The activating rat Ly49s5 receptor responds to increased levels of MHC class Ib molecules on Listeria monocytogenes-infected enteric epithelial cells

Authors


Abstract

We have investigated whether rat Ly49 receptors can monitor Listeria-infected intestinal epithelial cells through altered expression of MHC class I molecules. The rat colon carcinoma epithelial cell line CC531 infected with Listeria expressed higher levels of both classical and nonclassical MHC-I molecules. Reporter cells expressing the activating Ly49s5 receptor displayed increased stimulatory responses when incubated with Listeria-infected CC531 cells in vitro, which could be blocked with mAb 8G10 specific for nonclassical MHC-I molecules of the RT1u haplotype, but not with mAb OX18 reacting with classical MHC-I molecules in this haplotype. Similar responses were observed against IFN-γ-treated cells that also upregulated their expression of MHC-I molecules. Thus, the Ly49s5 receptor can respond to increased levels of nonclassical MHC-I molecules induced on target cells by either bacterial infection or cytokine stimulation. We furthermore found that splenic NK and NKT cells produced IFN-γ in response to Listeria-infected CC531 cells, and that this was not limited to Ly49-expressing cells, since similar levels of IFN-γ production were observed in Ly49+ and Ly49 NK cell subsets. Therefore, NK cells may recognize Listeria-infected cells through both MHC-I-dependent and -independent innate immune receptor systems.

Introduction

Listeria monocytogenes is a Gram-positive intracellular bacterial pathogen that may cause infections in immunodeficient individuals and animals 1. Listeriosis is usually contracted through contaminated food products 2. Listeria primarily infects macrophages and epithelial cells, but fibroblasts, endothelial cells and neurons are also susceptible 3. In humans, Listeria gains entry into intestinal epithelial cells via the interaction between the Listeria surface protein internalin A and the E-cadherin expressed by intestinal epithelial cells 1, 4.

NK cells are innate immune lymphocytes capable of recognizing and destroying a wide variety of cells, including malignant, virally infected, allogeneic, Ab-coated and stressed cells 5. NK cells may also provide antibacterial immunity 6, 7. The mechanisms by which NK cells recognize and eliminate cells infected with intracellular bacteria have been less well investigated and may involve complex interactions with other cells like antigen-presenting cells 8. Control of Listeria infection depends on a critical cytokine, IFN-γ, since mice deficient in IFN-γ or its receptor are particularly susceptible to infection even with small inocula of Listeria9, 10. Cellular components important for IFN-γ-mediated resistance may include NK cells, NKT cells and CD8+ T cells, all secreting IFN-γ in response to Listeria infection. IFN-γ activates macrophages necessary for limitation of the infection 11.

Here, we have focused our studies on the epithelial cell line, CC531, a colon carcinoma line obtained from WAG rats (MHC haplotype RT1u). Specifically, we investigated whether NK cell interactions with Listeria-infected CC531 cells may induce IFN-γ production. Listeria infection resulted in an increased expression of MHC class I molecules on CC531 cells, and overnight coincubation with spleen cells stimulated IFN-γ production in NK and NKT cells. Previous studies in rats suggested that NK cells play a central role in the early control of Listeria infection as rats depleted of NK cells showed increased splenic bacterial loads after systemic infection 12. Rat NK cells recognize MHC class I molecules both of the classical (Ia) and nonclassical (Ib) type 13, 14. We have previously shown that activating rat Ly49 receptors is triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, the RT1-CE/M/N region; whereas inhibitory Ly49 receptors may bind ligands encoded both within the classical and nonclassical MHC class I regions 15, 16. We have here focused on the activating Ly49s5 receptor that recognizes a nonclassical MHC class I ligand of the RT1u haplotype, which, we show here, is present on CC531 cells 13. Reporter cells expressing this receptor displayed increased activation when coincubated with Listeria-infected CC531 cells, and this activation could be completely blocked by mAb specific for nonclassical MHC class I molecules. This effect was not Listeria specific since IFN-γ treatment of CC531 cells yielded similar results.

The physiologic roles of activating Ly49 receptors in immunity to intracellular bacteria are unknown. This study, however, suggests that activation of NK cells through Ly49 stimulatory receptors may contribute to antibacterial immunity. However, Listeria also triggers NK cells through alternate mechanisms since NK-cell subsets void of Ly49 receptors also produced IFN-γ in response to Listeria infection.

Results

mAb 8G10 reacts with nonclassical class I molecules of the RT1u haplotype

Monoclonal antibody 8G10 specifically stained spleen cells from PVG.1U (RT1u-u-u) but not PVG.R23 (RT1u-av1-av1) rats. This suggests that 8G10 recognize nonclassical MHC class I molecules of the RT1u haplotype, as described previously 17. We did not observe any 8G10 staining of the other MHC congenic rat strains tested; PVG (RT1c), PVG.1N (RT1n), PVG.1L (RT1l) and PVG.1LV1 (RT1l-l-lv1) (Fig. 1). As expected, mAb AAS1 did not stain PVG.1U cells, but cells from all other PVG congenic rat strains tested. AAS1 stained PVG.R23 cells weakly, probably because it recognizes only nonclassical MHC class I molecules of this haplotype.

