D-2-Hydroxyglutarate Inhibits Calcineurin Phosphatase Activity to Abolish NF-AT Activation and IL-2 Induction in Stimulated Lymphocytes

Gliomas expressing mutant isocitrate dehydrogenases excessively synthesize d-2-hydroxyglutarate (D2HG), suppressing immune surveillance. A portion of this D2HG is released from these tumor cells, but the way environmental D2HG inhibits lymphocyte function is undefined. We incubated human PBLs or Jurkat T cells with D2HG at concentrations present within and surrounding gliomas or its obverse l-2-hydroxyglutarate (L2HG) stereoisomer. We quantified each 2HG stereoisomer within washed cells by N-(p-toluenesulfonyl)-l-phenylalanyl chloride derivatization with stable isotope-labeled D2HG and L2HG internal standards, HPLC separation, and mass spectrometry. D2HG was present in quiescent cells and was twice as abundant as L2HG. Extracellular 2HG rapidly increased intracellular levels of the provided stereoisomer by a stereoselective, concentration-dependent process. IL-2 expression, even when elicited by A23187 and PMA, was abolished by D2HG in a concentration-dependent manner, with significant reduction at just twice its basal level. In contrast, L2HG was only moderately inhibitory. IL-2 expression is regulated by increased intracellular Ca2+ that stimulates calcineurin to dephosphorylate cytoplasmic phospho-NF-AT, enabling its nuclear translocation. D2HG abolished stimulated expression of a stably integrated NF-AT-driven luciferase reporter that precisely paralleled its concentration-dependent inhibition of IL-2. D2HG did not affect intracellular Ca2+. Rather, surface plasmon resonance showed D2HG, but not L2HG, bound calcineurin, and D2HG, but not L2HG, inhibited Ca2+-dependent calcineurin phosphatase activity in stimulated Jurkat extracts. Thus, D2HG is a stereoselective calcineurin phosphatase inhibitor that prevents NF-AT dephosphorylation and so abolishes IL-2 transcription in stimulated lymphocytes. This occurs at D2HG concentrations found within and adjacent to gliomas independent of its metabolic or epigenetic transcriptional regulation.