NKp46 expression discriminates porcine NK cells with different functional properties

Authors

  • Kerstin H. Mair,

    1. Department for Pathobiology, Institute of Immunology, University of Veterinary Medicine Vienna, Austria
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  • Sabine E. Essler,

    1. Department for Pathobiology, Institute of Immunology, University of Veterinary Medicine Vienna, Austria
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  • Martina Patzl,

    1. Department for Pathobiology, Institute of Immunology, University of Veterinary Medicine Vienna, Austria
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  • Anne K. Storset,

    1. Department of Food Safety and Infection Biology, Norwegian School of Veterinary Science, Oslo, Norway
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  • Armin Saalmüller,

    Corresponding author
    1. Department for Pathobiology, Institute of Immunology, University of Veterinary Medicine Vienna, Austria
    • Correspondence Dr. Armin Saalmüller, Institute of Immunology, Department for Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria

      Fax: +43-1-25077-2791

      e-mail: armin.saalmueller@vetmeduni.ac.at

      Full Correspondence: Dr. Wilhelm Gerner, Institute of Immunology, Department for Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, AustriaFax: +43-1-25077-2791e-mail: wilhelm.gerner@vetmeduni.ac.at

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    • These authors contributed equally to this work.

  • and Wilhelm Gerner

    Corresponding author
    1. Department for Pathobiology, Institute of Immunology, University of Veterinary Medicine Vienna, Austria
    • Correspondence Dr. Armin Saalmüller, Institute of Immunology, Department for Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria

      Fax: +43-1-25077-2791

      e-mail: armin.saalmueller@vetmeduni.ac.at

      Full Correspondence: Dr. Wilhelm Gerner, Institute of Immunology, Department for Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, AustriaFax: +43-1-25077-2791e-mail: wilhelm.gerner@vetmeduni.ac.at

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    • These authors contributed equally to this work.


Abstract

So far little is known about natural killer (NK) cells in the pig due to the lack of NK cell-specific markers. In this study, we identified the activating receptor NKp46 (CD335) in swine with newly developed monoclonal antibodies (mAbs) for more detailed studies on NK cells in this species. The NKp46 mAbs showed a specific reactivity with a distinct population of perforin+CD2+CD3CD8α+CD16+ lymphocytes. In spleen and liver, an additional subset of CD8αdim/− lymphocytes with increased NKp46 expression was observed. Surprisingly, we could identify NKp46 cells with an NK cell phenotype in all animals analyzed. These lymphocytes showed comparable cytolytic activity against xenogeneic and allogeneic target cells as NKp46+ NK cells. In contrast, NKp46+ NK cells produced several fold higher levels of interferon-γ (IFN-γ) than the NKp46 cells after cytokine stimulation. Furthermore, an activation-dependent induction of NKp46 expression in formerly NKp46 cells after stimulation with interleukin-2 (IL-2), IL-12, and IL-18 could be shown. In summary, our data indicate that NKp46 is not expressed by all porcine NK cells and that NKp46 discriminates porcine NK cells differing in regard to cytokine production, which challenges the paradigm of NKp46 as a comprehensive marker for NK cells across different mammalian species.

Introduction

Natural killer (NK) cells are a highly specialized subpopulation of lymphocytes that play a crucial role in the early phase of immune responses. So far little is known about NK cells in swine. The phenotype of porcine NK cells was recently described as perforin+CD2+CD3CD4CD5CD6CD8α+CD8ßCD11b+CD16+ and it was observed that these cells exhibit natural cytotoxicity against NK-susceptible targets [[1-4]]. Likewise, the involvement of porcine NK cells has been reported in immune responses against parasitic and viral infections [[5-9]]. But since porcine NK cells share several phenotypic markers with other cell populations such as NKT cells, TCR-γδ T cells, and myeloid cells [[3, 4]], the precise detection, isolation, and more detailed functional characterization of porcine NK cells has been thus far difficult. Thus, the identification of a discrete and unifying marker for this cell population in swine would be highly beneficial.

NKp46 (CD335, NCR1), a member of the natural cytotoxicity receptor (NCR) family, is specifically expressed on NK cells, resting as well as activated. This activating receptor has been postulated to be a valid marker for NK cells in different species. This was demonstrated so far in humans [[10, 11]], monkeys [[12, 13]], rodents [[14-17]], cattle [[18]], and more recently in sheep [[19]]. Currently, NKp46 gained significant relevance in tumor surveillance [[20]], autoimmune diseases [[21]], and due to its recognition of haemagglutinins [[22, 23]] in influenza infections. Since the phenotypic definition of NK cells across mammalian species was introduced as CD3NKp46+ [[15]], the triggering receptor NKp46 might also be a good candidate molecule for the specific identification of NK cells in swine.

Porcine NKp46 was previously described as being encoded in the porcine leukocyte receptor complex on chromosome 6 [[24]]. The first expression analysis at the mRNA level revealed that the overall sequence and tissue distribution of porcine NKp46 was comparable with that of other species [[25]]. By the use of newly developed antibodies (Abs) against porcine NKp46, we were able to identify NKp46 on a distinct population of porcine lymphocytes with an NK cell phenotype. Detailed phenotypical studies revealed that, in contrast to other species, NKp46 does not seem to be expressed on all NK cell-defined lymphocytes in the pig. We found two discrete populations of NKp46+ and NKp46 cells that seem to account equally for NK cells as they showed comparable cytotoxic properties. Nevertheless, the two NK cell populations differed in regard to their interferon-γ (IFN-γ) production.

