Glycolytic requirement for NK cell cytotoxicity and cytomegalovirus control

Abstract

NK cell activation has been shown to be metabolically regulated in vitro; however, the role of metabolism during in vivo NK cell responses to infection is unknown. We examined the role of glycolysis in NK cell function during murine cytomegalovirus (MCMV) infection and the ability of IL-15 to prime NK cells during CMV infection. The glucose metabolism inhibitor 2-deoxy-ᴅ-glucose (2DG) impaired both mouse and human NK cell cytotoxicity following priming in vitro. Similarly, MCMV-infected mice treated with 2DG had impaired clearance of NK-specific targets in vivo, which was associated with higher viral burden and susceptibility to infection on the C57BL/6 background. IL-15 priming is known to alter NK cell metabolism and metabolic requirements for activation. Treatment with the IL-15 superagonist ALT-803 rescued mice from otherwise lethal infection in an NK-dependent manner. Consistent with this, treatment of a patient with ALT-803 for recurrent CMV reactivation after hematopoietic cell transplant was associated with clearance of viremia. These studies demonstrate that NK cell-mediated control of viral infection requires glucose metabolism and that IL-15 treatment in vivo can reduce this requirement and may be effective as an antiviral therapy.

Keywords: Immunology; NK cells.

Figure 1. Glucose metabolism blockade during activation decreases NK cell proliferation and cytotoxicity.

Purified NK cells were cultured for 72 hours in 100 ng/ml IL-15 without (black) or with 1 mM 2-deoxy-ᴅ-glucose (2DG, green). (A) Representative extracellular flux assay showing glycolytic rate as estimated by extracellular acidification rate (ECAR); glycolytic stress test shows baseline ECAR in glucose-free media and after addition of glucose, maximal ECAR after addition of oligomycin, and nonglycolytic acidification after 2DG. Summary data shows ECAR, oxygen consumption rate (OCR), and the OCR/ECAR ratio normalized to control (n = 4/group in 3 separate experiments, ratio paired t test performed on raw data). (B) Representative histogram shows CFSE dilution of proliferating NK cells. Summary graph shows the percentage of NK cells proliferated (n = 5 experiments, paired t test). (C) NK cells activated in IL-15 ± 2DG were assessed for their ability to kill Ba/F3-m157 target cells at different effector/target ratios (ratios represent Ly49H⁺ NK cells/targets), n = 8/group, 4 experiments, 2-way ANOVA. Data show mean ± SEM. *P < 0.05, ***P < 0.001.

Figure 2. In vitro treatment with 2DG decreases NK cell production of granzyme B and alters actin accumulation at the immunological synapse.

Purified NK cells were cultured for 72 hours in 100 ng/ml IL-15 with or without 1 mM 2-deoxy-ᴅ-glucose (2DG). (A) Representative flow histogram shows granzyme B (Gzmb) levels in naive (filled) and activated NK cells cultured without (black) or with (green) 1 mM 2DG. Summary data are normalized to mean fluorescence intensity (MFI) of naive NK cells (n = 9/group, 5 separate experiments, ratio paired t test performed on raw data). (B) NK cells were incubated at a 1:1 ratio with Ba/F3-m157 target cells for various times; conjugates were detected by flow cytometry. Representative flow cytometry shows CFSE⁺ and CellTrace Violet (CTV)⁺ conjugates, and conjugation curve shows percentage of CFSE⁺ cells conjugated (n = 2–3 technical replicates, representative of 3 separate experiments, 2-way ANOVA). (C–F) NK cells were incubated with Ba/F3-m157 cells for 40 minutes at a 2:1 ratio. (C and D) Fluorescence microscopy of NK:Ba/F3-m157 conjugates show convergence of granules (green, Gzmb), Ly49H accumulation at the synapse (yellow), polarization of the microtubule organizing center (MTOC, blue) toward the synapse, and actin accumulation (red). Scale bar: 2 μm. Representative images for the range of control and 2DG-treated NK cells are shown. (E) Quantification of actin accumulation by area × intensity of synapse – (NK + target) (n = 25–27 conjugates/group, 4 experiments, distribution analyzed by Kolmogorov-Smirnov test). (F) Quantification of MTOC-synapse distance in μm, not significant by 2-tailed Welch’s t test for means or Kolmogorov-Smirnov test (n = 58–60 conjugates/group, 5 experiments). For A and B, data show mean ± SEM. For E and F, the gray box displays median, 25th, and 75th percentiles. *P < 0.05, ***P < 0.001.

