Responsiveness to Influenza Vaccination Correlates with NKG2C-Expression on NK Cells.

Article Responsiveness to Influenza Vaccination Correlates with NKG2C-Expression on NK Cells Peggy Riese 1,*,†, Stephanie Trittel 1,†, Rishi D. Pathirana 2,3, Frank Klawonn 4,5, Rebecca J. Cox 2,3,6,†and Carlos A. Guzmán1,7,† 1Department of Vaccinology and Applied Microbiology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany; [email protected] (S.T.); [email protected] (C.A.G.) 2Department of Clinical Science, The Influenza Centre, University of Bergen, 5007 Bergen, Norway; [email protected] (R.D.P.); r[email protected] (R.J.C.) 3K.G. Jebsen Centre for Influenza Vaccine Research, University of Oslo, 0313 Oslo, Norway 4Department of Biostatistics, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany; [email protected] 5Department of Computer Science, Ostfalia University of Applied Sciences, 38302 Wolfenbuettel, Germany 6Department of Research and Development, Haukeland University Hospital, 5021 Bergen, Norway 7Centre for Individualized Infection Medicine, 30625 Hannover, Germany *Correspondence: peggy[email protected]; Tel.: +49-531-61814609; Fax: +49-531-61814699 †Authors contributed equally. Received: 30 April 2020; Accepted: 3 June 2020; Published: 5 June 2020   Abstract: Influenza vaccination often results in a large percentage of low responders, especially in high-risk groups. As a first line of defense, natural killer (NK) cells play a crucial role in the fight against infections. However, their implication with regard to vaccine responsiveness is insufficiently assessed. Therefore, this study aimed at the validation of essential NK cell features potentially associated with differential vaccine responsiveness with a special focus on NKG2Cand/or CD57-expressing NK cells considered to harbor memory-like functions. To this end, 16 healthy volunteers were vaccinated with an adjuvanted pandemic influenza vaccine. Vaccine responders and low responders were classified according to their hemagglutination inhibition antibody titers. A majority of responders displayed enhanced frequencies of NKG2C-expressing NK cells 7or 14-days post-vaccination as compared to low responders, whereas the expression of CD57 was not differentially modulated. The NK cell cytotoxic potential was found to be confined to CD56 dim CD16 + NKG2C-expressing NK cells in the responders but not in the low responders, which was further confirmed by stochastic neighbor embedding analysis. The presented study is the first of its kind that ascribes CD56 dim CD16 + NKG2C-expressing NK cells a crucial role in biasing adaptive immune responses upon influenza vaccination and suggests NKG2C as a potential biomarker in predicting pandemic influenza vaccine responsiveness. Keywords: influenza; vaccination; vaccine responsiveness; NK cells; NKG2C 1. Introduction Acute respiratory infections caused by the influenza virus are one of the major public health problems leading to high mortality rates worldwide, and consequently, to a large societal economic burden [ 1 ]. Occasionally, a novel virus arises, leading to worldwide spread and a pandemic. In the last decade, a number of pandemic influenza vaccines have received marketing authorization [ 2 ]. An increase in hemagglutination inhibition (HAI) antibody titer is commonly used to measure the response to the vaccine. However, antibody responses may be restricted in high-risk groups (i.e., Vaccines 2020,8, 281; doi:10.3390/vaccines8020281 www.mdpi.com/journal/vaccines Vaccines 2020,8, 281 2 of 18 the very young and the elderly, individuals with co-morbidities), resulting in a large percentage of low responders [3,4]. The generation of protective immunity requires the interplay between innate and adaptive immune cells. NK cells are described as crucial innate immune cells in the fight against influenza infections [ 5 ]. Recent findings in NK cell biology provide further evidence on specific functional features of NK cells, which highlight