Ketone Bodies Improve Human CD8+ Cytotoxic T-Cell Immune Response During COVID-19 Infection

Abstract

Severe COVID-19 is characterized by profound CD8⁺ T-cell dysfunction, which cannot be specifically treated to date. We here investigate whether metabolic CD8⁺ T-cell reprogramming by ketone bodies could be a promising strategy to overcome the immunoparalysis in COVID-19 patients. This approach was triggered by our recent pioneering study, which has provided evidence that CD8⁺ T-cell capacity in healthy subjects could be significantly empowered by a Ketogenic Diet. These improvements were achieved by immunometabolic rewiring toward oxidative phosphorylation. We here report similar strengthening of CD8⁺ T cells obtained from severely diseased COVID-19 patients: Flow cytometry and ELISA revealed elevated cytokine expression and secretion (up to + 24%) upon ketone treatment and enhanced cell lysis capacity (+ 21%). Metabolic analyses using Seahorse technology revealed upregulated mitochondrial respiratory chain activity (+ 25%), enabling both superior energy supply (+ 44%) and higher mitochondrial reactive oxygen species signaling. These beneficial effects of ketones might represent evolutionary conserved mechanisms to strengthen human immunity. Our findings pave the road for metabolic treatment studies in COVID-19.

Keywords: COVID-19; Ketogenic Diet (KD); SARS-CoV-2; T-cell immunometabolism; cytotoxic T cell; metabolic therapy; nutritional immunology.

FIGURE 1

Beta-hydroxybutyrate enhances human T-cell immune capacity during COVID-19. Human peripheral blood mononuclear cells (PBMC) were cultivated for 5 days in RPMI containing 80 mg/dl glucose (NC) and supplemented with 10 mM D/L-beta-hydroxybutyrate (BHB). T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. (A) Flow cytometric quantification of CD8⁺ T cells expressing intracellular Granzyme B (left), n = 15 individual patients. For better visualization, a histogram example of one patient is shown. (B) Mean fluorescence intensity (MFI) Granzyme B per cell, measured in CD8⁺ T cells (right), n = 15 individual patients. (C) Protein expression of TNFα/IFNγ/Perforin/Granzyme B in the supernatant of stimulated PBMC, n = 17/14/17/18 individual patients. (D) Relative CD8⁺ cell lysis activity as measured by calcein-fluorescence of isolated CD8⁺ T cells, n = 8 individual patients. Paired t-test or Wilcoxon matched-pairs signed rank test, as appropriate. *p < 0.05, **p < 0.01.

FIGURE 2

Beta-hydroxybutyrate shifts human T-cell metabolism toward oxidative phosphorylation enabling higher mROS production. Human peripheral blood mononuclear cells (PBMC) were cultivated for 5 days in RPMI containing 80 mg/dl glucose (NC) and supplemented with 10 mM D/L-beta-hydroxybutyrate (BHB). T-cell stimulation was performed through CD3/CD28 Dynabeads at a bead:cell ratio of 1:8. CD8⁺ T cells were isolated via magnetic cell separation. (A–D) Oxygen consumption rate [OCR] (A), basal (B), maximum (C) and spare respiratory capacity (D) were measured using a Seahorse HS mini Analyzer, n = 5 individual patient samples, each performed in 2–3 technical replicates. (E) Mitochondrial mass determined via MitoTracker green, indicated by MFI FITC in human CD8⁺ T cells, n = 12 individual patient samples. Histogram depicting exemplary change of MitoTracker green. (F) Quantification of mitochondrial superoxide production using MitoSOX, displayed as MFI PE in human CD8⁺ T cells, n = 11 individual patient samples. Histogram depicting exemplary change of MitoSOX fluorescence. *p < 0.05, **p < 0.01.