Stem-like exhausted CD8 T cells in pleural effusions predict improved survival in non-small cell lung cancer (NSCLC) and mesothelioma

Anti-tumor CD8 T cells are important for immunity but can become 'exhausted' and hence ineffective. Tumor-infiltrating exhausted CD8 T cells include less differentiated stem-like exhausted T (Tex ) cells and terminally exhausted T (Tex ) cells. Both subsets have been proposed as prognostic biomarkers in cancer patients. In this study, we retrospectively investigated their prognostic significance in patients with metastatic non-small cell lung cancer (NSCLC) and validated our findings in a mesothelioma cohort. Pre-treatment malignant pleural effusions (PEs) from 43 NSCLC (41 non-squamous, 2 squamous) patients were analyzed by flow cytometry. The percentages of Tex and Tex CD8 T cells were correlated with overall survival (OS) after adjusting for clinicopathological variables. Findings were validated using a mesothelioma cohort (n=49). Mass cytometry was performed on 16 pre-treatment PE samples from 5 mesothelioma and 3 NSCLC patients for T-cell phenotyping. Single-cell multi-omics analysis was performed on 4 pre-treatment PE samples from 2 NSCLC patients and 2 mesothelioma patients for analysis of the transcriptomic profiles, surface markers and T cell receptor (TCR) repertoire. Higher frequency of Tex was associated with significantly increased OS [median 9.9 3.4 months, hazard ratio (HR) 0.36, 95% CI: 0.16-0.79, P=0.01]. The frequency of Tex was not associated with OS. These findings were validated in the mesothelioma cohort (high low Tex , median OS 32.1 19.8 months, HR 0.31, 95% CI: 0.10-0.96, P=0.04). Detailed single-cell sequencing and mass cytometry profiling revealed that exhausted T cells from NSCLC expressed greater stem-likeness and less inhibitory markers than those from mesothelioma and that Tex cells also contained 'bystander' virus-specific T cells. This study demonstrates that PE CD8 Tex cell abundance is associated with better survival outcomes, and thus may be a useful prognostic biomarker.