Functional immuno-imaging of lymphocyte motility and cell interactions in lymph n

描述(申请人提供)：T淋巴细胞必须从血液进入淋巴结，通过与树突状细胞直接接触识别特定抗原，增殖、分化，离开淋巴结，并作为记忆细胞迁移到其他淋巴结或作为效应器迁移到组织。这些过程涉及运动性、细胞识别动力学和钙信号--这些事件直到最近才被人发现。双光子(2P)显微镜现在可以实时显示淋巴器官深处的活细胞，揭示了基础条件下和免疫反应期间优雅的细胞编排。T淋巴细胞中的离子通道调节钙信号的触发、强度和持续时间，导致基因表达和细胞增殖的下游变化。特别是，钙离子通道(Orai1)和K+通道(Kv1.3)正被开发为治疗自身免疫性疾病的免疫抑制靶点。该项目的总体目标是研究如何优化初始T细胞的默认抗原搜索策略，耐受性B细胞和调节性T细胞(Tregs)如何干扰初始T细胞的激活，以及T细胞中的离子通道如何成为体内免疫抑制的靶点。研究将基于人体和小鼠免疫细胞的2P成像，在免疫反应的实验模型中。在目标1中，将开发在完整组织中长期追踪细胞的新技术，并将其应用于另外三个子目标：(I)探索T细胞是否被局部和动态地吸引到DC；(Ii)通过耐受诱导来表征B细胞传递的基因治疗模型中的细胞相互作用；以及(Iii)调节T细胞对初始T细胞激活的抑制。目的2研究Kv1.3和Orai1在体内的作用，包括三个子目标：(1)开发两种新的人细胞免疫显像剂。(Ii)在人类和小鼠模型中，在使用Kv1.3通道的特定阻滞剂治疗期间，对在自身免疫性疾病中引起炎症和组织损伤的效应T细胞进行成像。(Iii)通过特异性抑制钙通道活性，探讨Orai1通道在体内的功能作用。总的来说，拟议的实验探索了调节免疫反应的分子和细胞机制，并代表了改进炎症性和自身免疫性疾病治疗的潜在治疗目标。 公共卫生相关性：我们使用双光子显微镜的研究揭示了体内免疫细胞在免疫反应期间的动态。这个项目的第一个目标是了解B细胞传递的治疗如何改变淋巴结中的细胞相互作用以诱导免疫耐受，以及调节T细胞如何防止自身免疫。第二个目标是研究通过阻断T淋巴细胞上的特定离子通道-钾通道Kv1.3和钙通道Orai1-介导的免疫抑制，用于治疗自身免疫性疾病。

DESCRIPTION (provided by applicant): To mount an immune response, T lymphocytes must home from blood into lymph nodes, recognize specific antigens by direct contact with dendritic cells, proliferate, differentiate, exit the lymph node, and migrate to other lymph nodes as memory cells or to tissues as effectors. These processes involve motility, cell recognition dynamics, and Ca2+ signaling - events that were hidden from view until fairly recently. Two-photon (2P) microscopy now permits real-time visualization of living cells deep within lymphoid organs, revealing an elegant cellular choreography under basal conditions and during an immune response. Ion channels in T lymphocytes regulate the triggering, intensity and duration of Ca2+ signaling leading to downstream changes in gene expression and cell proliferation. In particular, a Ca2+ channel (Orai1) and a K+ channel (Kv1.3) are being developed as immunosuppressive targets for treatment of autoimmune disorders. The overall goals of this project are to investigate how the 'default' antigen search strategy of naive T cells is optimized, how tolerogenic B cells and regulatory T cells (Tregs) interfere with naive T cell activation, and how ion channels in T cells can be targeted for in vivo immunosuppression. Studies will be based on 2P imaging of human and murine immune cells in experimental models of the immune response. In Aim 1, new techniques for long-term cell tracking in intact tissues will be developed, and applied in 3 further subaims: (i) To explore whether T cells are attracted locally and dynamically toward DCs; (ii) Characterization of cellular interactions in a model of B cell-delivered gene therapy by tolerance induction, and (iii) Regulatory T cell suppression of naive T cell activation. Aim 2 focuses on the in vivo action of Kv1.3 and Orai1, with three subaims: (i) Development of two novel preparations for human cell immunoimaging. (ii) Imaging of effector T cells that cause inflammation and tissue damage in autoimmune disorders during treatment with a specific blocker of Kv1.3 channels in both human and murine models. (iii) Exploration of the functional roles of the Orai1 channel in vivo by specific inhibition of Ca2+ channel activity. Collectively, the proposed experiments probe the molecular and cellular mechanisms that modulate immune responses, and which represent potential therapeutic targets for improved treatment of inflammatory and autoimmune diseases. PUBLIC HEALTH RELEVANCE: Our studies using two-photon microscopy reveal the dynamics of immune cells in vivo during an immune response. The first goal in this project is to understand how cellular interactions in the lymph node are modified by B cell-delivered therapy to induce immunological tolerance and by regulatory T cells that prevent autoimmunity. The second goal is to investigate immunosuppression mediated by blockade of specific ion channels in T lymphocytes - a potassium channel Kv1.3 and a calcium channel Orai1 - for treatment of autoimmune disorders.