Distinct Transcriptomic Signatures in Pulmonary Tissue Resident and Vascular “Resident-Like” T Cells in Nonhuman Primates

Abstract Intravascular staining (ivs) provides an innovative method for distinguishing tissue-resident lymphocytes from those within the organ’s vasculature, a distinction that is particularly important in highly vascularized organs such as the lung. Cells that stain positive for the infusion antibody (ivs+) have generally been considered lingering blood contaminants; however, recent studies have shown that these cells may not be as transitory as originally thought. Here, we utilized ivs staining to differentiate and isolate T cells from both the lung interstitium (ivs-) and lung vasculature (ivs+) of rhesus macaques (RhM). We then characterized and compared these T cell populations via single-cell RNA sequencing (scRNAseq) and flow cytometry. We found that ivs- T cells exhibit a core gene signature resembling that of tissue-resident memory T cells (Trms), whereas ivs+ T cells represent a distinct population with a unique phenotype and transcriptional profile. Notably, CD8+ T cells were enriched within the lung vasculature (ivs+ subset) and predominantly exhibited an effector phenotype, highlighted by increased cytotoxicity. Moreover, these pulmonary vascular “resident-like” CD8+ T cells (ivs+) demonstrated a gene expression profile enriched in transcripts typically associated with a tissue-resident phenotype, as well as cell adhesion markers and pathways involved in vascular/platelet interactions. In Simian Immunodeficiency Virus (SIV) infected RhMs, pulmonary ivs+ CD8+ T cells displayed enhanced effector responses compared to Trms. Our findings suggest that ivs+ T cells in the pulmonary vasculature represent a unique, “resident-like” population with potent effector functions, highlighting their potential significance in pulmonary immunity. Thawed PBMC, lung, and bronchial lymph node (BLN) cells were stained in 15 mL conical tubes with live/dead and surface cocktails with 2x the typical antibody concentration. Cells were washed in FACS buffer and resuspended in RPMI-1640 without phenol red supplemented with 10% FBS. Cells were sorted using fluorescence-activated sorting (FACS) to isolate CD3+ T cells from blood and BLN as well as CD3+ ivs- and CD3+ ivs+ cells from lung using the BD FACSAria (BD Bioscience) or MACSQuant Tyto (Miltenyi Biotec). CD3+ ivs- and CD3+ ivs+ cells from lung, as well as CD3+ T cells from blood and BLN, were processed for single-cell RNA sequencing (scRNAseq) following the 10x Genomics® Single Cell Protocols – Cell Preparation Guide for the “preparation of limited samples.”