Elucidating the role of APCs in immune tolerance during direct lymph node immunotherapy

项目摘要 多发性硬化症(MS)是一种衰弱的自身免疫性疾病，当免疫系统错误地识别 髓磷脂是外来的。髓磷脂是一种保护性基质，可以隔离中枢神经系统(CNS)中的神经元。当前的治疗方法 需要在患者的一生中持续治疗，不解决疾病的根本原因，并导致易感性 与感染有关。虽然最近的进展提高了治疗的靶向性，但即使是最新的单抗也无法 区分引起炎症的疾病细胞和健康的免疫细胞。在MS中，髓鞘反应性T细胞是 由淋巴结(LN)中的抗原提呈细胞(APC)激活，LN是协调免疫细胞相互作用的部位。APCS 树突状细胞(DC)、巨噬细胞和B细胞向T细胞递呈抗原、共刺激分子和极化细胞因子 和B细胞。在T细胞-APC相互作用期间存在的信号将T细胞极化为调节性的(例如，Treg)或 炎症(如TH1和TH17)细胞功能。在多发性硬化期间，髓鞘反应性免疫细胞迁移到中枢神经系统，在那里它们 分泌炎性细胞因子促进炎性细胞募集。我们的实验室显示了单个Intra LN(ILN) 微粒子(MP)治疗可永久逆转MS小鼠模型的瘫痪。这些微粒子包裹着一种髓鞘 抗原(髓鞘少突胶质细胞糖蛋白，MOG)和免疫调节分子，RAPA。这些国会议员的目的是 要太大，不能从LN中排出，而是作为仓库，随着MPS的降解而缓慢释放货物。尽管如此 尽管APC具有强大而持久的疗效，但其对这种治疗效果的贡献仍不清楚。该系统提供了一种 了解APC如何调节抗原特异性免疫反应的独特机会。APC提供关键信号 T细胞活化、增殖和分化所必需的，因此了解APC的调节是很重要的 信号。在获奖期间，我将测试ILN传递MOG/RAPA MPS驱动耐受性APC的假设 表型和功能。此外，我将测量APC的变化如何影响下游T细胞-APC的相互作用 并促进髓鞘特异性T细胞的调节功能。为了支持这一观点，我的初步研究显示 经MOG/Rapa MPS处理的小鼠的DC的活化水平低于用Empty处理的DC 国会议员。引人注目的是，经MOG/RAPA MPS处理的小鼠DC导致髓鞘特异性T细胞增殖水平降低 用流式细胞仪检测。这些结果表明，MOG/RAPA MP治疗改变了DC的功能和 这些细胞随后与髓鞘特异性T细胞的相互作用。受这些初步研究的启发，我将使用鼠标 MS模型实现两个目标：1)评估APC表型的变化以及MP和APC的组织定位 2)确定MPS对APC的功能变化及其下游对T细胞发育的影响。这些目标将 了解抗原前体细胞在建立抗原特异性耐受中所起的作用。除了我提出的 研究，我设计了一个全面的培训计划，帮助我朝着领导独立研究的目标前进 实验室。为F31奖励期计划的修订工作将在研究和培训机会方面都很有价值。

Project Summary Multiple Sclerosis (MS) is a debilitating autoimmune disease that occurs when the immune system incorrectly recognizes myelin as foreign. Myelin is a protective matrix that insulates neurons in the central nervous system (CNS). Current therapies require continual treatment over the patient’s life, do not address the underlying cause of disease, and causes susceptibility to infection. While recent advances have improved therapeutic targeting, even the newest monoclonal antibodies are unable to differentiate between disease causing inflammatory cells and healthy immune cells. In MS, myelin reactive T cells are activated by antigen presenting cells (APCs) in lymph nodes (LNs) – sites that coordinate immune cell interactions. APCs (dendritic cells (DCs), macrophages, and B cells) present antigens, costimulatory molecules, and polarizing cytokines to T and B cells. The signals present during T cell-APC interactions polarize T cells to either regulatory (e.g. TREG) or inflammatory (e.g. TH1 and TH17) cell functions. During MS, myelin reactive immune cells migrate to the CNS where they secrete inflammatory cytokines promoting inflammatory cell recruitment. Our lab has shown a single intra LN (iLN) microparticle (MP) treatment permanently reverses paralysis in a mouse model of MS. These MPs encapsulate a myelin antigen (myelin oligodendrocyte glycoprotein, MOG), and immune regulatory molecule, rapa. These MPs are designed to be too large to drain from the LN, instead serving as depots that slowly release the cargo as the MPs degrade. Despite this potent and durable efficacy, the contribution of APCs to this therapeutic impact remain unknown. This system provides a unique opportunity to understand how APCs regulate antigen specific immune responses. APCs provide critical signals required for T cell activation, proliferation, and differentiation, thus it is important to understand the regulation of APC signals. During the award period, I will test the hypothesis that iLN delivery of MOG/rapa MPs drives tolerogenic APC phenotype and function. In addition, I will measure how the changes in APCs impact downstream T cell-APC interactions and promote regulatory functions among myelin-specific T cells. In support of this idea, my preliminary studies revealed mice treated with MOG/rapa MPs had DCs that displayed lower levels of activation than DCs from mice treated with empty MPs. Strikingly, DCs from mice treated with MOG/rapa MPs caused lower levels of myelin specific T cell proliferation as measured by flow cytometry. These results indicate that MOG/rapa MP treatment alters the function of DCs and the subsequent interactions of these cells with myelin-specific T cells. Motivated by these pilot studies, I will use a mouse model of MS to carry out two aims: 1) Assess alterations in APC phenotype as well as MP and APC tissue localization and 2) Determine functional changes MPs exert on APCs and the downstream impact on T cell development. These aims will provide understanding about the role APCs play in establishing antigen-specific tolerance. In addition to my proposed research, I designed a holistic training plan that will help me advance toward my goal of leading an independent research lab. The revised work planned for the F31 award period will be valuable in terms of both research and training opportunities.