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Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK Cell Function

View Article: PubMed Central - PubMed

ABSTRACT

Cross-linking of FcγRIII (CD16) by immune complexes induces antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells, contributing to control of intracellular pathogens; this pathway can also be targeted for immunotherapy of cancerous or otherwise diseased cells. However, downregulation of CD16 expression on activated NK cells may limit or regulate this response. Here, we report sustained downregulation of CD16 expression on NK cells in vivo after intramuscular (but not intranasal) influenza vaccination. CD16 downregulation persisted for at least 12 weeks after vaccination and was associated with robust enhancement of influenza-specific plasma antibodies after intramuscular (but not intranasal) vaccination. This effect could be emulated in vitro by co-culture of NK cells with influenza antigen and immune serum and, consistent with the sustained effects after vaccination, only very limited recovery of CD16 expression was observed during long-term in vitro culture of immune complex-treated cells. CD16 downregulation was most marked among normally CD16high CD57+ NK cells, irrespective of NKG2C expression, and was strongly positively associated with degranulation (surface CD107a expression). CD16 downregulation was partially reversed by inhibition of ADAM17 matrix metalloprotease, leading to a sustained increase in both CD107a and CD25 (IL-2Rα) expression. Both the degranulation and CD25 responses of CD57+ NK cells were uniquely dependent on trivalent influenza vaccine-specific IgG. These data support a role for CD16 in early activation of NK cells after vaccination and for CD16 downregulation as a means to modulate NK cell responses and maintain immune homeostasis of both antibody and T cell-dependent pathways.

No MeSH data available.


Downregulation of NK cell CD16 expression after vaccination. (A,B) Increased plasma concentrations of anti-influenza IgG after (A) intramuscular (I.M, n = 17) but not after (B) intranasal vaccination (I. N, n = 18) up to 36 weeks after vaccination. Data represent median values with interquartile ranges. (C–F) Flow cytometric gating to assess the impact of vaccination on CD16 expression in CD56dim, CD57−, or CD57+ NK cell subsets, ex vivo. NK cells were gated as CD56+ CD3−(C) and then as CD56bright, CD56dimCD57− or CD56dimCD57+(D). (E,F) For determination of antigen-driven effects CD16+ CD56+ NK cells were gated among PBMC cultured with TIV and immune plasma (E) or immune plasma without TIV (F). Ex vivo analysis of CD16 expression at baseline (0), 2, 4, and 12 weeks after intramuscular (I. M) vaccination with TIV (G) or intranasal (I.N.) vaccination with LAIV (H). Impact on NK cell CD16 expression of culturing baseline PBMC with TIV (shaded bars) or without TIV (open bars) and either pre- (0) and post- (2) vaccination I.M. (I) or I.N. (J) plasma; data are shown for CD56dim, CD57−, and CD57+ NK subsets after 6 h of in vitro culture with TIV. Trend analysis was performed using a one-way repeated measures ANOVA. Paired comparisons between pre- and post-vaccination plasma were made using Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Figure 1: Downregulation of NK cell CD16 expression after vaccination. (A,B) Increased plasma concentrations of anti-influenza IgG after (A) intramuscular (I.M, n = 17) but not after (B) intranasal vaccination (I. N, n = 18) up to 36 weeks after vaccination. Data represent median values with interquartile ranges. (C–F) Flow cytometric gating to assess the impact of vaccination on CD16 expression in CD56dim, CD57−, or CD57+ NK cell subsets, ex vivo. NK cells were gated as CD56+ CD3−(C) and then as CD56bright, CD56dimCD57− or CD56dimCD57+(D). (E,F) For determination of antigen-driven effects CD16+ CD56+ NK cells were gated among PBMC cultured with TIV and immune plasma (E) or immune plasma without TIV (F). Ex vivo analysis of CD16 expression at baseline (0), 2, 4, and 12 weeks after intramuscular (I. M) vaccination with TIV (G) or intranasal (I.N.) vaccination with LAIV (H). Impact on NK cell CD16 expression of culturing baseline PBMC with TIV (shaded bars) or without TIV (open bars) and either pre- (0) and post- (2) vaccination I.M. (I) or I.N. (J) plasma; data are shown for CD56dim, CD57−, and CD57+ NK subsets after 6 h of in vitro culture with TIV. Trend analysis was performed using a one-way repeated measures ANOVA. Paired comparisons between pre- and post-vaccination plasma were made using Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Mentions: Data analysis was performed using FlowJo V10 (Tree Star). FACS gates set on unstimulated cells (medium alone or isotype controls) were applied across all samples and all conditions. Responses where the gated subset contained <100 events were excluded. CD57+ subsets were gated using an isotype-matched control reagent (mIgG1-eFluor450, eBioscience). Sample gating strategies are shown in Figure 1.


