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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2014 Oct 28;111(45):16118–16123. doi: 10.1073/pnas.1409485111

Modulation of CD112 by the alphaherpesvirus gD protein suppresses DNAM-1–dependent NK cell-mediated lysis of infected cells

Korneel Grauwet a,1, Claudia Cantoni b,c,d,1, Monica Parodi b, Andrea De Maria c,e,f, Bert Devriendt a, Daniela Pende e, Lorenzo Moretta d, Massimo Vitale e,2, Herman W Favoreel a,2,3
PMCID: PMC4234607  PMID: 25352670

Significance

Herpesviruses have developed fascinating mechanisms to evade elimination by key elements of the host immune system, allowing these pathogens to cause lifelong infections with periods of recurrent virus spread. Natural killer (NK) cells are central in the innate antiviral response. Here, we report that the gD glycoprotein of the alphaherpesviruses, pseudorabies virus and herpes simplex virus-2, displays previously uncharacterized immune evasion properties toward NK cells. Expression of the gD protein leads to degradation of CD112/nectin-2, a ligand for the NK-activating receptor DNAX accessory molecule 1 (DNAM-1). This impairs binding of DNAM-1 to the cell surface, thereby suppressing NK-mediated killing of virus-infected (or gD-transfected) cells. Identification of this previously unidentified immune evasion mechanism may contribute to the design of improved herpesvirus vaccines and herpesvirus-based therapeutic vectors.

Keywords: herpes, NK, DNAM-1, CD112, pseudorabies

Abstract

Natural killer (NK) cells are key players in the innate response to viruses, including herpesviruses. In particular, the variety of viral strategies to modulate the recognition of certain herpesviruses witnesses the importance of NK cells in the control of this group of viruses. Still, NK evasion strategies have remained largely elusive for the largest herpesvirus subfamily, the alphaherpesviruses. Here, we report that the gD glycoprotein of the alphaherpesviruses pseudorabies virus (PRV) and herpes simplex virus 2 (HSV-2) displays previously uncharacterized immune evasion properties toward NK cells. Expression of gD during infection or transfection led to degradation and consequent down-regulation of CD112, a ligand for the activating NK receptor DNAX accessory molecule 1 (DNAM-1). CD112 downregulation resulted in a reduced ability of DNAM-1 to bind to the surface of both virus-infected and gD-transfected cells. Consequently, expression of gD suppressed NK cell degranulation and NK cell-mediated lysis of PRV- or HSV-2–infected cells. These data identify an alphaherpesvirus evasion strategy from NK cells and point out that interactions between viral envelope proteins and host cell receptors can have biological consequences that stretch beyond virus entry.

Alphaherpesviruses constitute the largest subfamily of the herpesviruses, comprising closely related and important pathogens like herpes simplex virus (HSV) in man, pseudorabies virus (PRV) in pigs, and bovine herpesvirus 1 (BHV-1) in cattle.

Natural killer (NK) cells play a central role in the defense against viral infections and cancer development. Functional NK cells are of particular importance in preventing herpesviruses from causing aggravated disease, including life-threatening encephalitis for alphaherpesviruses like HSV and varicella-zoster virus (1–3). The significance of the NK cell response against herpesviruses is also reflected by the various mechanisms that these pathogens have evolved to evade or delay it (4). Indeed, for beta- and gammaherpesviruses, a variety of molecular mechanisms avoiding the NK-mediated antiviral activity have been defined (4–16). Remarkably and paradoxically, such mechanisms have remained largely elusive for the alphaherpesviruses (17).

Identifying and understanding these mechanisms is of particular relevance for alphaherpesviruses also in view of the potential therapeutic applications of HSV. Indeed, a limiting factor in HSV vector-based oncotherapy is the premature clearance of the viral vector by NK cells (18).

NK cell activity is regulated by an array of germline-encoded activating and inhibitory surface receptors capable of transducing signals upon engagement by their respective ligands (19, 20). The sum of these signals determines the outcome of NK cell effector responses including cytotoxicity against NK-sensitive targets (20). A variety of NK activating receptors are involved in recognition of virus-infected cells (18, 21–26). One of the important NK activating receptors is DNAX accessory molecule 1 (DNAM-1), which binds to CD112 (nectin-2) and CD155 (poliovirus receptor, PVR), whose expression can be induced in both virus infected and tumor cells (5, 26–28).