Figure 3. 2DG treatment in vivo causes decreased target clearance during MCMV infection.

Female WT C57BL/6 mice were infected with 1 × 10⁵ PFU murine cytomegalovirus (MCMV), treated daily with 2-deoxy-ᴅ-glucose (2DG) or PBS, and analyzed on day 2 after infection. (A) Equal numbers of labeled nontarget splenocytes and target m157-transgenic splenocytes were injected i.v., and target clearance in the spleen was measured 4 hours later (n = 7–12 individuals/group, 2 separate experiments). (B) The number and percentage of NK cells isolated from the spleen or liver (n = 7–8/group, 2 experiments). (C) Flow plots and mean fluorescence intensity (Gzmb MFI) measured by flow cytometry in Ly49H⁺ (solid) and Ly49H^- (hatched) NK cells from the spleen or liver (n = 4/group, representative of 2 experiments). (D) CD107a expression on splenocytes (n = 7–8/group, 2 experiments). Data show mean ± SEM. All statistical analyses are 1-way ANOVA with Tukey’s multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. 2DG treatment in vivo confers MCMV susceptibility.

Female WT C57BL/6 mice were infected with 1 × 10⁵ PFU murine cytomegalovirus (MCMV) and treated daily with 2-deoxy-ᴅ-glucose (2DG) or PBS. Viral copy number by quantitative PCR from the spleen and liver was measured on (A) day 2 or (B) day 4 after infection, shown as copies of MCMV ie1 gene/copies of β-actin × 1,000 (n = 3–8 individuals/group, 2 experiments, 1-way ANOVA on log-transformed data). (C) Total numbers of lymphocytes and NK cells from the spleen or liver at day 4 after infection. (D) Representative flow cytometry and summary data showing the percentage of BrdU⁺Ly49H⁺ NK cells (open) and Ly49H^– NK cells (hatched), assessed 3 hours after BrdU injection. For C and D, n = 7–8/group, 2 experiments, 1-way ANOVA with Tukey’s multiple comparison test. (E) Survival of uninfected mice compared with infected mice treated with 2DG or PBS (n = 20/group, 4 experiments, log-rank Mantel-Cox test). Data shown as mean ± SEM or survival. *P < 0.05, ***P < 0.001.

Figure 5. NK activation with ALT-803 rescues MCMV susceptibility caused by 2DG treatment.

(A) Female WT C57BL/6 mice were treated with 5 μg ALT-803 twice, NK cells were purified from their spleens, and a glycolytic stress test was performed. Representative graph showing baseline, normal (+glucose), and maximal (+oligomycin) extracellular acidification rate (ECAR) in control vs. ALT-803–treated mice. Average ECAR, oxygen consumption rate (OCR), and OCR/ECAR ratio are shown after glucose addition (n = 3 individuals/group, 2 separate experiments, 2-tailed t test). (B–E) ALT-803–treated mice were infected with 1 × 10⁵ PFU murine cytomegalovirus (MCMV) and treated with 2-deoxy-ᴅ-glucose (2DG) daily. (B) Clearance of m157-transgenic targets from the spleens of infected and uninfected mice ± ALT-803 and 2DG on day 2 after infection (n = 5/group, representative of 2 experiments, 1-way ANOVA). (C) The number and percentage of NK cells isolated from the spleens or livers of mice in B. (D) Mean fluorescence intensity of granzyme B (Gzmb MFI) in all NK cells from B. (E) Viral copy number measured by quantitative PCR from the spleens and livers collected in B, shown as copies of MCMV ie1 gene/copies of β-actin × 1,000. For C–E, n = 10/group, 2 experiments, 1-way ANOVA, using log-transformed data for E. (F) Mice were given an additional dose of ALT-803 2 days after infection and followed for 10 days. Survival of mice with no treatment (gray dashed line), 2DG treatment (black), ALT-803 treatment (blue), or ALT-803 with 2DG (green) (n = 10/group, 2 experiments, log-rank Mantel-Cox test). Data shown as mean ± SEM or survival. **P < 0.01, ***P < 0.001. Asterisks in A, B, and F indicate treatment with ALT-803.

