Supplementary MaterialsEMS86680_ReportingSummary. Tumour mutational burden (TMB) predicts immunotherapy outcome in non-small cell lung cancer (NSCLC), consistent with immune recognition of tumour neoantigens. However, persistent antigen exposure is detrimental for T cell function. How TMB affects CD4 and CD8 T cell differentiation in untreated tumours, and whether this affects patient outcomes is usually unknown. Here we paired high-dimensional flow cytometry, exome, single-cell and bulk RNA sequencing from patients with resected, untreated NSCLC to examine these associations. TMB was associated with compartment-wide T cell differentiation skewing, characterized by loss of TCF7-expressing BMS-345541 progenitor-like CD4 T cells, and an increased abundance of dysfunctional CD8 and CD4 T cell subsets, with significant phenotypic and transcriptional similarity to neoantigen-reactive CD8 T cells. A gene signature of redistribution from progenitor-like to dysfunctional says associated with poor survival in lung and other cancer cohorts. Single-cell characterization of these populations informs potential strategies for therapeutic manipulation in NSCLC. Tumour neoantigens are a key substrate for T cell-mediated recognition of cancer cells1. Neoantigen-specific T cells respond to immune checkpoint-blockade (ICB) and have been detected in the blood and tumours of patients with non-small cell lung (NSCLC)2,3 and other cancer types4. Although tumour mutational burden (TMB) predicts response to checkpoint blockade2,5,6, clinically evident tumours usually progress without therapy, suggesting functional impairment of anti-tumour T cell responses7,8. T cell activation is determined by antigen characteristics including abundance, physiochemical properties, MHC affinity and self-similarity9C11. In acute contamination and vaccination, optimal T cell stimulation results in differentiation from progenitor (e.g. naive, central memory) to effector and memory phenotypes, together with acquisition of diverse effector functions12. However, persistently high antigen load13C15 in cancer and chronic infections leads to continuous, or repetitive T cell receptor (TCR) stimulation, which induces transcriptional, epigenetic and metabolic changes that drive differentiation into dysfunctional says with progressively limited T cell effector functions16C18. Two broad says of functional impairment have been described in these settings. Firstly, T cell exhaustion (interchangeably referred to as dysfunction), which is characterized by expression of transcription factors such as TOX, high levels of co-inhibitory and co-stimulatory receptors, impaired cytokine production and replicative capacity19. Secondly, terminal differentiation which is characterized by a senescence phenotype including shortened telomeres signifying a history of cell division20, heightened sensitivity to apoptosis21, and expression of markers including CD57, KLRG1 and Eomes22,23. Whilst functional impairment is considered one endpoint of intratumour CD8 T cell differentiation, recent studies have highlighted the presence of progenitor-like CD8 T cells that respond to ICB and are characterized by expression of transcription factors TCF7 and LEF1 that regulate a gene expression programme conferring high proliferative capacity, self-renewal and the ability to repopulate more differentiated subsets following antigen re-exposure24C28. Less is known about Aviptadil Acetate dysfunctional and progenitor-like CD4 T cell says within the tumour microenvironment. In general, CD4 T cells play a central role in orchestrating adaptive immunity including initiation29 and maintenance of anti-pathogen CD8 responses30. In tumour models, optimal CD8 activity requires CD4 T cell help31 and human studies indicate a role for neoantigen specific CD4 responses in tumour control32,33. The role of antigen exposure on the relative balance and functional characteristics of tumour infiltrating CD4 and CD8 BMS-345541 subsets is usually unknown, and potentially relevant BMS-345541 to identify crucial targetable pathways restricting anti-tumour T cell function. To characterize how the T cell differentiation landscape in NSCLC is usually affected by TMB as a surrogate for antigenic weight, we BMS-345541 integrated high-dimensional flow cytometry, RNA and whole exome sequencing (WES) data from surgically resected, untreated, NSCLC specimens obtained from BMS-345541 patients in the Tracking Cancer Evolution through Therapy (TRACERx) 100 cohort34, along with bulk and single T cell RNA sequencing data from impartial cohorts. Diverse progenitor-like and dysfunctional CD4 and CD8 T cell populations identified by high-dimensional phenotyping of NSCLC TILs To characterize NSCLC tumour infiltrating lymphocytes (TILs) we performed 19 parameter flow cytometry on 41 tumour regions from 15 treatment-na?ve patients with stage IA-IIIA disease amongst the first 100 enrolled to the TRACERx study34. Thirteen patients had paired non-tumour adjacent (NTA) tissue (Extended Data Fig. 1A-B, Supplementary Table 1). Samples were selected on the basis of available paired WES and sufficient single-cell digest material. Clustering of viable CD3+ cells in tumour and NTA samples revealed 26 T cell subpopulations (Figures 1A-B). Visualization of the T cell differentiation scenery by UMAP35 dimension reduction revealed CD8 and.