Results

Generation of mAbs against porcine NKp46

We generated monoclonal antibodies (mAbs) against porcine NKp46 for more detailed studies of NK cells in the pig by immu-nizing mice with recombinant NKp46 protein. Supernatants of hybridoma cultures derived from single cells were screened for mAbs that specifically reacted with HEK293T cells transfected with a construct containing the entire sequence of porcine NKp46 with a FLAG-tag. A distinct co-staining was visible in HEK293T cells transfected with the NKp46-FLAG construct when labeled with supernatants containing anti-NKp46 mAbs and M2 anti-FLAG control Ab (Fig. 1A). The untransfected control and HEK293T cells transfected with empty plasmid showed no positive staining. After several screening rounds, we obtained three NKp46-specific mAbs derived from clonal hybridoma cultures (VIV-KM1, VIV-KM2, VIV-KM3). All of them were of IgG1 isotype and all showed comparable staining patterns on porcine peripheral blood lymphocytes (PBLs) and splenocytes. They also reflected the staining pattern obtained with serum from the immunized mouse that was used for the fusion procedure (Supporting Information Fig. 1A). By using the different mAbs in blocking assays with VIV-KM1 we investigated whether these three mAbs recognize different binding sites on porcine NKp46 (Supporting Information Fig. 1B). VIV-KM3 was not able to block the binding of VIV-KM1 at any concentration, thus most likely recognizing a different binding site on NKp46. VIV-KM2 was able to reduce binding of VIV-KM1 only in the highest concentration used and therefore might recognize a closely adjoining binding site.

Figure 1.

Recognition of porcine NKp46 by newly developed mAbs in lymphatic and non-lymphatic tissues in the pig. (A) For screening of reactive mAbs against porcine NKp46, HEK293T cells transfected with a NKp46-FLAG construct were stained with supernatants of single-cell hybridoma cultures, and M2 anti-FLAG Ab and analyzed by FCM. Cells without plasmid and HEK293T cells transfected with empty vector served as negative controls. (B) Immunoprecipitation of porcine NKp46 derived from biotinylated PBMCs with an NKp46-specific mAb (clone VIV-KM1) or an isotype-matched control Ab (control Ig) is shown. Biotinylated mass standards in kDa are displayed on the right. (C) Porcine PBMCs and lymphocytes derived from mediastinal lymph nodes, spleen, and liver were analyzed for NKp46 expression by three-color FCM. NKp46-specific mAbs (clone VIV-KM1) were used in combination with markers discriminating major lymphocyte populations. Cells were either gated on lymphocytes or on whole PBMCs for analysis of CD14 co-expression. Percentages of NKp46+ cells are indicated in the corresponding gates. Data shown are representative of experiments using cells from five different animals.

In order to investigate the molecular mass of porcine NKp46, immunoprecipitation of surface-biotinylated total peripheral blood mononuclear cells (PBMCs) with anti-NKp46 mAb was performed. A distinct band of ∼48 kDa was obtained (Fig. 1B), confirming data in other species where NKp46 is described with a molecular mass of approximately 46 kDa [[10, 14, 17, 18]].

NKp46 expression is correlated with an NK phenotype

The definition of NK cells in other species is outlined as CD3NKp46+ and hence NKp46 is an adequate and specific marker for this cell population. To confirm this dogma in swine, we investigated the surface expression of NKp46 on lymphocytes derived from blood, lymph node, spleen, and liver by flow cytometry (FCM) (Fig. 1C). The overall number of NKp46+ cells within PBLs ranged from 0.5 to 11% (Fig. 1C, and data not shown). NKp46+ cells in mediastinal lymph node (medLN) did not exceed 1% of the lymphocyte population in all animals tested. In spleen and especially in liver, the number of NKp46+ cells was considerably higher, ranging from 3 to 13% in the spleen and 13–35% in the liver. To get more information about the phenotype of cells expressing NKp46, anti-NKp46 mAbs were used in combination with markers discriminating major lymphocyte and monocyte populations in the pig. Monocytes (CD14+), B cells (CD79α+), TCR-γδ+ T cells, and CD4+ T-helper cells were negative for NKp46 in blood and all organs tested. In some animals, we observed a small but distinct population of CD3+NKp46+ cells (0.8–2.0%). All NKp46+ cells were positive for CD2 and partially NKp46+ cells expressed major histocompatibility complex (MHC) class II (swine leukocyte antigen-DR, SLA-DR) with individual differences (data not shown). Regarding the CD8α co-expression, a more hetero-geneous picture between the different organs was visible. In blood and lymph node, virtually all NKp46+ cells were positive for CD8α (3.4 and 0.5%, respectively). Surprisingly, spleen and liver showed a second population of NKp46+ cells with increased NKp46 expression that was correlated with a CD8αdim phenotype (3.2 and 15.6%) or a CD8α phenotype (3.8 and 14.0%, respectively).

