Specific in situ immuno-imaging of pulmonary-resident memory lymphocytes in human lungs

Duncan C. Humphries, Richard A. O'Connor, Hazel L. Stewart, Tom M. Quinn, Erin E. Gaughan, Beth Mills, Gareth O.S. Williams, James M. Stone, Keith Finlayson, Martine Chabaud-Riou, Florence Boudet, Kevin Dhaliwal, Vincent Pavot

Research output: Contribution to journalArticlepeer-review

2 Citations (SciVal)


Introduction: Pulmonary-resident memory T cells (TRM) and B cells (BRM) orchestrate protective immunity to reinfection with respiratory pathogens. Developing methods for the in situ detection of these populations would benefit both research and clinical settings. Methods: To address this need, we developed a novel in situ immunolabelling approach combined with clinic-ready fibre-based optical endomicroscopy (OEM) to detect canonical markers of lymphocyte tissue residency in situ in human lungs undergoing ex vivo lung ventilation (EVLV). Results: Initially, cells from human lung digests (confirmed to contain TRM/BRM populations using flow cytometry) were stained with CD69 and CD103/CD20 fluorescent antibodies and imaged in vitro using KronoScan, demonstrating it's ability to detect antibody labelled cells. We next instilled these pre-labelled cells into human lungs undergoing EVLV and confirmed they could still be visualised using both fluorescence intensity and lifetime imaging against background lung architecture. Finally, we instilled fluorescent CD69 and CD103/CD20 antibodies directly into the lung and were able to detect TRM/BRM following in situ labelling within seconds of direct intra-alveolar delivery of microdoses of fluorescently labelled antibodies. Discussion: In situ, no wash, immunolabelling with intra-alveolar OEM imaging is a novel methodology with the potential to expand the experimental utility of EVLV and pre-clinical models.

Original languageEnglish
Article number1100161
Number of pages13
JournalFrontiers in Immunology
Publication statusPublished - 10 Feb 2023

Bibliographical note

Data availability statement
The original contributions presented in the study are included in
the article/Supplementary Material. Further inquiries can be directed
to the corresponding authors.

Authors DH, MC-R, FB and VP were employed by
company Sanofi.
The remaining authors declare that the research was conducted in
the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
The authors declare that this study received funding from Sanofi.
The funder had the following involvement in the study: study design,
data analysis, decision to publish, and preparation of the manuscript.


  • fluorescence lifetime imaging
  • lung
  • optical endomicroscopy
  • resident memory B cells
  • resident memory T cells

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology


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