Interestingly, the viral gD envelope glycoproteins of certain human and animal alphaherpesviruses, including HSV-2, PRV, and BHV-1, interact with CD112 and/or CD155 to facilitate viral entry (29, 30). HSV-1 gD does not typically display substantial affinity for CD112, except for particular HSV-1 isolates, including some retrieved from patients with encephalitis (31).

In the current study, we demonstrate that the significance of these virus ligand-cellular receptor interactions can stretch beyond virus entry and can influence immune recognition. We report that expression of gD of PRV and HSV-2 reduces DNAM-1–mediated cell lysis by NK cells through suppression of CD112 levels in infected and transfected cells. The gD/CD112/DNAM-1 interplay identified here may have consequences for the development of medical applications ranging from vaccination to oncolytic virotherapy.

Results

Expression of PRV gD Reduces NK Cell-Mediated Killing.

To investigate a potential effect of alphaherpesvirus gD expression on NK-mediated lysis, the porcine PRV was used. PRV is commonly used as a model pathogen to study general aspects of alphaherpesvirus biology (32). In addition, gD of PRV has the unique capacity to bind both CD112 and CD155 (29, 30).

To investigate whether expression of PRV gD affects the susceptibility of cells to NK-mediated lysis, 293T cells were infected with wild-type (WT) PRV or an isogenic gDnull virus, coincubated with IL2-primed human NK cells and subsequently assessed for viability by flow cytometry. Fig. 1A shows that cells infected with WT PRV display lower susceptibility to NK cell-mediated lysis compared with cells infected with gDnull PRV. This difference was not due to possible differences in virus replication efficiency or MHC class I cell surface levels, as expression levels of other viral proteins (e.g., gB and gC) and MHC class I were similar for both viruses (Figs. S1A and S2A).

Fig. 1.

Fig. 1.

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Expression of PRV gD during infection or transfection reduces NK cell degranulation and NK-mediated cell lysis. (A and B) The 293T cells were mock infected or infected with WT or gDnull PRV (A) or mock transfected or transfected with PRV gD or empty vector (B) and subsequently incubated with IL-2–primed NK cells for 4 h at a target:effector ratio of 1:1. Viability of target cells was assessed by propidium iodide and flow cytometry. Data represent mean + SEM of three independent repeats (*P < 0.05). (C) Cell surface expression of CD107a on NK cells incubated for 4 h with 293T cells that were mock transfected or transfected with PRV gD or empty vector. Histograms show CD107a cell surface signal, with marker to discriminate positive cells. Bar graphs represent mean + SD of three independent repeats (**P < 0.01).

To confirm the inhibitory effect of gD expression on NK-mediated cell lysis, 293T cells were transfected with a gD-encoding vector or an empty vector and assayed for NK cell-mediated lysis. Again, expression of gD resulted in reduced susceptibility of cells to NK cell-mediated lysis (Fig. 1B). We next investigated whether this diminished susceptibility to NK-mediated cell lysis is reflected by a decreased ability of target cells to trigger NK cell degranulation. NK cells were coincubated with transfected 293T cells and then analyzed for surface expression of CD107a (i.e., a marker of degranulation) by flow cytometry (33), which showed that gD expression resulted in substantially reduced degranulation of NK cells (Fig. 1C).

In conclusion, the above data indicate that gD has the previously uncharacterized ability to interfere with NK-mediated cytotoxicity.

Expression of PRV gD Reduces Cell Surface Levels of CD112 and Leads to Reduced Binding of DNAM-1 and Reduced DNAM-1–Dependent NK-Mediated Cell Lysis.

