Figure 1.
Anti-cryptococcal activity of NK cells is greater in pH 6.6 compared to pH 7.4.
YT cells were co-cultured with different strains of Cryptococcus; C. gattii strain R265 (A), C. neoformans strain H99 (B), or C. neoformans strain B3501 (C). Primary human NK cells were co-cultured with B3501 (D) in different pH for 20 h. The reduction in CFU was calculated by deducting the number of experimental CFUt = 20 h (Cryptococcus cultured with NK cells) from the number of control CFUt = 20 h(Cryptococcus cultured alone). Each bar represents the mean of four replicates. ns, not significant, *, p value≤0.05, **, p value≤0.01 ***, p value≤0.001 compared to pH 7.4. Data are representative of three (A, C and D) or two (B) experiments. (E) CFSE-labeled K562 cells (tumor cells) were cultured either alone or with YT cells at 50∶1 E∶T ratio for 4 h. The number of K562 cells killed by YT cells was calculated as described in materials and methods. Each bar represents the mean of three replicates *, p value≤0.05 compared to pH 7.2. Data are representative of three experiments.
Figure 2.
Acidic pH does not increase the effector to target (E∶T) ratio.
(A) YT cells were cultured for 20 h in different pH (7.4, 7.2, 7.0, 6.8, and 6.6) and the numbers of viable YT cells were counted by light microscopy using trypan blue staining at the beginning and end of the incubation. Fold increase in the number of viable YT cells was calculated by dividing the number of viable YT cells at 20 h by the number of viable YT cells at t = 0. Each bar represents the mean of three replicates. Data are representative of three experiments. C. gattii strain R265 (B), C. neoformans strains H99 (C), or B3501 (D) were cultured in different pH (7.4, 7.2, 7.0, 6.8, 6.6) for 20 h. The number of viable organisms was detected by counting the CFU on agar plates. Each bar represents the mean of four replicates. *, p value≤0.05, ***, p value≤0.001 compared to the fold increase at pH 7.4. Data are representative of three experiments.
Figure 3.
Acidic pH does not increase the amount of active perforin within YT cells.
YT cells alone were cultured in complete media at pH 7.4 and 6.6 for 20 h. Cells were harvested at the beginning and end of incubation, made permeable and immunolabeled with either anti-perforin or isotype control antibodies and then analyzed by flow cytometry. Data are representative of three experiments.
Figure 4.
Acidic pH does not increase the number of conjugates between YT cells and C. neoformans.
TRITC-labeled YT cells were cultured with CFSE-labeled B3501 in pH 7.4 and pH 6.6 for up to 300 minutes. At the indicated times, samples were isolated and fixed in ice-cold PBS containing 0.5% formalin and data acquired immediately using flow cytometry. Cells were identified based on their labeling and the percentage of events that simultaneously contained both TRITC-labeled YT cells and CFSE-labeled B3501 was determined. At least 5000 NK cells and 300 conjugates were counted at each time point. Data are representative of three experiments.
Figure 5.
Cryptococcus-induced ERK1/2 signaling in YT cells is greater in pH 6.6 compared to pH 7.4.
(A) YT cells were left unstimulated (YT) or stimulated with B3501 (YT+C) at E∶T ratio of 1∶100 at 1, 2 and 5 minutes in pH 7.4 and 6.6. Immunoblotting was performed to detect dual Tyr/Thr phosphorylation of ERK1/2 (p-ERK1/2). Total ERK1/2 was used as a loading control. Data are representative of three experiments. YT, YT cells; C, Cryptococcus. (B) Densitometry of the bands in A was performed using LI-COR Odyssey instrument software and the fold increase in ERK1/2 phosphorylation was calculated by dividing the density of the stimulated bands with the density of unstimulated bands at each time point. Data are representative of three experiments. C. YT cells were pretreated for 2 h with 50 µM U0126 and anticryptococcal activity was compared with untreated and vehicle control (DMSO). ***, p<0.0001. Data are representative of three experiments.
Figure 6.
Cryptococcus-induced perforin loss in YT cells is greater in pH 6.6 compared to pH 7.4.
(A) YT cells were cultured alone or with live Cryptococcus (B3501 in upper panel and R265 in lower panel, E∶T, 1∶10) in pH 7.4 and 6.6 for 20 hours. Cells were harvested, made permeable and immunolabeled with either anti-perforin or isotype control antibodies. Data are representative of three experiments. (B) Reduction in perforin MFI was calculated by deducting the MFI of Cryptococcus stimulated YT from the MFI of unstimulated YT. (C) Same as A except for heat killed B3501. Data are representative of three experiments. (D) YT cells were pretreated for 2 h with 10 nM concanamycin A (B3501+YT+CMA) and anticryptococcal activity was compared with untreated group (B3501+YT). ***, p<0.0001. Data are representative of three experiments.
Figure 7.
NK cell mediated cryptococcal killing is proportional to the rate of replication of the organisms.
(A&B) H99ras1Δ and H99ras1Δ+RAS1 were cultured alone at 37°C for 20 h in pH 7.4 (A) and pH 6.6 (B). Fold increase in CFU was determined by dividing the CFUt = 20 h by CFUt = 0. (C&D) H99ras1Δ and H99ras1Δ+RAS1 were cultured with or without YT cells at 37°C for 20 h in pH 7.4 (C) and pH 6.6 (D). The CFU reduction was calculated as described in figure 1. Each bar represents the mean of four replicates. Data are representative of three experiments. (E&F) H99ras1Δ and H99ras1Δ+RAS1 were either cultured alone or with YT cells at 37°C for 20 h at pH 7.4 (E) and pH 6.6 (F) and the CFUs for each time point (t = 0 and t = 20 h) are shown. For H99ras1Δ, two different starting inocula are shown. Each bar represents the mean of four replicates. Data are representative of two experiments.
Figure 8.
Cryptococcomas contain numerous granzyme B positive, but very few perforin positive NK cells.
Case 1: The brain was removed at autopsy and fixed in formalin with subsequent sectioning and examination. Sections were taken from the cryptococcoma in the cerebral cortex and stained with mucicarmine (200×) (A); or stained with PAS and labeled with either anti-CD56 (400×) (B); anti-CD57 (600×) (C); anti-granzyme B (1000×) (D); or anti-perforin (E, 600×) antibody. Cryptococcus (open arrow) in association with labeled cells (solid arrows) are shown. Perforin positive cells (brown) in NK cell lymphoma in lung, shown as a positive control for perforin (F). Case 2: Wedge resection of lung cryptococcoma, fixed in formalin with subsequent sectioning, PAS staining and labeled with either anti-CD57 (G, 400×), anti-granzyme B (H, 1000×) or anti-perforin (I, 600×). No organisms are present in figure G. The organisms stain bright pink with PAS, whilst positive staining for antigens is dark brown.