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. 2015 Jul 27;146(2):292–300. doi: 10.1111/imm.12502

Indoleamine 2,3-dioxygenase depletes tryptophan, activates general control non-derepressible 2 kinase and down-regulates key enzymes involved in fatty acid synthesis in primary human CD4+ T cells

Theodoros Eleftheriadis 1,✉, Georgios Pissas 1, Georgia Antoniadi 1, Vassilios Liakopoulos 1, Ioannis Stefanidis 1
PMCID: PMC4582970  PMID: 26147366

Abstract

Indoleamine 2,3-dioxygenase (IDO) is expressed in antigen-presenting cells and exerts immunosuppressive effects on CD4+ T cells. One mechanism is through the inhibition of aerobic glycolysis. Another prerequisite for T-cell proliferation and differentiation into effector cells is increased fatty acid (FA) synthesis. The effect of IDO on enzymes involved in FA synthesis was evaluated in primary human cells both in mixed lymphocyte reactions in the presence or not of the IDO inhibitor 1-dl-methyl-tryptophan, and in stimulated CD4+ T cells in the presence or not of the general control non-derepressible 2 (GCN2) kinase activator tryptophanol (TRP). IDO or TRP inhibited cell proliferation. By assessing the level of GCN2 kinase or mammalian target of rapamycin complex 1 substrates along with a kynurenine free system we showed that IDO exerts its effect mainly through activation of GCN2 kinase. IDO or TRP down-regulated ATP-citrate lyase and acetyl coenzyme A carboxylase 1, key enzymes involved in FA synthesis. Also, IDO or TRP altered the expression of enzymes that control the availability of carbon atoms for FA synthesis, such as lactate dehydrogenase-A, pyruvate dehydrogenase, glutaminase 1 and glutaminase 2, in a way that inhibits FA synthesis. In conclusion, IDO through GCN2 kinase activation inhibits CD4+ T-cell proliferation and down-regulates key enzymes that directly or indirectly promote FA synthesis, a prerequisite for CD4+ T-cell proliferation and differentiation into effector cell lineages.

Keywords: acetyl coenzyme A carboxylase 1; ATP-citrate lyase; fatty acid; general control non-derepressible 2 kinase; indoleamine 2,3-dioxygenase; T cells

Introduction

During T-cell activation, rapidly proliferating T cells reprogramme their metabolic pathways from pyruvate oxidation via the Krebs’ cycle to the glycolytic, pentose-phosphate, and glutaminolytic pathways in order to fulfil the bioenergetic and biosynthetic demands of proliferation.1–3 This metabolic shift offers glucose metabolism intermediates for synthesis of new biomolecules required for rapidly proliferating T cells. It has also been confirmed that beyond clonal expansion, aerobic glycolysis is a prerequisite for T-cell differentiation into effector cell lineages.4,5

Although aerobic glycolysis during T-cell activation has been extensively studied, fatty acid (FA) synthesis plays a significant role as well. Recent studies showed that FA synthesis is up-regulated during activation of CD4+ T cells, enhancing their proliferation and promoting their differentiation into T helper type 17 cells instead of regulatory T cells.6 Fatty acid synthesis is also up-regulated upon CD8+ T-cell activation promoting effective clonal expansion by preventing the death of proliferating cells.7

Indoleamine 2,3–dioxygenase (IDO) catalyses tryptophan degradation along the kynurenine pathway. This enzyme is expressed in antigen-presenting cells and is up-regulated upon inflammation.8,9 In the inflammatory microenvironment IDO depletes l-tryptophan, leading to activation of general control non-derepressible 2 (GCN2) kinase and/or to inhibition of mammalian target of rapamycin complex 1 (mTORC1) in CD4+ T cells,10,11 resulting in a decrease in both clonal expansion and differentiation into effector T-cell lineages. Products along the kynurenine pathway contribute also to these IDO-induced effects by activating the aryl hydrocarbon receptor.12,13 The immunosuppressive properties of IDO have been confirmed in various animal models of autoimmune diseases and allotransplantation.14–19 In patients on haemodialysis, increased plasma IDO level has been related to a reduced response to vaccination,20 with patients’ plasma IDO level being negatively related to T-cell count.21