Given the ability of PRV gD to bind CD112 and CD155 (29, 30), we investigated whether expression of PRV gD compromises normal cell surface levels of CD112 and CD155 and, consequently, binding of the activating NK receptor DNAM-1. Fig. 2A shows that cell surface levels of CD112 are significantly reduced in cells infected with WT PRV but not with gDnull PRV. On the other hand, CD155 cell surface levels were not significantly reduced. To determine whether reduced cell surface levels of CD112 affects binding of DNAM-1 to the cell surface, binding of recombinant DNAM-1Fc was assessed by flow cytometry and was found to be significantly reduced on WT PRV-infected cells compared with cells infected with gDnull PRV (Fig. 2A). We also consistently noticed a reproducible, yet statistically nonsignificant, trend of increased DNAM-1Fc binding in cells infected with gDnull PRV compared with mock-infected cells (Fig. 2A).

Fig. 2.

Fig. 2.

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Expression of PRV gD reduces cell surface availability of CD112, DNAM-1Fc binding, and DNAM-1–dependent NK degranulation and cell lysis. (A and B) The 293T cells were infected with WT or gDnull PRV for 6 h (A) or transfected with PRV gD or empty vector for 48 h (B) and subsequently assessed by flow cytometry for cell surface expression of gD, CD112, and CD155 or for binding of recombinant DNAM-1Fc. Histograms show relative fluorescence intensities, with vertical lines indicating median fluorescence intensity. Dotted line histograms represent isotype-matched antibody control. Bar graphs represent mean fluorescence intensity ratios (MFIRs). Data represent mean + SEM of three independent repeats (**P < 0.01, ***P < 0.001). (C) Cells infected or transfected as in A and B were incubated for 4 h with IL2-primed NK cells at the indicated target:effector (T:E) ratios, in the absence or presence of the DNAM-1–blocking antibody F5. Viability of target cells was assessed by propidium iodide and flow cytometry, and percentage of DNAM-1–dependent NK-mediated killing was calculated. (Left and Middle) DNAM-1–mediated lysis of 293T cells. (Left) Results for cells infected with WT PRV (solid line), gDnull PRV (short dashed line), or mock infected (long dashed line). (Middle) Results for cells transfected with PRV gD (solid line), empty vector (short dashed line), or mock transfected (long dashed line). (Right) Cell surface expression of the degranulation marker CD107a on NK cells incubated with 293T cells transfected with PRV gD (gray bars) or empty vector (white bars) in the presence or absence of DNAM-1–blocking F5 mAb. Data represent mean and SEM of three independent repeats. Asterisks in Left and Middle indicate significant differences between WT PRV and gDnull PRV-infected samples or between empty vector and PRV gD-transfected samples (*P < 0.05, ***P < 0.001).

As shown in Fig. 2A, Left, gD protein was not completely absent from the surface of gDnull PRV-infected cells. Because the gD envelope protein is essential for virus entry, gDnull virus stocks were grown on gD-expressing cells to allow incorporation of gD in the viral envelope and render progeny virions infectious. Hence, these viruses do not carry the gD gene in their genome but do carry the gD protein in their envelope. Upon fusion of the viral envelope with the host plasma membrane during virus entry, gDnull PRV leaves the gD envelope proteins in the host cell membrane, thereby yielding the weak gD cell surface signal shown in Fig. 2A. In line with this, the gD signal observed in gDnull PRV-infected cells was already observed at 2 h postinoculation (hpi) (upon virus entry) and did not increase at later time points of infection. This also indicates that comparing WT PRV-infected cells with gDnull PRV-infected cells may underestimate any gD-mediated effects.

To be able to better address the magnitude of the gD-mediated effects, and to confirm our results, transfection assays were performed, which confirmed that expression of gD leads to substantially reduced levels of CD112 on the cell surface and reduced binding of DNAM-1Fc (Fig. 2B). In these assays, gD expression also led to significantly reduced cell surface levels of CD155, although the effect on CD155 was substantially less pronounced compared with the effect on CD112 (Fig. 2B).

To assess whether reduced binding of DNAM-1Fc also results in reduced susceptibility of cells to DNAM-1–dependent NK-mediated cell lysis, NK cytotoxicity assays were performed in the presence or absence of the DNAM-1 blocking antibody F5 and DNAM-1–dependent NK-mediated cell lysis was calculated. As shown in Fig. 2C, Left and Middle, cells infected with WT PRV or transfected with PRV gD showed lower susceptibility to DNAM-1–dependent NK-mediated cell lysis compared with cells infected with gDnull PRV or transfected with empty vector, respectively. In line with this, DNAM-1–dependent degranulation of NK cells was reduced when coincubated with PRV gD-transfected cells compared with cells transfected with empty vector (Fig. 2C, Right).

