Abstract
Immunometabolism plays a pivotal role in T cell fate decisions, yet its specific contribution to human Th17 differentiation remains incompletely understood. Th17 cells, a subset of CD4(+) T cells, are central to autoimmune pathogenesis through their secretion of pro-inflammatory cytokines. Elucidating the metabolic drivers of Th17 differentiation may reveal novel therapeutic targets. We investigated the role of mitochondrial activity in Th17 differentiation using an in vitro model with naïve human CD4(+) T cells. Single-cell metabolic profiling and functional assays were used to characterise metabolic changes during differentiation. Th17 cells exhibited a hyperpolarised mitochondrial membrane potential (ΔΨ) compared to non-Th17 cells. Hyperpolarised ΔΨ cells displayed increased metabolic activity and enhanced differentiation capacity. Metabolic profiling at 48 h revealed an early reliance on glycolysis, followed by a shift toward increased dependence on oxidative phosphorylation (OXPHOS) by 96 h. Gene expression analysis indicated early upregulation of TEFM, a mitochondrial transcription regulator, at 48 h. By 96 h, ΔΨ hyperpolarised cells exhibited a downregulation of DRP1 and MFN2, genes responsible for mitochondrial fission and fusion. Functionally, ΔΨ hyperpolarised cells expressed elevated activation markers (CD69, CD25) but also showed increased exhaustion markers (TIGIT, PD-1), indicating a link between high metabolic activity and exhaustion. Additionally, these cells triggered weaker NF-κB and AP-1 signalling and secreted lower levels of effector molecules (IFN-γ, Granzyme B) than ΔΨ depolarised cells. In conclusion, mitochondrial activity critically shapes Th17 differentiation. Although hyperpolarised ΔΨ cells exhibit greater activation, they are more prone to exhaustion and reduced effector function. These findings offer insights into Th17 metabolic regulation and its therapeutic potential in autoimmune diseases.