Indoleamine 2,3-dioxygenase decreases glucose influx in activated human T cells and inhibits aerobic glycolysis by affecting the expression of glucose transporter 1 and various glycolytic enzymes.22,23 However, the effect of IDO on FA synthesis upon T-cell activation has not been evaluated. The purpose of this study was to address the effect of IDO on key enzymes that directly or indirectly affect the availability of carbon atoms for FA synthesis in primary human CD4+ T cells (Fig.1). Moreover, the role of GCN2 kinase activation, mTORC1 inhibition or kynurenine pathway products in the IDO-induced effect on CD4+ T cells was evaluated.

Figure 1.

Figure 1

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Key enzymes involved in fatty acid (FA) synthesis. The first step in FA synthesis is the formation of malonyl-CoA by acetyl-CoA in the cytoplasm, a reaction that is catalysed by acetyl coenzyme A carboxylase 1 (ACC1). Cytoplasmic acetyl-CoA is formed by the action of ATP-citrate lyase (ACL) on cytoplasmic citrate. Besides malonyl-CoA formation, cytoplasmic acetyl-CoA is required for FA elongation. Cytoplasmic citrate is derived from the mitochondria. The carbon atoms for its formation through the Krebs’ cycle are derived from glucose metabolism or glutaminolysis. The enzymes pyruvate dehydrogenase (PDH) or lactate dehydrogenase-A (LDH-A) control the entry or not of pyruvate into the Krebs’ cycle. Glutaminase isoenzymes GLS1 and GLS2 play key roles in glutaminolysis.

For the purposes of our study the two-way mixed lymphocyte reaction (MLR) was used as a model of alloreactivity,24 along with the specific IDO inhibitor 1-dl-methyl-tryptophan (1-MT).17,25 In addition, an IDO-lacking system was tested to distinguish the effect of GCN2 kinase activation from the effect of kynurenine pathway products. Isolated CD4+ T cells were stimulated with anti-CD2, anti-CD3 and anti-CD28 antibodies in the presence or not of tryptophanol (TRP). Tryptophanol is a competitive inhibitor of the tryptophanyl-tRNA synthetase enzyme and by raising the pool of uncharged tRNA it acts as a pharmacological activator of GCN2 kinase.26

Materials and methods

Subjects

Blood samples were collected from eight unrelated healthy volunteers (four men, four women; 36 ± 8 years old). An informed consent was obtained from each individual enrolled in the study and the hospital ethics committee gave its approval to the study protocol.

Peripheral blood mononuclear cell and CD4+ T-cell isolation and culture

Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by Ficoll–Hypaque density gradient centrifugation (Histopaque 1077, Sigma-Aldrich, St Louis, MO) and counted by optical microscopy on a Neubauer plaque. Cell viability was assessed by trypan blue assay (Sigma-Aldrich).

The PBMCs were resuspended in RPMI-1640 medium with l-glutamine and 10 mm HEPES and supplemented with 10% fetal bovine serum (Sigma-Aldrich) and antibiotic–antimycotic solution (Sigma-Aldrich). Isolated PBMCs from the individuals were coupled to set up eight different MLRs.

