Distinct Regulation of Th17 and Th1 Cell Differentiation by Glutaminase-Dependent Metabolism

Abstract

Activated T cells differentiate into functional subsets with distinct metabolic programs. Glutaminase (GLS) converts glutamine to glutamate to support the tricarboxylic acid cycle and redox and epigenetic reactions. Here, we identify a key role for GLS in T cell activation and specification. Though GLS deficiency diminished initial T cell activation and proliferation and impaired differentiation of Th17 cells, loss of GLS also increased Tbet to promote differentiation and effector function of CD4 Th1 and CD8 CTL cells. This was associated with altered chromatin accessibility and gene expression, including decreased PIK3IP1 in Th1 cells that sensitized to IL-2-mediated mTORC1 signaling. In vivo, GLS null T cells failed to drive Th17-inflammatory diseases, and Th1 cells had initially elevated function but exhausted over time. Transient GLS inhibition, however, led to increased Th1 and CTL T cell numbers. Glutamine metabolism thus has distinct roles to promote Th17 but constrain Th1 and CTL effector cell differentiation.

Keywords: T cells; chromatin; glutaminase; glutamine; mTOR; metabolism.

Figure 1.. Activated T Cells Rely on Both Glucose and Glutamine to Sustain Cell Metabolism

(A) Metabolites extracted for mass spectrometry and presented as fold change from naive in T cells stimulated for 16 hr (S) or naive (N) conditions (one-way ANOVA). (B) Oxygen consumption rate (OCR) assayed from naive CD4 cells from wild-type (WT) mice stimulated for 3 days on αCD3/CD28, injected with drug described (top). OCR at time point 200 min (bottom, one-way ANOVA). (C and D) Abundance of metabolites (left, unpaired t test) and fractional labeling (right, one-way ANOVA) of stimulated CD4⁺ T cells in the presence of CB839 and ¹³C-glucose for glutaminolytic intermediates (C) and TCA intermediates (D). Also see Figure S1.

Figure 2.. Th1 and Th17 Cells Differ in Their Use of Glutaminolysis, and GLS Deficiency Is Distinct from Glutamine Deficiency

(A) Metabolite fold change from naive in wild-type CD4⁺ cells maintained in IL-7 (N) or differentiated for 5 days into Th1 (1), Th17 (17), or Treg (R) cells (one-way ANOVA). (B) Cytokine production from Th1 (top) and Th17 (bottom) differentiated T cells in the presence of glutamine (left), absence of glutamine (middle), or presence of GLS1-inhibitor CB839 (right). (C) Proliferation of cell trace violet (CTV)-labeled T cells stimulated and differentiated in Th1 or Th17 conditions with (black lines) or without (red lines) glutamine after 3 and 5 days of culture. (D) Same as in (C), but with vehicle (black lines) or CB839 (green lines). (E) Foxp3 expression in CD4 T cells activated in Th1- or Th17-skewing conditions in glutamine deficient (red, left) conditions or in the presence of CB839 (green, right). (F) Heatmap (left) and principle component analysis (right) of metabolites from Th1 and Th17 cells with or without CB839. Also see Figure S2 and Table S1.

Figure 3.. GLS Is Dispensable for T Cell Homeostasis but Constrains Development of a Th1-like Phenotype

