Early changes in the metabolic profile of activated CD8(+) T cells

Abstract

Background: Antigenic stimulation of the T cell receptor (TCR) initiates a change from a resting state into an activated one, which ultimately results in proliferation and the acquisition of effector functions. To accomplish this task, T cells require dramatic changes in metabolism. Therefore, we investigated changes of metabolic intermediates indicating for crucial metabolic pathways reflecting the status of T cells. Moreover we analyzed possible regulatory molecules required for the initiation of the metabolic changes.

Results: We found that proliferation inducing conditions result in an increase in key glycolytic metabolites, whereas the citric acid cycle remains unaffected. The upregulation of glycolysis led to a strong lactate production, which depends upon AKT/PKB, but not mTOR. The observed upregulation of lactate dehydrogenase results in increased lactate production, which we found to be dependent on IL-2 and to be required for proliferation. Additionally we observed upregulation of Glucose-transporter 1 (GLUT1) and glucose uptake upon stimulation, which were surprisingly not influenced by AKT inhibition.

Conclusions: Our findings suggest that AKT plays a central role in upregulating glycolysis via induction of lactate dehydrogenase expression, but has no impact on glucose uptake of T cells. Furthermore, under apoptosis inducing conditions, T cells are not able to upregulate glycolysis and induce lactate production. In addition maintaining high glycolytic rates strongly depends on IL-2 production.

Keywords: AKT/PKB; Aerobic glycolysis; Lactate; T-cell activation.

Fig. 1

Stimulation of CD8⁺ T cells with antibodies leads to rapid ATP consumption. Purified CD8⁺ T cells were treated with either soluble CD3/CD8 mAbs or OT-I tetramers (PMHC) for the indicated time periods. a Cellular ATP production was analyzed using the ATP-Assay Kit. n = 6 (b) AMPK activation was determined by Western blotting. Phospho-ERK staining was included to show effective stimulation. Total AMPK and actin are included as loading controls. c Quantification of AMPK phosphorylation within 60 min after stimulation, n = 3

Fig. 2

Stimulation of CD8⁺ T cells with tetramers leads to enhanced glycolysis with no significant change in the citric acid cycle. Purified CD8⁺ T cells were treated with soluble CD3/CD8 mAbs or OT-I tetramers for the indicated time periods. Samples were analyzed by MS coupled high-performance anion-exchange chromatography for intermediates of glycolysis – Fructose-1,6-bisphosphate (F-1,6-bp), 3-phosphoglycerate (3-PG) and pyruvate (a) and the citric acid cycle – a-ketoglutarate, malate and fumarate (b). n = 5

Fig. 3

Enhanced glycolysis after stimulation of CD8⁺ Tcells leads to production of lactate. Purified CD8⁺ T cells were stimulated with OT-I tetramers or soluble CD3/CD8 mAbs (a) or tetramers carrying the Q4H7 peptide (b) for the indicated time periods. Samples were analyzed for production of lactate. n = 4

Fig. 4

Inhibition of lactate production leads to decreased proliferation/IL-2 is necessary for lactate production. Purified CD8⁺ T cells were treated with OT-I tetramers for the indicated time periods. Samples were analyzed for proliferation (a) and lactate production (b) in presence of rotenone or oxamate. Samples were analyzed for lactate production in presence of IL-2 or anti-IL-2 (c). n = 3

Fig. 5

CTLA4-/- OT-I T cells showed no decrease in lactate production and IL-2 production and CTLA-4 mediated reduction of surface CD25 expression in WT CD8 T cells. Purified CD8+ T cells from CTLA-/- or CTL4+/+ mice were stimulated with OT-I tetramers for indicated time points, cells were analyzed for intracellular lactate concentrations (a), intracellular IL-2 concentrations by ELISA (b), and (c) Naïve (CD62Lhigh) CD8+ T cells from WT mice were stimulated with anti-CD3/CD28/CTLA-4 or anti-CD3/CD28/Isotype control-coupled microspheres. Surface expression of CD25 was detected by flow cytometry. Geomean numbers of CD25 fluorescence are depicted for the respective conditions

Fig. 6

Inhibition of AKT abrogates lactate production. Purified CD8⁺ T cells were treated with OT-I tetramers for the indicated time periods. Samples were analyzed for production of lactate in presence of the AKT-Inhibitor AKT 8 and the mTOR-inhibitor Rapamycin (a). Samples were analyzed for upregulation of LDH by Western blotting in the presence of 3 different AKT inhibitors (b). Expression of GLUT1 after tetramer stimulation in presence and absence of AKT inhibitor was analyzed via FACS (c) and calculated from 3 independent experiments (d). Glucose uptake was analyzed 24 h after stimulation of CD8⁺ T cells with tetramers in the absence and presence of AKT inhibitor and apigenine (e) and calculated from 4 independent experiments (f)