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Published in final edited form as: Nat Med. 2018 Jul 23;24(9):1330–1336. doi: 10.1038/s41591-018-0117-4

a–f, Imaging flow cytometry analysis of uptake of GzmB-AF488 (a,b) or GNLY-AF488 (c,d) on their own, or of GzmB-AF488 in the presence of unlabeled GNLY (e,f) by healthy donor (HD) unifected Retics (uRetics) and by uRetics and infected retics (iRetics) from acute untreated P. vivax patients (Pv). (a,c,e) show representative images, while (b,d,f) show mean ± SEM of 3 HD and Pv samples. g, Imaging flow cytometry images of Pv uRetic and iRetics incubated with GzmB-AF488 and GNLY-AF750, and stained for CD235a and with a Hoechst dye to stain parasite DNA. BF, bright field. This experiment was repeated three times with similar results. h–j, Effect of mβCD depletion of cholesterol in HD RBCs on binding of GNLY-AF488 on its own (n=4). Shown p values are in comparison to intact RBC. (h) and of GzmB-AF488 in the presence of unlabeled GNLY (n=3) (i); and on RBC lysis by increasing amounts of GNLY, assessed by LHD release (n=4). k,l, Staining of HD uRetics and of untreated Pv patient uRetics and iRetics with the cholesterol analog 25-NBD (n=3). Representative images are shown in (k) (n=5) and the proportion of cells with detectable 25-NBD fluorescence is shown in (l) (n=4). m,n, Cytolysis of Pv iRetics (m, n=5) or HD uRetics (n, n=4) after 1 hr incubation with GzmB ± GNLY ± PFN, assessed by LDH release. o, Effect of incubation of iRetics from 4 Pv patients for 1 hr with indicated cytotoxic granule proteins on parasite invasion of fresh Retics, assessed by Giemsa staining. p–r, Electron micrographs of iRetics that were untreated or incubated with GNLY ± GzmB. Higher magnification images after treatment with GNLY plus GzmB in (q) show parasitophorous vacuole membrane disruption ( ) and chromatin condensation ( ) (left); cytoplasmic vacuolization ( ) and dense granules ( ) (middle); and mitochondrial swelling ( ) (right). Higher magnification images of untreated cells (r) show intact digestive vacuole ( ), parasitophorous vacuole membrane ( ) and mitochondria ( ). These experiments were repeated three times with similar results (p,r). Graphs show mean ± SEM; statistics in (b,d,f,j,l–o) were analyzed by one-way ANOVA and in (h,i) by two-tailed non-parametric paired t-test at 95% CI.

Inline graphic — a–f, Imaging flow cytometry analysis of uptake of GzmB-AF488 (a,b) or GNLY-AF488 (c,d) on their own, or of GzmB-AF488 in the presence of unlabeled GNLY (e,f) by healthy donor (HD) unifected Retics (uRetics) and by uRetics and infected retics (iRetics) from acute untreated P. vivax patients (Pv). (a,c,e) show representative images, while (b,d,f) show mean ± SEM of 3 HD and Pv samples. g, Imaging flow cytometry images of Pv uRetic and iRetics incubated with GzmB-AF488 and GNLY-AF750, and stained for CD235a and with a Hoechst dye to stain parasite DNA. BF, bright field. This experiment was repeated three times with similar results. h–j, Effect of mβCD depletion of cholesterol in HD RBCs on binding of GNLY-AF488 on its own (n=4). Shown p values are in comparison to intact RBC. (h) and of GzmB-AF488 in the presence of unlabeled GNLY (n=3) (i); and on RBC lysis by increasing amounts of GNLY, assessed by LHD release (n=4). k,l, Staining of HD uRetics and of untreated Pv patient uRetics and iRetics with the cholesterol analog 25-NBD (n=3). Representative images are shown in (k) (n=5) and the proportion of cells with detectable 25-NBD fluorescence is shown in (l) (n=4). m,n, Cytolysis of Pv iRetics (m, n=5) or HD uRetics (n, n=4) after 1 hr incubation with GzmB ± GNLY ± PFN, assessed by LDH release. o, Effect of incubation of iRetics from 4 Pv patients for 1 hr with indicated cytotoxic granule proteins on parasite invasion of fresh Retics, assessed by Giemsa staining. p–r, Electron micrographs of iRetics that were untreated or incubated with GNLY ± GzmB. Higher magnification images after treatment with GNLY plus GzmB in (q) show parasitophorous vacuole membrane disruption ( ) and chromatin condensation ( ) (left); cytoplasmic vacuolization ( ) and dense granules ( ) (middle); and mitochondrial swelling ( ) (right). Higher magnification images of untreated cells (r) show intact digestive vacuole ( ), parasitophorous vacuole membrane ( ) and mitochondria ( ). These experiments were repeated three times with similar results (p,r). Graphs show mean ± SEM; statistics in (b,d,f,j,l–o) were analyzed by one-way ANOVA and in (h,i) by two-tailed non-parametric paired t-test at 95% CI.