Fig 1.
NK cells cultured with VZV are unresponsive to K562 stimulation.
(A) NK cells (CD3–CD56+) were FACS sorted from CD56+-selected lymphocytes following culture with mock or VZV inoculum, plus 200 U/ml IL-2. At 2 days pi isolated NK cells were then challenged with K562 cells in a calcein-AM release assay. Graph depicts means ± SEM for four donors. ****P < 0.0001 (repeated measures two-way ANOVA with Sidak correction). (B) Flow cytometry of degranulation (CD107a+) of NK cells (viable CD3–CD56+ cells) from PBMCs cultured with mock or VZV inoculum for 2 days, and stimulated with K562 cells with IL-2 or left unstimulated. VZV exposed or infected was determined by surface staining for VZV gE:gI. Graph shows frequency of specific degranulation against K562 cells for five donors. Symbols represent individual donors, and grey columns indicate mean. ***P < 0.001, ns = not significant (repeated measures one-way ANOVA with Sidak correction). (C) Flow cytometry of degranulation (CD107a+) or isotype control stain (Iso) of NK cells (viable CD3–CD56+ cells) from PBMCs mock cultured, exposed to VZV, or VZV infected for the times indicated, and stimulated with K562 cells with IL-2. Graph depicts mean frequency of degranulation (± SEM) for two donors. (D) Flow cytometry of VZV infection (gE:gI+) of NK cells from (C).
Fig 2.
Polyfunctional analysis of VZV cultured NK cells.
PBMCs were mock cultured, exposed to VZV, or VZV infected for 2 days and stimulated as specified for flow cytometry analysis of NK cells (viable CD3–CD56+ cells). (A) Degranulation (CD107a+) following stimulation with PMA and ionomycin (PMA/I). Graph shows specific degranulation with PMA/I stimulation for seven donors. Symbols represent individual donors, and grey columns indicate mean. ns = not significant (Friedman test with Dunn’s correction). (B) SPICE pie charts show the proportion of responses to different stimuli (listed left) based on combinations of detected CD69, CD107a, IFN-γ and TNF expression. Pie slices indicate the number of responses (0–4) (key, middle right). Arcs detail which markers were detected for each response (key, outer right). Data represent the means of three donors. (C) Histograms show CD69 expression for NK cells either unstimulated (above) or stimulated with PMA/I (below), for one representative donor (n = 6 donors).
Fig 3.
VZV infected NK cells produce less IFN-γ and TNF upon PMA/I stimulation.
PBMCs were mock cultured, exposed to VZV, or VZV infected for 2 days (A) or 1 day (B) and stimulated with PMA/I for flow cytometry analysis of IFN-γ (top panels) or TNF (bottom panels) expression by NK cells (viable CD3–CD56+ cells). Symbols represent individual donors, and grey columns indicate mean. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns = not significant (Repeated measures one-way ANOVA with Tukey correction (A) or Friedman test with Dunn’s correction (B)). Data are from nine donors (A) or seven donors (B).
Fig 4.
Profiling of cell surface receptor phenotype of VZV cultured NK cells.
PBMCs were mock cultured, exposed to VZV, or VZV infected untreated or with 200 U/ml IL-2 for 1 day. NK cells (viable CD3–CD56+ cells) were assessed by flow cytometry for cell surface receptor expression (A), and heatmaps show receptor expression as measured by percentage positive with hierarchical clustering for 3 donors (denoted A, B and C) (B). (C) Graph shows fold change over mock in median fluorescence intensity (MFI) for ubiquitously expressed receptors (n = 3). Symbols represent individual donors. Dotted line at y = 1 indicates point of variance from mock. Statistical analysis performed compared to mock. **P < 0.01, ***P < 0.001, ****P < 0.0001 (repeated measures two-way ANOVA with Dunnett’s correction).
Fig 5.
