Core Director, Assistant Professor University of Delaware Newark, Delaware, United States
Disclosure(s):
Arit Ghosh, PhD: No financial relationships to disclose
Introduction/Rationale: 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.
