S of NK cells positive for IFNc staining also increased in three out of five donors tested (Figure 5B), but minimal staining was observed compared to that seen in T cells (Figure 5C). Thus, unlike the bovine system, oenothein B-induced IFNc production was not restricted to the human NK cell population. In the bovine system, NK cells were primed to produce enhanced IFNc in response to IL-18. We tested whether the samecould be true for human NK cells. First, multi-color flow cytometry showed that IL-18 receptor was increased on oenothein B-treated human NK cells in two out of five donors (Figure 6A). Similarly, further analyses showed that in two, possibly three, of five human PBMC preparations, IFNc production was increased in oenothein B-primed, IL-18-treated human NK cells compared to cells treated with oenothein B or IL-18 alone (Figure 6B and 6C). We then tested if oenothein B could enhance IFNc production in response to the NK cell target leukemic cell line, K562. Others have shown that stimulation by K562 cells induces IFNc secretion by NK cells and that this response can be enhanced by the presence of a second stimuli [42]. Consistent with these reports, pretreatment of human PBMCs with oenothein B enhanced IFNc production in response to K562 cells compared to untreated PBMCs (Figure 7A) and NK cells were the major cell population primed by oenothein B for enhanced IFNc production (Figure 7B).Stimulation of Lymphocytes by Oenothein BFigure 8. Oenothein B directly primes purified human NK cells to produce IFNc. Human NK cells (56104 cells/well) were sorted and treated with 20 mg/ml oenothein B, 100 ng/ml rhu IL-18, both, or medium alone. After 24 hrs, soluble IFNc was measured by ELISA. The graph represents soluble IFNc levels in culture supernatant fluids from three separate experiments with three different donors. Error bars indicate SEM. Each sample was analyzed in triplicate. Significance was determined by One-way ANOVA with Bonferroni post-test. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0050546.gFigure 7. Priming of human NK cells to K562 cells by oenothein B. (A) Human PBMCs (105 cells/well) were treated with 20 mg/ml oenothein B or X-VIVO medium alone for approximately 24 hrs. Cells were then Tubastatin-A washed and co-cultured with or without K562 cells at effector:target (E:T) ratios of 10:1 and 1:1 for approximately 42 hrs. After incubation, soluble IFNc levels were measured by ELISA. The data represent pooled results from three donors and are expressed as mean +/2 SEM. Samples were analyzed in duplicate. Statistical significance was measured by Two-way ANOVA with Bonferroni post-test. (B) Human PBMCs (105 cells/well) were treated with 20 mg/ml oenothein B or X-VIVO medium alone for approximately 24 hrs. Cells were then washed and co-cultured with or without K562 cells at an effector:target (E:T) ratio of 1:1 for approximately 24786787 18 hrs. After incubation, HIV-RT inhibitor 1 custom synthesis brefeldin A was added to 18334597 the culture for 6 hrs. IFNc expression by NK cells (CD32/ CD56+), T cells (CD3+), and others (CD32/CD56-) was then measured by intracellular flow cytometry. The data represent pooled results from two donors and are expressed as mean +/2 SEM. Samples were analyzed in duplicate. Statistical significance was measured by One-way ANOVA with Bonferroni post-test. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0050546.gNK cell numbers and activity can vary significantly from donor to donor. To address whether the inconsistency seen in PBMC prepar.S of NK cells positive for IFNc staining also increased in three out of five donors tested (Figure 5B), but minimal staining was observed compared to that seen in T cells (Figure 5C). Thus, unlike the bovine system, oenothein B-induced IFNc production was not restricted to the human NK cell population. In the bovine system, NK cells were primed to produce enhanced IFNc in response to IL-18. We tested whether the samecould be true for human NK cells. First, multi-color flow cytometry showed that IL-18 receptor was increased on oenothein B-treated human NK cells in two out of five donors (Figure 6A). Similarly, further analyses showed that in two, possibly three, of five human PBMC preparations, IFNc production was increased in oenothein B-primed, IL-18-treated human NK cells compared to cells treated with oenothein B or IL-18 alone (Figure 6B and 6C). We then tested if oenothein B could enhance IFNc production in response to the NK cell target leukemic cell line, K562. Others have shown that stimulation by K562 cells induces IFNc secretion by NK cells and that this response can be enhanced by the presence of a second stimuli [42]. Consistent with these reports, pretreatment of human PBMCs with oenothein B enhanced IFNc production in response to K562 cells compared to untreated PBMCs (Figure 7A) and NK cells were the major cell population primed by oenothein B for enhanced IFNc production (Figure 7B).Stimulation of Lymphocytes by Oenothein BFigure 8. Oenothein B directly primes purified human NK cells to produce IFNc. Human NK cells (56104 cells/well) were sorted and treated with 20 mg/ml oenothein B, 100 ng/ml rhu IL-18, both, or medium alone. After 24 hrs, soluble IFNc was measured by ELISA. The graph represents soluble IFNc levels in culture supernatant fluids from three separate experiments with three different donors. Error bars indicate SEM. Each sample was analyzed in triplicate. Significance was determined by One-way ANOVA with Bonferroni post-test. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0050546.gFigure 7. Priming of human NK cells to K562 cells by oenothein B. (A) Human PBMCs (105 cells/well) were treated with 20 mg/ml oenothein B or X-VIVO medium alone for approximately 24 hrs. Cells were then washed and co-cultured with or without K562 cells at effector:target (E:T) ratios of 10:1 and 1:1 for approximately 42 hrs. After incubation, soluble IFNc levels were measured by ELISA. The data represent pooled results from three donors and are expressed as mean +/2 SEM. Samples were analyzed in duplicate. Statistical significance was measured by Two-way ANOVA with Bonferroni post-test. (B) Human PBMCs (105 cells/well) were treated with 20 mg/ml oenothein B or X-VIVO medium alone for approximately 24 hrs. Cells were then washed and co-cultured with or without K562 cells at an effector:target (E:T) ratio of 1:1 for approximately 24786787 18 hrs. After incubation, brefeldin A was added to 18334597 the culture for 6 hrs. IFNc expression by NK cells (CD32/ CD56+), T cells (CD3+), and others (CD32/CD56-) was then measured by intracellular flow cytometry. The data represent pooled results from two donors and are expressed as mean +/2 SEM. Samples were analyzed in duplicate. Statistical significance was measured by One-way ANOVA with Bonferroni post-test. *p,0.05, **p,0.01, ***p,0.001. doi:10.1371/journal.pone.0050546.gNK cell numbers and activity can vary significantly from donor to donor. To address whether the inconsistency seen in PBMC prepar.
