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

BACKGROUND Allogeneic hematopoietic stem cell transplantation (AlloHSCT) is a curative therapy for many hematological disorders including primary immunodeficiencies and relapsed/refractory leukemia. However, the therapeutic application of AlloHSCT is limited by acute graft-versus-host-disease (GVHD), where donor T cells recognize and destroy recipient tissues in the skin, liver, and gastrointestinal tract. GVHD occurs following 30-50% of allogeneic transplants and is a leading cause of non-relapse morbidity and mortality. Recent efforts to treat GVHD have improved but attempts to prevent GVHD remain an important area of ongoing investigation.

Following transplantation, alloreactive T cells exhibit heightened energy requirements that present a promising area for therapeutic intervention. Previously, we have shown that murine T cells upregulate activity of the cellular energy sensor AMPK-activated protein kinase (AMPK) during GVHD initiation and furthermore, that transfer of AMPK-deficient T cells increased recipient survival while preserving leukemia clearance and immune reconstitution. Whether these findings translate to human cells is unknown. Here, we report successful targeting of AMPK in primary human T cells and demonstrate clear similarities in phenotype between murine and human T cells lacking AMPK.

METHODS To delete AMPK, primary human T cells were isolated from healthy donors and electroporated with Cas 9 ribonuclear protein complexes targeting the AMPKa1 locus. Deletion of AMPK was assessed via DNA sequencing (TIDE analysis) and phosphorylation of downstream target Unc-51-like kinase (ULK1) by immunoblot. Oxidative metabolism and spare respiratory capacity (SRC) were determined via extracellular flux analysis (Agilent Seahorse). Mitochondrial transmembrane potential (TMRM) and cell phenotype (mitochondrial density, apoptosis) were evaluated by flow cytometry after cells stimulation. Intracellular cytokine expression was assessed following 72 hours in a mixed lymphocyte reaction (MLR), followed by re-stimulation with PMA/Ionomycin in the presence of Brefeldin A. AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients for 7 days for comparative analysis.

RESULTS CRISPR treatment of primary human T cells efficiently deleted AMPK as measured by both DNA sequencing (92.3% insertion/deletion frequency by TIDE analysis) and immunoblot analysis of downstream ULK1 phosphorylation. When re-stimulated in physiological glucose (5.5 mM), AMPK-deficient human T cells significantly decreased mitochondrial respiration (Fig. 1A) as measured by both maximal oxygen consumption (174.6 ± 27.73 vs 277.3 ± 22.88, p<0.0001) and spare respiratory capacity (SRC, 126.9 ± 22.78 vs 207.1 ± 19.68, p<0.0001). This result was strikingly similar to the metabolism of post-transplant murine T cells lacking AMPK, which exhibited a proportional decrease in SRC (-6.499 ± 5.156 vs 29.11 ± 7.064, p<0.0001) and maximal oxygen consumption (46.82 ± 10.11 vs 80.01 ± 6.946, p<0.0001). AMPK-deficient human T cells also experienced decreased cellular viability (43.4% vs 51.8%) as well as limited mitochondrial membrane potential (Fig. 1B); however, no differences in mitochondrial density were observed. Upon co-culture with allogeneic antigen presenting cells, AMPK-deficient human CD4⁺ T cells produced lower levels of the pro-inflammatory cytokines TNFa (97.7 MFI vs 209 MFI) and IL-2 ( 391 vs 454 MFI).

DISCUSSION Here, we demonstrate a method to effectively delete AMPK in human T cells, a modification which decreases oxidative metabolism identical to what is observed in AMPK-deficient murine T cells. Specifically, human T cells lacking AMPK decreased their maximal mitochondrial respiration and spare respiratory capacity, a result we attribute to decreased mitochondrial transmembrane potential without changes in mitochondrial mass. Together with decreased pro-inflammatory cytokine production, we predict these changes will limit the viability and function of alloreactive T cells in vivo. As such, future studies will assess the disease potential of AMPK-deficient human T cells in a modified model of xenogeneic GVHD.

No relevant conflicts of interest to declare.