The low, respectively lacking CD8α expression in the NKp46+ cells led us to the question, whether both NKp46+ populations are true porcine NK cells, since previous studies described these cells as being all CD8α+ [[3, 26]]. In order to characterize these two subpopulations of putative NK cells, a four-color FCM analysis was performed using the NK-discriminating markers CD3, CD8α, CD16, and perforin in combination with NKp46 (Fig. 2A). In lymphocytes derived from liver, the frequency of this CD8αdim/−NKp46+ subpopulation was approximately five times higher than that of the CD8α+NKp46+ subset. CD8αdim/−NKp46+ cells were CD3CD16+perforin+, therefore displaying a classical NK cell phenotype. The majority of CD8α+NKp46+ cells was CD3CD16+perforin+, thus also accounting for porcine NK cells. However, within this population, a clear CD3+ population with a more heterogeneous CD16 expression and perforindim/− pheno-type was observed. A similar phenotype of the two NKp46+ subpopulations was also observed in spleen (data not shown).

Figure 2.

NKp46 is differentially expressed on porcine NK cells. (A) CD8αdim/−NKp46+ and CD8α+NKp46+ cells were analyzed for their co-expression of the NK-cell discriminating markers CD3/CD16 or CD3/perforin by four-color FCM. Representative results for PBMCs and liver cells are shown. Numbers indicate percentage of cells within respective gates or quadrants. Data shown are representative of experiments using cells from five different animals. (B) Porcine CD3CD8α+ PBLs were analyzed for CD16 and perforin expression and defined as total NK cells (contour plots on the left). Based on this CD3CD8α+ expression, PBLs of 50 healthy 6–7 month old pigs were investigated for the proportion of NKp46+ cells (black columns) within the CD3CD8α+ NK population (white + black columns). The data of the animal presented in the contour plots are indicated by an asterisk. (A, B) NKp46+ cells were defined according to corresponding fluorescence-minus-one controls. NKp46-specific mAb clone VIV-KM1 was used.

NKp46 expression on porcine CD3CD8α+ NK cells

By analyzing PBLs, we confirmed that all CD3CD8α+ lymphocytes co-expressed CD16 and perforin (Fig. 2A), thus displaying a NK cell phenotype as described earlier [[3, 26]]. However, only a proportion of those cells stained positive for NKp46, indicating that NKp46+ and NKp46 NK cells exist in swine (Fig. 2B). This observation was confirmed by relative quantification in quantitative reverse transcription polymerase chain reaction (RT-qPCR) of NKp46 transcripts in fluorescence-activated cell sorter (FACS)-sorted CD3CD8α+NKp46+ and CD3CD8α+NKp46 NK cells. Compared with the relative expression ratio threshold (R) of 1 in non-NK cells (CD3CD8α lymphocytes), the relative quantity of NKp46 transcripts in CD3CD8α+NKp46 cells was only slightly different (R ≤ 1.7), while the ratio was significantly elevated in CD3CD8α+NKp46+ NK cells (R ≥ 30) (Supporting Information Fig. 2). To further prove this observation, we analyzed the distribution of CD3CD8α+NKp46 and CD3CD8α+NKp46+ NK cells in the blood of 50 healthy 6–7 month old pigs by FCM. A vast heterogeneity in regard to NK cell number (1.3–24.5% of PBLs) as well as the proportion of NKp46+ cells in the CD3CD8α+ NK population was observed (Fig. 2B, and Supporting Information Fig. 3A and B). We identified animals with nearly 100% NKp46+ cells within CD3CD8α+ NK cells, but also others displaying only about 15% NKp46+ cells within the CD3CD8α+ NK cell fraction. Nevertheless, in the majority of pigs, an approximately 50:50 distribution of NKp46 and NKp46+ NK cells was observed (Supporting Information Fig. 3B) with a tendency that the fraction of NKp46+ NK cells was slightly larger than the NKp46 cells.

In organs tested, CD3CD8α+NKp46+ and CD3CD8α+NKp46 cells also were observed. The proportion of NKp46+ and NKp46 NK cells was similar between different organs for each individual animal (data not shown). Only in mediastinal lymph node, the majority of CD3CD8α+ cells was NKp46+ (60–75%) in each animal analyzed. Additionally, in all animals tested NKp46 was only moderately expressed on NK cells isolated from PBLs in comparison to cells isolated from lymph nodes, spleen, and liver (Supporting Information Fig. 3C and Fig. 1C). Overall, the data so far indicated that not all porcine NK cells express NKp46.

NKp46+ as well as NKp46 NK cells are competent to kill

Phenotypically, CD3CD8α+NKp46 cells resemble NK cells due to their CD16 and perforin expression. To further prove that these cells are functional NK cells despite the lack of NKp46 expression, we performed killing assays with different target cells to analyze the cytotoxic potential of the NKp46+ and NKp46 NK cells. FACS-sorted NKp46 and NKp46+ CD3CD8α+ NK cells were stimulated with recombinant human (rhu) interleukin-2 (IL-2) overnight and co-cultured either with the human leukemia cell line K562 or one of the two porcine kidney cell lines, MAX, and PK-15, for four hours. Both NK cell populations efficiently lysed the xenogeneic cell line K562 and the two allogeneic cell lines MAX and PK-15 (Fig. 3A) and showed comparable killing properties to total CD3CD8α+ NK cells. No obvious difference could be observed between the two NKp46-defined populations, demonstrating that both have features of functional NK cells. Moreover, lysis of investigated target cells did not seem to be NKp46 dependent.

Figure 3.