In conclusion, expression of PRV gD leads to substantially reduced cell surface expression of CD112, reduced binding of DNAM-1Fc and reduced susceptibility of the cells to DNAM-1–dependent NK cell degranulation and NK-mediated cell lysis.

Expression of PRV gD Leads to Degradation of CD112.

To further assess the effect of PRV gD expression on CD112, total cell lysates were analyzed for CD112 protein levels by Western blotting. Blots were probed with antibodies specific for CD112, CD155, and gD as well as for gB and gC (infection controls) and tubulin (loading control). As shown in Fig. S1A, WT PRV-infected cells showed substantially reduced CD112 protein levels compared with mock-infected cells or cells infected with gDnull PRV, whereas CD155 protein levels were only marginally reduced. To determine whether CD112 degradation occurs via an acidification-dependent pathway, experiments were repeated in the presence of the acidification inhibitor BFLA-1 (Fig. S1B). This restored CD112 levels in WT PRV-infected cells to levels of mock-infected and gDnull PRV-infected cells. Degradation of CD112 was confirmed in gD transfection assays (Fig. S1C). These results indicate that expression of PRV gD leads to an acidification-dependent degradation of CD112.

Expression of PRV gD Leads to Degradation of CD112 in Porcine Cells and Reduced Cell Lysis by Porcine NK Cells.

The natural host of PRV is the pig. To better assess the potential biological significance of our findings, we investigated whether the gD-mediated effects that we observed also occur in porcine cells.

As a first step, we confirmed the expression of CD112, CD155, and DNAM-1 by RT-PCR on mRNA isolated from the porcine swine kidney (SK) cell line (CD112 and CD155) or from primary porcine NK cells (DNAM-1) (Fig. 3A). All PCR products were confirmed by sequencing (SI Materials and Methods).

Fig. 3.

Fig. 3.

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Expression of PRV gD in porcine cells leads to CD112 degradation and reduced killing by porcine primary NK cells. (A) mRNA expression, assessed by RT-PCR, of CD112 and CD155 in porcine SK cells and of DNAM-1 in primary porcine NK cells. (B and C) Porcine SK cells (B) or primary porcine epithelial cells (C) were mock infected or infected for 12 h with WT or gDnull PRV and subsequently analyzed by Western blotting for expression of CD112, gB, gC, gD, and tubulin. (D) SK cells were mock infected or infected with WT or gDnull PRV and subsequently incubated with primary porcine NK cells at a target:effector ratio of 1:25 for 4 h. Viability was assessed by flow cytometry using propidium iodide. Data represent mean + SEM of three independent repeats (***P < 0.001).

We then assessed whether expression of PRV gD in porcine cells reduced CD112 protein levels. Fig. 3B shows that SK cells infected with WT PRV virtually lack CD112 protein compared with mock-infected cells or cells infected with gDnull PRV. CD155 protein levels could not be assessed due to the lack of antibodies cross-reacting with porcine CD155. Protein levels of gB and gC served as infection controls and tubulin levels as loading control. These results were confirmed in primary porcine epithelial cells (Fig. 3C).

Finally, we investigated whether expression of PRV gD in porcine cells alters their susceptibility to lysis by primary porcine NK cells. As shown in Fig. 3D, SK cells infected with WT PRV showed significantly reduced susceptibility to lysis by primary porcine NK cells compared with cells infected with gDnull PRV. Hence, also in the porcine system, PRV infection leads to gD-dependent degradation of CD112 and reduced susceptibility to cell lysis by primary porcine NK cells.

Expression of HSV-2 gD Leads to Down-Regulation of CD112 and Reduced NK-Mediated Cell Lysis.