In the experiments with TRP, CD4+ T cells were retrieved from freshly isolated PBMCs. Non CD4+ T cells were indirectly magnetically labelled with a cocktail of biotin-conjugated monoclonal antibodies and were depleted using the CD4+ T-Cell Isolation Kit, Human (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Isolated CD4+ T cells were cultured in the same medium as the PBMCs. All cultures were performed at 37° in a humidified atmosphere containing 5% CO2.

l-Tryptophan consumption in two-way MLR

The MLRs were performed in 12-well plates for 7 days in the presence or not of 100 μm 1-MT. The this concentration of 1-MT was selected according to previous studies.22,23 The number of PBMCs for each member of the MLR couple was 5 × 105, reaching a total of 1 × 106 PBMCs in each well. After 7 days, supernatants from each MLR were collected. l-Tryptophan consumption was assessed in the supernatants by means of ELISA (BlueGene Biotech, Shanghai, China). The sensitivity of this ELISA kit is 1 ng/ml. l-Tryptophan concentration was also measured in the supplemented culture medium. Eight MLRs were performed in triplicates with the results for each MLR referring to the mean of the three measurements.

Cell proliferation in two-way MLR

Mixed lymphocyte reactions were performed in 96-well plates for 7 days in the presence or not of 100 μm 1-MT. The number of PBMCs from each member of the MLR couple was 5 × 104, summing it up to 1 × 105 PBMCs in total in each well. Cultures of 1 × 105 resting PBMCs per well were used as controls. At the end of the 7-day period, cell proliferation was assessed by Cell Proliferation ELISA (Roche Diagnostics, Indianapolis, IN) using bromodeoxyuridine labelling and immunoenzymatic detection according to the manufacturer’s protocol. Proliferation index was calculated as the ratio of the optical density derived from each MLR to the mean of the optical densities derived from the control resting PBMCs of the two members of each MLR pair. Eight MLRs were performed in triplicates with the results for each MLR referring to the mean of the three measurements.

Assessment of GCN2 kinase, mTORC1 activity and key enzymes involved in FA synthesis in isolated CD4+ T cells from the two-way MLRs

Eight MLRs were performed in 12-well plates for 7 days in the presence or not of 100 μm 1-MT, with the cell number of each PBMC population in the MLR context remaining the same as before. At the end of the 7-day period for which MLRs lasted, CD4+ T cells were isolated from the MLRs by negative selection using the CD4+ T Cell Isolation Kit, Human (Miltenyi Biotec GmbH).

Isolated CD4+ T cells were counted via optical microscopy on a Neubauer plaque and cell viability was determined by trypan blue assay. Equal numbers of T cells from each MLR were lysed using the T-PER tissue protein extraction reagent (Thermo Fisher Scientific, Rockford, IL) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich and Roche Diagnostics). Protein was quantified via Bradford assay (Sigma-Aldrich) and 10 μg from each sample was electrophoresed in an SDS polyacrylamide gel (Invitrogen, Life Technologies, Carlsbad, CA). Subsequently, proteins were transferred to PVDF membrane (Invitrogen, Life Technologies). Blots were incubated with the primary antibody for 16 hr, followed by secondary antibody (anti-rabbit IgG, horseradish peroxidase-linked antibody; Cell Signaling Technology, Danvers, MA) for 30 min. Benchmark pre-stained protein ladder (Invitrogen, Life Technologies) was used as a marker. Bands were visualized by enhanced chemiluminescent detection using the LumiSensor Plus Chemiluminescent HRP Substrate Kit (GenScript, Piscataway, NJ). In the case of re-probing PVDF blots, the previous primary and secondary antibody were removed by using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) according to the manufacturer’s protocol. Analysis was performed using the Image J software (National Institute of Health, Bethesda, MD).

