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 RNAseq data from human naïve CD4 cells cultured under either Th0 or Treg polarizing conditions for 72 hours (Ubaid 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 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 4 independent experiments, with 3-4 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 hours. CD25 and FOXP3 expression were analyzed by flow cytometry. Data represent mean ± SEM from 5 independent experiments, with 3-4 biological replicates per experiment. (F – H) Naïve murine CD4 cells were cultured with either scrambled or anti-PGAM ASOs for 72 hours under Treg polarizing conditions. RNA was isolated for RNAseq, followed by library size normalization and differential expression analysis. Shown are (F) PCA plot for scrambled and anti-PGAM ASOtreated cells, (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 hours. To assess Treg suppressive function, the polarized Tregs were cultured with naïve CD4 cells stimulated with CD3/CD28 stimulating antibodies for an additional 72 hours at the indicated ratios. Cell proliferation was measured by dilution of a cell proliferation dye. Data represent mean ± SEM from 4 biological replicates. (J) Analysis of a published scRNAseq dataset of tumor-infiltrating 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.

PGAM regulates Treg differentiation through control of de novo serine synthesis.

(A) Schematic diagram of the intersection of glycolysis and the de novo serine synthesis pathway via the PGAM substrate 3PG. Pharmacologic inhibitors are shown in red boxes. (B) Naïve murine CD4 cells were cultured under Treg polarizing conditions for 4 days and treated with vehicle or EGCG (20 μM) on day 1. Cells were then lysed and metabolites were extracted and analyzed via LC-MS. Data from 4 biological replicates. (C) The rate of glucose-derived serine synthesis from polarized murine Tregs or Th17 cells was measured by adding U13C-glucose to the culture media for 6 hours and quantifying the percentage of labeled/unlabeled serine via LC-MS. The mass isotopomer distribution (MID) of U13C-glucose-derived serine and percent contribution to the total serine pool is shown. Data represent mean ± SEM from 3 biological replicates. (D) Naïve murine CD4 cells were cultured under Treg polarizing conditions for 4 days. On day 1, the cells were treated vehicle or the indicated combinations of the PGAM inhibitor EGCG (10 μM) and the PHGDH inhibitor NCT-503 (10 μM). Treg polarization was assayed by flow cytometry. Data represent mean ± SEM from 5 independent experiments. (E) Naïve murine CD4 cells were polarized under Treg conditions for 4 days. On day 1 of polarization, cells were either sham-electroporated or supplemented with 3PG (1.5 mM) by electroporation and treated with vehicle or the PHGDH inhibitor NCT-503 (10 μM). Analysis was performed by flow cytometry. Data represent mean ± SEM from 5 independent experiments. (F) Naïve CD4 cells were cultured under Treg polarizing conditions with either scrambled or anti-PHGDH ASOs, and Treg generation was analyzed by flow cytometry. Data represent mean ± SEM from 4 independent experiments. (G) Naïve murine CD4 cells were cultured under Treg polarizing conditions for 4 days with either serine/glycine-free media or media containing 4 mM serine, followed by flow cytometric analysis. Data represent mean ± SEM from 4 independent experiments. *P < 0.05, **P < 0.01 by Mann-Whitney U test (F, G), Kruskal Wallis test with multiple hypothesis correction (D, E), or student’s t test with multiple hypothesis correction (C). Each independent experiment shown in D – G included 3-4 biological replicates. Panel A was created using BioRender.com.

Serine synthesis and dietary availability regulate Tregs in vivo and in disease states.

(A) C57BL/6 mice were fed either serine/glycine-free diet or control diet for 8 weeks, beginning immediately post-weaning. Peripheral Tregs from blood were quantified by flow cytometry as the percentage of CD45+ cells expressing FOXP3. Data represent mean ± SEM from 4-5 mice per group. (B – D) Mice were fed either serine/glycine-free diet or control diet for 7 days and then subjected to MOG35-55 EAE. Clinical scoring was performed by a blinded observer. Data represent mean ± SEM from 8 mice per group. Shown are (B) clinical scores over the course of the experiment, (C) day of neurologic symptom onset, and (D) peak clinical scores. (E – G) Publicly available RNAseq data from three different datasets was analyzed using SCFEA to model serine synthesis rates from mRNA levels of metabolic enzymes. (E) SCFEA from human naïve CD4 cells cultured under either Th0 or Treg polarizing conditions for 72 hours (Ubaid Ullah et al., 2018) predicts decreased serine synthesis in iTregs. (F) Predicted serine synthesis is increased in Tregs derived from cerebrospinal fluid of patients with treatment-naïve relapsing MS compared to controls (Schafflick et al., 2020). (G) Predicted serine synthesis is decreased in Tregs derived from prostate cancer versus control prostate (Camps et al., 2023). *P < 0.05, **P < 0.01, **** P<0.00001 by student’s t-test (A, E, F, G), Mann-Whitney U test (C, D), and two-way ANOVA with repeated measures (B).

