Ghosh Arindam, Meub Mara, Helmerich Dominic A, Weingart Julia, Eiring Patrick, Nerreter Thomas, Kortüm K Martin, Doose Sören, Sauer Markus
Department of Biotechnology and Biophysics, Biocenter, University of Würzburg, Würzburg, Germany.
Department of Internal Medicine II, University Hospital Würzburg, Würzburg, Germany.
Science. 2025 Jan 10;387(6730):eadq4510. doi: 10.1126/science.adq4510.
Elucidating the interaction between membrane proteins and antibodies requires whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) achieves molecular resolution but is restricted to two-dimensional imaging owing to long acquisition times. We have developed two-dye imager (TDI) probes that enable ~15-fold faster imaging. Combining TDI-DNA-PAINT and LLS microscopy on immunological B cells revealed the oligomeric states and interaction of endogenous CD20 with the therapeutic monoclonal antibodies (mAbs) rituximab, ofatumumab, and obinutuzumab. Our results demonstrate that CD20 is abundantly expressed on microvilli that bind mAbs, which leads to an antibody concentration-dependent B cell polarization and stabilization of microvilli protrusions. These findings could aid rational design of improved immunotherapies targeting tumor-associated antigens.
阐明膜蛋白与抗体之间的相互作用需要高时空分辨率的全细胞成像。晶格光片（LLS）显微镜提供快速的体积成像，但空间分辨率有限。基于DNA的纳米级形貌成像点积累（DNA-PAINT）可实现分子分辨率，但由于采集时间长而仅限于二维成像。我们开发了双染料成像仪（TDI）探针，可实现快约15倍的成像。将TDI-DNA-PAINT与LLS显微镜结合用于免疫B细胞，揭示了内源性CD20与治疗性单克隆抗体（mAb）利妥昔单抗、奥法木单抗和奥妥珠单抗的寡聚状态及相互作用。我们的结果表明，CD20在结合mAb的微绒毛上大量表达，这导致抗体浓度依赖性的B细胞极化和微绒毛突起的稳定。这些发现有助于合理设计针对肿瘤相关抗原的改进免疫疗法。
