NKG2D blockade impairs cytotoxic CD8+ tissue resident memory T cell accumulation and attenuates severity of chronic lung allograft dysfunction after transplantation

Chronic lung allograft dysfunction (CLAD) significantly limits long-term survival following lung transplantation. To identify potential targets for CLAD prevention, T cells from explanted CLAD lungs and lung-draining lymph nodes, as well as diseased and non-diseased controls were isolated and single-cell RNA sequencing and TCR sequencing were performed. TCR sequencing revealed a clonally expanded population of tissue resident memory (TRM) CD8+ T cells with high cytotoxic potential including upregulation KLRK1, encoding the co-receptor NKG2D. These cytotoxic CD8+ TRM accumulated around the CLAD airways and had 100-fold increase in clonal overlap with lung draining lymph nodes when compared to non-CLAD lungs. Using a murine model of orthotopic lung transplant, we confirmed that cytotoxic CD8+ TRM accumulation was due to chronic rejection and not transplant alone. Furthermore, blocking NKG2D in-vivo attenuated the airway remodeling following transplantation and diminished airway accumulation of CD8+ T cells. Our findings support NKG2D as a potential therapeutic target for CLAD, affecting cytotoxic CD8+ TRM accumulation. Surgically explanted lung and hilar lymph node (HLN) from patients were obtained (lung-transplant recipients with chronic lung allograft dysfunction undergoing re-transplantation, healthy donor controls without chronic lung disease who were deemed not to be candidates for organ transplantation, and patients with idiopathic pulmonary fibrosis undergoing lung transplantation). Individual samples were tagged with unique hashtag oligonucleotides (HTOs, Biolegend). Recipient-derived T cells from each of these samples were isolated via FACS-sorting for live singlets that were CD45+Lineage (CD20, CD19, CD56, CD14)-CD4 and/or CD8+pan-human leukocyte antigen (HLA)+recipient-specific HLA+ as previously described. Sorted cells were captured for single-cell RNA and T cell receptor (TCR) sequencing using the 10x Genomics 5’ assay. Libraries were sequenced using the Illumina platform. Alignment, filtering, barcode counting, and unique molecular identifier counting were performed using 10x Genomics CellRanger v5 and CellRanger VDJ pipelines. Please note that processed data files generated from both HTO and GEX samples are linked to the corresponding GEX sample records.