Restoration of cytotoxic activity of CTLs from pediatric patients with NP-C1 by HPβCD. (A) Chromium-51 (⁵¹Cr) release assay shows reduced cytotoxicity of CTLs from pediatric patients with NP-C1 at the effector-to-target (E:T) ratios indicated using MDA-MB-231 target cell. Average cytotoxic activity (95% confidence interval shown as shaded gray) of CTLs from 4 HDs. (B) Confocal immunofluorescence microscopy shows GzmB (green)/Prf (magenta) vesicles from an HD and PP4. DAPI is shown in blue. The Diameter of CGs was estimated by computational 3-dimensional re-creation (Imaris vesicles) based on GzmB staining of 2 HD samples and PP4. (C) TEM images show details of activated CTLs (scale bar, 2 μm) and zoomed-in autolysosome-like structures (scale bar, 500 nm). Number of autolysosome-like structures per cell (left), area of autolysosome-like structures per cell (middle), and number of dense-core granules per cell (n = 73 for HD control and n = 72 for PP4). (D) Confocal microscopy of activated CD8⁺ T cells shows vesicles containing unesterified cholesterol stained with filipin III. HD and PP4 cells were treated with 1 mM of HPβCD for 3 days. Filipin area per cell (right; analysis provided in supplemental Figure 9). (E) Quantification of immunological synapses between anti-HER2 CAR T cells and MDA-MB-231 targets that result in PI blush are shown. Immunological synapses were considered as any events with Ca²⁺ flux in CTLs (n = 35 HD and n = 19 PP4 synapses). (F) ⁵¹Cr release assay of activated HPβCD-treated CTLs killing MDA-MB-231 target cells shows a partial restoration of CTL cytotoxicity from PP4. Detailed description of microscopy and analysis is provided in the data supplement. **P < .01, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.