Enhancement of cytokine-driven NK cell IFN-γ production after vaccination of HCMV infected Africans

Abstract

Human cytomegalovirus (HCMV) infection drives the phenotypic and functional differentiation of NK cells, thereby influencing the responses of these cells after vaccination. NK cell functional differentiation is particularly advanced in African populations with universal exposure to HCMV. To investigate the impact of advanced differentiation on vaccine-induced responses, we studied NK-cell function before and after vaccination with Trivalent Influenza Vaccine (TIV) or diphtheria, tetanus, pertussis, inactivated poliovirus vaccine (DTPiP) in Africans with universal, lifelong HCMV exposure. In contrast to populations with lower prevalence of HCMV infection, no significant enhancement of NK-cell responses (IFN-γ, CD107a, CD25) occurred after in vitro re-stimulation of post-vaccination NK cells with TIV or DTPiP antigens compared to pre-vaccination baseline cells. However, both vaccinations resulted in higher frequencies of NK cells producing IFN-γ in response to exogenous IL-12 with IL-18, which persisted for up to 6 months. Enhanced cytokine responsiveness was restricted to less differentiated NK cells, with increased frequencies of IFN-γ⁺ cells observed within CD56^bright CD57^- , CD56^dim CD57^- NKG2C^- and CD56^dim CD57^- NKG2C⁺ NK-cell subsets. These data suggest a common mechanism whereby different vaccines enhance NK cell IFN-γ function in HCMV infected donors and raise the potential for further exploitation of NK cell "pre-activation" to improve vaccine effectiveness.

Keywords: DTPiP vaccine; Influenza vaccine; NK cells; NKG2C; Vaccination.

Figure 1

Age‐dependent differences in NK‐cell subsets. (A–F) Proportions of NK cells and subsets were determined ex‐vivo at baseline for three age‐defined groups (2–6, 20–30, 60–75 years). Proportions of (A) CD56⁺CD3⁻ NK cells within total lymphocytes and (B) CD56^bright cells within NK cells. Frequency of (C) CD57 and (D) NKG2C⁺ cells within CD56^dim NK cells. Expression of (E) NKG2A and (F) NKG2C within CD56/CD57‐defined NK cell subsets. Data are shown for 68 subjects. Boxes indicate median values with interquartile ranges and whiskers indicate 95th percentiles. Statistical analysis was performed on samples using (A–D) Kruskal–Wallis test, *p < 0.05, **p < 0.01, ***p < 0.001 and (E,F) using linear trend ANOVA with correction for multiple comparisons ****p < 0.0001.

Figure 2

No change in NK cell responses to TIV after vaccination. (A–F) Frequencies of IFN‐γ (A, D), CD107a⁺ (B, E) and CD25⁺ (C, F) NK cells were determined after in vitro stimulation of PBMC from (A–C) TIV vaccinated Gambians at baseline and 4, 12, and 24 weeks after vaccination. (D–F) Data from HCMV seropositive and seronegative UK subjects at baseline and 4 weeks post TIV vaccination. Cells were cultured in (LCC) low concentration of cytokines (rIL‐12: & rIL18) alone (open symbols) or LCC plus TIV vaccine antigen (closed symbols) (A–F). Data are shown for (A–C) 61 TIV vaccinated Gambian and (D–F) 52 UK subjects, comprising 19 HCMV+ and 33 HCMV‐ individuals. Dots represent individual values and horizontal lines represent the median frequencies for each group. Paired statistical analysis was performed using Wilcoxon signed‐rank test and comparisons between groups were made using Mann–Whitney U test.*p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001.

Figure 3

Vaccination does not enhance antibody‐dependent, TIV‐driven NK cell responses. (A‐C) Plasma IgG antibodies expressed as Arbitrary ELISA Units (AEU)/mL were measured by ELISA against TIV antigen for each age group. Dots indicate median antibody levels and bars represent IQR. Data are shown for 68 subjects. Repeated measures ANOVA was used to analyze the trend in antibody levels before and up to 24 weeks post‐vaccination *p < 0.05, ***p < 0.001; ****p < 0.0001. (D, E) Frequency of CD107a expressing NK cells at week 0 and week 4 post TIV vaccination of (D) Gambian and (E) UK donors. Cells were in medium alone (open symbols) or TIV antigen (closed symbols) in the presence of week 0 or week 4 autologous plasma. NK cells were gated as shown in Supporting Information Fig. 1H,K. Data are shown for 38 Gambian subjects, comprising 3 children, 17 adults and 18 elderly individuals (D) and for 17 TIV vaccinated UK adults (E). Paired statistical analysis was performed using Wilcoxon Signed rank test, *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4

NK cell IFN‐γ responses to exogenous cytokines are significantly enhanced post vaccination. (A‐C) Frequencies of NK cells expressing IFN‐γ after restimulation with IL‐12 + IL‐18 (HCC) before and after vaccination with TIV in (A) Gambian and (B) UK donors. (C) Age‐stratified (2–6, 20–30, 60–75 years) NK cell IFN‐γ responses of Gambians to cytokines at baseline and 4 weeks after TIV vaccination showing the percentage of individuals within each age group with enhanced responses post‐vaccination. (D, E) Frequencies of baseline CD57‐ NK cells across the entire cohort (D) or in children only (E), split according to ‘responder’ individuals, with increased HCC driven IFN‐γ responses and ‘non‐responder’. (F) Responses to HCC in Gambian adults before and 4 weeks post‐vaccination with DTPiP. All NK cells analysis was gated as shown in Supporting Information Fig. 1. Data from 65 subjects are shown for TIV vaccinated Gambians (A, C), and 52 UK donors (comprising 19 HCMV+, closed symbols and 33 HCMV‐ individuals, open symbols) and from 18 Gambian subjects for DTPiP vaccination (F). Dots represent the frequency of (A,B,C,F) IFN‐γ⁺ or (C, D) CD57⁻ NK cells for each subject and horizontal lines represent median frequency for each group. Statistical analysis of paired samples (A, C, F) was performed using Wilcoxon signed‐rank test and for nonpaired analysis with Mann–Whitney U test, *p < 0.05, **p < 0.01.

Figure 5

Enhancement of cytokine‐driven IFN‐γ production within CD56^bright, CD56^dimNKG2C⁻CD57⁻ and CD56^dimNKG2C⁺CD57⁻ NK cells after vaccination. (A) Frequencies of IFN‐γ producing NK cells after HCC stimulation of PBMC at baseline (0) and up to 24 weeks post TIV vaccination. (B) Gating strategy showing CD57 and NKG2C‐defined subsets. (C, D) Subset distribution for NK cell IFN‐γ production after HCC stimulation of pre (0 weeks) and post‐vaccination (4, 12, 24 weeks) samples from subjects receiving (C) TIV and (D) DTPiP. Dots represent the frequency of IFN‐γ⁺ NK cells for each subject and horizontal lines represent median frequency for each group. Statistical analysis was performed on paired samples using Wilcoxon signed‐rank test, *p < 0.05, **p < 0.01.