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

摘要 结构研究已经证实，自然产生的抗体可以广泛地与艾滋病毒包膜(Env)结合。 由其Fab臂(即“免疫复合物几何构型”)介导的表位和角度的范围。这会产生怎样的影响 抗体依赖的细胞毒性(ADCC)活性，特别是自然杀伤(NK)细胞活性尚不清楚。我们的 对抗体免疫复合体如何协调抗体受体的了解很少，这限制了人们的认识 基于信令(专门用于FC伽马RIIIa、FCRIIIa)。长期目标是获得更详细的 了解抗体如何招募Fc介导的细胞活性并开发新的策略来进行工程 抗体、药物和疫苗可以在体内以最大效力招募特定的效应器功能。这个 这项建议的目的是确定免疫复合体几何构型如何影响NK细胞抗HIV NK细胞免疫突触(NKIS)内免疫球蛋白G受体的时空动力学。我们的中心假设 抗体几何构型将调节FcRIIIa相互作用和与免疫复合物相关的adcc活性 几何图形。这项工作的基本原理是，尖端的显微镜观察将提供对 ADCC功能对HIV抗体治疗设计有直接影响，应用范围更广 人类疾病。我们的中心假设将在三个具体目标中得到检验：1)确定如何免疫 复杂几何结构对FcgRIIa相互作用的影响2)FcRIIa的单分子示踪 3)确定FcRIIIa的纳米尺度定位和信号转导通路 在ADCC期间的NKIS。我们将使用MINFLUX纳米显微镜的创新技术来实现这些目标， 超分辨率荧光显微镜技术，能够在2到3纳米的空间分辨率 以及活细胞中单分子的3维和亚毫秒级跟踪。仔细测量在 体外ADCC活性和Förster共振能量转移(FRET)测量也将补充我们的 MINFLUX观察并拓宽了对我们结果的解释。这些研究具有重要意义，因为它们 将为抗体效应器功能，特别是与NK细胞ADCC有关的功能建立分子基础，这可能 改进艾滋病毒的治疗方法。这项提案中建立的技术也将对审讯有用 抗体ADCC对其他病毒病原体起作用。我们研究的预期结果是描述 改变NK细胞ADCC活性的生物物理原理以及形成基础的分子力学 为这样的活动。这些发现将对人类健康产生重要影响，为 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 对NK细胞ADCC有基本了解。

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.