Figure 5.
HPβCD depletes lipid clusters from CGs, unlocking Prf dysfunction and restoring cytotoxic activity of Npc1-deficient CTLs. (A) Confocal immunofluorescence microscopy shows efficient GM1 clearance (as determined by cholera toxin B [CTxB] staining) in Npc1−/−.CL4 CTLs upon their treatment with 1 mM of HPβCD for 4 days; cells were costained with GzmB antibodies (green) and CTxB (magenta). Scale bar, 3 μm. Overlap coefficient was used to quantify the colocalization of GzmB and CTxB in HPβCD-treated and control cells. (B) Diameter of CGs (per cell) was estimated by computational 3-dimensional re-creation (Imaris vesicles) based on GzmB staining. (C) Total area of LC3-B+ vesicles per cell. (D) Chromium-51 (51Cr) release assay using CT-26 target cells shows the recovery of cytotoxic activity of HPβCD-treated Npc1−/−.CL4 CTLs to the level of Npc1+/+.CL4 CTLs. Values plotted are standardized to maximum killing observed in untreated Npc1+/+.CL4 CTLs at 10:1 effector-to-target (E:T) ratio and set at 100% (average cytotoxicity at 10:1 E:T ratio was 64.2% ± 16.4% [mean ± standard deviation of n = 3 independent experiments). Each value shown represents mean ± standard error of the mean (SEM) of n = 3 independent experiments. (E) Representative montage of confocal time-lapse microscopy shows the formation of the immunological synapse between CTLs and target cells. CTLs were labeled with calcium fluorophore Fluo-4AM, and the increase of green fluorescence indicated calcium(2) ion (Ca2+) flux and formation of the functional synapse by CTLs. Cells were incubated in the presence of 100 μM of propidium iodide (PI),21 and target cell membrane permeabilization was determined by the PI blush (red) in the cytosol of target cells. Day-5 activated Bl/6.OTI CTLs used here were transfected either with NT or Npc1-targeting guide RNA (Npc1 KO), as described in Methods. Ca2+ flux and PI uptake were observed at 4:30 and 5:30 minutes in NT, respectively; Ca2+ flux was observed at 2:30 minutes in Npc1 KO, but there was no PI uptake detected through to the end of the montage at 38:30 minutes. Quantification of immunological synapses that result in PI blush are shown on the right. Immunological synapses were considered as any events with Ca2+ flux in CTLs (n = 2 biological replicates per group; n = 70 NT and n = 88 Npc1-KO synapses). Scale bar, 10 μm. (F) (i) Live-cell 3-dimensional confocal microscopy shows time-dependent secretion of ALFA-tagged Prf from Npc1+/+.CL4 cells after their addition to CD3/28-coated coverslips. LifeAct mScarlet is shown in red, and Prf was detected by anti-ALFA Atto-488 nanobodies added to the media (shown in green). Scale bar, 2 μm. Enlarged image at 12.5 minutes is shown in supplemental Figure 6B. (ii) TIRF microscopy shows that that Npc1+/+ and Npc1−/− CTLs secrete similar amounts of Prf when added to anti-CD3/CD28–coated coverslips. Both cell types were sorted to achieve similar levels of expression of LifeAct mScarlet and Prf ALFA. Shown in each frame (separated by a white line) are 4 tiled images obtained at that time point. Scale bar, 10 μm. (iii) Quantitative analysis of Prf ALFA release per cell as described in supplemental Figure 6C; shown are combined results from 3 independent experiments (supplemental Movies 3 [empty vector control (EV)], 4 (Npc1+/+), and 5 (Npc1−/−). (G) Four-hour 51Cr release assay shows that ALFA Prf overexpression increases the activity of Npc1+/+.CL4 but not Npc1−/−.CL4 CTLs (each value represents mean ± SEM of 3 independent experiments). Detailed description of microscopy and analysis is provided in the data supplement. *P < .05, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

