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

总结 结构研究已经确定，天然引发的抗体可以在宽的范围内与HIV包膜（Env）结合。 表位的范围和由其Fab臂介导的角度（即“免疫复合物几何形状”）。这如何影响 抗体依赖性细胞毒性（ADCC）活性，特别是自然杀伤（NK）细胞的活性是未知的。我们 由于对抗体免疫复合物如何协调抗体受体缺乏了解， 基于信号传导（特别是Fc γ RIIIa、Fc γ RIIIa）。长期目标是获得更详细的 了解抗体如何募集Fc介导的细胞活性，并开发新的策略来工程化 可以在体内以最大效力募集特异性效应子功能的抗体、药物和疫苗。的 本提案的目的是确定免疫复合物几何结构如何影响NK细胞抗HIV的ADCC 和NK细胞免疫突触（NKIS）内的IgG受体时空动力学。我们的核心假设 抗体几何结构将调节Fc γ RIIIa相互作用和与免疫复合物相关ADCC活性 几何这项工作的基本原理是，尖端的显微镜观察将提供新的见解， ADCC功能对HIV抗体治疗设计具有直接影响，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进行测试：1）确定免疫力如何 复杂的几何结构影响ADCC期间的FcgRIIIa相互作用; 2）进行FcgRIIIa的单分子追踪 3）确定在ADCC期间NKIS内Fc区RIIIa和信号传导激酶的纳米级定位; NKIS在ADCC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 - 3纳米的空间分辨率， 和三维以及亚毫秒级的活细胞中单个分子的跟踪。仔细衡量， 体外ADCC活性和Förster共振能量转移（FRET）测量也将补充我们的研究。 MINFLUX观测和扩大我们的结果的解释。这些研究意义重大，因为它们 将建立抗体效应子功能的分子基础，特别是与NK细胞ADCC相关， 改进HIV治疗方法。本建议中确立的技术也将有助于询问 抗体ADCC功能的其他病毒病原体。我们研究的预期结果是 改变NK细胞ADCC活性的生物物理学原理以及形成基础的分子力学 对于这样的活动。这些发现将对人类健康产生重要影响， 设计改进的艾滋病毒抗体疗法，以及其他病毒和疾病，并将增加我们的 对NK细胞ADCC的基本认识。

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.