Fig 1.
Schematic structures and membrane orientation of NA-Fc chimeras.
A series of 4 plasmids were constructed to encode chimeric proteins consisting of the NA cytoplasmic tail, transmembrane signal-anchor and stalk region fused to the IgG1 Fc region. NA-Fc1 through NA-Fc4 differ only in the length of the extracellular stalk region and increases the distance of the Fc domain from the plasma membrane.
Fig 2.
PM21-NK cells effectively lyse ovarian cancer cells expressing the NA-Fc chimeras.
(A, B) SKOV3 ovarian cells were transfected with cDNAs encoding one of the four NA-Fc chimeras. Cell lines were stained with antibody to IgG1 Fc and sorted by flow cytometry. Naïve and transfected SKOV4cells were stained with antibody to Fc domain and percent Fc+ (panel A) and MFI (panel B) for surface expression was assayed by flow cytometry. (Panel C) PM21-NK cells were incubated with cultures of naïve SKOV3 cells or one of the four cell lines expressing NA-Fc for 45 mins at an E:T of 1:1. Percent cytotoxicity was determined using a flow based cytometric assay as described in Material & Methods. Data is representative of 4 independent experiments conducted with 2 different NK cell donors with triplicates and error bars represent SD. Data was analyzed using two-way ANOVA analysis. * and ** indicate p values of p<0.05 and p<0.01.
Fig 3.
Real time assay for PM21-NK cell killing of NA-Fc4 ovarian cancer cells.
SKOV3-NLR expressing NA-Fc4 were incubated with PM21-NK cells at an E:T ratio of 2.5:1. Red object count (ROC) was recorded on IncuCyte instrument at 2 h intervals and normalized to ROC at time 0 when PM21-NK cells were added to the target cells. (A) Phase and fluorescence images (10x) of naïve and NA-Fc4-expressing SKOV3 cultures at 30 h post addition of PM21-NK cells. The scale bar represents 400 μm. (B, C) Time dependent ROC curves of target cells + PM21-NK cells at an E:T ratio of 2.5:1. Panel B represents ROC of target alone samples; panel C represents ROC of indicated SKOV3 cells incubated with PM21-NK cells. Each data point represents an average of three samples with error bars as SD. Data was analyzed using two-way ANOVA test and by applying Dunett’s post hoc test when comparing with the control group. For all graphs, the adjusted p values after applying post hoc tests were *p<0.05, **p<0.01, ***p<0.001 when comparing naïve to NA-Fc cells. Stars indicate the point where the p-values first begins, and this statistical significance extends for later timepoints until a new star appears with a different p value.
Fig 4.
NA-Fc4 expression enhances PM21-NK cell killing of lung cancer cells.
A549-NLR cells were transduced with LV encoding NA-Fc4 and sorted for stable NA-Fc expression. Cells were plated and incubated with PM21-NK cells at different E:T ratios. ROC was recorded on IncuCyte instrument at 2 h intervals and normalized to ROC at time 0 when PM21-NK cells were added to the target cells. (A) Fc expression of stable A549 NA-Fc4 NLR cells was quantified using flow cytometry post sorting. (B) PM21-NK cells were co-incubated with target cells, their phase and fluorescence images (10x) were captured for NK cell Donor 1 at 30 h post addition of PM21-NK cells. The scale bar represents 400μm. (C, D) Time dependent red intensity curves of target cells + PM21-NK cells were recorded at an interval of 2 hrs at two different E:T ratios, 5:1 and 2.5:1 respectively. Panels (E, F) represent normalized ROC for naïve and NA-Fc4 cells when incubated with Donor 2 at two different E:T ratios of 5:1 and 1.25:1. Each data point represents an average of three samples with error bars as SD. Data was analyzed using two-way ANOVA test and by applying Dunett’s post hoc test when comparing with the control groups. For all graphs, the adjusted p values after applying post hoc tests were *p<0.05, **p<0.01, ***p<0.001. Stars indicate the point where the p-values first begin, and this statistical significance extends for later timepoints until a new star appears with a different p value.
Fig 5.
NA-Fc4 expression on target tumor cells enhances NK cell activity.
PM21-NK cells were incubated for 16 h with indicated naïve or NA-Fc4 expressing target cells at a 1:1 ratio. As controls, cells were left untreated or treated with PMA/Ionomycin drugs or a cocktail of cytokines known to induce NK cell cytokine secretion (Cyto). A) PM21-NK cells were then assessed by flow cytometry for cell surface CD107a expression as a measure of degranulation, or alternatively stained intracellularly for IFN-γ (panel B) or TNF-α (panel C). Values are a mean of three samples with error bars as SD. Data was analyzed using two-way ANOVA analysis. For all graphs *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 when comparing indicated groups of naïve and NA-Fc4.
Fig 6.
NA-Fc4 mediated enhancement of PM21-NK cell killing requires engagement of CD16 receptor and Fc ligand.