a formerly underestimated role during infection. Several studies revealed unexpected NK cell characteristics, like their continuous differentiation process and the impact of the education status on the magnitude of NK cell functionality [ 6 – 9 ]. Likewise, it was discovered that NK cells display adaptive immune features similar to T cells, including the generation of memory-like NK cells [ 10 , 11 ]. Several reports highlighted the occurrence of murine memory-like NK cells by (i) hapten-induced contact hypersensitivity, (ii) murine cytomegalovirus infection (MCMV) and (iii) cytokine stimulation [12–15]. Human memory-like NK cells were first described in vivo as a unique subset of NK cells expressing CD57 and NKG2C detected in a cohort of individuals infected with the human cytomegalovirus (HCMV) [ 16 – 18 ]. Subsequently, it was also demonstrated that human memory-like NK cells, characterized by the expression of NKG2C, could be induced in vitro by cytokine stimulation [ 19 , 20 ]. The CD94-NKG2C activating NK cell receptor binds to HLA-E and acts via the ITAM-bearing DAP12 signaling pathway [ 21 ]. CD57 is mainly expressed by terminally differentiated NK cells, which are suggested to have undergone clonal expansion following infection. CD57 + NK cells are described to harbor a lower proliferative capacity and to be less cytotoxic in response to cytokine stimulation, but show higher CD16-induced cytotoxicity [22]. Next to their newly discovered functional features, NK cells are well known to interact directly and indirectly with adaptive immune cells. Thus, it can be hypothesized that NK cells might be considered as relevant players in the initiation of adaptive immunity following influenza vaccination. Supporting evidence comes from a recent study, in which NK cell responsiveness following influenza vaccination was investigated. The results of this study demonstrated that NK cells with an intracellular immune memory, characterized by enhanced IFN γ secretion following antigen-specific re-stimulation, are generated following vaccination [ 23 ]. These NK cells displayed an increased internalization of the NKp46 receptor, which is known to interact with the influenza surface protein hemagglutinin (HA). However, despite this fragmentary evidence, there is still a considerable paucity of knowledge in this field. In this regard, NK cell subsets expressing CD57 and NKG2C have yet to be addressed. Thus, in the present study, the impact of the H1N1 vaccination on phenotypic and functional changes of NK cells expressing CD57 and NKG2C and their reciprocal influence on the vaccination efficacy was investigated. 2. Materials and Methods 2.1. Study Design Sixteen healthy volunteers (health care workers (HCWs)) were vaccinated with the pandemic influenza vaccine Pandemrix ® (split virion, inactivated; A/California/07/2009 (H1N1)v-like strain (X-179A), GlaxoSmithKline, Brentford, UK), adjuvanted with AS03 as part of a large clinical trial. Fourteen of the participants were female and two were male (one normal and one low-responder), and they were born between 1951 and 1987 with a median birth year of 1974 and 1969 for normaland low-responders, respectively. Other than three participants (normal responders), all participants received previous seasonal influenza vaccines. All participants provided written informed consent before inclusion in the study, which had ethical (Regional Committee for Medical Research Ethics (ethical approval number is 2009/1224, issued by REC west), Western Norway (REK Vest)) and regulatory (Norwegian Medicines Agency) approval and is registered at the National Institute for Health Database Clinical trials.gov (NCT01003288). Human subject rights were protected during the trial and the data analysis. Blood (clotted and Cell Preparation Tubes (CPTs)) was collected prior and 7-, 14-, 21and 180-days post-vaccination [ 24 ]. Peripheral blood mononuclear cells (PBMCs) were Vaccines 2020,8, 281 3 of 18 isolated from CPT tubes according to the manufacturer’s instructions and cryo-preserved in 90% fetal bovine serum (FBS)/10% dimethyl sulfoxide (DMSO) until further analysis. 