Cytotoxic defects in CTLs from adult patients with NP-C1 are in concordance with the increased formation of autolysosome-like structures. (A) Chromium-51 release assay of activated CTLs against MDA-MB-231 target cells at the effector-to-target (E:T) ratios indicated. Average of 8 HD CTL cytotoxicity assays is shown as shaded area (95% confidence interval). CTLs from patients with NP-C1 (P1-P3) display a spectrum of cytotoxic activities, from normal to severely impaired. (B) Confocal immunofluorescence microscopy shows GzmB (green) vesicles and DAPI (blue) in activated CTLs of HDs and patients with NP-C1. Diameter of CGs was estimated by the computational 3-dimensional reconstruction (Imaris vesicles) based on GzmB staining of CTLs from 2 HDs and 3 patients whose cells had distinctly different cytotoxic activity. (C) TEM images of activated CTLs (scale bar, 2 μm) and zoomed-in autolysosome-like structures (scale bar, 200 nm). Quantification of autolysosome-like structures per cell using n = 30 cells per HD or patient (middle). Quantification of the area of autolysosome-like structures based on GzmB staining (right). Detailed description of microscopy and analysis is provided in the data supplement. **P < .01, ***P < .001, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.