Fig 1: NK cell numbers and mitochondrial mass are increased in response to vaccination.a Schematic representation of vaccine schedule and blood draws to assess NK cell responses after vaccination. Arrows at Day 0 and Day 56 indicate the timing of prime and boost vaccines, respectively. Blood samples were taken prior to vaccination and at subsequent timepoints indicated by the Days (D) post-prime vaccine. b PBMCs were analysed for % CD69 expression on CD56dim and CD56bright NK cells by flow cytometry at baseline and at indicated timepoints post vaccination (n = 7). c Absolute peripheral blood NK cell counts before and after vaccination (n = 7). d Representative histograms, paired donor responses and summaries of relative fluorescence intensity of Mitotracker CMXRos and e ATP5B in CD56dim and CD56bright NK cells after vaccination. Data shown are normalised to baseline (Day 0) values (n = 6–8 dependent on the timepoint analysed). Paired donor responses are shown for d Day 0 and Day 56, e Day 0 and Day 1 (n = 7). In summary, graphs dots indicate individual donor responses, while lines represent the mean. f Representative histograms and summary of mean fluorescence intensity of CD98 in NK cells (n = 7). Bold timepoints indicate baseline samples taken on the day of prime (DO) or boost (D56) vaccination. Samples were compared by mixed-model with Bonferroni post hoc test. c n.s. not significant, *p < 0.05 and **p < 0.01.
Fig 2: Prior HCMV infection stratifies divergent CD56dim NK cell responses to vaccination.a Identification of HCMV seropositive donors in co-vaccinated group. HCMV specific IgG was quantified from pre-vaccination baseline serum by ELISA. The line indicates cut-off for a positive result (n = 13). b PBMC ex vivo ELISPOT responses to HCMV lysate at baseline and after vaccination. c–e Stratification of vaccine recipient responses based on HCMV seropositive (black circle) or negative (open circle) status. Each dot shows NK cells stimulated with IL-12/IL-15 overnight and measurement of NK cells expressing c IFN?, d granzyme B and e pS6 for CD56dim NK cells for pre- and post vaccination (Days 7 and 28). (HCMV-, n = 6 for c, d, n = 3 for e, HCMV+, n = 4). f Summary graphs of post-vaccination relative fluorescence intensity of Mitotracker CMXRos and g ATP5B in NK cells from HCMV serodiscordant donors normalised to baseline (Day 0) values (n = 7 HCMV-, n = 4 HCMV+). Representative histogram of ATP5B post vaccination. Samples were compared by two-way ANOVA with Sidek’s post hoc test. n.s. not significant, *p < 0.05 and **p < 0.01.
Fig 3: Prior CMV infection impacts ATP synthase expression in CD56bright NK cells in response to vaccination.a Summaries of IL-12 (30 ng)/IL-15 (100 ng) stimulated IFN?+, b granzyme B expression and c pS6 + CD56bright NK cells in vaccine recipients stratified by HCMV serostatus at pre- and post-vaccination timepoints (Days 7 and 28). (HCMV–, n = 6 for a, b, n = 3 for c, HCMV+, n = 4). Representative histograms and summaries of post-vaccination relative fluorescence intensity of d Mitotracker CMXRos and e ATP5B in NK cells from HCMV sero-discordant donors normalised to baseline (Day 0) values (n = 7 HCMV-, n = 4 HCMV+). Samples were compared by two-way ANOVA with Sidek’s post hoc test. n.s. not significant, *p < 0.05, **p < 0.01.
Fig 4: HCMV impacts on functional and metabolic responses of CD56dim NK cell subsets post vaccination.Summary of canonical and adaptive CD56dim NK cells responses at baseline and post-priming vaccination (Day 0, Day 7 and Day 28). Canonical (FceR?1+Syk+) or adaptive (FceR?1+Syk-, FceR?1-Syk+ and FceR?1-Syk-) cells were analysed as indicated for a ex vivo frequencies of NK subsets, b frequency of pS6+, c IFN?+ and d IFN? MFI in response to IL-12/IL-15 stimulation after vaccination (n = 4). e Summary of post-priming Mitotracker and f ATP5B MFI in canonical and adaptive CD56dim NK cells at baseline and Days 7 and 28 (n = 4). Error bars show SEM. a, e, f Samples were compared by mixed-model with Bonferroni post hoc test. b–d Samples were compared by two-way ANOVA with Sidek’s post hoc test. n.s. not significant.
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