Translational control shapes the suppressive output of tumour-infiltrating regulatory T cells by coupling metabolism to protein synthesis

Regulatory T cells (Tregs) are central mediators of immune evasion in cancer, but the mechanisms that shape their functional diversity within tumours remain poorly understood. While transcriptional heterogeneity among tumour-infiltrating Tregs has been described, the contribution of post-transcriptional mechanisms, including mRNA translation, to their functional identity remains largely unexplored. In this study, we introduce CITePuro-seq, a novel method that links single-cell transcriptomes with active protein synthesis rates, enabling simultaneous measurement of the transcriptional and mRNA translational state in individual cells. Using this approach together with ex vivo puromycin incorporation assays, spatial proteomics and functional cytokine assays, we characterized Tregs isolated from pleural mesothelioma (PM) and non-small cell lung cancer (NSCLC), two malignancies with distinct responses to immunotherapy. Mitochondrial density, cytokine secretion profiles and transcriptional dynamics were examined in relation to mRNA translational activity in order to dissect the metabolic and functional determinants of Treg heterogeneity. We found that Tregs from PM and NSCLC were transcriptionally distinct, yet equally suppressive. PM Tregs exhibited markedly higher translational activity compared to NSCLC Tregs. Mechanistically, mitochondrial density emerged as a key determinant of translational activation, linking respiratory capacity to protein synthesis and defining distinct metabolic niches within the tumour microenvironment. Higher translational activity directly determined cytokine secretion patterns, and in turn drove transcriptional reshaping associated with enhanced suppressive function. In particular, translationally active PM Tregs appeared to produce higher levels of cytokines, including IL-10, potentially establishing a self-reinforcing immunosuppressive network. Taken together, these findings identify protein synthesis as an important contributor to Treg heterogeneity in cancer. We suggest that mRNA translational activity is an important parameter in predicting immunotherapy outcomes, and position translational regulation as a potential therapeutic target in cancer immunology.