Wei Hsi-Ju, Letterio John J, Pareek Tej K
Department of Biochemistry, School of Medicine, Case Western Reserve University.
Department of Pediatrics, Division of Pediatric Hematology/Oncology, Case Western Reserve University; Angie Fowler Cancer Institute, Rainbow Babies & Children's Hospital, University Hospitals, Cleveland.
J Vis Exp. 2018 May 18(135):57637. doi: 10.3791/57637.
The immune system operates by maintaining a tight balance between coordinating responses against foreign antigens and maintaining an unresponsive state against self-antigens as well as antigens derived from commensal organisms. The disruption of this immune homeostasis can lead to chronic inflammation and to the development of autoimmunity. Dendritic cells (DCs) are the professional antigen-presenting cells of the innate immune system involved in activating naïve T cells to initiate immune responses against foreign antigens. However, DCs can also be differentiated into TolDCs that act to maintain and promote T cell tolerance and to suppress effector cells contributing to the development of either autoimmune or chronic inflammation conditions. The recent advancement in our understanding of TolDCs suggests that DC tolerance can be achieved by modulating their differentiation conditions. This phenomenon has led to tremendous growth in developing TolDC therapies for numerous immune disorders caused due to break in immune tolerance. Successful studies in preclinical autoimmunity murine models have further validated the immunotherapeutic utility of TolDCs in the treatment of autoimmune disorders. Today, TolDCs have become a promising immunotherapeutic tool in the clinic for reinstating immune tolerance in various immune disorders by targeting pathogenic autoimmune responses while leaving protective immunity intact. Although an array of strategies has been proposed by multiple labs to induce TolDCs, there is no consistency in characterizing the cellular and functional phenotype of these cells. This protocol provides a step-by-step guide for the development of bone marrow-derived DCs in large numbers, a unique method used to differentiate them into TolDCs with a synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid-difluoro-propyl-amide (CDDO-DFPA), and the techniques used to confirm their phenotype, including analyses of essential molecular signatures of TolDCs. Finally, we show a method to assess TolDC function by testing their immunosuppressive response in vitro and in vivo in a preclinical model of multiple sclerosis.
免疫系统通过在协调针对外来抗原的反应与维持针对自身抗原以及共生生物衍生抗原的无反应状态之间保持紧密平衡来运作。这种免疫稳态的破坏会导致慢性炎症和自身免疫性疾病的发展。树突状细胞（DCs）是先天性免疫系统的专职抗原呈递细胞，参与激活幼稚T细胞以启动针对外来抗原的免疫反应。然而，DCs也可分化为耐受性树突状细胞（TolDCs），其作用是维持和促进T细胞耐受性，并抑制促成自身免疫或慢性炎症性疾病发展的效应细胞。我们对TolDCs理解的最新进展表明，可通过调节其分化条件来实现DC耐受性。这一现象导致在开发针对因免疫耐受性破坏而引起的多种免疫疾病的TolDC疗法方面取得了巨大进展。在临床前自身免疫性小鼠模型中的成功研究进一步验证了TolDCs在治疗自身免疫性疾病中的免疫治疗效用。如今，TolDCs已成为临床上一种有前景的免疫治疗工具，可通过靶向致病性自身免疫反应同时保持保护性免疫完整来恢复各种免疫疾病中的免疫耐受性。尽管多个实验室提出了一系列诱导TolDCs的策略，但在表征这些细胞的细胞和功能表型方面尚无一致性。本方案提供了一个详细的指南，用于大量培养骨髓来源的DCs，这是一种独特的方法，可使用合成三萜类化合物2-氰基-3,12-二氧代齐墩果-1,9-二烯-28-酸-二氟丙基酰胺（CDDO-DFPA）将其分化为TolDCs，并介绍了用于确认其表型的技术，包括对TolDCs基本分子特征的分析。最后，我们展示了一种在多发性硬化症临床前模型中通过在体外和体内测试其免疫抑制反应来评估TolDC功能的方法。
