Differential Protein Synthesis Rates in Tregs, conventional T cells, and ex-Tregs following LPS Stimulation 2332109

Introduction Protein synthesis is a metabolically demanding process that differs among T cell subsets based on their functional states and suppressive capacity. While regulatory T cells (Tregs) are known to exhibit distinct metabolic profiles compared to conventional T cells (Tconv), the translational dynamics of ex-Tregs cells that have lost the classical Treg phenotype remain poorly characterized. Methods We employed a 10-color flow cytometry panel with puromycin incorporation to measure protein synthesis in human CD4+ T cell subsets during LPS-induced inflammation. Whole (human) blood was stimulated with LPS (TLR4 agonist, 24h) ± cycloheximide (CHX, translation inhibitor) and pulsed with puromycin to label nascent proteins. Results Tregs (CD3+CD4+CD25highCD127low), Tconv (CD3+CD4+CD25-CD127+), and ex-Tregs (CD3+CD4+CD25-CD127high). Tconv and ex-Tregs exhibited significantly higher puromycin incorporation (MFI: 1173±71 and 1209±68) compared to Tregs (557±52, p < 0.01). LPS modestly increased Tconv synthesis (1318±96 MFI) while Tregs remained quiescent. CHX reduced synthesis ∼60% across all subsets (p < 0.01), confirming translation-dependent incorporation. Memory phenotyping revealed Tconv and ex-Tregs were predominantly central memory (>85%), while Tregs showed heterogeneous distribution (25-45% naive). Treg frequencies remained stable (∼4-5% of CD4+) while ex-Tregs constituted ∼15%. Ex-Tregs exhibit translational profiles similar to Tconv compared to Tregs, which suggests functional reprogramming after CD25 loss. Conclusion Differential protein synthesis rates may reflect distinct metabolic requirements for suppressive versus effector functions during TLR4-mediated inflammation. These subset-specific translational differences may contribute to immune dysfunction in ribosomopathies such as Diamond-Blackfan anemia and Shwachman-Diamond syndrome. Single-cell RNA sequencing with ribosome profiling will be critical to define translational landscapes driving distinct T cell fates.