Pf781 Fatty Acid Oxidation and Mitochondrial Oxidative Phosphorylation Regulate T‐cell Effector Function During Graft‐versus‐host Disease

Abstract

Background:Upon activation, effector T‐cells preferably utilize glucose for rapid and extensive proliferation, and cytokine production. Until recently, it has been thought that extracellular fatty acid (FA) uptake and oxidation are severely reduced in alloreactive T‐cells; however, recent studies have suggested that lipid metabolism is rather increased in alloreactive T‐cells, and that metabolic pathways of FA can be a promising therapeutic target for GVHD.Aims:To determine the role of lipid metabolism in human alloreactive T‐cells, we investigated the metabolic changes in human T‐cells in vivo using xenogeneic GVHD models.Methods:NOG mice received 250cGy of total body irradiation (TBI) and were subsequently injected intravenously with human pan T‐cells. Under this condition, all mice developed severe GVHD and died within 2 weeks. Human T‐cells were harvested from GVHD target organs of mice at day 9 after transplantation. For the measurement of glucose and FA uptake by flow cytometry, cells were stained with fluorescent‐labeled deoxyglucose analogue (2‐NBDG) and long‐chain FA analogue (BODIPY 500/510 C12), respectively. PCR array and extracellular flux analysis were performed according to the manufacturer's instructions.Results:Glucose uptake was significantly increased in human T‐cells obtained from GVHD mice as expected. Extracellular FA uptake was also increased in human T‐cells of GVHD mice, and was associated with cell proliferation rate. Effector memory T‐cells followed by central memory T‐cells showed a higher FA uptake than did naive T‐cells. These findings were similarly observed in both CD4+ and CD8+ T‐cells. Extracellular uptake of FA as well as glucose in T‐cells was significantly higher in MHC+/+ GVHD mice compared with MHC−/− GVHD mice, suggesting that the recognition of host MHC molecules is the initial trigger for metabolic shift in alloreactive T‐cells. Quantitative real‐time PCR analysis demonstrated up‐regulation of mRNAs encoding the enzymes involved in FA transport including carnitine palmitoyltransferase (CPT1B), FA binding protein (FABP1–4, FABP6, and FABP7), and FA oxidation (FAO) pathway including acyl‐CoA synthase (ACSBG2) and acyl‐CoA dehydrogenase (ACAD9–11, ACADS, and ACADL) in human T‐cells of GVHD mice. Similarly, the expression of genes encoding the enzymes in triacylglycerol metabolism such as glycerol kinase (GK, GK2) and lipoprotein lipase (LPL) was up‐regulated in GVHD mice. Furthermore, the expression of genes associated with mevalonate pathways such as HMG‐CoA synthase (HMGCS1, HMGCS2), was also upregulated. These observations suggest that alloreactive T‐cells increase lipid hydrolysis, mitochondrial FA transport, and FAO, resulting in greater utilization of intra‐ and extracellular FA. Extracellular flux analysis demonstrated that mitochondrial FA transport blockade by specific CPT1 inhibitor, etomoxir, significantly decreased the maximum respiration and spare respiratory capacity in human T‐cells in MHC+/+ GVHD mice but not in MHC−/− GVHD mice. Even after glucose deprivation or inhibition of mitochondrial pyruvate transport by UK5099, the oxygen consumption rate was precipitously reduced by inhibition of CPT1, suggesting that increasing FAO supported oxidative phosphorylation (OXPHOS) in alloreactive T‐cells of GVHD mice.Summary/Conclusion:Our results clearly indicate that alloreactive T‐cells increase FA uptake, intra‐ and extracellular lipid metabolism, and OXPHOS, and suggest that lipid metabolic processes in T‐cells have potential as a therapeutic target for GVHD.image