Cancer immunotherapy has revolutionized oncology, yet only a fraction of patients respond. Our previous work revealed that T cells rapidly deploy translational machinery during activation within the tumor microenvironment, raising questions about how translational regulation affects T cell trajectories in tumors. Regulatory T cells (Tregs) - the master orchestrators of immune suppression - represent a critical barrier to effective anti-tumor immunity. Despite their crucial role, the translational landscape shaping Treg function within tumors remains largely unexplored.

We performed multifocal analysis of tumor infiltrating lymphocytes (TILs) translatomes in pleural mesothelioma (PM) and lung cancer (NSCLC). We developed CITePuro-seq technology to simultaneously capture, for the first time, single-cell translation and gene expression in human lymphocytes. Single-cell metabolic activity was analyzed using a microfluidic system with pH-sensitive fluorescence.

Our discovery reveals a striking translational divergence in TILs across tumor types: 1. CD4+ T cells exhibit dramatically higher translation rates in mesothelioma compared to lung cancer, with actively translating Tregs predominating in PM, 2. translation is not uniformly active in the tumor and 3) specific genes are controlled at the translational level. Transcriptomic analysis revealed significant heterogeneity in Treg populations between PM and NSCLC. Moreover PM-associated Tregs exhibited a distinct signature enriched for translational machinery. Patient-specific Treg profiles revealed two clusters with divergent signatures. Using CITePuro-seq, we associated single-cell RNA sequencing with direct measurement of translational activity. The superimposition of the the translational activity of CD4+ Tregs with their transcriptional profile leads to cluster highly suppressive cells and to define known and novel subtypes of Tregs by differential translation. Remarkably, our in vitro translational signature manifested in vivo in PM tumors, with tumor hypoxia emerging as a critical driver of Treg translational reprogramming and metabolic adaptation. Most surprisingly, Tregs with different translation levels exhibited functional versatility beyond immunosuppression, revealing an unexpected plasticity.

Our findings establish translation as the hidden regulator shaping T cell fate in the tumor microenvironment. The differences in translational potential across TILs reveal how tumor-specific factors reprogram cellular function independently of transcription. This demonstrates why purely transcription-based analyses can be misleading. Our work identifies translational control as a previously unrecognized checkpoint in TIL programming, suggesting a promising avenue for enhancing immunotherapy.