1032 Early remodelling of CD4 T cell differentiation in lung squamous carcinogenesis

Background Advanced lung cancer kills 90% of patients within a decade, underscoring an urgent need for improved early detection and prevention technologies. T cell responses are initiated and dysregulated during early carcinogenesis in vivo, providing a potential basis for immune-focused early cancer detection and interception. Here we examined cross-tissue T cell dynamics during human and murine lung squamous carcinogenesis to assess their utility in lung cancer early detection and immune-interception. Methods Over a decade ago we initiated a clinical autofluorescence bronchoscopy surveillance program at University College London, to longitudinally monitor preinvasie pulmonary neoplasia in respiratory patients with a smoking history. We deeply profiled the immune and genomic landscape in rarely analysed, laser-captured, preinvasive bronchial biopsies and paired PBMCs from over 100 patients using either spectral cytometry, scRNAseq, TCRseq and/or WES. In parallel, we generated and immune-profiled an N-nitroso-tris-chloroethylurea (NTCU)-induced mouse model that replicated the histopathology and immune landscape of progressive human central airway neoplasia. Results Our scRNAseq data demonstrate an extensive remodelling of MHCII signalling networks during lung squamous (LUSC) carcinogenesis; patterned by loss of MHCII on basal airway epithelium, gain on B-cells and cDC2s and concomitant expansion of highly suppressive Basic Leucine Zipper ATF-Like Transcription Factor-expressing (BATF+) Treg cells (BATF+Tregs). BATF+Treg signatures significantly increased during LUSC carcinogenesis in independent bulk RNAseq from over 100 biopsies and defined immunotherapy failure and truncated survival in stage I LUSC. We recapitulated these data in the NTCU mouse model, wherein BATF+ Tregs gradually accumulated in preinvasive and invasive lesions and could be intercepted via PI3Kδ inhibition, which prevented lung tumours in 50% of mice. BATF+Tregs in the tissue shared TCRs with activated Tregs in the blood during early-stage lung cancer, prompting us to profile PBMCs from 107 patients via high-dimensional cytometry and deep TCRseq. Activated Treg frequencies and global TCR metrics were comparable between healthy individuals and those with low grade pulmonary neoplasia. However, we observed a dramatic increase in circulating activated Tregs and cancer-associated or total clonal TCR expansion in patients with high grade neoplasia (50% average risk of LUSC development) and stage Ia lung cancer. Preliminary multivariate regression models showed that liquid immune metrics outperformed clinical variables in classification of high vs low grade neoplasia and forecasted significantly increased risk of future LUSC diagnosis. Conclusions These data provide a roadmap for harnessing CD4 T cell differentiation as a basis for the early detection and precision immune-interception of lung cancer. Ethics Approval All studies used are ethically approved, the PI is Professor Sam Janes. Reference numbers for approval are 1. REC 01/0148, REC 20/SC/0128, REC 18/SC/051, All participants gave informed consent.