Figure 4.

The R66G mutation in Sec61α confers resistance to mycolactone. (A) Primary mouse T cells were activated with anti-CD3/CD28 antibodies and then transduced with WT Sec61α or R66G-Sec61α before exposure to mycolactone (Myco) in resting or PMA/IO-stimulated conditions. (B) Differential effect of mycolactone (24 h at 400 nM) on the CD4 surface expression of WT Sec61α– or R66G-Sec61α–transduced (Zsgreen⁺) cells. Data are mean fluorescence intensity (MFI) from one of two independent experiments, which gave similar results. Ctrl, vehicle control; NT, nontransduced. (C) Dose-dependent effect of mycolactone on the CD62L surface expression of WT Sec61α– or R66G-Sec61α–transduced (Zsgreen⁺-gated) cells. (D) Effect of a 1-h pretreatment with increasing doses of mycolactone on the PMA/IO-induced production of IFN-γ by primary T cells transduced with WT Sec61α or R66G-Sec61α (Zsgreen⁺-gated) cells. (E) Bone marrow–derived macrophages were transduced with WT Sec61α or R66G-Sec61α before exposure to mycolactone in resting or LPS + IFN-γ–stimulated conditions. (F) Dose-dependent effect of mycolactone on the IFNGR1 surface expression of WT Sec61α– or R66G-Sec61α–transduced (Zsgreen⁺-gated) cells. (G) Dose-dependent effect of mycolactone on the LPS + IFN-γ–induced production of iNOS by WT Sec61α– or R66G-Sec61α–transduced (Zsgreen⁺-gated) cells. (C, D, F, and G) Data are mean fluorescence intensity or mean cell percentages ± SEM of triplicates, relative to vehicle controls. They are from one of two independent experiments, which gave similar results.