Detection and characterisation of lung-resident memory T cells in human and non-human primates

Abstract Rationale: Lung-resident memory T cells (TRM) play a critical role in protecting against respiratory infection. Maintained in a quiescent state, TRM are poised for rapid effector function upon reactivation with cognate antigen. Kronoscan is a clinic-ready, fibre-based, optical imaging system developed by the University of Edinburgh, capable of measuring both fluorescence intensity and lifetime. The aim of this study is to develop a staining protocol to enable the detection of human and non-human primate (NHP) pulmonary TRM populations in situ. Methods: Human lungs (deemed non-suitable for transplantation) following ex vivo lung perfusion (EVLP) and NHP lungs (cynomolgus macaque obtained from Charles River Laboratories) were enzymatically digested to achieve a single cell suspension. Cells were stained with cross-reactive antibodies prior to flow cytometric analysis. Fluorescence intensity and lifetime imaging was also performed using Kronoscan on MACs-enriched, antibody-stained, pulmonary T cells. Results: Human and NHP TRM were primarily identified using CD69 and CD103 and co-expressed a number of chemokine receptors (CXCR3, CCR10), adhesion molecules (CD49a, CD49d) and the immune checkpoint inhibitor PD-1. Differences in phenotype were observed between CD4+ and CD8+ TRM. TRM could be identified via fluorescence intensity and lifetime imaging using Kronoscan. Conclusions: A cross-reactive antibody panel capable of detecting human and NHP pulmonary TRM was successfully developed. Fibre-based fluorescence lifetime imaging may provide a valuable tool for assessing pulmonary TRM populations in situ, reflecting the level of immunity following infection and vaccination.