The primary antibodies used in Western blotting were specific for the substrate of GCN2 kinase eukaryotic initiation factor 2α phosphorylated at serine 51 (p-eIF2α) (Cell Signaling Technology, Danvers, MA) and the substrates of mTORC1 p70S6 kinase phosphorylated at threonine 389 (p-p70S6K) (Cell Signaling Technology) and eukaryotic translation initiation factor 4E-binding protein 1 phosphorylated at threonines 37/46 (p-4E-BP1) (Cell Signaling Technology). Specific antibodies against ATP-citrate lyase (ACL) (Cell Signaling Technology), acetyl coenzyme A carboxylase 1 (ACC1) (Biorbyt, Cambridge, UK) and its phosphorylated (and inactivated) at serine 79 form (p-ACC1) (Biorbyt) were used to address enzymes directly involved in FA synthesis. Antibodies against lactate dehydrogenase-A (LDH-A) (Cell Signaling Technology), pyruvate dehydrogenase (PDH) (Cell Signaling Technology) and its phosphorylated (and inactivated) at serine 293 form (p-PDH) (Biorbyt), glutaminase 1 (GLS1) (Acris Antibodies, San Diego, CA), and glutaminase 2 (GLS2) (Acris Antibodies) were used to assess enzymes that are indirectly involved in FA synthesis by controlling the availability of carbon atoms. All Western blot results were normalized to β-actin (Cell Signaling Technology).

Stimulation of CD4+ T cells in the presence or not of tryptophanol

CD4+ T cells retrieved from freshly isolated PBMCs were cultured without stimuli or stimulated with anti-CD2, anti-CD3 and anti-CD28 conjugated beads using the T-Cell activation/expansion kit (Miltenyi Biotec GmbH) in a bead to cell ratio of 1 : 2. Stimulated CD4+ T cells were cultured in the presence or not of 0·25 mm TRP. The above concentration of TRP was selected according to previous studies.10,23

Effect of tryptophanol on proliferation of stimulated CD4+ T cells

Proliferation of CD4+ T cells retrieved from freshly isolated PBMCs was determined by Cell Proliferation ELISA (Roche Diagnostics). CD4+ T cells that were resting, stimulated or stimulated in the presence of 0·25 mm TRP were cultured in 96-well plates (1 × 105/well) for 72 hr. T cells were derived from the blood of eight individuals and the experiments were performed in triplicates.

Effect of tryptophanol on GCN2 kinase activity and on enzymes involved in fatty acid synthesis

Resting, stimulated or stimulated in the presence of 0·25 mm TRP isolated CD4+ T cells were cultured in 12-well plates (1 × 106 cells/well) for 12 hr. Subsequently, Western blotting was performed using antibodies specific for p-eIF2α (Cell Signaling Technology), ACL (Cell Signaling Technology), ACC1 (Biorbyt), LDH-A (Cell Signaling Technology), PDH (Cell Signaling Technology), GLS1 (Acris Antibodies), GLS2 (Acris Antibodies) and β-actin (Cell Signaling Technology). Once again, experiments were performed in CD4+ T cells derived from the blood of eight individuals.

Statistical analysis

For comparison of means, paired-sample t-test or one-way repeated-measures analysis of variance was used. Results were expressed as mean ± standard deviation and a P < 0·05 was considered statistically significant.

Results

1-MT decreases l-tryptophan degradation in MLRs

As expected, in MLRs, the IDO inhibitor 1-MT decreased significantly the degradation of l-tryptophan. Indeed, in untreated MLRs l-tryptophan concentration was 2·73 ± 0·55 μg/ml, whereas in the presence of 1-MT it was 6·63 ± 0·89 μg/ml (P < 0·001) (Fig.2). l-Tryptophan concentration in the culture medium with 10% fetal bovine serum was measured to be 7·10 μg/ml.

Figure 2.

Figure 2

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The effect of 1-dl-methyl-tryptophan (1-MT) on l-tryptophan degradation. In mixed lymphocyte reactions (MLRs), 1-MT increased l-tryptophan concentration in the supernatants, indicating that indoleamine 2,3-dioxygenase accelerates l-tryptophan degradation. In untreated MLRs, l-tryptophan concentration was 2·73 ± 0·55 μg/ml, whereas in the presence of 1-MT it was 6·63 ± 0·89 μg/ml. l-Tryptophan concentration in the culture medium with 10% fetal bovine serum was measured to be 7·10 μg/ml. In MLRs, peripheral blood mononuclear cells from different individuals were cultured for 7 days. Error bars correspond to the standard error.

