Fig. 1: Tigecycline compromises human T cell activation and proliferation by inhibiting mitochondrial translation.

A Representative dose-response curves of tetracycline antibiotic cytotoxicity towards PBMCs (left; n = 5) and Jurkat T cells (right; n = 3) measured 72 h after treatment; mean ± SEM is presented. Data are normalized to DMSO treatment without antibiotics. B Representative dose-response curves for additional bacterial protein synthesis inhibitors on PBMCs for 72 h. n = 6 biological replicates; mean ± SEM. Data are shown relative to DMSO treatment without antibiotics. C ³⁵S metabolic labeling assay of mitochondrial (left) and cytosolic (right) translation in Jurkat T cells after 18 h treatment with Q/D, tigecycline, or doxycycline. One representative result from n = 3 is shown. D Western blot OXPHOS profiling in Jurkat T cells after treatment with Q/D, tigecycline, and doxycycline and PBMCs after treatment with tigecycline for 6 days. n = 2 biological replicates, one shown. E Seahorse Mito Stress Test of PBMCs cultured with or without tigecycline for 6 days after stimulation with anti-CD3/CD28 antibodies + IL-2 (10 ng/ml). OCR is reported as picomoles (pmol) of O₂ per min and normalized according to protein amount/well. n = 4 biological replicates, mean ± SEM. Oligo, oligomycin; FCCP, carbonyl cyanide-p-tri-fluoromethoxyphenylhydrazone; Rot, rotenone; AA, antimycin A. F CD25 expression (MFI) on CD4⁺ and CD8⁺ T cells 18 h after anti-CD3/CD28 activation + IL-2 (10 ng/ml) in the presence or absence of tigecycline. n = 3 biological replicates, mean ± SEM. An RM one-way ANOVA with Tukey’s multiple comparisons test was used to analyze the data. G T cell proliferation assessed by flow cytometry. PBMC T cells were stimulated with anti-CD3/CD28 beads + IL-2 (10 ng/ml) in the presence or absence of tigecycline. Proliferation was assessed by CTV dilution in live cells after 6 days (n = 5 blood donor PBMC samples). PBMCs were labelled with CTV prior to stimulation. H Percentage of live T cells in division four or later after antibiotic treatment; data from (G), mean ± SEM. An RM one-way ANOVA with Tukey’s multiple comparisons test was applied to analyze the data. I Proliferation of FACS-isolated CD45RA⁺CD27⁺ naïve, CD45RA^-CD27⁺ central memory, and CD45RA^-CD27^- effector memory CD4⁺ T cells from healthy blood donors after 6 days of stimulation with anti-CD3/CD28 beads and IL-2 (10 ng/ml). FACS-isolated cells were seeded in the presence or absence of tigecycline (from 2.5 to 10 μM) and proliferation assessed by CTV dilution in live cells (n = 3 blood donor PBMC samples). Representative dot plots from non-treated and treated samples are shown. The FACS gating strategy is shown in Supplementary Fig. 2G. J Percentage of live CD4⁺ T naïve cells and central memory cells in each division; data from (I); mean ± SEM. Tn: naïve T cells; Tcm: central memory T cells; Tem: effector memory T cells. Source data for all panels are provided as a Source Data file.