(A) Immunoblot (left) and genomic DNA (right) in isolated Pan T cells (CD4⁺ and CD8⁺) from GLS^fl/fl CD4-Cre⁺ (GLS KO) and littermate wild-type controls (WT). (B) Cell counts (left) and percent of total splenocytes (right) from WT and GLS KO animals. No significance versus wild-type, one-way ANOVA. (C and D) Flow cytometry analysis of T cell activation markers and cell size of CD4⁺ T cells (C) freshly isolated from WT and GLS KO T spleens or (D) activation markers and proliferation of WT and GLS KO CD4⁺ T cells activated on αCD3/CD28 over 48 hr. (E) Flow cytometry analysis of CD44 in CB839- or vehicle-treated T cells activated on αCD3/CD28 at day 5. (F–K) Naive CD4⁺ T cells activated without cytokines over 3 days, split with IL-2, then stimulated to measure cytokines on day 5. (F) Cytokine production of wild-type and GLS KO T cells. (G) Average percent total IFNγ⁺ producers (left), percent double positive IFNγ⁺IL2⁺ producers (middle), and the median fluorescence intensity (MFI) (right) of all IFNγ⁺ cells in (F) (unpaired t test). (H) Tbet protein expression in WT, GLS KO, and isotype control T cells. Representative of n = 2 experiments. (I–K) Same as in (F–H), except with GLS-inhibitor CB839 and vehicle. (L and M) CD8⁺ T cells from WT or GLS KO animals activated on αCD3/CD28 + IL2 for 5 days. (L) Expression of CD8⁺ granzyme B protein at day 5 (left) and average of granzyme B MFI signal (right) (Student’s t test, n = 3 replicates/group). (M) Tbet protein expression in WT, GLS KO, and isotype control (representative of n = 2 experiments). Also see Figure S3.

Figure 4.. GLS Specifies Th1 and Th17 Differentiation and Metabolism

(A–D) Naive CD4⁺ T cells from WT and GLS KO T cells differentiated in Th1-, Th17-, or Treg-skewing media over 5 days. (A) IFNγ and IL2 production in Th1-skewing conditions (top) and IL-17 production in Th17-skewing conditions (bottom). (B) Average percent change cytokine producers in Th1 and Th17 cells from WT (paired t test). (C) Transcription factor expression of Th1, Th17, and Treg cells in WT (black) and GLS KO (red). (D) Average percent change from WT of transcription factors (one-sample t test) in GLS KO T cells. (E–K) WT CD4⁺ T cells differentiated in Th1 or Th17 conditions in the presence of vehicle or CB839 over 5 days. (E) Percent of Th1 cells producing IFNγ, IL2, and TNFα at day 5 (unpaired Student’s t test; NS, no stim). (F) Median fluorescence intensity of inhibitory receptors (two-way ANOVA). (G) Fold change of metabolites from T cells differentiated in Th1 and Th17 conditions in the presence of CB839 relative to vehicle by mass spectrometry over 5 days. (H) ³H-2-deoxyglucose uptake in Th1- and Th17-skewed T cells at day 3 (left) and day 5 (right) (Student’s t test). (I) Extracellular acidification rate (ECAR) of Th1- and Th17-skewed T cells at day 5 as in (H). (J) Fold change of Tbet (Th1) or RORyt (Th17) protein levels and (K) cell size in CB839-treated cells normalized to vehicle from same experiment as (G). Also see Figure S4.

Figure 5.. Th17 and Th1 Cells Differentially Rely on GLS-Mediated ROS Neutralization and Production of α-Ketoglutarate to Maintain Chromatin

(A–D) WT CD4⁺ T cells differentiated in Th1 or Th17 conditions in the presence of vehicle or CB839 over 5 days. (A) Cytokine production in Th1 (top)- and Th17 (bottom)-skewing conditions dosed as indicated. (B) Average IFNγ⁺ only producers (left) and average IFNγ⁺IL2⁺ producers (right) as in (A). (C) Average protein expression of Tbet as in (A). (D) Average IL-17A producers in Th17-skewing media (left) and average RORyt expression (right) (one-way ANOVA). (E and F) Global H3K27 trimethylation normalized to total H3 by flow cytometry. (E) Average H3K27 trimethylation expression at day 3. (F) Same as (E), but at day 5 (Student’s t test). (G) Average cytokine producers of skewed CD4⁺ T cells in the presence of CB839, JMJD3-inhibitor GSKJ4, or CB839+GSKJ4 (CB+J4) at day 5 (one-way ANOVA). (H and I) WT CD4⁺ T cells differentiated in Th17 conditions as indicated. (H) Percent IL17A⁺ producers (left) and protein expression of RORyt (right). (I) Average expression of H3K27me3 normalized to total H3 as in (H) (one-way ANOVA). (J) Number of loci with more (blue circles) and less accessible (orange circles) chromatin peaks with CB839 as determined by ATAC-seq. (K) Example ATAC-seq traces of IFNγ in Th1 and IL17 gene locus in Th17-skewing conditions. Also see Figure S5.