Cytolytic properties of NKp46-defined NK cell populations. (A) FACS-sorted CD3CD8α+NKp46 and CD3CD8α+NKp46+ NK cells were stimulated with rhu IL-2 overnight and used in a 4-h cytotoxic assay with K562 (top), MAX (middle), and PK-15 (bottom) cells as target cells at four different E:T ratios, 24:1, 12:1, 6:1, and 3:1. FACS-sorted CD3CD8α+ NK cells (total NK cells) served as controls. Data shown are from one animal and are representative of five independent experiments with three different animals. NKp46-specific mAb clone VIV-KM1 was used. (B) FACS-sorted CD3CD8α+ NK cells were either stimulated with rhu IL-2 overnight (bottom) or left in medium (top) and used in a 4-h redirected lysis assay with FcγR+ P815 as target cells. Effector cells were pre-incubated either with unspecific isotype-matched Ab or mAb against CD16 or NKp46 (clone VIV-KM3). Data shown are from one animal and are representative of eight independent experiments with six different animals. (C) NKp46-mediated killing in relation to CD16-mediated lysis (set to 100%, after subtraction of spontaneous lysis) at an E:T ratio of 24:1. The asterisk indicates the dataset that is displayed in (B).

To investigate the role of NKp46 as activating receptor in the pig, we tested the activating properties of NKp46 in redirected lysis assays with the murine FcγR+ P815 cell line. Unstimulated, FACS-sorted CD3CD8α+ NK cells only showed a moderate killing activity when stimulated with anti-NKp46 mAb (12% at an E:T ratio of 24:1) in contrast to CD16 stimulation, where 34% specific lysis was achieved (Fig. 3B). Upon stimulation with rhu IL-2, specific lysis increased to 57% upon triggering of NKp46 but likewise reached an intermediate level compared with unspecific isotype-matched control Ab (40%) and CD16-mediated lysis (78%). When comparing NKp46-mediated lysis to CD16-mediated lysis (set to 100% lysis, Fig. 3C), triggering of NKp46 in unstimulated NK cells never exceeded CD16-induced killing (13–50%) in all animals tested. However, upon cytokine stimulation, all animals showed elevated cytotoxic properties after NKp46 triggering. Two of six animals analyzed even reached the same level as with CD16. Reduced redirected lysis upon NKp46 triggering might be due to the lower NKp46 expression on NK cells compared with that of CD16 and the fact that, contrary to NKp46, CD16 is expressed on all porcine NK cells. Nevertheless, our data indicated that NKp46 has a role as an activating receptor as described for other species.

NKp46+and NKp46 NK cells differ in IFN-γ production

Since IFN-γ is a key player in NK cell response, we wanted to investigate if IFN-γ production of the two NK cell populations resembles their comparable killing properties. Therefore, we stimulated FACS-sorted CD3CD8α+NKp46 and CD3CD8α+NKp46+ NK cells for 3 days with rhu IL-2 + recombinant porcine (rpo) IL-12 + rpo IL-18 since we could previously show that this combination of cytokines is superior in stimulating IFN-γ production in porcine NK cells [[26]]. Subsequently, supernatants of these cultures were analyzed by enzyme-linked immunosorbent assay (ELISA) for IFN-γ production. Even though NKp46 NK cells produced considerable amounts of IFN-γ, NKp46+ cells showed a strongly enhanced IFN-γ production (3.6- to 10-fold, n = 4) in comparison to NKp46 NK cells, thus indicating that NKp46+ NK cells have an increased capacity to produce this cytokine (Fig. 4A). To confirm this data, total PBMCs were either stimulated with rhu IL-2 + rpo IL-12 + rpo IL-18 for 24 h or left in medium only and accumulation of intracellular IFN-γ was analyzed in combination with NKp46 expression on gated CD3CD8α+ NK cells. As expected, unstimulated cells did not show any IFN-γ production. After cytokine stimulation, NKp46+ cells clearly showed an increased IFN-γ production whereas NKp46 cells only showed a marginal cytokine production (Fig. 4B). In all animals tested, NKp46+ NK cells accounted for at least 90% of IFN-γ-producing NK cells.

Figure 4.

IFN-γ production of NKp46-defined porcine NK cells. (A) FACS-sorted CD3CD8α+NKp46 and CD3CD8α+NKp46+ NK cells were stimulated with rhu IL-2, rpo IL-12, and rpo IL-18 for 3 days. Thereafter, supernatants were collected and tested for IFN-γ production in ELISA. Total PBMCs and FACS-sorted CD3CD8α+ NK cells served as controls. Data are displayed as the mean of duplicates + SD. (B) Intracellular cytokine staining for IFN-γ production in CD3CD8α+NKp46 and CD3CD8α+NKp46+ NK cells within PBMCs after 24 h in vitro stimulation with rhu IL-2, rpo IL-12, and rpo IL-18. Unstimulated cells served as control group (Medium). Numbers indicate percentage of cells within respective gates or quadrants. Data shown are from one animal and are representative of experiments with (A) four or (B) five different animals. (A, B) NKp46-specific mAb clone VIV-KM1 was used.

