Extended Data Fig. 10: By dual action, IspH prodrugs expand and activate Vγ9Vδ2 T cells and reduce the emergence of antibiotic resistant bacteria.

a, Top, uninfected (UI) human PBMCs or those co-infected with E. coli analysed for expansion of CD3⁺Vγ9TCR⁺ (γδ) T cells and compared to untreated (UT) or TPP-, prodrug (C23.07–TPP)- or kanamycin (Kan)-treated PBMCs. Middle, bottom, gated γδ T cell populations analysed for cytotoxic granule proteins granulysin (GNLY) and perforin (middle) or cell surface markers of T cell activation CD69 and HLA-DR (bottom). Representative of 4 independent experiments (4 donors). Percentage of Vγ9⁺ T cells from CD3⁺ population and the percentage of Vγ9⁺ T cells with increased expression of granulysin, perforin, CD69 and HLA-DR were plotted in the respective graphs. Data are mean ± s.e.m. **P < 0.05, ***P < 0.001, the remainder are not significant; one-way ANOVA relative to untreated sample. b, Uninfected human PBMCs or those co-infected with V. cholerae (top) or M. smegmatis (bottom) were analysed for expansion of CD3⁺Vγ9TCR⁺ (γδ) T cells and compared to untreated or TPP-, prodrug (C23.07–TPP)- or kanamycin-treated PBMCs (n = 4 biological replicates). Percentage of Vγ9⁺ T cells from the CD3⁺ population were plotted in the respective graphs. Data are mean ± s.e.m. ***P < 0.001, rest not significant, calculated by one-way ANOVA relative to untreated sample. c, Human PBMCs co-infected with kanamycin-resistant E. coli or V. cholerae can kill neither on their own. Addition of C23.07–TPP kills both V. cholerae and E. coli (n = 2 biological and 3 technical replicates). Data are mean ± s.e.m. ***P < 0.001, ns, not significant; unpaired Student’s t-test relative to untreated samples. d, γδ T cell depletion from human PBMCs is verified by treating depleted (γδ⁻) and undepleted human PBMCs treated with 10 μM HMBPP and 50 ng ml⁻¹ IL-15. Representative of 4 independent experiments (4 donors). Percentage of Vγ9⁺ T cells from the CD3⁺ population on different days were plotted in the respective graphs. Data are mean ± s.e.m. ***P < 0.001 comparing γδ depleted and undepleted PBMCs calculated by unpaired t-test. e, f, Multidrug-resistant clinical isolates of Vibrio (e) and Klebsiella (f) grown for 18 serial passages in media (RPMI+ 10% human serum) containing DAIA prodrug (C23.28–TPP) or conventional antibiotics (hygromycin (Hyg) or streptomycin (Strep)) gradually develop resistance when measured by CFU (top). Similar serial passages in presence of human PBMC inhibit the development of resistance against the DAIA prodrug but not against hygromycin or streptomycin. Passages in γδ depleted (γδ⁻) PBMCs show higher antibiotic resistance against the DAIA prodrug. (n = 3 technical replicates). Data are mean ± s.e.m. ***P < 0.001, NS, not significant; unpaired Student’s t-test. g, C57BL/6 mice infected with V. cholerae are treated with TPP or the DAIA prodrug C23.28–TPP and monitored daily from day 2 post-infection for survival (n = 10 mice per group). h, Vibrio load in different organs at the experimental endpoint measured as CFU mg⁻¹ (n = 10 mice with 3 technical replicates), comparing changes in bacterial CFU in C57BL/6b mice after C23.28–TPP treatment. Data are mean ± s.e.m. ***P < 0.001; unpaired Student’s t-test, relative to TPP-treated mice. i, Hu-mice injected i.p. with HMBPP at different concentrations show dose-dependent expansion of γδ T cells but not αβ T cells in blood taken every day for a week (n = 2 mice per group).

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