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. 2019 Sep 15;203(6):1609-1618.
doi: 10.4049/jimmunol.1801648. Epub 2019 Aug 19.

Influenza Vaccination Primes Human Myeloid Cell Cytokine Secretion and NK Cell Function

Helen R Wagstaffe  1 , Harry Pickering  2 , Joanna Houghton  2 , Jason P Mooney  1   3 , Asia-Sophia Wolf  1   4 , Natalie Prevatt  2 , Ron H Behrens  2 , Martin J Holland  2 , Eleanor M Riley  1   3 , Martin R Goodier  5
Affiliations

Influenza Vaccination Primes Human Myeloid Cell Cytokine Secretion and NK Cell Function

Helen R Wagstaffe et al. J Immunol. .

Abstract

Cytokine-induced memory-like (CIML) NK cells generated in response to proinflammatory cytokines in vitro and in vivo can also be generated by vaccination, exhibiting heightened responses to cytokine stimulation months after their initial induction. Our previous study demonstrated that in vitro human NK cell responses to inactivated influenza virus were also indirectly augmented by very low doses of IL-15, which increased induction of myeloid cell-derived cytokine secretion. These findings led us to hypothesize that IL-15 stimulation could reveal a similar effect for active influenza vaccination and influence CIML NK cell effector functions. In this study, 51 healthy adults were vaccinated with seasonal influenza vaccine, and PBMC were collected before and up to 30 d after vaccination. Myeloid and lymphoid cell cytokine secretion was measured after in vitro PBMC restimulation with low-dose IL-15, alone or in combination with inactivated H3N2 virus; the associated NK cell response was assessed by flow cytometry. PBMC collected 30 d postvaccination showed heightened cytokine production in response to IL-15 compared with PBMC collected at baseline; these responses were further enhanced when IL-15 was combined with H3N2. NK cell activation in response to IL-15 alone (CD25) and H3N2 plus IL-15 (CD25 and IFN-γ) was enhanced postvaccination. We also observed proliferation of less-differentiated NK cells with downregulation of cytokine receptors as early as 3 d after vaccination, suggesting cytokine stimulation in vivo. We conclude that vaccination-induced "training" of accessory cells combines with the generation of CIML NK cells to enhance the overall NK cell response postvaccination.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1. Influenza vaccination enhances IL-15 stimulated cytokine production
Baseline (white bars) and 30 day post-vaccination (grey bars) PBMC samples were cultured with medium (n=10), 0.75ng/ml IL-15 alone (n=26), inactivated H3N2 (n=10) or H3N2 + IL-15 (n=26) for 18 hours and culture supernatants were collected. Concentrations (pg/ml) of GM-CSF (a), IL-10 (b), IL-1β (c), IFN-α2 (d), IL-12(p70) (e), TNFα (f), and IFN-γ (g) in supernatant were determined by Luminex. Graphs show one dot per donor, with a bar representing median value. Comparisons between vaccination time points were made using Wilcoxon signed-rank test. *p < 0.05, **p < 0.01, *** p < 0.001.
Figure 2
Figure 2. Enhanced NK cell responses to IL-15 post-vaccination
Baseline (white bars) and 30 day post-vaccination (grey bars) PBMC samples from 2015-2016 TIV (n=37) or 2017-2018 QIV (n=14) vaccinated individuals (all donors were combined for analysis), were stimulated in vitro with medium alone, IL-15 alone, H3N2 or H3N2 + IL-15. IFN-γ, CD107a and CD25 expression of CD56+CD3- NK cells was measured after 6 (IFN-γ, CD107a) or 18 hours (CD25) by flow cytometry. Plots show the gating strategy for IFN-γ (a), CD107a (b) and CD25 expression (c) from one representative donor at baseline. Numbers shown are the percentage of total NK cells positive for each marker. IFN-γ (d), CD107a (e) in response to IL-15 and H3N2 + IL-15 and CD25 in response to all 4 conditions (f) is shown at baseline and 30 days post-vaccination. IFN-γ (g), CD107a (h) and CD25 (i) in response to H3N2 + IL-15 attributed to NK cell differentiation subsets defined by CD56 and CD57 expression is also shown. Plots show one dot per donor with a bar representing median percentage. Comparisons between vaccination time points were made using Wilcoxon signed-rank test and between culture conditions by one-way ANOVA with Dunn’s correction for multiple comparisons. *p < 0.05, **p < 0.01, ****p < 0.0001.
Figure 3
Figure 3. Vaccine induced enhancement of polyfunctional NK cell responses requires IL-15 co-stimulation
Percentages of double positive IFN-γ+CD25+ (a), CD107a+IFN-γ+ (b) and CD107a+CD25+ (c) NK cells at baseline (white bars) and 30 days post vaccination (grey bars) were determined by flow cytometry after 18 hour stimulation with medium alone, IL-15 alone, H3N2 or H3N2 + IL-15 (n=51). Plots are one dot per donor with a bar representing median percentage. Comparisons between vaccination time points were made using Wilcoxon signed-rank test and between culture conditions by one-way ANOVA with Dunn’s correction for multiple comparisons. *p < 0.05, ****p < 0.0001.
Figure 4
Figure 4. Post-vaccination NK cell responses are dependent on IL-12
Baseline (white bars) and 30 day post-vaccination (grey bars) PBMC were stimulated in vitro with IL-15 alone or H3N2 + IL-15 in the presence of an anti-IL-12 blocking antibody for 18 hours, (a) percentage of IFN-γ+ and CD25+IFN-γ+ double positive NK cells were determined by flow cytometry (n=51). Linear regression analysis showing correlation between IFN-γ+ and CD25+IFN-γ+ double positive NK cells and IL-12(p70) concentration measured by Luminex in response to H3N2 + IL-15 (no IL-12 blocking) at 18 hours both before (b) and after (c) vaccination. Percentage of lineage negative (CD3-CD56-CD19-) CD14-CD11c+ mDCs expressing IL-12(p40) was determined by flow cytometry (gating strategy shown in d) in response to IL-15 alone or H3N2+IL-15 (e). Graphs show median percentage with 95% confidence interval or box and whisker plots with 10th-90th percentile. Goodness of fit was determined using r2 and significance was determined by Pearson coefficient as p value below 0.05. Comparisons between culture conditions and vaccination time points were made using Wilcoxon signed-rank test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Figure 5
Figure 5. Altered ex-vivo NK cell phenotype and cytokine receptor expression early after TIV vaccination
NK cell phenotype was measured after 2015-2016 TIV vaccination (n=37) at baseline, day 3, day 7 and day 30 post-vaccination. Percentage of CD56bright and CD56dim NK cells (a), Ki67 (b), IL-12Rβ2 (c) and CD25 MFI (d) were determined and attributed to NK cell differentiation subsets defined by CD56 and CD57 expression. Graphs show box and whisker plots with 10th-90th percentile, comparison across vaccination visits was determined by one-way ANOVA with Dunn’s correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p< 0.001, **** p < 0.0001.
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