Fig 1.
Human NK cell subsets respond to infection with WNV in a time-dependent manner.
PBMCs from healthy young subjects (n = 6) were incubated with medium alone (mock) or infected with WNV (MOI = 1) for 8, 24, and 48 h. The samples were labeled with fluorescence-conjugated antibodies against CD3, CD19, CD14, CD56, CD16 and CD57 and analyzed by flow cytometry. (A) Manual gating strategy to define NK cell subsets. Total NK cells, CD56brightCD16-, CD56dimCD16-, CD56dimCD16+CD57-, and CD56dimCD16+CD57+ were assessed for surface expression of CD107a (B) and intracellular production of MIP-1β (C) and IFN-γ (D). Means ± s.e.m, multiple t tests.
Fig 2.
Downregulation of activating receptors in NK cells in response to WNV infection.
PBMCs were incubated with medium alone (mock) or infected with WNV (MOI = 1) for 24 h. The samples were labeled with metal-conjugated antibodies and analyzed by mass cytometry. (A) Manual gating strategy to define NK cell subsets. CD56brightCD16- (B) and CD56dimCD16- (C) NK cell subsets were compared between mock and WNV-infected groups for expression of NK cell inhibitory receptor NKG2A and activating receptors NKG2D, NKp30, NKp44, and NKp46 (n = 56). Paired Wilcoxon tests.
Table 1.
Cohort demographics.
Fig 3.
Expression levels of NKG2D ligands in PBMCs with infection of WNV in vitro.
PBMCs were incubated with medium alone (mock) or infected with WNV (MOI = 1) for 24 h. Samples from all subjects (n = 56) were compared by qPCR between mock and WNV-infected groups for expression of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6, paired Wilcoxon tests.
Fig 4.
Robust functional response to WNV by NK cells from younger and older healthy subjects.
PBMCs from young (n = 20) and old (n = 14) healthy subjects were incubated with medium alone (mock) or infected with WNV (MOI = 1) for 24 h. (A) Total NK cells from mock and WNV-infected groups of each WNV subject were compared by mass cytometry for surface expression of CD107a and production of production of MIP-1β, perforin, IFN-γ, GM-CSF, and TNF. Mock-WNV comparisons, paired Wilcoxon tests. Mock-mock and WNV-WNV comparisons, Mann-Whitney U tests. (B) Four NK cell subsets from young and old healthy subjects were compared for induction level of IFN-γ by subtracting baseline of the mock group from the WNV-infected group. Mann-Whitney U tests.
Fig 5.
Robust functional response to WNV by NK cells from asymptomatic and symptomatic WNV subjects.
PBMCs from asymptomatic (n = 10) and symptomatic (n = 12) WNV subjects were incubated with medium alone (mock) or infected with WNV (MOI = 1) for 24 h. (A) Total NK cells from mock and WNV-infected samples of each WNV subject were compared by mass cytometry for surface expression of CD107a and production of MIP-1β, perforin, IFN-γ, TNF, and GM-CSF. Mock-WNV comparisons, paired Wilcoxon tests. Mock-mock and WNV-WNV comparisons, Mann-Whitney U tests. (B) Four NK cell subsets from asymptomatic and symptomatic WNV subjects were compared for induction of IFN-γ by subtracting baseline of the mock group from the WNV-infected group. Mann-Whitney U tests. (C) Samples from all four groups of subjects (n = 56) were compared by qPCR for baseline expression of MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, and ULBP6. Mann-Whitney U tests. P < 0.05 = *; P < 0.01 = **; P < 0.001 = ***.
Fig 6.
Decreased frequency of immature NK cells in subjects with a history of symptomatic WNV infection.
(A) Frequency of total NK cells in asymptomatic (n = 10) and symptomatic (n = 12) WNV subjects. (B-D) The NK dataset from asymptomatic and symptomatic subjects was analyzed by automated hierarchical clustering. Distinguishing clusters between the two groups were identified using SAM model (q < 0.01). (B) Stratifying clusters (yellow circles) including distinguishing clusters between the two groups (blue circles) and abundance of cells within the identified distinguishing clusters. (C) Expression of CD56, CD16, NKG2A and CD57 of stratifying clusters. (D) Histograms to characterize cluster features, with red outline indicating the expression pattern in the cluster that differed between groups and the blue outline indicating the level of staining in the population as a whole.