HIV antibodies and NK cell ADCC: nanometer-scale tracking of immune synapse dynamics.

摘要 结构研究已经证实，自然引发的抗体可以在很宽的范围内与 HIV 包膜 (Env) 结合。 由其 Fab 臂介导的表位和角度范围（即“免疫复合物几何结构”）。这如何影响 抗体依赖性细胞毒性（ADCC）活性，尤其是自然杀伤（NK）细胞的抗体依赖性细胞毒性（ADCC）活性尚不清楚。我们的 由于对抗体免疫复合物如何协调抗体受体了解甚少，知识受到限制 基于信号传导（特别是 Fc gamma RIIIa、FcRIIIa）。长期目标是获得更详细的信息 了解抗体如何招募 Fc 介导的细胞活性并开发新的策略来设计 可以在体内发挥最大效力的特定效应功能的抗体、药物和疫苗。的 该提案的目的是确定免疫复合物的几何形状如何影响 NK 细胞 ADCC 对抗 HIV NK 细胞免疫突触 (NKIS) 内的 IgG 受体时空动态。我们的中心假设 抗体几何结构将调节与免疫复合物相关的 Fc-RIIIa 相互作用和 ADCC 活性 几何。这项工作的基本原理是尖端显微镜观察将为我们提供新的见解 ADCC 功能对 HIV 抗体治疗设计产生直接影响，应用范围更广 的人类疾病。我们的中心假设将在三个具体目标上进行检验：1）确定免疫的方式 复杂的几何形状影响 ADCC 过程中 FcgRIIIa 相互作用； 2) 对Fc-RIIIa进行单分子追踪 ADCC 期间在 NKIS 内； 3) 确定 Fc-RIIIa 和信号激酶的纳米级定位 ADCC 期间的 NKIS。我们将利用 MINFLUX 纳米显微镜的创新技术来实现这些目标，这是一种 超分辨率荧光显微镜技术，在 2- 活细胞中单分子的三维以及亚毫秒追踪。仔细测量 体外 ADCC 活性和 Förster 共振能量转移 (FRET) 测量也将补充我们的 MINFLUX 观察并拓宽了我们结果的解释。这些研究意义重大，因为它们 将为抗体效应功能建立分子基础，特别是与 NK 细胞 ADCC 相关的功能，这可以 改善艾滋病毒的治疗方法。本提案中建立的技术也可用于询问 抗体 ADCC 对其他病毒病原体具有作用。我们研究的预期结果是表征 改变 NK 细胞 ADCC 活性的生物物理原理以及构成基础的分子力学 对于这样的活动。这些发现将为人类健康产生重要影响，为人类健康提供合理依据。 设计针对艾滋病毒以及其他病毒和疾病的改进抗体疗法，并将增加我们的 NK细胞ADCC的基本了解。

SUMMARY Structural studies have established that naturally elicited antibodies can bind to HIV envelope (Env) over a wide range of epitopes and angles mediated by their Fab arm (i.e. “immune complex geometry”). How this affects antibody dependent cellular cytotoxicity (ADCC) activity, especially by Natural Killer (NK) cells is unknown. Our knowledge is limited by a poor understanding of how antibody immune complexes orchestrate antibody receptor based signaling (specifically for Fc gamma RIIIa, FcRIIIa). The long-term goal is to acquire a more detailed understanding of how antibodies recruit Fc mediated cellular activity and to develop novel strategies to engineer antibodies, drugs, and vaccines that can recruit specific effector functions with maximal potency in vivo. The objective of this proposal is to determine how immune complex geometry impacts NK cell ADCC against HIV and IgG receptor spatiotemporal dynamics within the NK cell immune synapse (NKIS). Our central hypothesis is that antibody geometry will modulate FcRIIIa interaction and ADCC activity in relation to immune complex geometry. The rationale for this work is that cutting-edge microscopy observations will provide new insight into ADCC function with immediate impacts on HIV antibody therapeutic design with applications to a broader range of human diseases. Our central hypothesis will be tested in three specific aims: 1) Determine how immune complex geometry influences FcgRIIIa interaction during ADCC; 2) Perform single molecule tracking of FcRIIIa within the NKIS during ADCC; 3) Determine nanometer-scale localization of FcRIIIa and signaling kinases within the NKIS during ADCC. We will pursue these aims using the innovative technique of MINFLUX nanoscopy, a super-resolution fluorescence microscopy technique that is capable of 1- to 3-nm spatial resolutions in both 2- and 3-dimensions as well as sub-millisecond tracking of single molecules in live cells. Carefully measured in vitro ADCC activity and Förster resonance energy transfer (FRET) measurements will also complement our MINFLUX observations and broaden the interpretation of our results. These studies are significant because they will establish a molecular basis for antibody effector function, especially in relation to NK cell ADCC, that could improve therapeutics for HIV. The techniques established in this proposal will also be useful for interrogating antibody ADCC function for other viral pathogens. The expected outcome of our studies is the characterization of biophysical principles that alter NK cell ADCC activity as well as the molecular mechanics that form the basis for such activity. These findings will have an important impact on human health by offering a rational basis for designing improved antibody therapeutics for HIV, as well as other viruses and diseases, and will increase our basic understanding of NK cell ADCC.