NKp46 can be induced in NKp46 NK cells by cytokine stimulation

The observation that NKp46 seems not to be expressed on all porcine NK cells raised the question whether NKp46 and NKp46+ porcine NK cells are distinct subpopulations. Initial experiments showed that NKp46 can be up-regulated on NK cells after in vitro stimulation of total PBMCs with the cytokine combination of rhu IL-2, rpo IL-12, and rpo IL-18 (data not shown). To test the possibility of NKp46 induction in NKp46 negative cells, porcine CD3CD8α+ NK cells were FACS sorted into NKp46 and NKp46+ cells. After stimulation with rhu IL-2 + rpo IL-12 + rpo IL-18 for 3 days, cells were restained for NKp46 expression. Indeed, an NKp46-expressing population could be identified in microcultures of previous NKp46 cells (Fig. 5). This was also indicated by a 15-fold increase in the mean fluorescence intensity (MFI) of NKp46 in the former negative population (218 versus 3416, respectively). Furthermore, even the NKp46 positive NK cells showed an increase in the average MFI of NKp46 (3.7-fold, 3708 versus 13,800, respectively), demonstrating an up-regulation of NKp46 expression upon cytokine stimulation.

Figure 5.

Induction of NKp46 expression in NKp46 porcine NK cells. FACS-sorted CD3CD8α+NKp46 and CD3CD8α+NKp46+ NK cells were in vitro stimulated with rhu IL-2, rpo IL-12, and rpo IL-18 for 3 days, and re-stained for NKp46 expression after harvest. Histograms show the NKp46 expression profile of freshly sorted (d0) and stimulated (d3) cells. Dotted line grey histograms represent respective isotype-matched negative controls. Numbers indicate the MFI of total cells displayed in respective histograms. Data shown are from one animal and are representative of experiments with five different animals. For experiments on d0 and d3, NKp46-specific mAb clone VIV-KM1 was used.

Discussion

NK cells have been phenotypically defined as CD3NKp46+ lymphocytes and the activating receptor NKp46 is considered as a specific NK marker in all mammals studied so far [[15]]. In this study, we investigated NKp46 expression on porcine NK cells. Our experiments showed that the phenotypeof NKp46-expressing cells in the pig resembled NKp46+ lymphocytes in other species since they did not express surface markers of γδ T cells, B cells, T-helper cells, and monocytes [[10, 14], [17-19]]. NKp46+ cells in swine co-expressed CD2, perforin, and CD16 and the majority was negative for CD3 in peripheral blood and all organs tested, which correlates with the so far described phenotype of porcine NK cells [[1-4]]. Nonetheless, we could detect a small but distinct population of CD3+NKp46+ cells. Although it is postulated that NKp46 is exclusively expressed on NK cells, it was previously described that particular non-NK cell subsets can express NKp46, like NK-like γδ T cells [[15, 27, 28]] and NKT cells [[29]]. Therefore, CD3+NKp46+ cells may represent NKT cells in the pig since they lack γδ-TCR and CD3+CD16+perforin+ porcine T cells were described before [[3]].

However, in contrast to other species, our results revealed that not all porcine NK cells express NKp46. When investigating CD3CD8α+-defined NK cells in PBLs for their NKp46 expression, we observed that only a proportion of these cells was positive for NKp46. Nevertheless, NKp46 cells within the CD3CD8α+ population showed comparable CD16 and perforin expression and therefore phenotypically resembled NK cells. Furthermore, CD3CD8α+NKp46 cells showed comparable spontaneous lytic activity against xenogeneic and allogeneic targets to NKp46+ cells, thus providing evidence that, independent of their NKp46 expression, both populations represent functional NK cells. Killing of our tested target cells seemed to be independent of NKp46 triggering and up to date we have not encountered NKp46-dependent target cells for the pig. The fact that CD3CD8α+NKp46 cells have comparable negligible mRNA levels compared with those of non-NK cells further strengthen the observation of NKp46 NK cells in the pig.

To our knowledge, NKp46 NK cells have not been reported before, but our findings of NKp46+ and NKp46 NK cells in the pig might resemble the observed expression patterns of NKp46 in some human individuals were NKp46bright and NKp46dull populations could be observed [[30]] and likewise two NK cell subpopulations on the basis of NKp46 expression were described. Noteworthy, data in human tonsils and lymph node have been reported where the majority of NK cells was negative for NK-discriminating markers, including NKp46 [[31]]. More recently, it was reported that a very minor CD3CD56+ human NK cell subset exists in blood that displays only low-density expression of NKp46 or even lacks this marker [[32]]. Additionally, we observed a rather moderate surface expression of NKp46 on PBLs in all animals tested. A speculation that this moderate reactivity with peripheral NK cells is based on the low affinity of the mAbs generated against NKp46 can be overruled by the fact that especially in liver and spleen, NKp46+ NK cells showed a two-fold higher surface antigen expression.

