# Glycolytic flux sustains human Th1 identity and effector function via STAT1 glycosylation

## TL;DR

Glycolysis helps human Th1 cells function properly by modifying a protein called STAT1, which is important for immune responses.

## Contribution

The study reveals that glycolytic flux sustains Th1 identity and function through STAT1 glycosylation, a novel mechanism in human immunity.

## Key findings

- Glycolytic activity is elevated in human Th1 cells compared to non-polarized cells.
- Glycolysis sustains STAT1 phosphorylation and IFNγ production independently of JAK1/2 or SHP2.
- O-glycosylation of STAT1 is essential for Th1 differentiation and effector function.

## Abstract

Glycolysis sustains STAT1 activity via STAT1 O-glycosylation, which is essential for human Th1 cell differentiation and IFNgamma production by maintaining STAT1 S727 phosphorylation.

T helper (Th) cell lineages are linked to metabolism, but precise mechanisms in human Th1 cells remain unclear. We addressed this question by in vitro stimulation and CRISPR/Cas9-mediated gene editing. Metabolic profiling revealed enhanced glycolytic activity in Th1 versus non-polarized cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, lactate secretion, NADH abundance, and elevated glycolysis-dependent anabolic activity. Inhibition of glycolysis reduced IFNγ production and STAT1 phosphorylation independent of JAK1/2 or SHP2 activity and STAT1 abundance, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. O-glycosylation of STAT1 via O-glycosyltransferase was pivotal in modulating STAT1 activity. Pharmaceutical O-glycosyltransferase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation sites at Ser499 and Thr510 diminished STAT1 Ser727 phosphorylation and IFNγ synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells.