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. 2015 Mar;89(5):2906-17.
doi: 10.1128/JVI.03489-14. Epub 2014 Dec 24.

Natural killer cells can inhibit the transmission of human cytomegalovirus in cell culture by using mechanisms from innate and adaptive immune responses

Zeguang Wu  1 , Christian Sinzger  1 , Johanna Julia Reichel  2 , Marlies Just  1 , Thomas Mertens  3
Affiliations

Natural killer cells can inhibit the transmission of human cytomegalovirus in cell culture by using mechanisms from innate and adaptive immune responses

Zeguang Wu et al. J Virol. 2015 Mar.

Abstract

Human cytomegalovirus (HCMV) transmission within the host is important for the pathogenesis of HCMV diseases. Natural killer (NK) cells are well known to provide a first line of host defense against virus infections. However, the role of NK cells in the control of HCMV transmission is still unknown. Here, we provide the first experimental evidence that NK cells can efficiently control HCMV transmission in different cell types. NK cells engage different mechanisms to control the HCMV transmission both via soluble factors and by cell contact. NK cell-produced interferon gamma (IFN-γ) suppresses HCMV production and induces resistance of bystander cells to HCMV infection. The UL16 viral gene contributes to an immune evasion from the NK cell-mediated control of HCMV transmission. Furthermore, the efficacy of the antibody-dependent NK cell-mediated control of HCMV transmission is dependent on a CD16-158V/F polymorphism. Our findings indicate that NK cells may have a clinical relevance in HCMV infection and highlight the need to consider potential therapeutic strategies based on the manipulation of NK cells.

Importance: Human cytomegalovirus (HCMV) infects 40% to 100% of the human population worldwide. After primary infection, mainly in childhood, the virus establishes a lifelong persistence with possible reactivations. Most infections remain asymptomatic; however, HCMV represents a major health problem since it is the most frequent cause of infection-induced birth defects and is responsible for high morbidity and mortality in immunocompromised patients. The immune system normally controls the infection by antibodies and immune effector cells. One type of effector cells are the natural killer (NK) cells, which provide a rapid response to virus-infected cells. NK cells participate in viral clearance by inducing the death of infected cells. NK cells also secrete antiviral cytokines as a consequence of the interaction with an infected cell. In this study, we investigated the mechanisms by which NK cells control HCMV transmission, from the perspectives of immune surveillance and immune evasion.