1-MT increases cell proliferation in MLRs

Treatment of MLRs with the IDO inhibitor 1-MT increased cell proliferation significantly. Bromodeoxyuridine assay revealed a proliferation index of 2·92 ± 0·21 in MLRs, whereas 1-MT significantly increased the proliferation index to 5·61 ± 1·19 (P < 0·001) (Fig.3a).

Figure 3.

Figure 3

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The effect of 1-dl-methyl-tryptophan (1-MT) or of tryptophanol in cell proliferation. In mixed lymphocyte reactions (MLRs), 1-MT increased cell proliferation significantly (a). Activation of CD4+ T cells by anti-CD2, anti-CD3 and anti-CD28 results in profound cell proliferation. The addition of the general control non-derepressible 2 (GCN2) kinase activator tryptophanol (TRP) attenuates cell proliferation in activated CD4+ T cells (b). In MLRs, peripheral blood mononuclear cells from different individuals were cultured for 7 days. The culture of the CD4+ T cells retrieved from freshly isolated peripheral blood mononuclear cells lasted 72 hr. Error bars correspond to the standard error.

1-MT decreases GCN2 kinase activity, leaves mTORC1 activity unaffected and up-regulates enzymes involved in fatty acid synthesis in isolated CD4+ T cells from MLRs

In the CD4+ T cells of 1-MT-treated MLRs, GCN2 kinase activity assessed by the phosphorylation of its substrate eIF2α was decreased by a factor of 0·58 ± 0·095 (P < 0·001) (Fig.4a,b).

Figure 4.

Figure 4

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The effect of 1-dl-methyl-tryptophan (1-MT) on general control non-derepressible 2 (GCN2) kinase activity, mammalian target of rapamycin complex 1 (mTORC1) activity and on the expression of key enzymes involved in fatty acid (FA) synthesis in CD4+ T cells isolated from mixed lymphocyte reactions (MLRs). Eight MLRs were performed in the presence or not of the indoleamine 2,3-dioxygenase (IDO) inhibitor 1-MT. After the 7-day period that MLRs lasted, CD4+ T cells were retrieved from the MLRs, cellular proteins were extracted and the levels of the evaluated proteins were determined by means of Western blotting. The results of four experiments are depicted in (a). 1-MT decreased GCN2 kinase activity, assessed by the phosphorylation state of its substrate eIF2α, whereas mTORC1 activity, assessed by the phosphorylation state of its substrates p70S6K and 4E-BP, was unaffected. 1-MT increased the levels of ATP-citrate lyase (ACL) and acetyl coenzyme A carboxylase 1 (ACC1), but decreased the level of phosphorylated (inactivated) ACC1. 1-MT up-regulated lactate dehydrogenase A (LDH-A), but it had no profound effect on pyruvate dehydrogenase (PDH) or on its phosphorylated (inactivated) form. Finally, 1-MT up-regulated both glutaminase isoenzymes, GLS1 and GLS2. All these effects of 1-MT are depicted in (b). Error bars correspond to the standard error, ns indicates P ≥ 0·05, *P < 0·05 and **P < 0·001.

In contrast, 1-MT did not affect mTORC1 activity, as the phosphorylation of its substrate p70S6K remained stable, altered only by a factor of 0·98 ± 0·15 (P = 0·718). In accordance, the level of the mTORC1 substrate p-4E-BP1 remained unaffected in the presence of IDO inhibitor, altered only by a factor of 1·02 ± 0·16 (P = 0·677) (Fig.4a,b).

In the same cellular context, 1-MT markedly increased the expression of ACL and ACC1 by a factor of 1·81 ± 0·46 (P = 0·002) and 1·52 ± 0·22 (P < 0·001), respectively. In contrast, 1-MT significantly decreased the level of the inactivated p-ACC1 by a factor of 0·74 ± 0·12 (P = 0·001) (Fig.4a,b).