2.2. Humoral Immune Responses The HAI titers in serum samples pre-vaccination and 7-, 14-, 21-, 90and 180-days post-vaccination were determined by a HAI assay using the X179A virus. The assay was performed with 0.7% turkey red blood cells, as described previously [ 24 ]. The titers analyzed at days 0 and 90 were used to define responders and low responders. Vaccinees with a 4-fold seroconversion or a titer increase >40 were considered as responders. Human cytomegalovirus (CMV)-specific IgG antibodies were assessed using the Alinity i instrument (Abbott). 2.3. Cellular Immune Responses PBMCs were thawed and 1 × 10 6 to 4 × 10 6 cells/sample were re-stimulated for 16 h in complete RPMI 1640 (Gibco, supplemented with 10% FCS, 5% Penicillin/Streptomycin and 5% Glutamine) containing the vaccine formulation with a final concentration of 4 µ g hemagglutinin (HA)/mL split virus vaccine (kindly provided by GlaxoSmithKline, Belgium). Unstimulated samples were incubated for the same time in complete RPMI without the vaccine formulation. Brefeldin A and monensin were added to all samples after 5 h of incubation. Cells were collected and stained for flow cytometric analysis. Surface marker staining was performed for 20 min at 4 ◦ C. The following antibodies were used diluted in PBS: CD56 (PE-Cy7, clone B159, BD, Franklin Lakes, NJ, USA), CD3 (V450, clone UCHT1, BD), CD14 (Pacific Blue, clone M5E2, BD), CD19 (V450, clone HIB19, BD Horizon), CD16 (APC-H7, clone 3G8, BD Pharmingen), NKG2C (PE, clone 134591, R&D Systems, Minneapolis, MN, USA), CD57 (APC, clone HCD57, BioLegend, San Diego, CA, USA), Live/Dead (Fixable Blue, Invitrogen, Carlsbad, CA, USA). The expression of CD107a was used as a correlate of degranulation. To this end, the anti-CD107a antibody (PE-Cy5, clone eBioH4A3, eBioscience, San Diego, CA, USA) was added to the culture. The secretion of IFN γ (Alexa Fluor 700, clone B27, BioLegend) was detected by intracellular staining using Cytofix/Cytoperm solution (BD Biosciences). Samples were acquired at a BD Fortessa flow cytometer and analyzed using FlowJo (FlowJo, LLC, Ashland, OR, USA). Unstained, single stained (one antibody/sample) as well as fluorescence-minus-one (FMO) samples were used as controls for the acquisition as well as the subsequent analysis. Statistical differences were determined by the GraphPad Prism software. 2.4. Stochastic Neighbor Embedding (SNE) Analysis Flow cytometry data of responders and low responders derived preand 7-days post-vaccination were imported into FlowJo (version 9) and compensation channel values were extracted for the following parameters: CD56, CD16, NKG2C, CD57, CD107a and IFN γ . Up to 10,000 values were extracted per vaccinee and time point and then pooled for responders and low responders. By using the R package “tsne”, a t-distributed SNE analysis using Barnes–Hut implementation was performed and the resulting data were plotted with intensities for the depicted markers (RStudio version 3.2.1, RStudio, Inc., Boston, MA, USA), as described earlier [25,26]. 2.5. Statistical Analysis GraphPad Prism (version 6.0 for Windows, GraphPad Software, La Jolla, CA, USA) was used for the statistical assessment (unpaired low-parametric Mann–Whitney or Kruskal–Wallis test and Spearman correlation). Values of p≤0.05 were considered significant. Vaccines 2020,8, 281 4 of 18 3. Results 3.1. Influenza Vaccination Leads to Enhanced Frequencies of NKG2C-Expressing NK Cells NK cells are characterized by the intensity of CD56 expression and the co-expression of CD16 and can be thereby divided into different functional subsets (see gating strategy, Figure S1). The impact of pandemic vaccination on the distribution of blood NK cell subsets was assessed by flow cytometry at various time points. A slightly reduced frequency of total NK cells in vaccinated individuals was