Glycolysis and glutaminolysis cooperatively control T cell function by limiting metabolite supply to N-glycosylation

Abstract

Rapidly proliferating cells switch from oxidative phosphorylation to aerobic glycolysis plus glutaminolysis, markedly increasing glucose and glutamine catabolism. Although Otto Warburg first described aerobic glycolysis in cancer cells >90 years ago, the primary purpose of this metabolic switch remains controversial. The hexosamine biosynthetic pathway requires glucose and glutamine for de novo synthesis of UDP-GlcNAc, a sugar-nucleotide that inhibits receptor endocytosis and signaling by promoting N-acetylglucosamine branching of Asn (N)-linked glycans. Here, we report that aerobic glycolysis and glutaminolysis co-operatively reduce UDP-GlcNAc biosynthesis and N-glycan branching in mouse T cell blasts by starving the hexosamine pathway of glucose and glutamine. This drives growth and pro-inflammatory T_H17 over anti-inflammatory-induced T regulatory (iTreg) differentiation, the latter by promoting endocytic loss of IL-2 receptor-α (CD25). Thus, a primary function of aerobic glycolysis and glutaminolysis is to co-operatively limit metabolite supply to N-glycan biosynthesis, an activity with widespread implications for autoimmunity and cancer.

Keywords: N-acetylglucosamine; N-glycosylation; T cell; Warburg effect; cell biology; glutaminolysis; glycolysis; immunology; mouse.

Figure 1.. N-glycan branching controls T_H17 versus iTreg cell fate.

(A) Fructose 6-phosphate and glutamine may be metabolized by glycolysis and glutaminolysis, respectively, or enter the hexosamine pathway to supply UDP-GlcNAc to the Golgi branching enzymes Mgat1, 2, 4 and 5. HK: hexokinase, GPI: glucose-6-phosphate isomerase, PFK1: phosphofructokinase1, LDH: Lactate dehydrogenase, GFPT: glutamine-fructose-6-phosphate transaminase. (B–H) Flow cytometry (B,E–H), Western blot (C) and LC-MS/MS (D) analysis of purified mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 for 4 days (B,E–H) or 3 days (C,D) with T_H17 inducing conditions (TGFβ+IL-6+IL-23) or as indicated. PFK1-L (liver), PFK1-P (platelet), PFK1-M (muscle). (G) Co-incubation with doxycycline in vitro. (H) Doxycycline treatment in vivo, with Mgat1^f/ftetO-Cre⁺ROSA^rtTA cells in right panel gated on L-PHA⁻ population. (B,E–H) gated on CD4⁺. (B,D–H) Unpaired two tailed t-test with Welch’s (E) and Bonferroni corrections (B,E). **p<0.01; ***p<0.001. Data are mean ± s.e.m of triplicate cultures and representative of n ≥ 3 experiments. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.002

Figure 1—figure supplement 1.. N-glycan branching controls T_H17 versus iTreg cell fate.

(A–F) Flow cytometry (A,B,E,F), real-time qPCR (C) and UDP-GlcNAc LC-MS/MS analysis (D) of purified mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 under T_H17 inducing conditions (TGFβ+IL-6+IL-23) for 3 days (D) or 4 days (A,B,E,F) or as indicated (C). (A) gated on CD4⁺IL-17A^- or CD4⁺IL-17A⁺ as indicated. (B,E,F), gated on CD4. (B) Lactose (50 mM) is an inhibitor of galectin binding to branched N-glycans. (A,B,F) Unpaired two tailed t-test (B) with Bonferroni corrections (A,F). *p<0.05; **p<0.01; ***p<0.001. Data are mean ± s.e.m and n = 3. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.003

Figure 1—figure supplement 2.. N-glycan branching controls T_H17 versus iTreg cell fate.