Figure 6.. GLS Inhibition Alters Gene Expression to Sensitize Th1 Cells to IL2 Activation of mTORC1

(A) Top 200 modified genes from RNA-seq compared to vehicle (Log2Fold > 0.5, p < 0.05) in Th1 (left) and Th17 (right). (B and C) Phospho-S6 expression on day 5 in Th1 and Th17 conditions as indicated with or without CB839 or IL2 at concentrations shown (ng/mL) at day 3 (Student’s t test). (D) Cytokine production in Th1-skewing conditions in the presence of vehicle (top) or CB839 (bottom) after 5 days, under no IL2 conditions or with IL2 + mTOR inhibitor rapamycin added on day 3. (E) Phospho-S6 protein expression (left), average pS6 MFI (middle), percent IFNγ⁺IL2⁺ or IL2⁺ cells (right) in CD4 T cells in Th1-skewing conditions and infected with control- or PIK3IP1-expressing retrovirus with CB839-treatment. (middle, Student’s t test; right, two-way ANOVA). (F) Protein expression of phospho-S6 (left) and IFNγ (right) in activated Cas9-transgenic CD4⁺ T cells transduced with retrovirus-containing control guide RNA or guide RNAs targeting PIK3IP1. (G and H) Wild-type CD4⁺ T cells activated and treated with PIK3IP1 antibody or IgG control antibody over 3 days. Protein expression of phospho-S6 (left) and average MFI of pS6 (right, one-way ANOVA). (H) Protein expression of activation markers of control or PIK3IP1 antibody-treated T cells upon stimulation (no CB839). Also see Figure S6 and Table S2.

Figure 7.. GLS Is Essential for T Cell-Mediated Inflammation, but Transient Inhibition Can Augment T Cell Responses

(A–C) Airway inflammation in cGvHD following transfer of WT or GLS KO T cells. (A) Hematoxylin- and eosin-stained lung sections focusing on bronchioles. (B) Average histopathological scores from sections from (A) (unpaired t test). (C) Percent cytokine producers from peripheral lymph node cells stimulated with PMA/ionomycin for 5 hr from GvHD mice (BM, n = 8; WT, n = 5; KO, n = 7; unpaired t test). (D) Bodyweights from T cell adoptive transfer inflammatory bowel disease (IBD) model in which RAG1 KO mice injected with WT and GLS KO naive CD4⁺ T cells and induced for IBD with piroxicam (^#p < 0.05, ^‡p < 0.01, two-way ANOVA; WT, n = 6; GLS KO, n = 8; data presented as SEM). (E and F) T cells from WT and GLS KO infected to express CAR T cell constructs and injected into recipient mice. CAR- no infection; Δ, m19-delta-ζ; 28-ζ, m19–28-ζ. (E) CD19+ B cells per μL of blood at day 14 (left) and day 28 (right). (F) Same as in (E), but at day 42 (one-way ANOVA). (G) CAR T cell numbers on day 28 following transfer of CAR T cells treated with vehicle or CB839 prior to transfer to recipient mice (WT no CAR, n = 2 animals; all others, n = 5–6 animals; one-way ANOVA). (H) Number of CD19⁺ Eμ-Myc lymphoma cells after 48 hr culture with indicated ratios of CAR T cells (Student’s t test). (I) Counts of total CD8⁺ cells in response to hgp100_25–33-expressing vaccinia virus collected from tail vein after indicated time (two-way ANOVA). (J) Counts of total CD8⁺ T cells in spleen (left) and lymph node (right) after 38 days and re-challenge with hgp100_25–33-expressing vaccinia virus (Vehicle, n = 5 animals; GLS inhibitor, n = 4 animals; two-way ANOVA). Also see Figure S7.