Ramgopal, A. | bioRxiv Science

bioRxiv2023

AMPK Drives Both Glycolytic and Oxidative Metabolism in T Cells During Graft-versus-host Disease

Allogeneic T cells reprogram their metabolism during acute graft-versus-host disease (GVHD) in a process reliant on the cellular energy sensor AMP-activated protein kinase (AMPK). Deletion of AMPK in donor T cells limits GVHD but still preserves homeostatic reconstitution and graft-versus-leukemia (GVL) effects. In the current studies, murine T cells lacking AMPK decreased oxidative metabolism at early timepoints post-transplant and were also unable to mediate a compensatory increase in glycolysis following inhibition of the electron transport chain. Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and following expansion in vivo in a modified model of GVHD. Immunoprecipitation of proteins from day 7 allogeneic T cells, using an antibody specific to phosphorylated AMPK targets, recovered lower levels of multiple glycolysis-related proteins including the glycolytic enzymes aldolase, enolase, pyruvate kinase M (PKM), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Functionally, murine T cells lacking AMPK exhibited impaired aldolase activity following anti-CD3/CD28 stimulation and a decrease in GAPDH activity on day 7 post-transplant. Importantly, these changes in glycolysis correlated with an impaired ability of AMPK KO T cells to produce significant amounts of interferon gamma (IFN{gamma}) upon antigenic re-stimulation. Together these data highlight a significant role for AMPK in controlling oxidative and glycolytic metabolism in both murine and human T cells during GVHD and endorse further study of AMPK inhibition as a potential target for future clinical therapies. KEY POINTSAMPK plays a key role in driving both and oxidative and glycolytic metabolism in T cells during graft-versus-host disease (GVHD) Absence of AMPK simultaneously impairs both glycolytic enzyme activity, most notably by aldolase, and interferon gamma (IFN{gamma}) production

immunology↗

bioRxiv2022

Overexpression of AMPKγ2 increases AMPK signaling to augment human T cell metabolism and function

T cell-based cellular therapies benefit from a product with reduced differentiation and enhanced oxidative metabolism. Methods to achieve this balance without negatively impacting T cell expansion or impairing T cell function have proven elusive. AMP-activated protein kinase (AMPK) is a cellular energy sensor which promotes mitochondrial health and improves oxidative metabolism. We hypothesized that increasing AMPK activity in human T cells would augment their oxidative capacity, creating an ideal product for adoptive cellular therapies. Lentiviral transduction of the regulatory AMPK{gamma}2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates (OCR), higher maximal OCR, and augmented spare respiratory capacity. These changes were accompanied by increased mitochondrial density and elevated expression of proteins involved in mitochondrial fusion. AMPK{gamma}2-transduction also increased T cell glycolytic activity. This combination of metabolic reprogramming enhanced in vitro T cell expansion while promoting memory T cell yield. Finally, when activated under decreasing glucose conditions, AMPK{gamma}2-transduced T cells maintained higher levels of both proliferation and inflammatory cytokine production. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPK{gamma}2 can improve the expansion and function of human T cells for subsequent use in adoptive cellular therapies. Key pointsLentiviral Transduction of AMPK{gamma}2 increases oxidative metabolism in human T cells AMPK{gamma}2 transduction enhances in vitro proliferation without inducing exhaustion AMPK{gamma}2-transduced T cells function better under low glucose conditions

immunology↗