observed during the first 14 days after vaccination ( ≈ 6% at day 0 to ≈ 5.4% at day 7 and 4.5% at day 14) (Figure 1a). The marginally diminished frequency remained relatively stable over the observation period until day 180 post-vaccination ( ≈ 5% at day 21 and ≈ 4.8% at day 180). The division of NK cells into primarily cytokine secreting CD56 bright and highly cytotoxic CD56 dim subsets revealed a decrease in the CD56 dim NK cell frequencies, especially of CD56 dim CD16 + cells ( ≈ 48% at day 0 to ≈ 34% at day 7), whereas CD56 bright NK cell frequencies were not affected (Figure 1b). These data suggest that vaccination-induced modulation mainly affects CD56 dim CD16 + NK cells, which are described to hold a higher cytotoxic but lower cytokine secreting capability as compared to CD56 bright NK cells [ 27 ]. Human memory-like NK cells based either on the expression of CD57 and NKG2C or NKG2C alone were recently described to exert amplified recall responses upon CMV infection or to be induced after cytokine stimulation (IL-12, IL-15 and IL-18), respectively [ 13 , 17 , 20 ]. The analysis of CD56 dim CD16 + NK cells with regard to CD57 and NKG2C expression after pandemic vaccination revealed that the changes in NK cell frequencies were mainly restricted to the NKG2C-expressing subsets. Increased frequencies of both CD57 − NKG2C + (from ≈ 2% at day 0–9% at day 7 and ≈ 8% at day 14) and CD57 + NKG2C + NK cell subsets (from ≈ 1% at day 0–4% at day 7 and ≈ 7% at day 14) were detected at days 7 and 14 post-vaccination (orange and red) in samples derived from 8 out of 10 vaccine responders (marked with an arrow, Figure 1c). The most striking differences were observed at day 7 and 14 post-vaccination, with significantly elevated levels of both CD57 − NKG2C + and CD57 + NKG2C + NK cells, whereas the frequency of CD57 + NKG2C − NK cells was not affected (Figure 1d). Within the NKG2C-expressing subsets, high ratios comparing postand pre-vaccination values were detected ( ≥ 1.5, red data points) while the CD57 + NKG2C − NK cell subset showed ratios >1 (blue data points), but not ≥ 1.5. The analysis of NK cells either expressing NKG2C or CD57 confirmed the increased frequency of NKG2C but not CD57 expression (Figure 1e). The data suggest that adjuvanted pandemic vaccination induces changes in the subset of NKG2C-expressing NK cells, which in turn can be potentially involved in determining the outcome of vaccination. While the frequency of NKG2C-expressing NK cells is described to be connected with CMV sero-positivity, no such correlation was found here (Figure S2a) [ 16 , 17 ]. Furthermore, no differences between the mean CMV titer of low and normal responders was observed (Figure S2b). The correlation analysis of the CMV titer and the fold change of the HAI titer or the age of the participants also did not yield any significant relation (Figure S2c). Likewise, the fold change of the HAI titer did not correlate with the age of the vaccinees (Figure S2d). However, in normal but not in low responders, the frequency of NKG2C + CD57 + NK cells at day 7 post-vaccination showed a significant negative correlation with the age of the vaccinees (Figure S2e). These findings highlight that the hypothesized relation between NKG2C+NK cells and the vaccination outcome is not dependent on the CMV sero-status, thus supporting publications stating that CMV infections do not affect influenza vaccine efficacy [28]. Vaccines 2020,8, 281 5 of 18 Vaccines 2020, 8, 281 5 of 18 Figure 1. Cont. Vaccines 2020,8, 281 6 of 18 Vaccines 2020, 8, 281 6 of 18 Figure 1. Influenza vaccination affects the frequency of NKG2C-expressing natural killer (NK) cells. Peripheral blood mononuclear cells (PBMCs) isolated from vaccinated individuals prior to vaccination and at the indicated time points post-vaccination (dpv = days post-vaccination) were stained for the surface markers CD56, CD3, CD16, NKG2C and CD57. (a) Frequencies of total CD3−CD56+ NK cells and (b) of CD3-CD56bright, CD3−CD56dim