![Murine Npc1−/−.CL4 CTLs have reduced cytotoxic activity. (A) Chromium-51 (51Cr) release assay at the effector-to-target (E:T) ratios indicated shows >85% reduction in Npc1−/−.CL4 cytotoxic activity compared with Npc1+/+.CL4 CTLs, because equal killing efficiency was observed at 5:1 and 0.75:1 E:T ratios, respectively (shown with a dotted line). Values plotted are standardized to maximal killing observed in Npc1+/+.CL4 at 10:1 E:T ratio and set at 100% (average cytotoxicity at 10:1 E:T ratio was 54% ± 13% [mean ± standard deviation of n = 5 independent experiments]). Each value shown represents mean ± standard error of the mean (SEM; n = 5). (B) Npc1 gene was knocked out in primary CTLs by CRISPR/Cas9 (supplemental Figure 2A) 51Cr release assay using day-6 activated CTLs transfected on day 0 with nontargeting guide RNA (NT) and Npc1-targeting guide RNA (Npc1 KO), and target EL-4 cells at the E:T ratios indicated shows decreased cytotoxic activity of Npc1-KO cells. Values plotted are standardized to maximal killing observed in NT at 10:1 E:T ratio and set at 100% (average cytotoxicity at 10:1 E:T ratio was 82.6% ± 21.7% [mean ± SEM of n = 3 independent experiments]). (C) Confocal immunofluorescence microscopy shows colocalization of GzmB (green) and LAMP-1 (magenta). GzmB/Lamp1 colocalization (n = 3 biological replicates per genotype; right), mean biological replicates (inset), and number of CGs estimated by computational 3-dimensional re-creation (Imaris vesicles) based on GzmB staining. (D) Degranulation assay was conducted by coincubating CD8+ T cells with CT-26 targets in the presence or absence of the cognate antigenic peptide. CD8+ T-cell degranulation was assessed by measurement of LAMP-1 (CD107a) externalization on CD8+ T cells. Summary of degranulation assays using n = 5 Npc1+/+ and n = 9 Npc1−/− mice. (E) Total internal reflection fluorescence (TIRF) microscopy reveals that day-4 Npc1−/− CTLs release more GzmB than Npc1+/+ cells, when added to anti-CD3/CD28–coated coverslips. GzmB (blue) was detected by anti-GzmB Alexa 647; both cell types were sorted to achieve the same levels of expression of LifeAct mScarlet (red). Shown in each frame (separated by a white line) are 4 tiled images obtained at that time point. Scale bar, 10 μm. Full time course is shown in supplemental Figure 3 and in supplemental Movies 1 and 2. Detailed description of microscopy and analysis is provided in the data supplement. **P < .01. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/139/12/10.1182_blood.2021013477/3/bloodbld2021013477f1.png?Expires=1736049009&Signature=vHXsyGIz5ISmV9000KEn6w8K622O902e3~NlaN91UaL7MFoZqpaLKumvr~e63srnbyT2-acxshm9-18ox2dNluqo9cfnkgDCi0Ayn6UQ0Ee0ZQ3JhfNYkbhSEOxiOrL~IQ1SYQYl37A-TvMN6H8wth1tGIGbrsMUbyEPLrb3TVrOZelus6MAimhrnPN56fo2crajM8mp4C4gli2Jx-wMsNTtdL6vQDt~WjhML3MMY-KVo81dUDRLX5vMrqzTHN6-qdevpqRpWbiPaCoSTiDOqOr1btQDVMiZRgRkH59Sdy4SAz6xKcF6efpU75DH3R6ulYsCOUl9hxSw1i7xEBgUjQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Murine Npc1−/−.CL4 CTLs have reduced cytotoxic activity. (A) Chromium-51 (51Cr) release assay at the effector-to-target (E:T) ratios indicated shows >85% reduction in Npc1−/−.CL4 cytotoxic activity compared with Npc1+/+.CL4 CTLs, because equal killing efficiency was observed at 5:1 and 0.75:1 E:T ratios, respectively (shown with a dotted line). Values plotted are standardized to maximal killing observed in Npc1+/+.CL4 at 10:1 E:T ratio and set at 100% (average cytotoxicity at 10:1 E:T ratio was 54% ± 13% [mean ± standard deviation of n = 5 independent experiments]). Each value shown represents mean ± standard error of the mean (SEM; n = 5). (B) Npc1 gene was knocked out in primary CTLs by CRISPR/Cas9 (supplemental Figure 2A) 51Cr release assay using day-6 activated CTLs transfected on day 0 with nontargeting guide RNA (NT) and Npc1-targeting guide RNA (Npc1 KO), and target EL-4 cells at the E:T ratios indicated shows decreased cytotoxic activity of Npc1-KO cells. Values plotted are standardized to maximal killing observed in NT at 10:1 E:T ratio and set at 100% (average cytotoxicity at 10:1 E:T ratio was 82.6% ± 21.7% [mean ± SEM of n = 3 independent experiments]). (C) Confocal immunofluorescence microscopy shows colocalization of GzmB (green) and LAMP-1 (magenta). GzmB/Lamp1 colocalization (n = 3 biological replicates per genotype; right), mean biological replicates (inset), and number of CGs estimated by computational 3-dimensional re-creation (Imaris vesicles) based on GzmB staining. (D) Degranulation assay was conducted by coincubating CD8+ T cells with CT-26 targets in the presence or absence of the cognate antigenic peptide. CD8+ T-cell degranulation was assessed by measurement of LAMP-1 (CD107a) externalization on CD8+ T cells. Summary of degranulation assays using n = 5 Npc1+/+ and n = 9 Npc1−/− mice. (E) Total internal reflection fluorescence (TIRF) microscopy reveals that day-4 Npc1−/− CTLs release more GzmB than Npc1+/+ cells, when added to anti-CD3/CD28–coated coverslips. GzmB (blue) was detected by anti-GzmB Alexa 647; both cell types were sorted to achieve the same levels of expression of LifeAct mScarlet (red). Shown in each frame (separated by a white line) are 4 tiled images obtained at that time point. Scale bar, 10 μm. Full time course is shown in supplemental Figure 3 and in supplemental Movies 1 and 2. Detailed description of microscopy and analysis is provided in the data supplement. **P < .01. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.