Azithromycin (AZM) inhibits T-cell proliferative and cytotoxic functions by impeding energetic boost from glycolysis. (A) Sorted CD3⁺ cells from healthy-donor (HD) peripheral mononuclear blood cells were stained with carboxyfluorescein succinimidyl ester (CFSE) and treated in vitro with AZM or control (CTRL) for 24 hours before activation with anti-CD3/CD28 beads. Two days after activation, cells were retrieved from the incubator and analyzed by means of flow cytometry. CFSE staining in CD4⁺ and CD8⁺ T-cell subsets are shown in histograms (representative results) and boxplots (each dot is the median value of 3 technical replicates). Results are from 3 independent experiments with 6 independent HDs. (B) Anti-CD19 chimeric antigenic receptor (CAR) T cells were cultured with AZM or CTRL for 24 hours and co-cultured with luciferase expressing CD19⁺ NALM6 cell lines overnight. Cell lysis was quantified by means of luminescence. A representative result is shown as a dot plot, and pooled 6 independent experiments are depicted in boxplots. (C) Cell energy metabolism overview and corresponding glycolysis and oxidative phosphorylation (OXPHOS) assays from HD sorted CD4⁺ and CD8⁺ cells after 24 hours of incubation with AZM or dimethylsulfoxide (CTRL). Extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) were measured at time of activation with anti-CD3/CD28 antibody complexes and then glycolysis was inhibited by 2-deoxyglucose (2-DG). Dot plots show representative results and boxplots pooled results from 6 independent experiments. All P values were computed by means of 2-sided matched-pair Wilcoxon rank test.