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Figures

FIG 1
FIG 1
Kinetics of HCMV transmission in different cell types. (A) Fibroblasts infected by clinical isolates and laboratory strain TB40/E were cocultured with a 2,000-fold excess of uninfected fibroblasts, endothelial cells, or epithelial cells for 2, 3, 4, and 5 days. Monolayers were fixed at the indicated times, and newly infected cells were monitored by HCMV IEA staining. The numbers of infected cells per focus were counted. One dot represents the number of infected cells of an individual focus. Bars indicate mean values of all foci. (B) Representative infectious foci of clinical isolate 1 and TB40/E in fibroblasts, endothelial cells, and epithelial cells are shown. The presence of HCMV IEA (green fluorescence) indicates infected cells, and cell nuclei are stained in blue (DAPI). *, the foci could not be counted due to the high infection rate in fibroblasts; **, days of coculture.
FIG 2
FIG 2
NK cells efficiently inhibit HCMV transmission in fibroblasts and endothelial and epithelial cells. (A) Fibroblasts infected by the clinical isolates and laboratory strain TB40/E were cocultured with a 2,000-fold excess of uninfected fibroblasts, endothelial cells, or epithelial cells for 4 days. NK-92 cells were added to the cocultures immediately at different E:T ratios. Monolayers were fixed at the indicated times, and newly infected cells were identified by HCMV IEA staining. The numbers of infected cells per focus were counted. One dot represents the number of infected cells of an individual focus. Bars indicate mean values of all foci. (B) Thawed PBMCs were cultured using NK cell medium with IL-2 for 20 h, and then NK cells were negatively purified from PBMCs. Fibroblasts infected by the clinical isolate 1 and TB40/E were cocultured with a 2,000-fold excess of uninfected fibroblasts, endothelial cells, or epithelial cells for 3 days. Primary NK cells were added to the focus expansion assay immediately at different E:T ratios with IL-2-containing medium. Infectious foci were monitored by HCMV IEA staining.
FIG 3
FIG 3
IL-2 enhances the inhibition of HCMV transmission by primary NK cells. (A) Thawed PBMCs were cultured using NK cell medium with or without IL-2 for 20 h. Next, NK cells were negatively selected from PBMCs. TB40/E-infected fibroblasts were cocultured with a 2,000-fold excess of uninfected fibroblasts for 3 days. Purified NK cells were added to the focus expansion assay immediately at different E:T ratios with or without IL-2-containing medium. Monolayers were fixed at the indicated times, and newly infected cells were monitored by HCMV IEA staining. The numbers of infected cells per focus were counted. One dot represents the number of infected cells of an individual focus. Bars represent mean values of all foci from three donors tested. *, P < 0.05. (B) The concentrations of IFN-γ in supernatants from indicated cultures were tested by ELISA. Uninf., uninfected; Inf., infected. “0.25*” indicates that the same amount of NK cells was used.
FIG 4
FIG 4
Cell contact and soluble factors both contribute to the control of HCMV transmission by NK cells. Thawed PBMCs were cultured using NK cell medium with IL-2 for 20 h, and then NK cells were negatively selected from PBMCs. TB40/E-infected fibroblasts were cocultured with a 2,000-fold excess of uninfected fibroblasts for 3 days. Purified NK cells were added to the focus expansion assay from the beginning at an E:T ratio of 0.25 with IL-2-containing medium. Primary NK cells were either added to the cocultures directly or kept separated from fibroblasts using a Transwell filter system. Specific blocking MAb against IFN-γ or an isotype antibody as a control was added at the beginning of cocultures as indicated. Monolayers were fixed, and newly infected cells were monitored by HCMV IEA staining. The numbers of infected cells per focus were counted. One dot represents the number of infected cells of an individual focus. Bars indicate mean values of all foci.
FIG 5
FIG 5
NK cell-containing supernatants reduce HCMV production. (A) Supernatants were collected from the indicated cocultures and filtered through a 0.1-μm-pore filter to remove cell-free virus. Uninf., uninfected; Inf., infected; Ab, antibody; Iso., isotype antibody control. (B) Fibroblasts were infected by TB40/E (MOI, 7.5) for 30 min and washed with PBS. Thereafter, supernatants were added to infected fibroblasts from 30 min postinfection (p.i.) until 24 h p.i., from 24 to 96 h p.i., or from 96 to 120 h p.i., and then infection rates were determined by HCMV IEA staining. IEA staining is shown by green fluorescence and MCP staining is red at late times infection, and cell nuclei are stained in blue (DAPI). The infection rates (IEA/DAPI) are indicated by the numbers. The MCP-to-DAPI surface area ratios are also indicated by the numbers. (C) Fibroblasts were infected by TB40/E (MOI, 7.5) for 30 min and washed with PBS. Thereafter, supernatants were added to the infected fibroblasts from 24 to 96 h p.i. or from 96 to 120 h p.i. Then 100 μl of 8-fold-diluted supernatants containing cell-free virus was transferred to uninfected fibroblasts. After 30 min of incubation, cells were washed with PBS, and the infection rates (IEA/DAPI) were determined after 24 h.
FIG 6
FIG 6
HCMV gene UL16 modulates the control of HCMV transmission by NK cells. (A) Thawed PBMCs were cultured using NK cell medium with IL-2 for 20 h, and then NK cells were negatively selected from PBMCs. Fibroblasts infected with BAC4 or BAC4ΔUL16 were cocultured with a 2,000-fold excess of uninfected fibroblasts for 3 days. Purified NK cells were added to the focus expansion assay from the beginning at an E:T ratio of 0.25 with IL-2-containing medium. Specific blocking MAb against IFN-γ or an isotype antibody as a control was added immediately as indicated. Monolayers were fixed, and newly infected cells were monitored by HCMV IEA staining. The numbers of infected cells per focus were counted. One dot represents the number of infected cells of an individual focus. Bars represent mean values of all foci from four donors tested. (B) The concentration of IFN-γ in supernatants from indicated cultures were tested by ELISA. (C) Fibroblasts infected with AD169 or AD169ΔUL16GFP were used in the focus expansion assay. Infected cells were monitored by live imaging of GFP or IEA staining after fixation. HCMV antibodies from a commercial Ig preparation (Ig) at a 1:20 dilution were used as a positive control.
FIG 7
FIG 7
CD16 receptor-158V/F polymorphism contributes to the efficacy of HCMV antibody-dependent NK cell inhibition of cell-to-cell virus transmission. (A) The levels of CD16 expression on three NK-92 cell lines are shown. (B) One-hundred-microliter cell-free suspensions of TB40/E with or without (w/o) HCMV antibodies (Abs) were used in neutralization experiments on fibroblasts. Virus suspensions and indicated HCMV antibodies (HCMV-positive serum used at a 1:10 final dilution, HCMV antibodies from a commercial Ig preparation [Ig] used at a 1:200 final dilution) were incubated for 30 min and then used to inoculated the cells. The infection rates were assessed after 24 h of incubation. The presence of HCMV IEA (red fluorescence) indicates infected fibroblasts, and cell nuclei are stained in blue (DAPI). (C) Fibroblasts were infected after 3 days and incubated with the Ig preparation followed by FITC–anti-human IgG staining. The presence of HCMV IEA (red fluorescence) indicates infected cells, human IgG binding on fibroblasts is shown by green fluorescence, and nuclei are stained in blue (DAPI). (D) Fibroblasts infected by clinical isolate 1 were cocultured with a 2,000-fold excess of uninfected fibroblasts for 3 days. HCMV antibody preparations (HCMV-positive [Pos.] and -negative [Neg.] sera used at a 1:10 final dilution, Ig preparation used at a 1:200 final dilution) were added at the beginning of cocultures in the presence or absence of the different NK-92 cell lines at an E:T ratio of 0.25. Monolayers were fixed at the indicated times, and newly infected cells were monitored by HCMV IEA staining. The numbers of infected cells per focus were counted. One dot represents the number of infected cells of an individual focus. Bars indicate mean values of all foci.
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