T32 Research Fellow, PGY-6 University of Pittsburgh Medical Center Pittsburgh, Pennsylvania, United States
Disclosure(s):
Allyson Lieberman, MD, PhD: No financial relationships to disclose
Introduction/Rationale: Identifying tissue resident memory T cells (TRM) in human lungs has traditionally relied on imprecise surrogate markers of tissue retention, markers that can change based on activation state. Using ex vivo lung perfusion (EVLP), we can identify circulating and tissue resident T cells in a functional manner. Better knowledge of differential gene regulation in TRM will allow for improved analysis of their role in various disease states, as well as identify potential therapeutic targets.
Methods: Human lungs declined for organ donation were ventilated and attached to a normothermic EVLP system for 2-4 hours (2 experiments with 2 human lungs each). A biotinylated CD45 antibody was instilled into the perfusate to label circulating cells for the final 20 minutes; tissue-resident cells were identified as those cells “protected” from the circulating antibody. Immune cells were isolated and then stained with oligo-tagged streptavidin as well as oligo-tagged antibodies against several traditional TRM markers in order to perform simultaneous single cell RNA sequencing and CITE-seq.
Results: UMAP clustering of scRNA-seq data identified 13 discrete T cell clusters with differential proportions of labeled vs. protected cells. Clusters corresponding to canonical CD4+ and CD8+ TRM populations contained almost no labeled cells. Differential gene analysis of protected vs labeled CD8+ T cells show that functionally defined CD8+ TRM have a gene expression signature similar, but not identical, to canonical marker-defined TRM. Of particular interest is upregulation of galectin-3 (LGALS3), a member of the lectin family previously implicated in inflammatory and fibrotic disease.
Conclusion: EVLP allows for effective functional identification of tissue resident T cells in the human lung, which overlap with but are not identical to TRM defined by canonical surface markers. Future directions include kinetics of antibody labeling, validation of differentially regulated gene targets, and analysis of TCR sequencing data.
