Deletion of AMPK Reduces Oxidative Metabolism in Gvhd-Causing Human T Cells

Background Following allogeneic transplantation, alloreactive T cells exhibit heightened metabolic requirements that represent a promising area for therapeutic intervention. We have shown previously that during acute graft-versus-host-disease (aGVHD), murine T cells upregulate activity of the cellular energy sensor AMP-activated protein kinase (AMPK). Importantly, transfer of allogeneic T cells deficient in AMPK (AMPK KO) increased recipient survival while still preserving leukemia clearance. In the current studies, we demonstrate a novel role for AMPK in regulating glycolysis, with significant therapeutic implications for the prevention and treatment of aGVHD. Results AMPK KO T cells recovered on day 7 post-transplant were placed on a Seahorse metabolic analyzer, where they exhibited decreased oxidative metabolism and impaired glycolytic compensation following inhibition of the electron transport chain (Fig. 1A). 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 and GAPDH (Fig. 1B). Functionally, T cells lacking AMPK exhibited impaired aldolase activity following anti-CD3/CD28 stimulation, with decreased GAPDH activity in day 7 post-transplant T cells. Importantly, these changes in glycolysis correlated with an impaired ability of AMPK KO T cells to produce significant amounts of IFNγ upon antigenic re-stimulation and similar glycolytic changes were seen in human T cells following AMPK removal by CRISPR targeting. Conclusion AMPK plays a role in directing both oxidative and glycolytic metabolism in murine and human T cells during GVHD. The current data suggest a mechanism for this post-transplant decrease in glycolysis through the novel observation that a set of glycolysis-related enzymes are immunoprecipitated together at much lower rates from AMPK KO T cells. These findings support a model where AMPK phosphorylation provides a nidus for important interactions among glycolytic enzymes. Important to our understanding of GVHD, diminished GAPDH activity in AMPK KO T cells also tracks with decreased IFNγ production, suggesting that GAPDH freed from glycolysis may instead sequester IFNγ transcripts and thereby limit protein translation. These results offer a mechanistic explanation for the decreased severity of GVHD seen following transplantation of AMPK KO T cells and provide a strong rationale for further development of AMPK inhibition as a clinically translatable strategy for GVHD prevention and treatment.