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.

AMPK agonism optimizes the in vivo persistence and anti-leukemia efficacy of chimeric antigen receptor T cells.

BACKGROUND: Chimeric antigen receptor T cell (CART) therapy has seen great clinical success. However, up to 50% of leukemia patients relapse and long-term survivor data indicate that CART cell persistence is key to enforcing relapse-free survival. Unfortunately, ex vivo expansion protocols often drive metabolic and functional exhaustion, reducing in vivo efficacy. Preclinical models have demonstrated that redirecting metabolism ex vivo can improve in vivo T cell function and we hypothesized that exposure to an agonist targeting the metabolic regulator AMP-activated protein kinase (AMPK), would create CARTs capable of both efficient leukemia clearance and increased in vivo persistence. METHODS: CART cells were generated from healthy human via lentiviral transduction. Following activation, cells were exposed to either Compound 991 or DMSO for 96 hours, followed by a 48-hour washout. During and after agonist treatment, T cells were harvested for metabolic and functional assessments. To test in vivo efficacy, immunodeficient mice were injected with luciferase+ NALM6 leukemia cells, followed one week later by either 991- or DMSO-expanded CARTs. Leukemia burden and anti-leukemia efficacy was assessed via radiance imaging and overall survival. RESULTS: Human T cells expanded in Compound 991 activated AMPK without limiting cellular expansion and gained both mitochondrial density and improved handling of reactive oxygen species (ROS). Importantly, receipt of 991-exposed CARTs significantly improved in vivo leukemia clearance, prolonged recipient survival, and increased CD4+ T cell yields at early times post-injection. Ex vivo, 991 agonist treatment mimicked nutrient starvation, increased autophagic flux, and promoted generation of mitochondrially-protective metabolites. DISCUSSION: Ex vivo expansion processes are necessary to generate sufficient cell numbers, but often promote sustained activation and differentiation, negatively impacting in vivo persistence and function. Here, we demonstrate that promoting AMPK activity during CART expansion metabolically reprograms cells without limiting T cell yield, enhances in vivo anti-leukemia efficacy, and improves CD4+ in vivo persistence. Importantly, AMPK agonism achieves these results without further modifying the expansion media, changing the CART construct, or genetically altering the cells. Altogether, these data highlight AMPK agonism as a potent and readily translatable approach to improve the metabolic profile and overall efficacy of cancer-targeting T cells.

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.

Enhanced visible-light photocatalytic activity of perylene diimide (PDI) supramolecular nanorods with Pt QDs deposited in situ.

Well-dispersed Pt quantum dots (QDs) were the first to be successfully deposited onto a PDI supramolecular nanorods surface via a simple in situ chemical reduction. Under visible light irradiation, Pt QDs/PDI composites displayed excellent photocatalytic property in the degradation of phenol. The optimum 1 wt% Pt QDs/PDI composite was found to be 6.2 times greater than pure PDI supramolecular nanorods for the degradation rate constant (k). The enhanced photocatalytic performance can be attributed to the rapid transfer and efficient separation of photogenerated carriers, originating from the effective trapping and transporting of electrons by Pt QDs. At the same time, Pt QDs were also loaded as active sites during the photocatalytic reaction. Moreover, the 1 wt% Pt QDs/PDI composite was found to have high photocatalytic stability and cycle utilization, suggesting its great potential in the area of water environmental purification.