Although PRV gD can bind both CD112 and CD155 (29, 30), our findings suggest that its protective effect against NK-mediated cell lysis is mainly due to its ability to bind and modulate CD112. As a consequence, our findings on PRV may have a more general relevance, as the gD proteins of other alphaherpesviruses, most notably HSV-2, also interact with CD112 (30). Therefore, we investigated whether HSV-2 gD also affects cell surface levels of CD112, DNAM-1Fc binding, and susceptibility of cells to NK-mediated cell lysis. Fig 4A shows that infection of 293T cells with WT HSV-2 resulted in reduced cell surface expression of CD112 and in reduced binding of DNAM-1Fc, compared with mock-infected cells or cells infected with gDnull HSV-2. These results were also confirmed in the human U87 malignant glioblastoma (U87-MG) cell line (Fig. S3).

Fig. 4.

Fig. 4.

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Expression of HSV-2 gD in 293T cells leads to CD112 down-regulation, decreased cell surface binding of DNAM-1Fc, and reduced NK-mediated cell lysis. (A) The 293T cells were mock infected or infected with WT or gDnull HSV-2 for 24 h and subsequently assessed by flow cytometry for cell surface expression of CD112 and CD155 or for binding of recombinant DNAM-1Fc. Histograms show relative fluorescence intensities, with vertical lines indicating median fluorescence intensity. Dotted line histograms represent isotype-matched antibody control. Bar graphs represent mean fluorescence intensity ratios (MFIRs). Data represent mean + SEM of three independent repeats (*P < 0.05, **P < 0.01, ***P < 0.001). (B) The 293T cells were mock infected or infected with WT or gDnull HSV-2 and subsequently incubated for 4 h with IL2-primed NK cells at a target:effector ratio of 1:2.5. Viability of target cells was assessed by propidium iodide staining and flow cytometry, and percentage of NK-mediated killing was calculated. Data represent mean + SEM of three independent repeats (*P < 0.05).

In line with these results, 293T cells infected with WT HSV-2 showed significantly reduced susceptibility to NK-mediated cell lysis compared with cells infected with gDnull HSV-2 (Fig. 4B). Like for PRV, flow cytometric analysis of viral protein gB and MHC class I confirmed that these parameters were similar for both WT and gDnull HSV-2 infected cells, thus ruling out that changes in viral replication or virus-induced modulation of MHC class I expression could account for the differences in susceptibility to NK cell lysis (Fig. S2B).

Again, similar to PRV, we consistently noticed a trend of increased DNAM-1 ligand expression and increased DNAM-1Fc binding in cells infected with gDnull HSV-2 compared with mock-infected cells (Fig. 4A).

In conclusion, also in HSV-2 infection, expression of gD results in CD112 down-regulation, reduced DNAM-1Fc binding, and reduced susceptibility of cells to NK-mediated cell lysis.

Discussion

In the current report, we describe that the gD glycoprotein of PRV and HSV-2 displays previously uncharacterized immune evasion properties toward NK cells, thereby identifying an NK evasion strategy of alphaherpesviruses. Expression of gD leads to reduced cell surface availability and degradation of its cellular ligand CD112 (nectin-2), thereby reducing binding of the activating NK cell receptor DNAM-1 and, consequently, decreasing NK-mediated lysis of gD-expressing cells.

Interestingly, the UL141 protein of the betaherpesvirus human cytomegalovirus (HCMV) also targets DNAM-1 ligands to protect infected cells from NK cell-mediated lysis. UL141 sequesters CD155 in the endoplasmic reticulum, and, similar to what we observe for PRV and HSV-2 gD, also leads to CD112 degradation (8, 34). However, unlike what we observe for gD, transfection of UL141 does not affect CD112, implicating an additional, not yet identified viral protein in this process (34). UL141 of HCMV does not display amino acid sequence similarity to gD of alphaherpesviruses. This points to convergent evolution in the herpesvirus family, with alpha- and betaherpesviruses that have developed evolutionary distinct methods to interfere with DNAM-1 ligand expression. This underscores the recent view that the activating NK receptor DNAM-1 represents a serious threat that different viruses need to circumvent (26).

