Directing Function at the Natural Killer Cell Secretory Immunological Synapse

自然杀伤细胞分泌免疫突触的指导功能

• 批准号: 9243188
• 负责人: Jordan Scott Orange
• 金额: $ 39.26万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9243188
• 关键词: Acoustics; Actins; Activated Natural Killer Cell; Advanced Malignant Neoplasm; Area; Automobile Driving; Biological; Cancerous; Cell Polarity; Cell Therapy; Cells; Cellular biology; Clinical; Collaborations; Cytoplasmic Granules; Cytoskeleton; Dangerousness; Disease; Distal; Effectiveness; Environment; Enzymes; Event; F-Actin; Flow Cytometry; Gene Targeting; Health; Human; Image; Immune; Immunobiology; Immunologics; Infection; Intermediate Filaments; Kinetics; Laboratories; Lead; Lytic; Microscopy; Microtubule-Organizing Center; Microtubules; Modeling; Motor; Movement; Myosin ATPase; Natural Killer Cells; Pathway interactions; Pediatric Hospitals; Plant Roots; Positioning Attribute; Process; Proteins; Publishing; Regulation; Reporting; Research; Resolution; Role; SNAP receptor; Secretory Cell; Side; Signal Transduction; Synapses; Synaptic Membranes; System; Testing; Texas; Therapeutic; Tissues; Translating; Tumor Escape; Ultrasonography; Vimentin; Work; base; cancer immunotherapy; cell killing; cell motility; cytotoxic; cytotoxicity; dynamic system; experimental study; extracellular; immunological synapse; inhibitor/antagonist; innovation; insight; killings; nanoscale; novel; novel therapeutics; pathogen; programs; public health relevance; receptor; success

 DESCRIPTION (provided by applicant): Natural Killer (NK) immune cells are essential to human health, performing key roles in surveillance and killing of cancerous and infected cells. Activated NK cells harness and deliver lytic granule-contained, cytotoxic proteins through a specialized area of cell-cell contact (immunological synapse) onto the offending cell. Over the past decade, our laboratory has greatly advanced the understanding of how NK cells use the precisely regulated steps constituting directed secretion to achieve cytotoxic function and control. In this program renewal, we build on our past discoveries to ask critical questions at the root of NK cell biology. In Aim 1, we ask why multiple pathways to degranulation exist and whether these can be exploited to match specific clinical scenarios. In the previous program, we reported that lytic granules converge to the microtubule organizing center (MTOC) prior to secretion. We hypothesize that convergence focuses an NK cell's killing effect on a single diseased cell (minimizing collateral damage to healthy cells), whereas dispersion promotes wider destruction in environments full of targets. Here, we test these hypotheses using cutting-edge, high- and super- resolution microscopy in conjunction with the only immunologically-dedicated ultrasound guided acoustic trap microscopy (UGATm) cell manipulation system available in the U.S. We will determine whether receptor blocking and/or inhibitors can be used to "fine tune" convergence to match specific disease contexts like extracellular pathogen infection or advanced cancer. In Aim 2, we define a new cooperative motility mechanism for degranulation. In the previous program, we discovered that granules are dynamic even after delivery to the synapse, and that degranulation occurs through a nanoscale mesh of actin pores. Here, we define the dynamics and movement mechanisms of both synaptic granules and the actin meshwork using advanced super-resolution microscopy. We hypothesize that these two independent systems utilize dynamics to promote pore finding, efficient degranulation, and ultimately cytotoxicity. This dual-dynamic paradigm would be new to cell biology and would set the stage for therapeutic strategies to enhance NK cell killing by promoting granule access to the synapse. Finally, in Aim 3, we investigate a new role for Vimentin and the intermediate filament (IF) cytoskeleton in the establishment of synapse polarity. Vimentin IF, like synaptic actin, granules, and the MTOC, are dynamic, but intriguingly, they show reverse polarity, localizing to the opposite side of the NK cell. Here, using high-throughput imaging flow cytometry, super-resolution microscopy, inhibitors, and gene targeting, we will test the hypotheses that IF respond to activating signals, establish the distal NK cell pole, and are critical for synapse polarity. This will be the first demonstration of IF as a means to cell polariy. Based on our success in the previous program, we expect the proposed experiments to lead to key insights into directed secretion for NK cell cytotoxicity. We further expect the paradigms that emerge to have broad biological significance and direct applicability to immunological and cell-based therapies.

 描述（由申请人提供）：自然杀伤（NK）免疫细胞对人类健康至关重要，在监视和杀死癌细胞和感染细胞方面发挥关键作用。活化的NK细胞通过细胞-细胞接触的专门区域（免疫突触）将含有溶解颗粒的细胞毒性蛋白质驾驭并递送到攻击细胞上。在过去的十年中，我们的实验室大大推进了对NK细胞如何使用构成定向分泌的精确调节步骤来实现细胞毒性功能和控制的理解。在这个项目更新中，我们以过去的发现为基础，在 NK细胞生物学的基础。在目标1中，我们问为什么存在多种脱颗粒途径，以及这些途径是否可以用于匹配特定的临床场景。在以前的计划中，我们报道了溶解颗粒收敛到微管组织中心（MTOC）分泌前。我们假设，收敛集中在一个单一的病变细胞的NK细胞的杀伤作用（最大限度地减少对健康细胞的附带损害），而分散促进在充满目标的环境中更广泛的破坏。在这里，我们使用尖端的高分辨率和超分辨率显微镜结合美国唯一的免疫专用超声引导声阱显微镜（UGATm）细胞操作系统来测试这些假设。我们将确定受体阻断和/或抑制剂是否可以用于“微调”会聚，以匹配特定的疾病背景，如细胞外病原体感染或晚期癌症。在目标2中，我们定义了一个新的合作动力机制脱粒。在之前的项目中，我们发现颗粒即使在传递到突触后也是动态的，并且脱粒通过肌动蛋白孔的纳米级网格发生。在这里，我们定义的动态和运动机制的突触颗粒和肌动蛋白网络使用先进的超分辨率显微镜。我们假设，这两个独立的系统利用动力学，以促进孔的发现，有效的脱粒，并最终细胞毒性。这种双重动力学范式对细胞生物学来说是新的，并将为通过促进颗粒进入突触来增强NK细胞杀伤的治疗策略奠定基础。最后，在目标3中，我们研究了波形蛋白和中间丝（IF）细胞骨架在建立突触极性中的新作用。波形蛋白IF，像突触肌动蛋白，颗粒和MTOC一样，是动态的，但有趣的是，它们显示相反的极性，定位于NK细胞的相对侧。在这里，使用高通量成像流式细胞术，超分辨率显微镜，抑制剂和基因靶向，我们将测试的假设，IF响应激活信号，建立远端NK细胞极，是突触极性的关键。这将是IF作为细胞极化手段的第一次演示。基于我们在以前的项目中的成功，我们期望所提出的实验能够对NK细胞细胞毒性的定向分泌产生关键的见解。我们还期望， 出现具有广泛的生物学意义和直接适用于免疫和基于细胞的治疗。