To investigate the role of porcine NKp46 as triggering receptor, we employed redirected lysis assays with the FcγR+ cell line P815. Data in other species showed that triggering with the NKp46 mAb results in a vast increase in killing by IL-2-activated NK cells [[10, 12, 17-19, 30]], even at low effector-to-target cell ratios. In human, triggering via anti-NKp46 mAb resulted in comparable lytic activity to triggering with mAb against the Fcγ-low-affinity receptor CD16 [[10, 12, 30, 33]], whereas in cattle NKp46-mediated killing led to a stronger cytotoxic response [[34]]. Similarly, we could observe NKp46-mediated killing in IL-2-stimulated porcine NK cell cultures. In non-stimulated NK cells, only moderate triggering activity of NKp46 was observed. This is in agreement with data of freshly isolated, resting human NK cells, where engagement of NKp46 induced only reduced lysis but could be augmented by cytokine stimulation [[33]]. The fact that not all NK cells express NKp46, but instead display a uniformly high CD16 surface expression, could explain the reduced killing activity induced by anti-NKp46 mAb in comparison to anti-CD16 mAb. Additionally, the overall surface expression of NKp46 is considerably lower than that of CD16. Thus, high efficient cross-linking of NKp46 might be hampered and therefore insufficient to induce higher cytolytic activity. Comparable results were seen in humans exhibiting NKp46dull NK cell populations. Only in NKp46bright NK cells, the anti-NKp46-mediated lysis paralleled CD16-mediated killing, whereas NKp46dull NK cells showed reduced triggering capacity [[30]]. Yet, it cannot be ruled out that the reduced triggering via NKp46 might be due to the fact that the anti-NKp46 mAbs used do not bind strongly to an activating epitope on the receptor, thus leading only to a reduced triggering activity. Although we have tested different mAbs recognizing distinct binding sites on porcine NKp46 (Supporting Information Fig. 1B), none of them was able to induce stronger cytolytic activity.

After demonstrating that CD3CD8α+NKp46 lymphocytes exhibit natural cytotoxicity and represent NK cells, we measured the IFN-γ production of the two NKp46-defined NK cell populations. Our data demonstrated that in swine the NKp46+ NK cells are the main IFN-γ producers and thus revealed a functional difference between NKp46+ and NKp46 NK cells. To our knowledge, no direct correlation of NKp46 expression and IFN-γ production has been reported in other species. However, differences in IFN-γ production can be assigned to distinct NK subsets. The human CD56bright NK cell subset is more potent in producing IFN-γ as well as other regulatory cytokines than CD56dim NK cells that exhibit elevated cytotoxic functions [[35, 36]]. Additionally, the more abundant IFN-γ producing CD56bright NK cells are described to show somewhat higher NKp46 expression levels [[37, 38]]. Among other functional differences, CD27high murine NK cells produce considerably higher amounts of IFN-γ compared with the CD27low subset [[39]]. Additionally the co-expression of the chemokine receptor CXCR3 on CD27bright NK cells provides a suitable marker for the IFN-γ producing NK cell subset in mice [[40]]. In cattle, a NK cell subset with markedly higher IFN-γ production was defined according to CD2 expression since CD2−/low cells show a higher IFN-γ production [[41]].

Our findings of NKp46 NK cells in the pig and the generally moderate NKp46 expression on porcine NK cells in blood raised the question whether NKp46 expression can be induced or up-regulated. Remarkably, we could demonstrate up-regulation of NKp46 expression in the former negative population after cytokine stimulation, thus revealing the possibility to convert NKp46 NK cells into the NKp46+ phenotype. Although it was reported that human NKp46 was not modified by cytokine stimulation in vitro [[42]], the influence of cytokines on the expression level of NKp46 was previously mentioned for human and other species. IL-2 was described to up-regulate NKp46 on human NK cells [[33, 43]] and to induce NKp46 on NKp46 NK cells in tonsils and lymph node [[31]]. Furthermore, it was demonstrated that NKp46 expression could be modulated by phagocytes in regard to CD56 co-expression [[44]]. Induction of NKp46 in former negative cells has also been reported in cattle for TCR-γδ T cells that expressed NKp46 upon stimulation with IL-12, IL-15, and IL-18 [[27]]. First evidence for up-regulation of porcine NKp46 was currently given on mRNA level after IL-12, IL-15, or IL-18 stimulation [[45]]. The fact that NKp46 seems not to be expressed on all porcine NK cells but simultaneously can be up-regulated after in vitro stimulation raises the question whether NKp46+ and NKp46 porcine NK cells are distinct subpopulations, or if porcine NKp46 cells may represent NK cells in a distinct differentiation stage. Studies for a more detailed characterization of NKp46 NK cells with the final aim to get more insight into the role of the activating receptor NKp46 are on the way.

Furthermore, an additional population of CD8dim/− lymphocytes with increased NKp46 expression was observed in spleen and liver. Especially in the liver, the frequency of this CD3CD8αdim/−NKp46high population was about five times higher than the “conventional” CD3CD8α+NKp46+ NK cells and thus accounted for the majority of NKp46+ cells in this organ. These data expand the knowledge of the phenotype of porcine NK cells that have been described so far as CD3CD8α+ lymphocyte subset [[3, 4, 26]]. Due to the enhanced expression of the activating receptor NKp46, those cells might play a special role in the immune surveillance of those organs.

Overall, the results of this work indicate that porcine NK cells take an exceptional position among the mammalian species in regard to their NKp46 expression pattern. Particularly the paradigm describing NKp46 as a unifying marker for mammalian NK cells is challenged by the fact that functional NKp46 NK cells exist in swine.

