Characterising endogenous CD8+ T cell responses in non-small cell lung cancer using single-cell and spatial transcriptomics

Despite recent improvements in detection and treatment, lung cancer remains the leading cause of global cancer-related mortality and overall response rates to current therapies are low. Previous studies have identified populations of pre-exhausted tumour specific CD8+ T cells that expand following PD-1 immune checkpoint blockade to reinvigorate the anti-tumour immune response in responding patients but have not identified alternative populations that could be targeted in non-responding patients. Inspired by these studies and our prior observation of PD-1- CD8+ T cells as a rare subset of tumour infiltrating lymphocyte, we hypothesised that the PD-1- compartment may contain non-exhausted tumour-specific CD8+ T cells specific for germline encoded tumour antigens. Using single-cell RNA & TCR sequencing of CD8+ tumour infiltrating lymphocytes from non-small cell lung cancer, enriching for PD-1- cells, we identify a novel population of PD-1- NK-like CD8+ T cells. Highly cytotoxic, non-exhausted, Trm-like, and clonally expanded, this population possesses characteristics indicative of tumourspecificity and enhanced tumour-lytic potential. We show that NK-like CD8+ T cells are detectable in independent non-small cell lung cancer cohorts by CITE-seq and multicolour flow cytometry and further demonstrate that the most expanded clone within our dataset is likely specific for a germline-encoded tumour-associated antigen. Additionally, we conduct the first detailed characterisation of spatial CD8+ T cell heterogeneity within non-small cell lung cancer using NanoString Technologies’ GeoMx® DSP. We demonstrate that true tumour infiltrating CD8+ T cells are phenotypically distinct and enriched in exhausted and actively cycling cells. Furthermore, we identify the integrin subunits a1, aE, aV, and b1 as potential mediators of tumour infiltration.