(A–C) Flow cytometry of purified mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 under T_H17 inducing conditions (TGFβ+IL-6+IL-23) or as indicated for 4 days. Gated on CD4⁺. (C) Unpaired two tailed with Bonferroni corrections. *p<0.05; **p<0.01; ***p<0.001. Data are mean ± s.e.m and n = 3. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.004

Figure 2.. N-glycan branching controls T_H17 versus iTreg cell fate via IL-2Rα (CD25).

(A–F) Flow cytometry (A–C,E,F) and Western blot (D) analysis of mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 under T_H17-inducing conditions (TGFβ+IL-6+IL-23) for 3 days (D) or 4 days (A–C,E,F); cells gated on CD4⁺. (A,F) Doxycycline treatment in vivo with Mgat1^f/ftetO-Cre⁺ROSA^rtTA cells gated on L-PHA⁻ population. (C) CD25 MFI in Mgat5^tetOROSA^rtTA CD4⁺ T cells normalized to ROS^rtTA CD4⁺ T cells, both treated in vitro with doxycycline. (E,F) ratio of IL-17A⁺ to FoxP3⁺ CD4⁺ cells. (A,B,E,F) Unpaired two-tailed t-test with Welch’s (A,E) and Bonferroni corrections (E,F). (C) Linear regression. *p<0.05; **p<0.01; ***p<0.001. Data are mean ± s.e.m of triplicate cultures and representative of n ≥ 3 experiments. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.005

Figure 2—figure supplement 1.. N-glycan branching controls T_H17 versus iTreg cell fate via IL-2Rα (CD25).

(A–D,F–H) Flow cytometry of purified mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 under T_H17 inducing conditions (TGFβ+IL-6+IL-23) or as indicated for 4 days. (D) Co-incubation with doxycycline in vitro. (A–C,D,F–H) gated on CD4⁺. (A,B) Mice treated with doxycycline in vivo, Mgat1^f/ftetO-Cre⁺ROSA^rtTA gated on L-PHA^- cells. (A) Gated on CD4⁺IL-17A^- or CD4⁺IL-17A⁺ as indicated. (B) Endocytosis analyzed by flow cytometry for CD25 internalization. Internalized percentage (MFI (internalized) / MFI (total) × 100%), normalized to control. (D) ELISA analysis of supernatant from purified mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 under T_H17-inducing conditions (TGFβ+IL-6+IL-23) as indicated for 4 days. (A,B,E) Unpaired two-tailed t-test with Welch’s (A) and Bonferroni corrections (E). *p<0.05; **p<0.01; ***p<0.001. Data are mean ± s.e.m and n = 3, except n = 2 for (B). MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.006

Figure 3.. Glycolysis promotes T_H17 over iTreg cell fate by inhibiting N-glycan branching.

(A–C) Ratio of the oxygen consumption rate (OCR) to the extracellular acidification rate (ECAR) of purified splenic CD4⁺ T cells at rest or activated with anti-CD3+anti-CD28 for 2 days with T_H17 cytokines (TGFβ+IL-6+IL-23) or as indicated. (D–G) Flow cytometry analysis of purified mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 for 4 days under T_H17-inducing conditions (TGFβ+IL-6+IL-23). (F (right panel), G) ratio of IL-17A⁺ to Foxp3⁺ CD4⁺ T cells, with Mgat1^f/ftetO-Cre⁺ROSA^rtTA cells gated on L-PHA^- population. *p<0.05; **p<0.01; ***p<0.001. NS, not significant. (A–G), Unpaired two-tailed t-test with Welch's (B,C,F,G) and Bonferroni correction (A–C,F,G). Data are mean ± s.e.m and n = 3. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.007

Figure 3—figure supplement 1.. Glycolysis promotes T_H17 over iTreg cell fate by inhibiting N-glycan branching.