Material and methods

Cell culture

The myeloma cell line SP2/0, human leukemia cell line K562 [[46]], murine FcγR+ mastocytoma cell line P815, porcine kidney cell lines MAX [[47]] and PK-15 [[48]], as well as isolated porcine PBMCs were cultivated in RPMI 1640 with stable glutamine supplemented with 10% (v/v) heat-inactivated FCS, 100 IU/mL penicillin, and 0.1 mg/mL streptomycin. Medium for sorted NK cells was additionally supplemented with 1 mM sodium pyruvate, non-essential amino acids, and 50 μM 2-mercaptoethanol. HEK293T cells were cultivated in Dulbecco's modified Eagle's medium (DMEM) with stable glutamine supplemented with 10% (v/v) FCS, 1 mM sodium pyruvate, and 25 μg/mL Gentamicin. For detachment of adherent cells, Trypsin–EDTA (ethylenediamine tetraacetic acid) was applied. Where indicated, PBMCs and NK cells were cultured in the presence of either 100 IU/mL rhu IL-2 (Roche, Vienna, Austria) overnight, or 30 IU/mL rhu IL-2 in combination with 25 ng/mL rpo IL-12 and 100 ng/mL rpo IL-18 (both R&D Systems, Minneapolis, MN, USA) for 3–4 days.

Isolation of porcine lymphocytes

Blood and organs were obtained from 6 to 7-month-old healthy pigs from an abattoir. Animals were subjected to electric high voltage anaesthesia followed by exsanguination. This procedure is in accordance to the Austrian Animal Welfare Slaughter Regulation. PBMCs were isolated using density gradient centrifugation (Lymphocyte Separation Medium, density: 1.077 g/mL) as described elsewhere [[49]]. Lymphocytes from spleen and medLN were isolated as decribed previously [[50]]. Isolation of intrahepatic lymphocytes was performed according to a protocol described elsewhere [[51]].

Generation of monoclonal antibodies

For generation of mAbs against porcine NKp46, a protocol according to the development of mAbs against bovine NKp46 [[18]] was used. RNA of total PBMCs was used for the production of cDNA with SuperScriptTM First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The extracellular part of porcine NKp46 (29–792 nt, NCBI accession number AB516285.1, [[25]]) was amplified by RT-PCR using Pfu DNA polymerase (Promega, Madison, WI, USA) and gene-specific primers modified with restriction overhangs for HindIII and BamHI. PCR products were cloned into the mammalian expression vector pMIg1 containing the sequence of the hinge and Fc region of murine IgG2b (kindly provided by H.-C. Aasheim, The Norwegian Radium Hospital, Oslo, Norway, [[52]]). HEK293T cells were transiently transfected with the expression construct using Lipofectamine (Invitrogen) according to manufacturer's instructions in a 175-cm2 flask. Supernatant containing the recombinant NKp46-Fcγ2b fusion protein was collected for 1 week and applied to Protein G column for protein purification. Female, 6-week-old BALB/c mice were immunized with 50 μg of recombinant protein in combination with 50 μg Lipopeptide Adjuvant (EMC microcollections, Tübingen, Germany) subcutaneously (s.c.) for four times in intervals of 3 weeks. During the week prior to fusion, antigen without adjuvant was applied intraperitoneally (i.p.) on three consecutive days. Animal experiments with mice were approved by the institutional ethics committee, the Advisory Committee for Animal Experiments (§12 of Law for Animals Experiments, Tierversuchsgesetz—TVG) and the Federal Ministry for Science and Research (bmwf GZ 68.205/0115-BrGT/2005). Generation of spleen-cell derived hybridoma cells was achieved according to Köhler and Milstein [[53]]. For screening of Ab-producing hybridoma cells, HEK293T cells were transiently transfected with an expression construct containing whole porcine NKp46 without the signal sequence (112–972 nt) with a N-terminal FLAG-tag (mammalian expression vector pFLAG-CMV1, Sigma-Aldrich, Vienna, Austria). Screening was performed by FCM using hybridoma cell supernatants in combination with M2 anti-FLAG control Ab (Sigma-Aldrich). Positively reacting cells were subcloned to obtain monoclonal cell cultures.

Immunoprecipitation

PBMCs were biotinylated using Sulfo-NHS-LC-Biotin (Thermo Scientific, Pierce, Vienna, Austria) following manufacturer's instructions. Immunoprecipitation was performed using Direct IP Kit (Thermo Scientific) according to a protocol provided by the manufacturer. For Ab coupling, 8 μg of either anti-porcine NKp46 mAb VIV-KM1 or unspecific isotype-matched control Ab (mouse-IgG1, Dianova, Hamburg, Germany) were used. Precipitates were desalted and concentrated by centrifugation using Vivaspin columns 500 (3000 MWCO) before mixed with sample buffer (Thermo Scientific). After boiling for 5 min at 96°C, samples were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer onto a polyvinylidene fluoride membrane by semi-dry blotting and blocking with 5% (w/v) skim milk in tris-buffered saline (TBS) (20 mM Tris, 0.5 M NaCl, pH 7.4), the membrane was incubated with streptavidin-horseradish peroxidase (HRP) (Roche, 1:2000 in 1% (w/v) Casein-TBS with Tween 20 (TBST)) for 1 h. Detection was carried out with enhanced chemiluminescence (ECL) Western Blotting Substrate chemoluminescence kit (Thermo Scientific) and developed on Amersham Hyperfilm MP autoradiography film (GE Healthcare, Little Chalfont, UK).

