Fig 1: Phosphoglycerate mutase (PGAM) regulates Treg differentiation and suppressive function.. (A – C) Analysis of publicly available transcriptomics and proteomics data reveals upregulation of PGAM expression in human iTregs and ex vivo Tregs. (A) Publicly available RNA sequencing (RNA-seq) data from human naïve CD4 cells cultured under either Th0 or Treg polarizing conditions for 72 hr (Ullah et al., 2018) was analyzed, and genes belonging to the Gene Ontology (GO) term ‘Glycolysis and Gluconeogenesis’ were plotted. (B) Differential expression of ‘Glycolysis and Gluconeogensis’ GO genes in ex vivo Tregs vs. total CD4 cells derived from healthy donor peripheral blood mononuclear cells (PBMCs) (Schafflick et al., 2020). GAPDH was removed for scaling purposes. (C) Differential expression of cytosolic glycolytic enzymes in ex vivo Tregs vs. conventional CD4 T cells derived from healthy donor PBMCs, analyzed from publicly available proteomics data (Procaccini et al., 2016). (D) Naïve murine CD4 cells were cultured under Treg polarizing conditions for 4 days and treated with either EGCG (20 μM) or vehicle on day 1. Treg polarization based on CD25 and FOXP3 expression was analyzed by flow cytometry. Data represent mean ± SEM from four independent experiments, with three to four biological replicates per experiment. (E) Naïve murine CD4 cells were treated with either scrambled or Pgam-specific antisense oligonucleotides (ASOs) and cultured under Treg polarizing conditions for 72 hr. CD25 and FOXP3 expression were analyzed by flow cytometry. Data represent mean ± SEM from five independent experiments, with three to four biological replicates per experiment. (F – H) Naïve murine CD4 cells were cultured with either scrambled or anti-Pgam ASOs for 72 hr under Treg polarizing conditions. RNA was isolated from unsorted cells for RNA-seq, followed by library size normalization and differential expression analysis. Shown for scrambled versus anti-Pgam ASO-treated cells are (F) PCA plot, (G) Gene Set Enrichment Analysis (GSEA) using MSigDB Hallmark gene sets, and (H) volcano plot of genes associated with T cell function. Data from 3 biological replicates. (I) Naïve murine CD4 cells were cultured under Treg polarizing conditions with either scrambled or anti-Pgam ASOs for 72 hr. To assess Treg suppressive function, the polarized Tregs were cultured with naïve CD4 cells stimulated with CD3/CD28 stimulating antibodies for an additional 72 hr at the indicated ratios. Cell proliferation was measured by dilution of a cell proliferation dye. Data represent mean ± SEM from four biological replicates. (J) Analysis of a published scRNA-seq dataset of tumor-infiltrating Tregs (TIL-Tregs) (Dykema et al., 2023) shows that PGAM1 is overexpressed in the most highly suppressive subpopulation, out of proportion to proximal rate-limiting glycolytic enzymes. *p<0.05, **p<0.01, ***p<0.0001 by Mann-Whitney U Test (D, E) or two-way ANOVA with multiple comparisons testing (I). P-values in (A), (B), and (F – H) were derived from Wald’s test after False Discovery Rate correction and median-of-ratios normalization via DESeq2.