Further in line with this view, our data and data by others indicate that cells may trigger increased cell surface expression of DNAM-1 ligands as a response to virus infection, which is subsequently counteracted by viral proteins like gD of PRV/HSV-2, UL141 of HCMV, and Nef/Vpu of HIV. Indeed, mutant viruses that lack these respective viral immune evasion proteins all show higher cell surface expression of DNAM-1 ligands/higher DNAM-1 binding, compared with mock-infected cells (8, 35) (Figs. 2A and 4A). Also, productive infection of cells with the gammaherpesvirus Epstein–Barr virus is associated with up-regulation of the DNAM-1 ligand CD112 (25). Although the underlying mechanism of such virus-induced cellular response remains to be investigated, the cellular DNA damage response (DDR) may play a role, as many viruses, including herpesviruses and retroviruses, activate the DDR, and DDR activation has been reported to trigger DNAM-1 ligand up-regulation (26, 36–38).

Our data also underscore the conservation of the DNAM-1 receptor activity over different species, as we showed that PRV gD-mediated interference with CD112 and consequent reduced killing by NK cells was also observed in porcine cells. Although the porcine genome was known to encode a DNAM-1 homolog, our RT-PCR data for the first time demonstrate the expression of DNAM-1 in porcine NK cells. Hence, this aspect of the immune system appears well conserved over different species, including humans, other primates, pigs, and mice (39, 40).

The biological consequences of the suppressive effect of gD on DNAM-1 function may not be limited to NK cells. DNAM-1 is also expressed on a variety of other immune cells, including T cells, monocytes/macrophages, platelets, and a subset of B lymphocytes (24, 41). In most cells, DNAM-1 is involved in cellular activation. For instance, in cytotoxic T cells DNAM-1 contributes to the cytolytic activity against tumor cells expressing CD112 and CD155 (42).

Blocking of endosomal acidification with an inhibitor of vacuolar H+ATPases prevented gD-mediated degradation of CD112, implicating the involvement of endosomal degradation. These results are in line with earlier reports showing HSV-1 gD-induced endosomal degradation of nectin-1 (CD111) (43) and suggest conserved routes of internalization and subsequent degradation for different nectin members. It will be interesting to further characterize these virus-induced internalization/degradation routes of CD112 and other nectins in future research, as this may also shed new light on the physiological role and regulation of nectins. Indeed, CD112 internalization has, to our knowledge, up to now only been reported and studied during spermatogenesis (44).

Our identification of a gD-mediated alphaherpesvirus NK cell evasion mechanism may have consequences for the design of future vaccines. For betaherpesviruses, indeed, a mutant murine cytomegalovirus with increased sensitivity to NK cells displayed improved vaccine properties (45). At the same time, our findings may have consequences for the design of herpes-based therapeutic vectors. Specifically, the use of attenuated HSV-1–based oncolytic vectors for glioblastoma therapy is limited by premature clearance of the viral vector by NK cells of the recipient (18). We observed that expression of PRV gD or HSV-2 gD in the U87-MG glioblastoma cell line reduces cell surface CD112 and DNAM-1 binding (Fig. S3). However, unlike PRV gD and HSV-2 gD, HSV-1 gD typically does not display significant affinity for CD112 (30). Thus, chimeric HSV-1 oncolytic vectors, expressing HSV-2/PRV gD (fragments), may possibly display reduced susceptibility to NK cell-mediated clearance. In support of the potential of such an approach, a vesicular stomatitis virus-based oncolytic vector expressing the DNAM-1–interfering HCMV protein UL141 displayed increased oncolytic potential through vector-mediated inhibition of NK cells (46).

In conclusion, the gD glycoprotein of different alphaherpesviruses (PRV and HSV-2) displays previously uncharacterized immune evasion properties, by reducing the susceptibility of infected cells to NK cell-mediated lysis through down-regulation of the cellular DNAM-1 ligand CD112. This represents a previously unidentified aspect of the pathogenetically important interplay between alphaherpesviruses and NK cells. Interactions between herpesvirus envelope glycoproteins and cellular receptors have been predominantly studied in the context of their role during virus entry. Our current data point out that these interactions can also have profound implications for other aspects of virus biology and pathogenesis, including evasion of the host immune system.