(A) NK cells from donors 1 or 2 were stained with antibodies to CD56 and CD16. They were gated for CD56 to determine the percent of NK cell population expressing CD16. (B) Target A549-NLR cells were stained with antibody to IgG1 Fc domain to quantify surface Fc expression. (C) Target naïve A549-NLR cells or NA-Fc4 expressing cells were treated with Fc blocking antibody or isotype control antibody prior to incubation with PM21-NK cells at an E:T ratio of 5:1. (D) Similarly, PM21-NK cells were treated with anti-CD16 antibody or isotype control prior to incubation with either of the target cells at an E:T of 5:1. For both experiments, ROC was recorded every 2 h and images were captured on IncuCyte. Values are a mean of 3 replicates and error bars represent SD. Data was analyzed using two-way ANOVA test and by applying Tukey’s post hoc test when comparing with the indicated groups. For all graphs, the adjusted p values after applying post hoc tests were **p<0.01, ***p<0.001 and ****p<0.001. Stars indicate the point where the p-values first begin, and this statistical significance extends for later timepoints until a new star appears with a different p value.
Fig 7.
Complement-mediated lysis of tumor cell lines is not enhanced by NA-Fc4 expression.
Naïve A549-NLR (panel A) and SKOV3-NLR (panel B) cells as well the corresponding cell lines that stably express NA-Fc4 were left untreated or incubated with 20% normal human serum (NHS) or heat-inactivated NHS (HI). As positive controls for complement lysis, cells were alternatively infected with PIV5 and treated as above. ROC was recorded every 2 h on IncuCyte.
Fig 8.
Lentiviral delivery of NA-Fc4 enhances the susceptibility of naïve A549 lung and SKOV3 ovarian tumor cells to PM21-NK cell mediated lysis.
(A, B) A549-NLR and SKOV3-NLR cells were transduced with LV expressing either NA-Fc4 or BFP as a control. Two days post transduction, transgene expression was quantified using flow cytometry by staining all the samples with IgG Fc. (C, D) The naïve target cells or cells transduced with LV-BFP or LV NA-Fc4 were co-incubated with PM21-NK cells at an E:T ratio of 2.5:1. ROC was recorded every 2 h on IncuCyte. Results are representative of two independent experiments conducted with two different NK cell donors in triplicate with error bars representing SD. Data was analyzed using one-way ANOVA & two-way ANOVA test and by applying Dunett’s post hoc test when comparing the indicated control group. For all graphs, adjusted p values after applying post hoc tests were **p<0.01, ***p<0.001 and ****p<0.0001. Stars indicate the point where the p-values first begin, and this statistical significance extends for later timepoints until a new star appears with a different p value.
Fig 9.
Lentiviral delivery of Fc protein enhances the susceptibility of naïve A375 melanoma and H1299 lung tumor cells to PM21-NK cell mediated lysis.
(A, B) A375-NLR and H1299-NLR cells were transduced with LV expressing NA-Fc4 or BFP as a control. Two days post transduction, transgene expression was quantified using flow cytometer by staining all the samples with IgG Fc antibody. (C, D) The target cells naïve, LV BFP or LV NA-Fc4 were co-incubated with PM21-NK cells at an E:T ratio of 2.5:1. ROC was recorded every 2 h on IncuCyte. These experiments are a representative of two independent experiments conducted with two different NK cell donors in triplicates with error bars representing SD. Data was analyzed using one-way ANOVA & two-way ANOVA test and by applying Dunett’s post hoc test when comparing with the indicated control group. For all graphs, the adjusted p values after applying post hoc tests were *p<0.05, **p<0.01 and ***p<0.001. Stars indicate the point where the p-values first begin, and this statistical significance extends for later timepoints until a new star appears with a different p value.
Fig 10.
Lentiviral delivery of NA-Fc4 enhances PM21-NK cell mediated killing of lung cells persistently infected with PIV5 mutant virus.
(A) P/V PI-NLR A549 cells were transduced with LV expressing NA-Fc4 or BFP as a control. Two days post transduction, transgene expression was quantified using flow cytometry. The untreated P/V PI cells stained with Fc antibody were used as controls. (B-D) The target A549 cells were acutely infected with P/V mutant, persistently infected A549 cells (P/V PI), P/V PI transduced with LV BFP or LV NA-Fc4 were plated and co-incubated with PM21-NK cells at an E:T ratio of 1.25:1. ROC was recorded every 2 h on IncuCyte. (B) Phase and red fluorescent images (10x) were captured at 32 h post addition of NK cells donor 2. The scale bar represents 400μm. (C, D) ROC were recorded on IncuCyte when each of the samples were co-incubated with NK cells Donor 1 and Donor 2. Curves represent data for acutely-infected cells (brown), P/V PI cells (black), or P/V PI cells transduced to express BFP (blue) or NA-Fc4 (red). Each data point is an average of 3 replicate samples with error bars representing SD. Data was analyzed using two-way ANOVA test and by applying Dunett’s post hoc test when comparing with the indicated groups. For both the graphs, the adjusted p values after applying post hoc tests were *p<0.05. Stars indicate the point where the p-values first begin, and this statistical significance extends for later timepoints until a new star appears with a different p value.