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

Abstract

Introduction: Pulmonary-resident memory T cells (T_RM) and B cells (B_RM) 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 T_RM/B_RM 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 T_RM/B_RM 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.

Keywords: fluorescence lifetime imaging; lung; optical endomicroscopy; resident memory B cells; resident memory T cells.

Figure 1

Characterisation of Human and NHP Pulmonary T_RM. T_RM from BAL and/or tissue digests were compared with peripheral T cells. (A) Identification of human pulmonary CD4⁺ and CD8⁺ T_RM. T_RM (live, CD3⁺, CD4⁺/CD8⁺, CD62-L^-) were identified based on their expression of CD69 and CD103. (B) Identification of NHP CD4⁺ and CD8⁺ T_RM. (C) Proportion of CD69⁺ and CD69⁺CD103⁺ T_RM in human and NHP lung. (D) Ki-67 staining in human CD4⁺ and CD8⁺ T_RM. (E) Ki-67 staining in NHP CD4⁺ and CD8⁺ T_RM. Human and NHP samples were concatenated for analysis. Data represented as representative flow cytometry plots or compiled frequencies with mean ± SEM (C) from human lungs (n=15), BAL (n=9) and NHP lung (n=5).

Figure 2

Characterisation of Human and NHP Pulmonary B_RM. B_RM were isolated from BAL and/or tissue digests and compared with peripheral B cells. (A) Identification of human pulmonary B_RM. B_RM (live, CD62L^-, B220⁺, CD20⁺ (lower panels)) were identified based on their expression of CD27 and CD69. (B) Identification of NHP pulmonary B_RM. (C) Proportion of B_RM in human and NHP lung. (D) Human and NHP B_RM surface marker expression. Human and NHP samples were concatenated for analysis. Surface marker expression was compared to B cells isolated from peripheral blood (PBMC). Data represented as representative flow cytometry plots, compiled frequencies with mean ± SEM or histograms from human lungs (n=9), BAL (n=7) and NHP lung (n=5).

Figure 3

Human and NHP Immunofluorescence T_RM/B_RM Detection. Frozen lung sections or BAL cells were stained using tyramide signal amplification. (A) Human and NHP T_RM detection in lung. CD4⁺ and CD8⁺ T_RM were detected based on their expression of CD69 and CD103. (B) Human and NHP B_RM detection in lung. B_RM were detected based on their expression of CD20, CD27 and CD69. Images taken at x400 magnification. Green arrows represent CD4⁺ T_RM and red arrows represent CD8⁺ T_RM (A), and white arrows represent B_RM (B).

Figure 4

In Vitro Human T_RM and B_RM Detection using KronoScan and Panoptes. (A) Cells from human lung tissue digests were stained with T_RM or B_RM antibodies and imaged in vitro using KronoScan imaging system and Panoptes multifunctional fibre. (B) Human T_RM detection. T_RM were detected via expression of CD69 and CD103. Blue arrow indicates CD69⁺ cells and yellow arrow points CD69⁺CD103⁺ T_RM. (C) Human B_RM detection. Blue arrow indicates CD69⁺ cells and yellow arrow points CD69⁺ CD20⁺ B_RM. Representative images from n=3 experiments.

Figure 5

In Situ Detection of Pre-labelled T_RM/B_RM in Human Lung. (A) Pre-labelled human pulmonary T_RM and B_RM in suspension were instilled into the alveolar space and detected using KronoScan and Panoptes during human EVLV. (B) Baseline lung imaging prior to pre-labelled cell instillation. (C) In situ imaging of pre-labelled CD69⁺ cells. Lung tissue digest cells were stained with fluorescent CD69 and CD103 antibodies prior to instillation into the alveolar space using Panoptes. Cells were imaged with KronoScan. Blue arrow indicates CD69⁺ cells. (D) In situ imaging of pre-labelled CD69⁺CD103⁺ T_RM. Yellow arrow indicates CD69⁺CD103⁺ T_RM. (E) In situ imaging of pre-labelled B_RM. Lung tissue digest cells were stained with fluorescent CD20 and CD69 antibodies prior to instillation to the alveolar space. Blue arrow indicates CD69⁺ cells and yellow arrow points CD69⁺CD20⁺ B_RM. Representative images from n=3 experiments.

Figure 6

In Situ Detection of T_RM/B_RM in Human Lung following Antibody Delivery. (A) T_RM or B_RM antibodies were instilled into the alveolar space via Panoptes fibre and imaged with KronoScan. (B) Baseline lung imaging prior to antibody delivery. (C) In situ detection of CD69⁺ cells. CD69 and CD103 antibodies were instilled into the alveolar space to detect T_RM. (D) In situ detection of CD69⁺CD103⁺ T_RM. (E) In situ detection of B_RM. CD20 and CD69 antibodies were instilled into the alveolar space to detect B_RM. Blue arrow indicates CD69⁺ cells and yellow arrow points CD69⁺CD20⁺ B_RM. Representative images from n=3 experiments.