Figure 6. ALT-803 rescue of 2DG-treated mice requires NK cells, and ALT-803 priming can eliminate susceptibility to MCMV caused by mTOR inhibition.

(A) Mice were either depleted of NK cells using α-NK1.1 or treated with IgG2a; they were given 2 doses of ALT-803 and infected with 1 × 10⁵ PFU murine cytomegalovirus (MCMV). 2-Deoxy-ᴅ-glucose (2DG) was given every 24 hours, and mice were euthanized on day 2 for assessment of (B) clearance of m157-transgenic targets from the spleen and (C) viral copy number from the spleen and liver; they were measured by quantitative PCR and log-transformed (n = 10 individuals/group, 2 separate experiments, 2-tailed t test). (D) To assess susceptibility, an additional NK depletion was given at day 3 (red, n = 30); control mice were treated with IgG2a (black, n = 23). Mice were followed for 10 days (3 separate experiments). (E) Mice were infected with MCMV, given daily i.p. injections of 1.5 mg/kg rapamycin (green) or vehicle (black), and monitored for susceptibility (n = 10/group, 1 experiment). (F) ALT-803 was given to MCMV-infected, rapamycin-treated mice on the same schedule as for 2DG-treated mice, with doses at –3, –1, and +2 days relative to infection (n = 10/group, 1 experiment). Data show mean ± SEM or survival in D–F analyzed by log-rank Mantel-Cox test. ***P < 0.001. Asterisks in A and D indicate treatment with ALT-803.

Figure 7. 2DG decreases human NK cell cytotoxicity.

Human NK cells were isolated from healthy donors, enriched using RosetteSep, and cryopreserved. Cells were later thawed and activated in 100 ng/ml human IL-15 for 24 hours in various concentrations of 2DG. (A) NK cells were washed and cocultured with K562 targets for 4 hours at various E:T ratios; representative killing assay shown (n = technical duplicate, representative of 6 donors across 4 experiments, mean with replicates shown). (B and C) NK cells from control conditions and 20–40 mM 2DG were cultured at a 2:1 ratio with K562 cells and imaged after 40 minutes. (B) Quantification of actin accumulation by area × intensity of synapse – (NK + target) (n = 41–43 conjugates/group, 1 experiment). (C) Quantification of distance from the microtubule organizing center (MTOC) to synapse in μm (n = 67–61 conjugates/group, 2 experiments). Gray boxes show median, 25th, and 75th percentiles. Neither B nor C was significant by 2-tailed t test or Kolmogorov-Smirnov test.

Figure 8. ALT-803 treatment of CMV reactivation in a posthematopoietic cell transplant patient.

(A) The patient’s course of cytomegalovirus (CMV) reactivation after hematopoietic cell transplant (HCT), with periods of viremia detectable by quantitative PCR assay shown as orange bars. Posttransplant immunosuppression is shown as a blue bar. Years 1–4 after HCT marked. (B) During reactivation event 5, CMV levels responded to treatment with ALT-803, as indicated by arrows. Points below limit of detection (dashed line) at 137 copies/ml are rounded up. (C) To assess IFN-γ production by the patient NK cells, peripheral blood mononuclear cells (PBMC) were collected and cryopreserved before ALT-803 treatment or 7–14 days after the listed ALT-803 dose. PBMC were thawed and stimulated with 10 ng/ml IL-12 and 100 ng/ml IL-18 for 16 hours; they were then washed and plated for an additional 5 hours (n = 4 technical replicates, 1 experiment, shown as mean ± SEM, statistical significance in a 1-way ANOVA compared with the quantity of cells before dose 1 shown above relevant column). *P < 0.05, ***P < 0.001.