Figure 4. Direct induction of endosomal antigen release and impaired release in CGD.

(a) Confocal micrographs of a DC expressing VAMP8-KillerRed (green) and incubated with OVA conjugated to Alexa fluor 647 (OVA-AF647; red). BF: bright field. Scale bar, 10 μm. (b) Linoleamide alkyne lipid peroxidation assay as in fig. 1i, but now for exposed or non-exposed DCs with or without expression of KillerRed. (c) CCF4 endosomal antigen leakage assay as in fig. 2b, but now for exposed or non-exposed DCs with or without expression of KillerRed. (d–e) Similar as panels (b,c) but now for HEK293T cells. (f) p47phox and gp91phox expression in lymphocytes from three p47phox-/- (CGD1-3; blue) and one gp91phox-/- (CGD4; green) CGD patients by Western blot (GAPDH: loading control). (g) CCF4 endosomal antigen release assay as in fig. 2b, but now with DCs derived from monocytes from the CGD patients from panel (d) (Ctrl: DCs from healthy donors). (h) Uptake of BSA conjugated to Alexa fluor 488 (BSA-AF488) by DCs of CGD patients, presented as percentage BSA-AF488-positive cells and MFI. Results show individual results of at least 3 donors/experiments. (i) Model scheme of NOX2-induced antigen leakage from endosomes. Superoxide anions produced by NOX2 and protons provided by the V-ATPase form H₂O₂ and hydroxyl radicals (Fenton reaction). These ROS induce lipid peroxidation, which disrupts endosomal membranes and causes the release of antigen (purple spheres) into the cytosol.