Regarding the enzymes involved indirectly in FA synthesis by controlling the availability of carbon atoms, treatment of MLRs with 1-MT increased significantly the expression of LDH-A in CD4+ T cells by a factor of 1·68 ± 0·36 (P = 0·001), but it left the expression levels of PDH and p-PDH unaffected (0·90 ± 0·09, P = 0·050 and 0·99 ± 0·15, P = 0·886). Finally, 1-MT increased GLS1 and GLS2 expression in CD4+ T cells by a factor of 2·00 ± 0·66 (P = 0·004) and 1·39 ± 0·13 (P < 0·001), respectively (Fig.4a,b).

Tryptophanol decreases cell proliferation in isolated CD4+ T cells

Stimulation of CD4+ T cells with anti-CD2, anti-CD3 and anti-CD28 resulted in profound cell proliferation, with a proliferation index of 5·56 ± 0·99 (P < 0·001). Co-treatment with TRP decreased the proliferation index by half (2·67 ± 0·33, P < 0·001, compared with activated CD4+ T cells) (Fig.3b).

CD4+ T-cell stimulation up-regulates enzymes involved in fatty acid synthesis, whereas tryptophanol activates GCN2 kinase and down-regulates the above enzymes

Stimulation of CD4+ T cells with anti-CD2, anti-CD3 and anti-CD28 did not alter significantly GCN2 kinase activity assessed by the level of its substrate p-eIF2α. The level of p-eIF2α increased by a factor of 1·49 ± 1·18 (P = 0·284). In contrast, co-treatment with TRP increased GCN2 kinase activity significantly, as p-eIF2α rose by a factor of 3·01 ± 2·14 (P = 0·003, compared to activated CD4+ T cells) (Fig.5a,b).

Figure 5.

Figure 5

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The effect of tryptophanol (TRP) on general control non-derepressible 2 (GCN2) kinase activity and on the expression of key enzymes involved in fatty acid (FA) synthesis in activated CD4+ T cells. CD4+ T cells were retrieved from freshly isolated peripheral blood mononuclear cells and activated with anti-CD2, anti-CD3 and anti-CD28 in the presence or not of TRP. After 12 hr, protein was extracted and the levels of the evaluated proteins were determined by means of Western blotting. The results of four of the eight performed experiments are depicted in (a). CD4+ T-cell activation did not affect GCN2 kinase activity, assessed by the phosphorylation state of its substrate eIF2α. ATP-citrate lyase (ACL), acetyl coenzyme A carboxylase 1 (ACC1), lactate dehydrogenase A (LDH-A), pyruvate dehydrogenase (PDH), glutaminase isoenzymes GLS1 and GLS2 were all increased. The addition of TRP upon CD4+ T-cell activation increased GCN2 kinase activity and decreased the elevation of ACL, ACC1, LDH-A, GLS1 and GLS2, but the level of PDH remained unaffected. All these effects of TRP are depicted in (b). Error bars correspond to the standard error, ns to P ≥ 0·05, *P < 0·05 and **P < 0·001.

Concerning the enzymes ACL and ACC1, activation of CD4+ T cells increased their expression significantly by a factor of 2·27 ± 0·98 (P = 0·008) and 1·70 ± 0·35 (P = 0·001). However, co-treatment with TRP attenuated these increments. Specifically, ACL increased by only a factor of 1·28 ± 0·61 (P = 0·001 compared with activated CD4+ T cells) and ACC1 by only a factor of 1·12 ± 0·33 (P < 0·001, compared with activated CD4+ T cells) (Fig.5a,b).

The level of LDH-A increased in stimulated CD4+ T cells by a factor of 1·97 ± 0·63 (P = 0·003), whereas co-treatment with TRP attenuated this increase by a factor of 1·41 ± 0·18 (P = 0·015 compared with activated CD4+ T cells). PDH was up-regulated in activated CD4+ T cells by a factor of 1·42 ± 0·22 (P = 0·001). Tryptophanol did not alter this expression any further because it enhances PDH level by a factor of 1·37 ± 0·09 (P = 0·378, compared with activated CD4+ T cells) (Fig.5a,b).