and CD3−CD56dimCD16+ NK cell subpopulations. Diagrams show the connected column mean with 95% confidence interval. (c) Frequencies of NK cell populations characterized by the expression of CD57 and NKG2C. Columns represent individual data points. (d) CD57and NKG2C-expressing CD56dimCD16+ subpopulations depicted as the ratio of cell frequencies detected at the indicated time points post-vaccination and the frequencies detected prior vaccination (day 0). Diagrams are depicted as scatter plots with bars of individual assigned data points. (e) CD56dimCD16+NKG2C+ and CD56dimCD16+CD57+ NK cells displayed as frequencies and as the ratio to the day before vaccination. Columns represent individual data points; diagrams are depicted as scatter plots with bars of individual assigned data points. Arrows indicate donors with vaccine-induced immunological changes. Asterisks denote significant values as calculated by unpaired and non-parametric Kruskal–Wallis test. * p ≤ 0.05. Letters in panels (d, e) indicate single responders. Figure 1. Influenza vaccination affects the frequency of NKG2C-expressing natural killer (NK) cells. Peripheral blood mononuclear cells (PBMCs) isolated from vaccinated individuals prior to vaccination and at the indicated time points post-vaccination (dpv =days post-vaccination) were stained for the surface markers CD56, CD3, CD16, NKG2C and CD57. ( a ) Frequencies of total CD3 − CD56 + NK cells and ( b ) of CD3 − CD56b right , CD3 − CD56 dim and CD3 − CD56 dim CD16 + NK cell subpopulations. Diagrams show the connected column mean with 95% confidence interval. ( c ) Frequencies of NK cell populations characterized by the expression of CD57 and NKG2C. Columns represent individual data points. ( d ) CD57and NKG2C-expressing CD56 dim CD16 + subpopulations depicted as the ratio of cell frequencies detected at the indicated time points post-vaccination and the frequencies detected prior vaccination (day 0). Diagrams are depicted as scatter plots with bars of individual assigned data points. ( e ) CD56 dim CD16 + NKG2C + and CD56 dim CD16 + CD57 + NK cells displayed as frequencies and as the ratio to the day before vaccination. Columns represent individual data points; diagrams are depicted as scatter plots with bars of individual assigned data points. Arrows indicate donors with vaccine-induced immunological changes. Asterisks denote significant values as calculated by unpaired and non-parametric Kruskal–Wallis test. * p ≤ 0.05. Letters in panels ( d , e ) indicate single responders. Vaccines 2020,8, 281 7 of 18 3.2. NK Cells of Influenza Vaccination Responders and Low Responders Display Differences in NKG2C and CD57 Expression The inefficacy of influenza vaccines, characterized by the varying occurrence of low responders, is a persisting problem. To classify normal and low responders following pandemic vaccination, the HAI titer of each vaccinee was evaluated at days 0 and 90 post-vaccination (Figure S3). The total frequencies of NK cells derived from normal and low responders were compared, as well as the expression of CD57 and NKG2C. Reduced frequencies of CD56 dim CD16 + NK cells were observed in both response groups at 7-days post-vaccination, as compared to day 0. However, the decrease was less profound in normal responders ( ≈ 30% reduction for responders and ≈ 46% reduction for low responders) (Figure 2a). These findings were consistent with the assessed ratios of NK cell frequencies (day 7/day 0). The assessment of CD57 expression prior to vaccination demonstrated differences in its basal expression by NK cells derived from normal and low responders (day 0). However, the ratio of CD56 dim CD16 + CD57 + NK cell frequencies (day 7/day 0) revealed no vaccine-induced effect on CD57 expression (ratios ≈ 1 for both responders and low responders (Figure 2b). The analysis of CD56 dim CD16 + NKG2C + NK cells revealed an increased frequency at day 7 post-vaccination in normal responders that was not observed in low responders (Figure 2c). The ratio of CD56 dim CD16 + NKG2C + NK cell frequencies (day 7/day 0) supports this finding by displaying a higher ratio for normal responders ( ≈ 6) as compared to low responders ( ≈ 1). This difference is further highlighted by ratios mainly ≥ 1.5 (red dots) detected for NKG2C-expression by CD16 + NK cells derived from normal responders. This indicates that responders show increased frequencies of NKG2C + expression at day 7 post-vaccination as compared to day 0, whereas NKG2C expression in low responders remains largely unaffected. To address whether the observed increased frequency of NKG2C + NK cells is due to the specific antigen (HA) re-stimulation, an individual analysis of single donors was performed. These data revealed that a significant number of responders displayed an HA-induced surface expression of NKG2C that was not observed in the group of low responders (Figure 2d, ratio (HA/unstimulated (NS)) >1=blue, ≥1.5 =red ). Interestingly, the individuals responding to HA re-stimulation with enhanced expression of NKG2C already displayed a higher basal expression. With regard to the HA-induced surface expression of CD57, neither vaccine responders nor low responders displayed a strong modulation, as indicated by ratios (HA/unstimulated) >1 but not ≥ 1.5 (Figure 2e). These findings suggest that NKG2C-expressing NK cells bias vaccine responsiveness and might serve as a determinant of responsiveness towards influenza vaccination. Vaccines 2020,8, 281 8 of 18 Vaccines 2020, 8, 281 8 of 18 Figure 2. Phenotypic analysis of NK cells derived from normal and low responders to the pandemic influenza vaccination. Flow cytometric analysis of frozen PBMCs isolated from vaccinated individuals classified into normal responders (black dots) and low responders (white dots; a-c). Frequencies and ratios of (a) CD56dimCD16+, (b) CD56dimCD16+CD57+ and (c) CD56dimCD16+NKG2C+ NK cells. Diagrams are depicted as scatter dot plots indicating the mean by a horizontal line. Ratios were derived from cell frequencies detected at day 7 post-vaccination and the frequencies detected prior to vaccination (day 0) depicted as scatter plots with bars. Ratios of unstimulated (NS) and HAre-stimulated (HA) (d) CD56dimCD16+NKG2C+ and (e) CD56dimCD16+CD57+ NK cells derived from normal and low responders. Blue dots depict ratios >1 and red dots depict values ≥ 1.5. 3.3. CD107a Expression is Confined to CD56dimCD16+NKG2C-Expressing NK Cells in Responders but not in Low Responders To dissect whether, in addition to the phenotypic alterations, the functionality of CD56dim NK cells in normal and low responders also differs, CD16 expression, CD107a expression and IFNγ secretion were addressed ex vivo. Independently of the responsiveness to vaccination, differences in the functionality of CD16and CD16+ NK cells within the CD56dim subset were detected (Figure 3a,b). The analysis of the NK cell degranulation capacity revealed that within the group of responders, CD56dimCD16− NK cells showed no changes in the frequency of CD107a-expressing cells after vaccination (Figure 3a). In contrast, CD56dimCD16+ NK cells derived from responders exhibited an enhanced expression of CD107a peaking at day 7 post-vaccination. Low responders showed similar functional differences between CD56dimCD16and CD16+ NK cells (Figure 3a). CD56dimCD16+ NK cells showed a higher frequency of CD107a-expressing cells at day 7 post-vaccination as compared to day 0. The comparison of normal and low responders further revealed that responders harbor a lower frequency of CD56dimCD16+CD107a+ NK cells at day 7 post-vaccination (≈4%) as compared to low responders (≈12%) (Figure 3a). Figure 2. Phenotypic analysis of NK cells derived from normal and low responders to the pandemic influenza vaccination. Flow cytometric analysis of frozen PBMCs isolated from vaccinated individuals classified into normal responders (black dots) and low responders (white dots; a-c). Frequencies and ratios of ( a ) CD56 dim CD16 + , ( b ) CD56 dim CD16 + CD57 + and ( c ) CD56 dim CD16 + NKG2C + NK cells. Diagrams are depicted as scatter dot plots indicating the mean by a horizontal line. Ratios were derived from cell frequencies detected at day 7 post-vaccination and the frequencies detected prior to vaccination (day 0) depicted as scatter plots with bars. Ratios of unstimulated (NS) and HA-re-stimulated (HA) ( d ) CD56 dim CD16 + NKG2C + and ( e ) CD56 dim CD16 + CD57 + NK cells derived from normal and low responders. Blue dots depict ratios >1 and red dots depict values ≥1.5. 