Glycolysis maintains effector function of lung Th17 TRM cells in precision cut lung slices

Vaccination with Klebsiella pneumoniae OmpX and the Th17 adjuvant LTA1 elicits lung CD4⁺ tissue-resident memory (TRM) Th17 cells that provide serotype-independent protection against K. pneumoniae, but vaccine efficacy declines as TRM numbers wane. To define the mechanisms sustaining these cells, we developed an ovalbumin (OVA) model with tetramer enrichment to track antigen-specific CD4⁺ TRM cells over time. Serial single-cell RNA sequencing on days 30, 90, and 184 after LTA1/OVA immunization revealed persistent glycolytic signatures across all time points, while ornithine decarboxylase 1 (Odc1), although required for Th17 differentiation in vitro, was dispensable for TRM maintenance in vivo. Inhibition of glycolysis with 2-deoxy-D-glucose impaired IL-17A production both in vivo and in precision-cut lung slice (PCLS) cultures which preserve functional TRM cells ex vivo. These findings identify glycolysis as a key metabolic pathway sustaining lung CD4⁺ Th17 TRM effector function and highlight PCLS as an ex vivo model.