HPβCD depletes lipid clusters from CGs, unlocking Prf dysfunction and restoring cytotoxic activity of Npc1-deficient CTLs. (A) Confocal immunofluorescence microscopy shows efficient GM1 clearance (as determined by cholera toxin B [CTxB] staining) in Npc1−/−.CL4 CTLs upon their treatment with 1 mM of HPβCD for 4 days; cells were costained with GzmB antibodies (green) and CTxB (magenta). Scale bar, 3 μm. Overlap coefficient was used to quantify the colocalization of GzmB and CTxB in HPβCD-treated and control cells. (B) Diameter of CGs (per cell) was estimated by computational 3-dimensional re-creation (Imaris vesicles) based on GzmB staining. (C) Total area of LC3-B+ vesicles per cell. (D) Chromium-51 (51Cr) release assay using CT-26 target cells shows the recovery of cytotoxic activity of HPβCD-treated Npc1−/−.CL4 CTLs to the level of Npc1+/+.CL4 CTLs. Values plotted are standardized to maximum killing observed in untreated Npc1+/+.CL4 CTLs at 10:1 effector-to-target (E:T) ratio and set at 100% (average cytotoxicity at 10:1 E:T ratio was 64.2% ± 16.4% [mean ± standard deviation of n = 3 independent experiments). Each value shown represents mean ± standard error of the mean (SEM) of n = 3 independent experiments. (E) Representative montage of confocal time-lapse microscopy shows the formation of the immunological synapse between CTLs and target cells. CTLs were labeled with calcium fluorophore Fluo-4AM, and the increase of green fluorescence indicated calcium(2) ion (Ca2+) flux and formation of the functional synapse by CTLs. Cells were incubated in the presence of 100 μM of propidium iodide (PI),21 and target cell membrane permeabilization was determined by the PI blush (red) in the cytosol of target cells. Day-5 activated Bl/6.OTI CTLs used here were transfected either with NT or Npc1-targeting guide RNA (Npc1 KO), as described in Methods. Ca2+ flux and PI uptake were observed at 4:30 and 5:30 minutes in NT, respectively; Ca2+ flux was observed at 2:30 minutes in Npc1 KO, but there was no PI uptake detected through to the end of the montage at 38:30 minutes. Quantification of immunological synapses that result in PI blush are shown on the right. Immunological synapses were considered as any events with Ca2+ flux in CTLs (n = 2 biological replicates per group; n = 70 NT and n = 88 Npc1-KO synapses). Scale bar, 10 μm. (F) (i) Live-cell 3-dimensional confocal microscopy shows time-dependent secretion of ALFA-tagged Prf from Npc1+/+.CL4 cells after their addition to CD3/28-coated coverslips. LifeAct mScarlet is shown in red, and Prf was detected by anti-ALFA Atto-488 nanobodies added to the media (shown in green). Scale bar, 2 μm. Enlarged image at 12.5 minutes is shown in supplemental Figure 6B. (ii) TIRF microscopy shows that that Npc1+/+ and Npc1−/− CTLs secrete similar amounts of Prf when added to anti-CD3/CD28–coated coverslips. Both cell types were sorted to achieve similar levels of expression of LifeAct mScarlet and Prf ALFA. Shown in each frame (separated by a white line) are 4 tiled images obtained at that time point. Scale bar, 10 μm. (iii) Quantitative analysis of Prf ALFA release per cell as described in supplemental Figure 6C; shown are combined results from 3 independent experiments (supplemental Movies 3 [empty vector control (EV)], 4 (Npc1+/+), and 5 (Npc1−/−). (G) Four-hour 51Cr release assay shows that ALFA Prf overexpression increases the activity of Npc1+/+.CL4 but not Npc1−/−.CL4 CTLs (each value represents mean ± SEM of 3 independent experiments). Detailed description of microscopy and analysis is provided in the data supplement. *P < .05, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; ns, not significant.

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