(A) Flow cytometry analysis of purified splenic CD4⁺ T cells activated with anti-CD3+ anti-CD28 for 2 days with T_H17 cytokines and as indicated for 1 hr with 2-NBDG. (B) Oxygen Consumption Rate (OCR) and the Extracellular Acidification Rate (ECAR) of purified splenic CD4⁺ T cells at rest or activated with anti-CD3+anti-CD28 for 2 days with/without T_H17 cytokines (TGFβ+IL-6+IL-23) as indicated. (C–G) Flow cytometry (C,D,F,G) and Western blot (E) analysis of purified mouse splenic CD4⁺ T-cells activated with anti-CD3+anti-CD28 under T_H17-inducing conditions (TGFβ+IL-6+IL-23). Gated on CD4⁺ T cells. *p<0.05; **p<0.01; ***p<0.001. (A–D,F,G) Unpaired two-tailed t-test with Welch's (B–D) and Bonferroni correction (A–D). Data are mean ± s.e.m and n = 3. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.008

Figure 4.. Oxidative phosphorylation promotes iTreg generation by enhancing N-glycan branching.

(A–F) Flow cytometry of purified splenic CD4⁺ T cells activated with anti-CD3+anti-CD28 and TGFβ1 for 4 days; gated on CD4⁺. (A), Mgat1^f/ftetO-Cre⁺ROSA^rtTA cells gated on L-PHA^- population from mice treated with doxycycline in vivo. *p<0.05; **p<0.01; ***p<0.001. (A–F) Unpaired two-tailed t-test with Bonferroni (C–F) and Welch’s (C,E,F) correction. Data are mean ± s.e.m and n = 3. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.009

Figure 4—figure supplement 1.. Oxidative phosphorylation promotes iTreg generation by enhancing N-glycan branching.

(A,B) Flow cytometry of purified splenic CD4⁺ T cells activated with anti-CD3+anti-CD28 and TGFβ1 for 4 days; gated on CD4⁺. (A) Mgat1^f/ftetO-Cre⁺ROSA^rtTA cells gated on L-PHA^- population from mice treated with doxycycline in vivo. *p<0.05; **p<0.01; ***p<0.001. (B) Unpaired two-tailed t-test; data are mean ± s.e.m. n = 3. DOI: http://dx.doi.org/10.7554/eLife.21330.010

Figure 5.. Glycolysis promotes T cell growth by inhibiting N-glycan branching.

(A–C) Flow cytometry of purified splenic CD4⁺ T cells activated with anti-CD3 for 3 days, gated on CD4⁺. (B,C) Proliferation measured by CFSE dilution. *p<0.05; **p<0.01; ***p<0.001. (A) Unpaired two-tailed t-test with Bonferroni correction. Data are mean ± s.e.m of triplicate cultures and representative of n = 3 experiments. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.011

Figure 5—figure supplement 1.. Glycolysis promotes T cell growth by inhibiting N-glycan branching.

Flow cytometry of CFSE-labeled splenic CD4⁺ T cells activated with anti-CD3 for 3 days, gated on CD4⁺. n = 3. DOI: http://dx.doi.org/10.7554/eLife.21330.012

Figure 6.. Glutaminolysis promotes T_H17 over iTreg cell fate by inhibiting N-glycan branching.

(A–J) Flow cytometry of purified splenic CD4⁺ T cells activated with anti-CD3+anti-CD28 under T_H17-inducing conditions (TGFβ+IL-6+IL-23) (A,B,E–J) or in the presence of TGFβ (C,D). Gated on CD4⁺. *p<0.05; **p<0.01; ***p<0.001. (A–D,G–J) Unpaired one-tailed t-test with Welch's (G,I,J) and Bonferroni (B,D,G–J) correction. Data are mean ± s.e.m and n = 3. MFI, mean fluorescence intensity. DOI: http://dx.doi.org/10.7554/eLife.21330.013

Figure 6—figure supplement 1.. Glutaminolysis promotes T_H17 over iTreg cell fate by inhibiting N-glycan branching.

(A,B) Flow cytometry of purified splenic CD4⁺ T cells activated with anti-CD3+anti-CD28 under T_H17-inducing conditions (TGFβ+IL-6+IL-23) (A) or in the presence of TGFβ (B). Gated on CD4⁺. n = 3. DOI: http://dx.doi.org/10.7554/eLife.21330.014