Serine inhibits Treg polarization by contributing to one-carbon metabolism and methylation of Treg-associated genes.

(A) Schematic diagram of serine entry into one-carbon metabolism and the methyl donor cycle. Pharmacologic inhibitors are shown in red boxes. (B) Naïve murine CD4 cells were cultured under Treg polarizing conditions for 72 hours with either vehicle or SHIN1 (SHMT1/2 inhibitor, 1 μM) added at day 0 of culture. Treg polarization was assayed by flow cytometry. Data represent mean ± SEM from 4 independent experiments, with 3-4 biological replicates per experiment. (C) Naïve murine CD4 cells were cultured in serine/glycine-free media, media containing 4 mM serine, or media with serine plus SHIN1 (1 μM). Treg polarization was assayed by flow cytometry. Data represent mean ± SEM from 3-4 biological replicates per condition. (D) Naïve murine CD4 cells were cultured in methionine-free media supplemented with vehicle, 1 mM methionine, or 4 mM serine, and FOXP3 expression was assayed by flow cytometry. Data represent mean ± SEM from 3 biological replicates. (E) Naïve murine CD4 cells were cultured in serine/glycine-free media for 48 hours under Treg polarizing conditions and then for 12 hours with either unlabeled serine or U13C-serine. The mass isotopomer ratio of carbon-labeled methylcytosine (m+1) / unlabeled methylcytosine is shown. Data represent mean ± SEM from 3 biological replicates. (F) Naïve CD4 cells were cultured under Treg polarizing conditions with either serine/glycine-free media or 4 mm serine. Methylation at the FOXP3 TSDR was assayed by pyrosequencing. The composite methylation was calculated as the percent methylation for each of the 4 CpG sites measured in the assay normalized to the serine/glycine-free condition. Data derived from 3 biological replicates per condition. (G) Naïve CD4 cells were cultured under Treg polarizing conditions with either scrambled or anti-PGAM ASOs. Methylation at the FOXP3 TSDR was assayed by pyrosequencing. The composite methylation was calculated as the percent methylation at each of the 4 CpG sites measured in the assay normalized to scrambled control. Data derived from 3 biological replicates per condition. (H) Naïve murine CD4 cells were cultured in serine/glycine-free media, media containing 4 mM serine, or media with serine plus SHIN1 (1 μM). Bisulfitetreated DNA was sequenced using a Treg-specific next generation sequencing panel, and percent methylation of key sites was calculated. Data derived from 3 biological replicates per condition. (I) Naïve CD4 cells were cultured under Treg polarizing conditions with either vehicle or 3-Deazaadenosine (3DZA, AHCY inhibitor, 5 μM). Treg polarization was assayed by flow cytometry. Data represent mean ± SEM from 4 independent experiments, with 3-4 biological replicates per experiment. (J) Naïve CD4 cells were polarized to Tregs with either vehicle or 3DZA and then cultured with CD4 cells stimulated with CD3/CD28 to evaluate Treg suppressive function. CD4 cell proliferation was measured by flow cytometry. Data represent mean ± SEM from 3 biological replicates. (K) Naïve CD4 cells were cultured under Treg polarizing conditions with either vehicle or 3DZA, and 106 cells were then transferred into RAG -/- mouse recipients along with 107 activated CD4 cells. Mice were weighed at the specified times and weights are shown relative to the weight at the day of injection. Data represent mean ± SEM from 5 mice per group. *P < 0.05, **P < 0.01 by Mann-Whitney U test (B, F, G, I), one-way ANOVA with Tukey’s multiple comparisons testing (C, D), two-way ANOVA with Dunnett’s multiple comparison testing (H), student’s t-test of average final weight (K), or student’s t test (J). Panel A was created using BioRender.com.