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

Cellular therapies are currently employed to treat a variety of disease processes. For T cell-based therapies, success often relies on the metabolic fitness of the T cell product, where cells with enhanced metabolic capacity demonstrate improved in vivo efficacy. AMP-activated protein kinase (AMPK) is a cellular energy sensor which combines environmental signals with cellular energy status to enforce efficient and flexible metabolic programming. 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 AMPKγ2 subunit stably enhanced intrinsic AMPK signaling and promoted mitochondrial respiration with increased basal oxygen consumption rates, higher maximal oxygen consumption rate, and augmented spare respiratory capacity. These changes were accompanied by increased proliferation and inflammatory cytokine production, particularly within restricted glucose environments. Introduction of AMPKγ2 into bulk CD4 T cells decreased RNA expression of canonical Th2 genes, including the cytokines interleukin (IL)-4 and IL-5, while introduction of AMPKγ2 into individual Th subsets universally favored proinflammatory cytokine production and a downregulation of IL-4 production in Th2 cells. When AMPKγ2 was overexpressed in regulatory T cells, both in vitro proliferation and suppressive capacity increased. Together, these data suggest that augmenting intrinsic AMPK signaling via overexpression of AMPKγ2 can improve the expansion and functional potential of human T cells for use in a variety of adoptive cellular therapies.

Immunometabolism in haematopoietic stem cell transplantation and adoptive cellular therapies.

PURPOSE OF REVIEW: Controlling T cell activity through metabolic manipulation has become a prominent feature in immunology and practitioners of both adoptive cellular therapy (ACT) and haematopoietic stem cell transplantation (HSCT) have utilized metabolic interventions to control T cell function. This review will survey recent metabolic research efforts in HSCT and ACT to paint a broad picture of immunometabolism and highlight advances in each area. RECENT FINDINGS: In HSCT, recent publications have focused on modifying reactive oxygen species, sirtuin signalling or the NAD salvage pathway within alloreactive T cells and regulatory T cells. In ACT, metabolic interventions that bolster memory T cell development, increase mitochondrial density and function, or block regulatory signals in the tumour microenvironment (TME) have recently been published. SUMMARY: Metabolic interventions control immune responses. In ACT, efforts seek to improve the in-vivo metabolic fitness of T cells, while in HSCT energies have focused on blocking alloreactive T cell expansion or promoting regulatory T cells. Methods to identify new, metabolically targetable pathways, as well as the ability of metabolic biomarkers to predict disease onset and therapeutic response, will continue to advance the field towards clinically applicable interventions.

Fatty acid oxidation in immune function.

Cellular metabolism is a crucial determinant of immune cell fate and function. Extensive studies have demonstrated that metabolic decisions influence immune cell activation, differentiation, and cellular capacity, in the process impacting an organism's ability to stave off infection or recover from injury. Conversely, metabolic dysregulation can contribute to the severity of multiple disease conditions including autoimmunity, alloimmunity, and cancer. Emerging data also demonstrate that metabolic cues and profiles can influence the success or failure of adoptive cellular therapies. Importantly, immunometabolism is not one size fits all; and different immune cell types, and even subdivisions within distinct cell populations utilize different metabolic pathways to optimize function. Metabolic preference can also change depending on the microenvironment in which cells are activated. For this reason, understanding the metabolic requirements of different subsets of immune cells is critical to therapeutically modulating different disease states or maximizing cellular function for downstream applications. Fatty acid oxidation (FAO), in particular, plays multiple roles in immune cells, providing both pro- and anti-inflammatory effects. Herein, we review the major metabolic pathways available to immune cells, then focus more closely on the role of FAO in different immune cell subsets. Understanding how and why FAO is utilized by different immune cells will allow for the design of optimal therapeutic interventions targeting this pathway.

AMPK drives both glycolytic and oxidative metabolism in murine and human T cells during graft-versus-host disease.

ABSTRACT: Allogeneic T cells reprogram their metabolism during acute graft-versus-host disease (GVHD) in a process involving the cellular energy sensor adenosine monophosphate (AMP)-activated protein kinase (AMPK). Deletion of AMPK in donor T cells limits GVHD but still preserves homeostatic reconstitution and graft-versus-leukemia effects. In the current studies, murine AMPK knock-out (KO) T cells decreased oxidative metabolism at early time points posttransplant and lacked a compensatory increase in glycolysis after inhibition of the electron transport chain. Immunoprecipitation using an antibody specific to phosphorylated targets of AMPK determined that AMPK modified interactions of several glycolytic enzymes including aldolase, enolase, pyruvate kinase M, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), with enzyme assays confirming impaired aldolase and GAPDH activity in AMPK KO T cells. Importantly, these changes in glycolysis correlated with both an impaired ability of AMPK KO T cells to produce significant amounts of interferon gamma upon antigenic restimulation and a decrease in the total number of donor CD4 T cells recovered at later times posttransplant. Human T cells lacking AMPK gave similar results, with glycolytic compensation impaired both in vitro and after expansion in vivo. Xenogeneic GVHD results also mirrored those of the murine model, with reduced CD4/CD8 ratios and a significant improvement in disease severity. Together these data highlight a significant role for AMPK in controlling oxidative and glycolytic metabolism in both murine and human T cells and endorse further study of AMPK inhibition as a potential clinical target for future GVHD therapies.

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 (IFNγ) 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 POINTS: AMPK 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 (IFNγ) production.