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. 2013 Jul;87(13):7717-25.
doi: 10.1128/JVI.01096-13. Epub 2013 May 1.

Human cytomegalovirus-induced NKG2C(hi) CD57(hi) natural killer cells are effectors dependent on humoral antiviral immunity

Zeguang Wu  1 , Christian Sinzger, Giada Frascaroli, Johanna Reichel, Carina Bayer, Li Wang, Reinhold Schirmbeck, Thomas Mertens
Affiliations

Human cytomegalovirus-induced NKG2C(hi) CD57(hi) natural killer cells are effectors dependent on humoral antiviral immunity

Zeguang Wu et al. J Virol. 2013 Jul.

Abstract

Recent studies indicate that expansion of NKG2C-positive natural killer (NK) cells is associated with human cytomegalovirus (HCMV); however, their activity in response to HCMV-infected cells remains unclear. We show that NKG2C(hi) CD57(hi) NK cells gated on CD3(neg) CD56(dim) cells can be phenotypically identified as HCMV-induced NK cells that can be activated by HCMV-infected cells. Using HCMV-infected autologous macrophages as targets, we were able to show that these NKG2C(hi) CD57(hi) NK cells are highly responsive to HCMV-infected macrophages only in the presence of HCMV-specific antibodies, whereas they are functionally poor effectors of natural cytotoxicity. We further demonstrate that NKG2C(hi) CD57(hi) NK cells are intrinsically responsive to signaling through CD16 cross-linking. Our findings show that the activity of pathogen-induced innate immune cells can be enhanced by adaptive humoral immunity. Understanding the activity of NKG2C(hi) CD57(hi) NK cells against HCMV-infected cells will be of relevance for the further development of adoptive immunotherapy.

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Figures

Fig 1
Fig 1
NKG2Chi CD57hi NK cells are associated with HCMV seropositivity. (A) Freshly isolated PBMCs from HCMV-seropositive (n = 35) and -seronegative (n = 24) healthy donors were stained with PerCP–Cy5.5–anti-CD3 (UCHT1), APC–anti-CD56 (B159), PE–anti-CD57 (NK-1), and AF488-NKG2C (134591) antibodies. After gating on lymphocytes, the percentages of NKG2Chi CD57hi NK cells gated on CD3− CD56dim (left), NKG2Chi CD57hi NKT cells gated on CD3+ CD56+ (middle), and NKG2Chi CD57hi T cells gated on CD3+ CD56− (right) are shown. Statistical significance is indicated at the top (Mann-Whitney U test). The horizontal lines represent the mean for each group. (B) Results of representative four-color staining to identify NKG2Chi CD57hi NK cells from 3 HCMV-seropositive donors and 1 seronegative donor are shown.
Fig 2
Fig 2
Expression of inhibitory and activating receptors on NKG2Chi CD57hi NK cells (A) and their degranulation to HCMV-infected autologous macrophages (B) and K562 cells (C). (A) Expression of CD158b, LIR-1, NKG2A, NKp46, 2B4, DNAM-1, NKG2D, and CD16 on NKG2Chi CD57hi NK cells and NKG2C-negative NK cells from NKG2Chi CD57hi NK cell-positive donors. The paired circles from one donor are connected by a line. Percentages (CD158b, LIR-1, and NKG2A) or geometric mean fluorescence intensities (gMFI) (NKp46, 2B4, DNAM-1, NKG2D, and CD16) are shown. (B) Thawed PBMCs (1 × 106) were cocultured with TB40/E-infected macrophages (1 × 105) or uninfected macrophages for 48 h. Then, surface expression of CD107a on NK cells was assessed 5 h after the addition of anti-CD107a. (C) Thawed PBMCs (1 × 106) were cultured for 48 h and afterwards cocultured with K562 cells (1 × 105) for 5 h in the presence of anti-CD107a, and then surface CD107a expression was assessed. (A, B, and C) The percentages of positive cells were determined on NKG2Chi CD57hi NK cells and NKG2C-negative NK cells. The nonparametric Wilcoxon signed rank sum test was used to compare NKG2Chi CD57hi NK cells with NKG2C-negative NK cells matched from the same donor.
Fig 3
Fig 3
Establishment of HCMV antibody-dependent NK cell-mediated responses to infected autologous macrophages. (A) Two hundred-microliter cell-free suspensions of TB40/E with or without HCMV antibodies were used in neutralization experiments on macrophages. Virus suspensions and indicated Ig preparations (autologous HCMV-antibody-positive plasma/serum with 1:10 dilution or Ig pool with 1:200 dilution) were incubated for 30 min and then inoculated onto the cells. The infection rates were assessed after 24 h of incubation. The presence of HCMV IEA (red fluorescence) indicates infected macrophages, and cell nuclei are stained in blue (DAPI). The infection rates are indicated in red numbers. Original magnification, ×40. (B) Macrophages were used for staining 3 days postinfection. Methanol-fixed macrophages were incubated with the indicated Ig preparations, followed by FITC–anti-human IgG staining. The presence of HCMV IEA (red fluorescence) indicates infected macrophages, human IgG binding on macrophages is shown by green fluorescence, and nuclei are stained in blue (DAPI). Original magnification, ×100. (C) Thawed PBMCs (1 × 106) were cocultured with TB40/E-infected macrophages (1 × 105) for 48 h. Then, surface expression of CD107a and IFN-γ production of NK cells was assessed after incubation with the indicated HCMV-specific antibodies (seronegative plasma and serum used as controls) and anti-CD107a for an additional 5 h. The results of one representative experiment from an HCMV-seropositive donor without NKG2Chi CD57hi NK cells are shown.
Fig 4
Fig 4
NKG2Chi CD57hi NK cells are highly responsive to HCMV antibody-dependent stimulation. (A) PBMCs (1 × 106) were cocultured with TB40/E-infected macrophages (1 × 105) for 48 h. Then, surface expression of CD107a, CD16, and IFN-γ production of NK cells was assessed after 5 h in the absence (left) or presence (right) of autologous plasma (1:10 dilution) and anti-CD107a. The indicated percentages of positive cells were determined as percentage of NKG2Chi CD57hi NK cells and NKG2C-negative NK cells. (B) CD16 and CD107a expression and IFN-γ production of the NKG2Chi CD57hi NK cell subset. First, NKG2Chi CD57hi NK cells were gated. Then, the percentages of positive cells were determined as percentage of CD16-positive and CD16-negative NK cells (top and middle) or as percentage of IFN-γ-positive and IFN-γ-negative NK cells (bottom). (A, B) The results of one representative experiment of four from NKG2Chi CD57hi NK cell-positive donors are shown.
Fig 5
Fig 5
NKG2C induces degranulation of NKG2Chi CD57hi NK cells and augments degranulation through CD16 stimulation. (A) Representative staining of redirected degranulation against antibody-coated P815 cells. Thawed PBMCs (1 × 106) were cocultured with P815 cells (1 × 105) which had been precoated with the isotype control MAb (IgG1), anti-NKG2C MAb, or/and anti-CD16 MAb for 4 h. The percentages of CD107a-positive cells were determined on NKG2Chi CD57hi NK cells and NKG2C-negative NK cells. (B) Percentages of CD107a-positive cells on NKG2Chi CD57hi NK cells and NKG2C-negative NK cells from different donors are shown. The paired circles from one donor are connected by a line.
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