Fig. 3: BKX-MSCs display reduced immunosuppressive capacity in vivo.

a Generation of BKX-MSCs by selective expansion of apoptosis-resistant MSCs following multiple rounds of treatment with BH3-mimetic drugs (1.25 μM x 2 rounds, then 10 μM x 2 rounds). b Nontargeted MSCs and BKX-MSCs were treated with 1.25 μM or 10 μM BH3-mimetic drugs, for 2 or 24 h, then lysed and analysed for BAX and BAK protein levels by Western blotting. GAPDH served as the control for equal protein loading. Untreated MSCs and HeLa cells served as positive controls, and BAX/BAK double knockout (DKO) HeLa cells served as negative control. Data representative of two independent experiments. c Activated caspase-3 levels in CTV-labelled MSCs or BKX-MSCs re-isolated from mouse lungs 10 min and 1 h after i.v. injection. Data representative of two independent experiments. d OVA-sensitised mice received MSCs or BKX-MSCs prior to OVA challenge. OVA-specific DLN cell proliferation, measured by CFSE dilution (UNS n = 12; SEN n = 12; MSC n = 12; BKX-MSC n = 11) and MTS bioreduction (UNS n = 4; SEN n = 5; MSC n = 6; BKX-MSC n = 6). Data expressed mean ± SEM. p values by one-way ANOVA (Tukey’s post hoc test); ns not significant. e Number of eosinophils (n = 5 mice per group) and AMs (n = 6 mice per group) in the lungs, and DLN OVA-specific IL-5 and IL-13 production (UNS n = 6; SEN n = 6; MSC n = 8; BKX-MSC n = 7). Data expressed as mean ± SEM, p values by one-way ANOVA (Tukey’s post hoc test); ns, not significant. f Measurement of RI (UNS n = 5; SEN n = 9; MSC n = 12; BKX-MSC n = 10) and Cdyn (UNS n = 4; SEN n = 9; MSC n = 12; BKX-MSC n = 10) in response to increasing doses of methacholine on Day 12. UNS = unsensitized mice; SEN = OVA-sensitised mice; MSC = OVA-sensitised mice that received MSCs; BKX-MSC = OVA-sensitised mice that received BKX-MSCs. Data expressed mean ± SEM, p values by two-way ANOVA (Tukey’s post hoc test), compared with SEN. g Lung sections were stained with H&E and PAS to analyse for pulmonary inflammation and mucus production, respectively. Magnification 20x, scale bar = 100 μm. Histological images were representative of 5 mice per group. h Mean daily clinical scores of EAE mice that received PBS, MSCs or BKX-MSCs on Days 1, 3 and 5 (arrows indicate days of MSC injections). Data expressed as mean ± SEM, six mice per group. p value by Kruskal-Wallis (Dunn’s post hoc test). i CNS sections were stained with H&E and LFB to assess infiltrating inflammatory cells and demyelination respectively. Representative of 6 mice per group. Scale bar = 300 μm. j MOG-specific LN cell proliferation of EAE mice that received PBS, MSCs or BKX-MSCs and euthanised on Day 9 (n = 6 mice per group) and 29 (NAÏVE n = 3; EAE n = 4; MSC n = 6; BKX-MSC n = 6). Data expressed as mean ± SEM, p values compared to PBS group (one-way ANOVA, Tukey’s post hoc test). k Percentage of CD45⁺Ly6G⁻Ly6C^hi monocytes and CD45⁺Ly6G⁺Ly6C^int neutrophils in the blood on Day 9. Data expressed as mean ± SEM, n = 6 mice per group. p values by one-way ANOVA (Tukey’s post hoc test). Source data are provided as a Source Data file.