Both glutaminase isoenzymes, GLS1 and GLS2, were up-regulated in stimulated CD4+ T cells by a factor of 2·17 ± 0·21 (P < 0·001) and 2·51 ± 1·44 (P = 0·021), respectively. Tryptophanol attenuated these increments as GLS1 was found to be up-regulated by a factor of 1·66 ± 0·25 (P < 0·001, compared with activated CD4+ T cells) and GLS2 by a factor of 1·07 ± 0·12 (P = 0·021, compared with activated CD4+ T cells) (Fig.5a,b).

Discussion

Various experimental models revealed that IDO is a key immunomodulatory enzyme that controls CD4+ T-cell proliferation, apoptosis and differentiation into effector cell lineages.8,9,14–19 Revelation of the method of IDO action would lead to a better understanding of immune system regulation that could be of clinical significance.

Three main mechanisms by which CD4+ T cells are affected by IDO-bearing antigen-presenting cells have been proposed. The first two correspond to IDO-induced l-tryptophan depletion in the local microenvironment. Different groups of investigators support that l-tryptophan depletion is sensed by GCN2 kinase, which is activated,10 or by mTORC1, which is inhibited.11 In the MLRs of this study, IDO decreased l-tryptophan concentration and activated GCN2 kinase, the latter being assessed by the level of phosphorylation of its substrate eIF2α. On the contrary, mTORC1 activity, assessed by the phosphorylation of its substrates p70S6K and 4-E-BP1, remained unaffected. This is in accordance with studies showing that mTORC1 is sensitive to the depletion of certain amino acids – more precisely to depletion of leucine, isoleucine, valine and possibly arginine, but not of tryptophan.27

Other studies suggest that kynurenine, a product of l-tryptophan degradation, by activating the transcription factor aryl hydrocarbon receptor contributes to the immunomodulatory effects of IDO.12,13 In the present study, addition of GCN2 kinase activator TRP in CD4+ T cells retrieved from freshly isolated PBMCs, a system lacking IDO-bearing antigen-presenting cells and consequently kynurenine free, recapitulated the results obtained by the MLRs. The latter system also suggests that GCN2 kinase activation alone, in the absence of mTORC1 inhibition, is adequate to induce the effects of IDO observed in the MLRs. Collectively the above results suggest that the main mechanism by which IDO affects CD4+ T cells is through l-tryptophan depletion and GCN2 kinase activation.

As expected,8,9 in MLRs the IDO inhibitor 1-MT enhanced cell proliferation, and in activated CD4+ T cells the GCN2 kinase activator TRP reduced cell proliferation. During T-cell proliferation and differentiation into effector cell lineages, aerobic glycolysis and glutaminolysis are the predominant mechanisms of energy production in contrast to pyruvate oxidation via the Krebs’ cycle.1–5 Indoleamine 2,3-dioxygenase has been shown to inhibit both aerobic glycolysis and glutaminolysis,22,23 a potential immunomodulatory mechanism of IDO. However, besides glycolysis and glutaminolysis, FA synthesis is also up-regulated during activation of CD4+ T cells, enhancing their proliferation and promoting their differentiation into T helper type 17 cells instead of regulatory T cells.6 The effect of IDO on FA synthesis in T cells has not been studied yet. Nevertheless, amino acid deprivation through GCN2 kinase activation has been shown to decrease FA synthesis in the liver of experimental animals.28 Our results confirmed that IDO may exert its immunosuppressive effect by decreasing the expression of key enzymes involved in FA synthesis. In the context of MLRs, 1-MT increased the expression of ACL and ACC1, whereas it decreased p-ACC1, the inactivated form of ACC1. Hence, IDO down-regulates ACC1, which converts acetyl-CoA to malonyl-CoA in the cytoplasm, the first step for FA synthesis.29 Cytoplasmic acetyl-CoA, required for FA elongation, is derived from cytoplasmic citrate by the action of ACL,29 the expression of which was also found to be down-regulated by IDO. These results were recapitulated by the GCN2 kinase activator TRP in activated CD4+ T cells. In MLRs, IDO up-regulated the phosphorylated inactivated form of ACC1, further inhibiting the capacity for FA synthesis. This phosphorylation may be mediated by AMP-activated protein kinase,30 but this remains to be elucidated. ACC1 has been shown to play a central role in clonal expansion and differentiation of CD4+ and CD8+ T cells.6,7 Interestingly, through induction of p53, IDO has been shown to affect many, but not all, enzymes of glycolysis, restraining the metabolic shift towards aerobic glycolysis that takes place upon T-cell activation.23 We performed experiments using the p53 inhibitor pifithrin-α.31 No effect of p53 on ACL or ACC1 expression was detected (data not shown).