(A) PBMCs were cultured with mock infected inoculum or inoculum infected with HSV-1 expressing GFP under different viral promoters (ICP47, ICP6 or gB as specified) for 1 day. Infection of NK cells (viable CD3–CD56+ cells) was determined by flow cytometry detection of GFP. (B) Frequency of NK cell infection with HSV-1 GFP-pICP47 performed as in (A), for seven donors. Symbols represent individual donors, and bar indicates mean.
Fig 6.
HSV-1 culture of NK cells reduces responsiveness to K562 stimulation and infection blocks IFN-γ expression.
PBMCs were mock cultured, exposed to HSV-1, or HSV-1 infected for 1 day and stimulated as specified for flow cytometry analysis of NK cells (viable CD3–CD56+ cells). (A) SPICE pie charts show the proportion of responses to different stimuli (listed left) based on combinations of detected CD69, CD107a, IFN-γ and TNF expression. Arcs detail which markers were detected for each response (key right). Data represent the means of three donors. (B) Degranulation (CD107a+) following stimulation with K562 cells with IL-2 (top panel) or PMA/I (bottom panel). Graphs show specific degranulation for specified stimulus for three donors. (C) IFN-γ and TNF expression following stimulation with PMA/I. Graphs show frequencies of positive cells for three donors. Symbols represent individual donors, and grey columns indicate mean. *P < 0.05, ns = not significant (Friedman test with Dunn’s correction).
Fig 7.
VZV inhibition of NK cell function is contact-dependent.
(A) PBMCs were either mock cultured, exposed to VZV, or VZV infected (top panel), or separated from the inoculum by a Transwell membrane (bottom panel) for 1 day. PBMCs were challenged with K562 cells with IL-2 or left unstimulated (inset panels) and NK cells (viable CD3–CD56+ cells) assessed for degranulation (CD107a+) by flow cytometry. Graph shows specific degranulation against K562 cells for three donors. Symbols represent individual donors, and grey columns indicate mean. ns = not significant (two-tailed Wilcoxon test). (B) PBMCs were either separated from VZV inoculum by a Transwell membrane or treated with supernatant harvested from PBMCs cocultured with VZV inoculum. Flow cytometry plots show surface staining for VZV gE:gI on NK cells (viable CD3–CD56+ cells). (C) PBMCs were treated for 1 day with supernatant harvested from PBMCs cocultured with VZV inoculum, and challenged with K562 cells with IL-2 or left unstimulated (inset panels) and NK cells (viable CD3–CD56+ cells) assessed for degranulation (CD107a+) by flow cytometry. Data are representative of n = 3 donors (Transwell separated) (A & B) or n = 2 donors (supernatant treated) (B & C).
Fig 8.
VZV culture interferes with NK cell signalling.
(A) Simplified diagram of the general NK cell signalling pathway leading to degranulation, as well as the inhibitory action of SHP-2. Examined signalling molecules are indicated with an orange outline. (B-D) PBMCs were mock cultured, exposed to VZV, or VZV infected in the presence of 200 U/ml IL-2 for 1 day and either left unstimulated or stimulated with K562 cells for 2, 5, 10 or 30 min as specified. Phosphorylation of signalling markers in NK cells (CD3–CD56+ cells) was detected by flow cytometry. (B) Heatmap of phosphorylated signalling marker median fluorescence intensity (MFI) fold increase. (C) Histogram shows phospho–SLP-76 expression for NK cells unstimulated (above) and after 10 min stimulation with K562 cells (below). (D) Histogram shows phospho–ERK1/2 expression for NK cells unstimulated (left) and after 10 min stimulation with K562 cells (right). MFI values are indicated on the top left of the histogram. Data are representative of three donors. ERK1/2, extracellular signal-regulated kinase 1 and 2; Grb2, growth factor receptor-bound protein 2; LAT, linker for activation of T cells; MEK1/2, mitogen-activated or extracellular signal-regulated protein kinase kinase 1 and 2; PI3K, phosphatidylinositol 3-kinase; PKC-θ, protein kinase C-θ; PLC-γ, phospholipase C-γ; SHP-2, Src homology phosphatase 2; SLP-76, SH2 domain-containing leukocyte phosphoprotein of 76 kD.