Materials and Methods

Viruses, cells, antibodies, reagents, infections, transfections, Western blot, RT-PCR, and statistical analysis were (performed as) described before, and are listed in SI Materials and Methods.

Flow Cytometric Analysis.

Cells were collected, incubated on ice with primary mouse antibodies or recombinant DNAM-1Fc (10 µg/mL), and subsequently washed and incubated on ice with R-phycoerythrin (R-PE) labeled goat anti-mouse or anti-human secondary R-PE antibodies (Invitrogen), respectively. Analysis was performed on 20,000 living cells with a FACSAria III and FACSDiva software (BD BioSciences) using Sytoxblue dye staining (Invitrogen).

NK Cells.

Human NK cells were isolated from peripheral blood mononuclear cells (PBMC) using the RosetteSepTM NK Cell Enrichment kit (Stemcell Technologies), cultured in the presence of 100 units/mL huIL2 (Chiron) as described before (47) and used within 3 wk. Primary porcine NK cells were isolated from porcine PBMC by negative MACS depletion and a FACS purification step using antibodies against porcine CD172, CD3, CD4, and CD8α, essentially as described before (48, 49) and primed for 18 h with 40 units/mL recombinant huIL2 (Invitrogen). CD16 expression on sorted cells confirmed ≥98% NK purity.

Cytolytic and Degranulation Assays.

A flow cytometric propidium iodide/carboxyfluorescein succinimidyl ester-based assay was used to quantify the NK-mediated lytic activity against infected or transfected target cells, essentially as described before (50). Viability of 5,000 target cells was evaluated by flow cytometry using propidium iodide (Invitrogen). The percentage of NK-mediated lysis was calculated using the formula: (%dead targetNK − %dead targetspont)/(%dead targetmaximum − %dead targetspont) (48). NK cell degranulation was assessed by flow cytometry using a FITC-labeled CD107a-specific antibody as described before (22). For DNAM-1–dependent cell killing/degranulation assays, cytotoxicity, or expression of the CD107a degranulation marker was evaluated in the absence or presence of the DNAM-1–blocking IgM antibody F5 (10 µg/mL) (51).

Supplementary Material

Supplementary File
pnas.201409485SI.pdf (320.3KB, pdf)

Acknowledgments

We thank C. Van Waesberghe, S. Brabant, H. Vereecke, and L. Sys for excellent technical assistance; R. Cooman for animal management; P. Spear and R. Longnecker (Northwestern University), T. Mettenleiter and B. Klupp (Friedrich Loeffler Institut), D. Schols (Rega Institute), R. Eisenberg, G. Cohen, and C. Krummenacher (University of Pennsylvania), H. Nauwynck and E. Cox (Ghent University), and A. Brun (Laboratoire Institut Français de la Fièvre Aphteuse) for reagents; and Federica Bozzano and Francesco Marras (University of Genoa) for their involvement in experiments related to, although not included in, this paper. This research was supported by grants from the Special Research Fund of Ghent University (Grants 01J29110, 01J11611, and 01G01311) and Fonds voor Wetenschappelijk Onderzoek Vlaanderen (F.W.O.-Vlaanderen) (Grants 1.5.077.11N and G.0835.09), as well as an international mobility grant from F.W.O.-Vlaanderen and Hercules Foundation Grant AUGE-035. This research was also supported by grants from Associazione Italiana per la Ricerca sul Cancro Investigator Grant Project 4725 (to L.M.), IG Project 10225 (to L.M.), IG Project 5282 (to M.V.), and Special Project “5X1000” 9962 (to L.M.); Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR)-Fondo Investimenti Ricerca di Base 2003 Project RBLA039LSF-001 (to L.M.), MIUR-Programmi di Ricerca scientifica di Rilevante Interesse Nazionale 2008 Project 2008PTB3HC_005 (to L.M.), Ministero della Salute: RF2006 - Ricerca Ordinaria (RO) strategici 3/07 (to L.M.), and RO strategici 8/07 (to M.V.); and Ministero della Salute Grant “5X1000” 2011 (to M.V.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission. L.W.E. is a guest editor invited by the Editorial Board.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1409485111/-/DCSupplemental.

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