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

描述（由申请人提供）：为了产生免疫反应，T淋巴细胞必须从血液中返回淋巴结，通过与树突状细胞直接接触识别特定抗原，增殖，分化，退出淋巴结，并作为记忆细胞或作为效应器迁移到其他淋巴结。这些过程包括运动、细胞识别动力学和Ca2+信号——直到最近才被发现。双光子（2P）显微镜现在可以实时可视化淋巴器官深处的活细胞，揭示基础条件下和免疫反应期间优雅的细胞舞蹈。T淋巴细胞中的离子通道调节Ca2+信号的触发、强度和持续时间，导致下游基因表达和细胞增殖的变化。特别是，Ca2+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，以及如何靶向T细胞中的离子通道进行体内免疫抑制。研究将基于免疫反应实验模型中人类和小鼠免疫细胞的2P成像。在目标1中，将开发完整组织中长期细胞跟踪的新技术，并将其应用于3个进一步的子目标：(i)探索T细胞是否被局部和动态地吸引到dc；（ii）通过耐受性诱导的B细胞传递基因治疗模型中细胞相互作用的表征，以及（iii）调节性T细胞抑制初始T细胞激活。Aim 2主要研究Kv1.3和Orai1在体内的作用，包括三个子目标：(i)开发两种用于人类细胞免疫成像的新型制剂。（ii）在人类和小鼠模型中，在使用Kv1.3通道的特异性阻滞剂治疗期间，在自身免疫性疾病中引起炎症和组织损伤的效应T细胞成像。（iii）通过特异性抑制Ca2+通道活性来探索Orai1通道在体内的功能作用。总的来说，提出的实验探讨了调节免疫反应的分子和细胞机制，并代表了改善炎症和自身免疫性疾病治疗的潜在治疗靶点。

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.