FCM and antibodies

Cells resuspended in PBS containing 10% (v/v) porcine plasma were labeled for FCM analysis as described earlier [[50]]. The following primary Abs were used for cell surface staining: anti-NKp46 (IgG1, clone VIV-KM1), Alexa647-conjugated anti-NKp46 (IgG1, clone VIV-KM1), anti-FLAG (IgG1, clone M2, Sigma-Aldrich), anti-CD2 (IgG2a, clone MSA4), anti-CD3 (IgG2b, clone BB23 8E6, Southern Biotech, Birmingham, AL, USA), Alexa405-conjugated anti-CD3 (IgG1, clone PPT3), anti-CD4 (IgG2b, clone 74-12-4), anti-CD8α (IgG2a, clone 11/295/33), anti-CD8β (IgG2a, clone PG164A, VMRD, Pullman, WA, USA), anti-CD14 (IgG2a, clone Tük4, Serotec, Raleigh, NC, USA), anti-CD16 (IgG1, clone G7, Serotec), anti-TCR-γδ (IgG2b, PPT16) and SLA-DR (IgG2a, clone MSA3). All non-commercial mAbs were produced in-house [[54]]. Where indicated, these Abs had been purified and covalently conjugated to Alexa-fluorochromes by the use of respective conjugation kits (Invitrogen). For all experiments, appropriate isotype-matched control Abs as well as fluorescent dye-conjugated control Abs were used. The following anti-mouse, isotype-specific fluorescent dye-conjugated Abs were used as second-step reagents: anti-IgG1-Alexa647, anti-IgG2a-Alexa488, anti-IgG2b-Alexa488 (Invitrogen), anti-IgG1-PE, anti-IgG2a-PE (Southern Biotech). To discriminate between live and dead cells, Fixable Aqua Dead Cell Stain Kit (Invitrogen) was used according to manufacturer's protocol. If Abs with the same isotype were used in combination, sequential staining of the two mAbs was performed. Unconjugated primary Ab was used in a first step, followed by isotype-specific dye-conjugated Ab. After secondary incubation, free binding sites of mouse-isotype specific Ab were blocked by whole mouse IgG molecules (2 μg per sample, Jackson ImmunoResearch, Suffolk, UK) followed by a further incubation step with fluorochrome-conjugated primary mAbs. Co-staining with VIV-KM1 and M2-anti-FLAG Ab was performed using Zenon labeling Kit Alexa Fluor647 for IgG1 (Invitrogen) according to manufacturer's protocol.

For intracellular staining with the mAbs, anti-IFN-γ-PE (IgG1, clone P2G10, BD Biosciences, San Jose, CA, USA), anti-Perforin-PE (IgG2b, clone δG9, BD Biosciences), and anti-CD79αcy-PE (IgG1, clone HM57, Dako, Glostrup, Denmark) cells were fixed and permeabilized as described elsewhere [[55]]. For IFN-γ labeling, Brefeldin A (GolgiPlug, BD Biosciences) was added to cells at a final concentration of 1 μg/mL, 4 h prior to harvest.

FCM analyses were performed on FACSCanto II or FACSAria (BD Biosciences), respectively. Data of at least 1 × 105 lymphocytes per sample were recorded. Data were analyzed with FACSDiva (Version 6.1.3, BD Biosciences) and FlowJo software (Version 7.6.3., Tree Star, Ashland, OR, USA). Box plots were created by SigmaPlot software (Version 11.0, Systat Software Inc., Erkrath, Germany).

Fluorescence-activated cell sorting of NK cells

For sorting of CD3CD8α+, CD3CD8α+NKp46, and CD3CD8α+NKp46+ NK cells, PBMCs were labeled with primary Abs against CD3, CD8α, and NKp46 as described above. As secondary Abs, anti-IgG1-PE (Southern Biotech), anti-IgG2a-Alexa647 (Invitrogen), and anti-IgG2b-Alexa488 (Invitrogen) were used. For all washing steps, PBS containing 5% (v/v) FCS and 2 mM EDTA was used. Sorting was performed on a FACSAria (BD Biosciences). Purity of sorted cell populations was ≥99.5% for CD3CD8α+ NK cells and 95–99% for NKp46 and NKp46+ NK cells.

Cytotoxic assays

Cytotoxic activity of sorted NK cells was assessed by FCM as described elsewhere [[26]]. Briefly, effector cells were stimulated with or without rhu IL-2 overnight and co-cultured with DIOC18 (Sigma-Aldrich)-labeled target cells for 4 h at different E:T ratios (24:1, 12:1, 6:1, and 3:1). For redirected lysis assays, effector cells were incubated with 1 μg/mL of anti-CD16, anti-NKp46, or isotype-matched control Ab for 30 min at 37°C prior to addition of target cells.

Analysis of IFN-γ production by ELISA

Supernatants of stimulated PBMCs or FACS-sorted NK cells were collected and tested with Swine IFN-γ ELISA Kit (Invitrogen) according to manufacturer's protocol. Optical density (OD) was measured at 450/620 nm with an ELISA reader (Tecan, Sunrise, Crailsheim, Germany).

Acknowledgements

The authors thank Barbara Rütgen, Maria Stadler, Sandra Groiß, and Katharina Reutner for general technical support and Georg E. Mair and Ralf Steinborn, VetCore, VetOmics/Genomics Unit for performance of RT-qPCR experiments. Kerstin Mair and part of this work was funded by the Ph.D. program “Host Pathogen Interaction in the Pig,” University of Veterinary Medicine Vienna, Austria.

Conflict of interest

The authors declare no financial or commercial conflict of interest.

Abbreviations
FCM

flow cytometry

medLN

mediastinal lymph node

NCR

natural cytotoxicity receptor

rhu

recombinant human

rpo

recombinant porcine

Ancillary