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

描述（由申请人提供）：在特发性炎性肌病中，疾病发病机制在很大程度上仍不明确。然而，我们最近发表的工作表明，不同的NOD和C57 BL/6同源株与鼠组氨酰-tRNA合成酶（Jo-1）的免疫产生肌肉和肺部炎症类似的抗合成酶综合征的功能。本申请中概述的实验方法通过树突状细胞（DC）表型的功能操作来扩展这些早期研究，以定义有助于疾病表达的Jo-1特异性免疫应答的特征。特异性目的1采用不同的体外细胞因子制剂和成熟刺激物来产生能够偏向于靶向该抗原的Th 1、Th 2或Th 17免疫应答的Jo-1负载的DC。随后用在限定条件下培养的Jo- 1脉冲的DC免疫NOD和C57 BL/6同源小鼠，与Jo-1/CFA免疫相比，将突出抗原特异性T细胞极化对组织炎症的影响。器官浸润淋巴细胞从DC免疫的幼稚小鼠的连续转移将牢固地建立Jo-1特异性自身免疫反应和抗合成酶综合征之间的关系。平行测量CFSE标记的T细胞增殖反应和来自DC免疫小鼠的脾和器官引流淋巴结的抗原激发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Th 17和/或Th 1极化的Jo-1特异性T细胞反应联系起来。相比之下，在特异性目标2中使用替代性细胞因子调节将产生能够阻断疾病诱导并且更重要地，在现有疾病中重建耐受性的Jo-1负载的“致耐受性”DC。补充体内评估Jo-1脉冲致耐受性DC免疫在疾病预防中的作用，体外transwell混合和抗体阻断实验将确定无反应性、旁观者抑制和接触介导的抑制对抗原特异性调节性T细胞功能的相对贡献。这些研究提供的机制见解将有助于确定适用于未来人体临床试验的免疫靶点和治疗方法。公共卫生相关性：该项目采用抗原脉冲树突状细胞的功能操作来定义Jo-1特异性T细胞在炎症性肌病小鼠模型中的作用。这种方法将阐明疾病起始/延续的机制，并建立抗原特异性树突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，其中全身免疫抑制是唯一的治疗选择。

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.