions will highlight the impact of antigen-specific T cell polarization on tissue inflammation in comparison to Jo-1/CFA immunization. Adoptive transfer of organ-infiltrating lymphocytes from DC-immunized to naive mice will firmly establish the relationship between Jo-1-specific autoimmune responses and the anti-synthetase syndrome. Parallel measurement of CFSE-labeled T cell proliferative responses and intracellular cytokine staining of antigen-challenged lymphocytes derived from spleen and organ-draining lymph nodes of DC-immunized mice will further link the observed tissue phenotype to Th17 and/or Th1-polarized, Jo-1-specific T cell responses. In contrast, use of alternative cytokine conditioning in Specific Aim 2 will generate Jo-1-loaded "tolerogenic" DCs capable of blocking disease induction and, more importantly, re-establishing tolerance in existing disease. Complementing the in vivo assessment of Jo-1-pulsed tolerogenic DC immunization in disease preventioNot applicable.melioration, in vitro transwell mixing and antibody blocking experiments will define the relative contribution of anergy, bystander suppression, and contact-mediated inhibition to antigen-specific regulatory T cell function. The mechanistic insight provided by these studies will help to define immunologic targets and therapeutic approaches applicable to future human clinical trials. PUBLIC HEALTH RELEVANCE: This project employs functional manipulation of antigen-pulsed dendritic cells to define the role of Jo-1-specific T cells in a murine model of inflammatory myopathy. This approach will clarify mechanisms of disease initiation/perpetuation and establish the feasibility of antigen- specific, dendritic cell-mediated tolerance induction. The latter objective is relevant to the treatment of multiple autoimmune diseases for which global immunosuppression is the only therapeutic option.