Sustained Immune Complex-Mediated Reduction in CD16 Expression after Vaccination Regulates NK Cell Function
Downregulation of NK cell CD16 expression after vaccination. (A,B) Increased plasma concentrations of anti-influenza IgG after (A) intramuscular (I.M, n = 17) but not after (B) intranasal vaccination (I. N, n = 18) up to 36 weeks after vaccination. Data represent median values with interquartile ranges. (C–F) Flow cytometric gating to assess the impact of vaccination on CD16 expression in CD56dim, CD57−, or CD57+ NK cell subsets, ex vivo. NK cells were gated as CD56+ CD3−(C) and then as CD56bright, CD56dimCD57− or CD56dimCD57+(D). (E,F) For determination of antigen-driven effects CD16+ CD56+ NK cells were gated among PBMC cultured with TIV and immune plasma (E) or immune plasma without TIV (F). Ex vivo analysis of CD16 expression at baseline (0), 2, 4, and 12 weeks after intramuscular (I. M) vaccination with TIV (G) or intranasal (I.N.) vaccination with LAIV (H). Impact on NK cell CD16 expression of culturing baseline PBMC with TIV (shaded bars) or without TIV (open bars) and either pre- (0) and post- (2) vaccination I.M. (I) or I.N. (J) plasma; data are shown for CD56dim, CD57−, and CD57+ NK subsets after 6 h of in vitro culture with TIV. Trend analysis was performed using a one-way repeated measures ANOVA. Paired comparisons between pre- and post-vaccination plasma were made using Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
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Figure 1: Downregulation of NK cell CD16 expression after vaccination. (A,B) Increased plasma concentrations of anti-influenza IgG after (A) intramuscular (I.M, n = 17) but not after (B) intranasal vaccination (I. N, n = 18) up to 36 weeks after vaccination. Data represent median values with interquartile ranges. (C–F) Flow cytometric gating to assess the impact of vaccination on CD16 expression in CD56dim, CD57−, or CD57+ NK cell subsets, ex vivo. NK cells were gated as CD56+ CD3−(C) and then as CD56bright, CD56dimCD57− or CD56dimCD57+(D). (E,F) For determination of antigen-driven effects CD16+ CD56+ NK cells were gated among PBMC cultured with TIV and immune plasma (E) or immune plasma without TIV (F). Ex vivo analysis of CD16 expression at baseline (0), 2, 4, and 12 weeks after intramuscular (I. M) vaccination with TIV (G) or intranasal (I.N.) vaccination with LAIV (H). Impact on NK cell CD16 expression of culturing baseline PBMC with TIV (shaded bars) or without TIV (open bars) and either pre- (0) and post- (2) vaccination I.M. (I) or I.N. (J) plasma; data are shown for CD56dim, CD57−, and CD57+ NK subsets after 6 h of in vitro culture with TIV. Trend analysis was performed using a one-way repeated measures ANOVA. Paired comparisons between pre- and post-vaccination plasma were made using Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Mentions: Data analysis was performed using FlowJo V10 (Tree Star). FACS gates set on unstimulated cells (medium alone or isotype controls) were applied across all samples and all conditions. Responses where the gated subset contained <100 events were excluded. CD57+ subsets were gated using an isotype-matched control reagent (mIgG1-eFluor450, eBioscience). Sample gating strategies are shown in Figure 1.

View Article: PubMed Central - PubMed

ABSTRACT

Cross-linking of Fc&gamma;RIII (CD16) by immune complexes induces antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells, contributing to control of intracellular pathogens; this pathway can also be targeted for immunotherapy of cancerous or otherwise diseased cells. However, downregulation of CD16 expression on activated NK cells may limit or regulate this response. Here, we report sustained downregulation of CD16 expression on NK cells in vivo after intramuscular (but not intranasal) influenza vaccination. CD16 downregulation persisted for at least 12&thinsp;weeks after vaccination and was associated with robust enhancement of influenza-specific plasma antibodies after intramuscular (but not intranasal) vaccination. This effect could be emulated in vitro by co-culture of NK cells with influenza antigen and immune serum and, consistent with the sustained effects after vaccination, only very limited recovery of CD16 expression was observed during long-term in vitro culture of immune complex-treated cells. CD16 downregulation was most marked among normally CD16high CD57+ NK cells, irrespective of NKG2C expression, and was strongly positively associated with degranulation (surface CD107a expression). CD16 downregulation was partially reversed by inhibition of ADAM17 matrix metalloprotease, leading to a sustained increase in both CD107a and CD25 (IL-2R&alpha;) expression. Both the degranulation and CD25 responses of CD57+ NK cells were uniquely dependent on trivalent influenza vaccine-specific IgG. These data support a role for CD16 in early activation of NK cells after vaccination and for CD16 downregulation as a means to modulate NK cell responses and maintain immune homeostasis of both antibody and T cell-dependent pathways.

No MeSH data available.