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. Therapeutic monoclonal antibodies (mAbs) are used in chronic lymphocytic leukemia immunotherapies to target the receptor CD20 at the plasma membrane of immunological B cells. Although they have been in clinical use for several years, the molecular binding mechanisms of mAbs to endogenous CD20 are unclear, as is how antibody binding activates the immune system to kill B cells. Ghosh et al. developed a method for fast volumetric fluorescence imaging with high spatiotemporal resolution that reveals the accumulation of CD20/mAb complexes on the microvilli of polarized B cells. The results show that the classification criterion of anti-CD20 mAbs based on their receptor cross-linking efficiency needs revision. Such high-end imaging techniques should help in the development of improved immunotherapies. ?Stella M. Hurtley
%0 Journal Article
%1 ghoshdecoding
%A Ghosh, Arindam
%A Meub, Mara
%A Helmerich, Dominic A.
%A Weingart, Julia
%A Eiring, Patrick
%A Nerreter, Thomas
%A Kortüm, K. Martin
%A Doose, Sören
%A Sauer, Markus
%B Science
%D 2025
%I American Association for the Advancement of Science
%J Science
%K doose home sauer
%N 6730
%P eadq4510--
%R 10.1126/science.adq4510
%T Decoding the molecular interplay of CD20 and therapeutic antibodies with fast volumetric nanoscopy
%U https://doi.org/10.1126/science.adq4510
%V 387
%X 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. Therapeutic monoclonal antibodies (mAbs) are used in chronic lymphocytic leukemia immunotherapies to target the receptor CD20 at the plasma membrane of immunological B cells. Although they have been in clinical use for several years, the molecular binding mechanisms of mAbs to endogenous CD20 are unclear, as is how antibody binding activates the immune system to kill B cells. Ghosh et al. developed a method for fast volumetric fluorescence imaging with high spatiotemporal resolution that reveals the accumulation of CD20/mAb complexes on the microvilli of polarized B cells. The results show that the classification criterion of anti-CD20 mAbs based on their receptor cross-linking efficiency needs revision. Such high-end imaging techniques should help in the development of improved immunotherapies. ?Stella M. Hurtley
@article{ghoshdecoding,
abstract = {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. Therapeutic monoclonal antibodies (mAbs) are used in chronic lymphocytic leukemia immunotherapies to target the receptor CD20 at the plasma membrane of immunological B cells. Although they have been in clinical use for several years, the molecular binding mechanisms of mAbs to endogenous CD20 are unclear, as is how antibody binding activates the immune system to kill B cells. Ghosh et al. developed a method for fast volumetric fluorescence imaging with high spatiotemporal resolution that reveals the accumulation of CD20/mAb complexes on the microvilli of polarized B cells. The results show that the classification criterion of anti-CD20 mAbs based on their receptor cross-linking efficiency needs revision. Such high-end imaging techniques should help in the development of improved immunotherapies. ?Stella M. Hurtley},
added-at = {2025-02-04T12:44:14.000+0100},
author = {Ghosh, Arindam and Meub, Mara and Helmerich, Dominic A. and Weingart, Julia and Eiring, Patrick and Nerreter, Thomas and Kortüm, K. Martin and Doose, Sören and Sauer, Markus},
biburl = {https://www.bibsonomy.org/bibtex/28bed8135870015c190c1a4377a125117/reichert},
booktitle = {Science},
comment = {doi: 10.1126/science.adq4510},
doi = {10.1126/science.adq4510},
interhash = {35a37a74fe6cedd99d48746088db5ad0},
intrahash = {8bed8135870015c190c1a4377a125117},
journal = {Science},
keywords = {doose home sauer},
number = 6730,
pages = {eadq4510--},
publisher = {American Association for the Advancement of Science},
timestamp = {2025-02-04T14:06:47.000+0100},
title = {Decoding the molecular interplay of CD20 and therapeutic antibodies with fast volumetric nanoscopy},
url = {https://doi.org/10.1126/science.adq4510},
volume = 387,
year = 2025
}