Cytoplasmic citrate is derived from the mitochondria, where it is synthesized through the Krebs’ cycle. Consequently, enzymes that control carbon atom entry into the Krebs’ cycle ultimately control the availability of carbon atoms for FA synthesis. These carbon atoms may be provided by glucose. However, treatment of MLRs with 1-MT showed that IDO actually reduced the expression of LDH-A, which converts pyruvate to lactate, whereas it left PDH and its inactivated form p-PDH unaffected. PDH is responsible for conversion of pyruvate to acetyl-CoA into the mitochondria and entry into the Krebs’ cycle. PDH is phosphorylated and inactivated by PDH kinase.32 Moreover, activation of CD4+ T cells increased LDH-A and PDH, whereas co-treatment with TRP, which at least in part imitates the action of IDO, decreased LDH-A expression leaving the PDH level unaffected. At first glance these results seem unexpected because they indicate that IDO on the one hand decreased the expression of enzymes directly involved on FA synthesis, while on the other hand increasing the availability of glucose-derived carbon atoms for FA synthesis by reducing the expression of LDH-A, which diverts pyruvate metabolism away from the Krebs’ cycle. However, during T-cell activation, IDO has been shown to decrease the glucose influx into the cells and to decelerate glycolysis at many points upstream of the pyruvate.22,23 Hence, because of the limited amounts of pyruvate formed, the role of glucose-derived carbon atoms for FA synthesis may be negligible.

During T-cell activation, cells rely on glutaminolysis to preserve the carbon atom supply for the Krebs’ cycle.3 Indeed, activation of CD4+ T cells resulted in increased expression of the key enzymes of the glutaminolysis pathway GLS1 and GLS2. Co-treatment of activated CD4+ T cells with TRP reduced the levels of both glutaminase isoenzymes. In MLRs, the IDO inhibitor 1-MT increased GLS1 and GLS2 expression. Considering that through glutaminolysis and the reverse of two Krebs’ cycle reactions that convert α-ketoglutarate (α-KG) to citrate into the mitochondria carbon atoms may become available for FA synthesis,33 our results indicate that IDO decreases the availability of carbon atoms for FA synthesis by reducing the levels of GLS1 and GLS2.

In conclusion, IDO depletes l-tryptophan, activates GCN2 kinase and down-regulates key enzymes directly or indirectly involved in FA synthesis in primary human CD4+ T cells. This may contribute to IDO-induced inhibition of CD4+ T-cell proliferation and differentiation into effector cell lineages.

Authors’ contribution

T.E. and I.S. designed the study, T.E. and G.P. performed the experiments, T.E., G.P., G.A, V.L. and I.S. interpreted the results and contributed to the writing of the manuscript.

Funding

None, the project was supported by the resources of our department.

Acknowledgments

None.

Disclosures

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References

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