Skip to main content
Dryad

Decoding the molecular interplay of endogenous CD20 and therapeutic antibodies with fast volumetric nanoscopy

Data files

Nov 04, 2024 version files 26.97 GB

Select up to 11 GB of files for download

Abstract

Elucidating the interaction between membrane proteins and antibodies requires fast whole-cell imaging at high spatiotemporal resolution. Lattice light-sheet (LLS) microscopy offers fast volumetric imaging but suffers from limited spatial resolution. DNA-PAINT achieves molecular resolution but is practically restricted to two-dimensional imaging due to long acquisition times. Here, we introduce two-dye imager (TDI) probes, manifesting negligible background and amplified fluorescence signal upon transient binding, enabling ~15-fold faster imaging. Using a combination of TDI-DNA-PAINT and LLS microscopy on B cells, we reveal the oligomeric states and interaction of endogenous CD20 with type I and II therapeutic monoclonal antibodies (mAbs) rituximab (RTX), ofatumumab (OFA), and obinutuzumab (OBZ), respectively, unperturbed by surface effects. Our results demonstrate that CD20 is abundantly expressed on microvilli that are concatenated by mAb binding accompanied by a concentration-dependent B cell polarization and stabilization of microvilli protrusions. These findings, we believe, will aid rational design of improved immunotherapies targeting tumor-associated antigens.