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.

The Protective Effects of Calcineurin on Pancreatitis in Mice Depend on the Cellular Source.

BACKGROUND & AIMS: Calcineurin is a ubiquitously expressed central Ca2+-responsive signaling molecule that mediates acute pancreatitis, but little is known about its effects. We compared the effects of calcineurin expression by hematopoietic cells vs pancreas in mouse models of pancreatitis and pancreatitis-associated lung inflammation. METHODS: We performed studies with mice with hematopoietic-specific or pancreas-specific deletion of protein phosphatase 3, regulatory subunit B, alpha isoform (PPP3R1, also called CNB1), in mice with deletion of CNB1 (Cnb1UBC△/△) and in the corresponding controls for each deletion of CNB1. Acute pancreatitis was induced in mice by administration of caerulein or high-pressure infusion of radiocontrast into biliopancreatic ducts; some mice were also given intraductal infusions of an adeno-associated virus vector that expressed nuclear factor of activated T -cells (NFAT)-luciferase into pancreas. Pancreas, bone marrow, liver, kidney, heart, and lung were collected and analyzed by histopathology, immunohistochemistry, and immunoblots; levels of cytokines were measured in serum. Mouse and human primary pancreatic acinar cells were transfected with a vector that expressed NFAT-luciferase and incubated with an agent that blocks interaction of NFAT with calcineurin; cells were analyzed by immunofluorescence. Calcineurin-mediated neutrophil chemotaxis and reactive oxygen species production were measured in neutrophils from mice. RESULTS: Mice with hematopoietic-specific deletion of CNB1 developed the same level of local pancreatic inflammation as control mice after administration of caerulein or infusion of radiocontrast into biliopancreatic ducts. Cnb1UBC△/△ mice or mice with pancreas-specific deletion of CNB1 developed less severe pancreatitis and reduced pancreatic inflammation after administration of caerulein or infusion of radiocontrast into biliopancreatic ducts compared with control mice. NFAT was activated in pancreas of Swiss Webster mice given caerulein or infusions of radiocontrast into biliopancreatic ducts. Blocking the interaction between calcineurin and NFAT did not reduce pancreatic acinar cell necrosis in response to caerulein or infusions of radiocontrast. Mice with hematopoietic-specific deletion of CNB1 (but not mice with pancreas-specific deletion of CNB1) had reduced infiltration of lung tissues by neutrophils. Neutrophil chemotaxis and production of reactive oxygen species were decreased after incubation with a calcineurin inhibitor. CONCLUSIONS: Hematopoietic and neutrophil expression of calcineurin promotes pancreatitis-associated lung inflammation, whereas pancreatic calcineurin promotes local pancreatic inflammation. The findings indicate that the protective effects of blocking or deleting calcineurin on pancreatitis are mediated by the source of its expression. This information should be used in the development of strategies to inhibit calcineurin for the prevention of pancreatitis and pancreatitis-associated lung inflammation.

Deletion of AMPK minimizes graft-versus-host disease through an early impact on effector donor T cells.

Allogeneic hematopoietic stem cell transplantation is a viable treatment for multiple hematologic diseases, but its application is often limited by graft-versus-host disease (GVHD), where donor T cells attack host tissues in the skin, liver, and gastrointestinal tract. Here, we examined the role of the cellular energy sensor AMP kinase (AMPK) in alloreactive T cells during GVHD development. Early posttransplant, AMPK activity increased more than 15-fold in allogeneic T cells, and transplantation of T cells deficient in both AMPKα1 and AMPKα2 decreased GVHD severity in multiple disease models. Importantly, a lack of AMPK lessened GVHD without compromising antileukemia responses or impairing lymphopenia-driven immune reconstitution. Mechanistically, absence of AMPK decreased both CD4+ and CD8+ effector T cell numbers as early as day 3 posttransplant, while simultaneously increasing regulatory T cell (Treg) percentages. Improvements in GVHD resulted from cell-intrinsic perturbations in conventional effector T cells as depletion of donor Tregs had minimal impact on AMPK-related improvements. Together, these results highlight a specific role for AMPK in allogeneic effector T cells early posttransplant and suggest that AMPK inhibition may be an innovative approach to mitigate GVHD while preserving graft-versus-leukemia responses and maintaining robust immune reconstitution.