3.3. CD107a Expression Is Confined to CD56 dim CD16 + NKG2C-Expressing NK Cells in Responders but Not in Low Responders To dissect whether, in addition to the phenotypic alterations, the functionality of CD56 dim NK cells in normal and low responders also differs, CD16 expression, CD107a expression and IFN γ secretion were addressed ex vivo. Independently of the responsiveness to vaccination, differences in the functionality of CD16 − and CD16 + NK cells within the CD56 dim subset were detected (Figure 3a,b). The analysis of the NK cell degranulation capacity revealed that within the group of responders, CD56 dim CD16 − NK cells showed no changes in the frequency of CD107a-expressing cells after vaccination (Figure 3a). In contrast, CD56 dim CD16 + NK cells derived from responders exhibited an enhanced expression of CD107a peaking at day 7 post-vaccination. Low responders showed similar functional differences between CD56 dim CD16 − and CD16 + NK cells (Figure 3a). CD56 dim CD16 + NK cells showed a higher frequency of CD107a-expressing cells at day 7 post-vaccination as compared to day 0. The comparison of normal and low responders further revealed that responders harbor a lower frequency of CD56 dim CD16 + CD107a + NK cells at day 7 post-vaccination ( ≈ 4%) as compared to low responders (≈12%) (Figure 3a). Vaccines 2020,8, 281 9 of 18 Vaccines 2020, 8, 281 10 of 18 Figure 3. Functional differences characterize NK cells isolated from normal and low responders. Functional flow cytometric analysis of frozen PBMCs isolated from vaccinated individuals classified into normal and low responders. Frequencies of (a) CD107a+CD56dim and (b) IFNγ+CD56dim NK cells with regard to CD16 expression (CD16− white/CD16+ black). Ratio of CD107a-expressing (c) CD57−NKG2C+, (d) CD57+NKG2C+ and (e) CD57+NKG2C− CD56dimCD16+ NK cells isolated from responders and non-responders on the indicated days postand pre-vaccination (day 0) depicted as scatter plots with bars indicating the mean with 95% confidence interval. (f) Frequency of CD56dimCD16+ NK cells expressing CD107a (red solid), NKG2C+CD57− (black solid), NKG2C+CD57+ (green), NKG2C−CD57+ (black dashed) shown for individual responders and (g) non-responders. Asterisks denote significant values as calculated by unpaired and non-parametric Mann–Whitney test. * p ≤ 0.05, ** p ≤ 0.01. Figure 3. Functional differences characterize NK cells isolated from normal and low responders. Functional flow cytometric analysis of frozen PBMCs isolated from vaccinated individuals classified into normal and low responders. Frequencies of ( a ) CD107a + CD56 dim and ( b ) IFN γ+ CD56 dim NK cells with regard to CD16 expression (CD16 − white/CD16 + black). Ratio of CD107a-expressing ( c ) CD57 − NKG2C + , ( d ) CD57 + NKG2C + and ( e ) CD57 + NKG2C − CD56 dim CD16 + NK cells isolated from responders and non-responders on the indicated days postand pre-vaccination (day 0) depicted as scatter plots with bars indicating the mean with 95% confidence interval. ( f ) Frequency of CD56 dim CD16 + NK cells expressing CD107a (red solid), NKG2C + CD57 − (black solid), NKG2C + CD57 + (green), NKG2C − CD57 + (black dashed) shown for individual responders and ( g ) non-responders. Asterisks denote significant values as calculated by unpaired and non-parametric Mann–Whitney test. * p≤0.05, ** p≤0.01. Vaccines 2020,8, 281 16 of 18 24. 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