Fig. 5: Inhibition of ATG4B-delipidation activity sustains MHC class II antigen presentation. | Nature Communications

Fig. 5: Inhibition of ATG4B-delipidation activity sustains MHC class II antigen presentation.

From: Oxidation inhibits autophagy protein deconjugation from phagosomes to sustain MHC class II restricted antigen presentation

Fig. 5

Macrophages transduced with the indicated lentiviruses encoding Flag, Flag-ATG4Bwt, or Flag-ATG4BC78S were investigated. A Transduced macrophages were stimulated with Candida (C.) albicans extract for 6 h and the expression of LC3B was analyzed by western blot. Bar graph shows the level of LC3B-II expression normalized to vinculin. All conditions are normalized to each unstimulated condition (US), which is set to 1. Bars represent mean ± SD of three independent experiments, Mann–Whitney test, two-tailed. B Transduced macrophages were stimulated with C. albicans extract-coated beads for 6 h and then stained for LC3 (red). Representative images from three independent experiments, scale bars 5 µm, and inserts are zoomed 5× from white-framed regions of the original images. Bar graph shows the percentage of cells with or without LAPosomes. Bars represent the mean ± SD from three independent experiments, and each symbol represents a single experiment. One-way ANOVA test. C Experimental outline of MHC class II antigen-presentation assay. We created this diagram using the free online platform Smart Servier Medical Art. D ELISA assays on supernatants of a C. albicans-specific CD4+ T-cell clone cocultured with MHC class II matched macrophages transduced with the indicated Flag tagged constructs. Transduced macrophages were pulsed with C. albicans extract. Time indicated below the graph corresponds to the resting time of the pulsed macrophages before coculture. Scatter plot represents the IFNγ production normalized to unstimulated conditions (US) from five independent experiments and only values above the detection limit are displayed. Each symbol represents a single experiment. Ratio paired Student’s t test, two-tailed. E ELISA on supernatants of whole blood memory CD4+ T cells with autologous macrophages transduced with the indicated Flag tagged constructs. Transduced macrophages were pulsed with C. albicans extract. Time indicated below the graph corresponds to the resting time of the pulsed macrophages before coculture. Scatter plot represents the IL-17A production normalized to unstimulated conditions (US) from five independent experiments with five different donors and each symbol represents a biological replicate. Kruskal–Wallis test. F Macrophages transduced with the indicated lentiviruses were stimulated with C. albicans extract for 4 h. The macrophages were washed and then incubated for 18 h without stimuli or directly analyzed by FACS. Scatter plot from four independent experiments and each symbol represents a different experiment. Source data are provided as a Source Data file.

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