Figure 1.

mAb 8G10 reacts with nonclassical MHC-I molecules of the RT1u haplotype. Spleen cells from the MHC congenic rat strains PVG (RT1c-c-c, stapled line), PVG.1N (RT1n-n-n, thin line), PVG.1U (RT1u-u-u, dark gray) and PVG.23 (RT1u-av1-av1, thick line) were stained with mAb 8G10 and AAS1 and analyzed by flow cytometry. Data are representative of two independent experiments.

Listeria-infected CC531 cells upregulate expression of classical and nonclassical MHC class I

Listeria primarily infects epithelial cells and macrophages. We infected the epithelial colon carcinoma cell line CC531 (RT1u haplotype) with Listeria and infection efficiency was confirmed by cytospin analysis (Fig. 2A). Furthermore, Listeria-infected CC531 cells increased their expression of both classical and nonclassical MHC class I molecules as detected by staining with mAb OX18 and 8G10, respectively (Fig. 2B and C). Listeria did not induce expression of MHC class II molecules as determined by staining with mAb OX6 (data not shown). As a positive control, we stimulated uninfected CC531 cells with IFN-γ. This induced an increased expression of MHC class I molecules similar to that induced by Listeria (Fig. 2B and C). No change in CC531 staining with the isotype control mAb TIB96 and STOK6 was observed.

Figure 2.

Listeria infects epithelial CC531 cells and induces upregulation of both classical and nonclassical MHC-I molecules. (A) Giemsa-stained cytospin of Listeria-infected CC531 cells after 48 h. Cells were approximately 20 μm in diameter. (B) FACS plots displaying mAb OX18 (anti-classical MHC-I) and 8G10 (anti-nonclassical MHC-I) staining of uninfected (thin line), Listeria-infected (upper panel, thick line) and IFN-γ stimulated CC531 cells (lower panel, thick line). Secondary Ab alone (stapled line). One representative experiment is shown. (C) MFI±SEM of CC531 cells treated as described above (data from three experiments). We observed an increased MHC-I expression on infected and IFN-γ-treated CC531 cells as compared with uninfected CC531 cells, p<0.05 (Student's t-test).

The activating Ly49s5 receptor recognizes Listeria-infected CC531 cells

We have previously shown that Ly49s5 recognizes ligand(s) encoded within the nonclassical region of the RT1u haplotype 13. In order to investigate whether Ly49s5 recognizes Listeria-infected CC531 cells, we generated two reporter T-cell lines: BWZ-Ly49s5 and BWN-Ly49s5 (Materials and methods). BWZ-Ly49s5 cells express the full-length Ly49s5 receptor together with the human DAP12 adapter molecule. Receptor stimulation leads to cellular activation through the DAP12 adapter, akin to Ly49 receptor signalling in NK cells. BWN-Ly49s5 cells express a chimeric Ly49s5-CD3ζ molecule and, hence, signal through the CD3ζ adapter, akin to TCR signalling. We found that both Ly49s5 reporter cells responded well to uninfected CC531 cells. Listeria infection of the CC531 cells induced an enhanced response (Figs. 3 and 4) by both reporter cell lines. These responses could be efficiently blocked with mAb 8G10 but not the isotype-matched control mAb STOK6 (Figs. 3 and 4 and data not shown). In some experiments, addition of mAb OX18 seemed to weakly reduce reporter cell activation (Fig. 4B and C), but similar results were observed with the isotype control mAb TIB96, suggesting that this was an unspecific effect. These results confirm that Ly49s5 recognizes a nonclassical MHC class I molecule of the RT1u haplotype. This ligand is most likely encoded by the RT1-CE region since we, in other experiments, have shown that mAb 8G10 binds to RT1-CE-encoded molecules in this haplotype, whereas OX18 does not (data not shown). OX18, on the other hand, binds to gene products encoded both within the classical MHC-I region (RT1-A) (Fig. 2B) and also some more telomeric-encoded MHC-I molecules such as RT-BM1 18 and RT1-M n some haplotypes 19. Furthermore, reporter cell activation correlated with the level of nonclassical MHC class CC531 cells as determined by FACS analysis (Fig. 2B). Activation of the Ly49s5 reporters was not a specific effect of Listeria infection as IFN-γ treatment of CC531 cells yielded similar results (Figs. 3 and 4). Addition of mAb 8G10 also efficiently blocked the recognition of IFN-γ-treated CC531 cells. As a control, we tested BWZ reporter cells stably transfected with Ly49s4 and human DAP12. Ly49s4 does not recognize ligands encoded by the RT1u haplotype, and these reporter cells failed to respond to IFN-γ-treated or Listeria-infected CC531 cells (data not shown).

Figure 3.

BWZ-Ly49s5 reporter cells recognize nonclassical MHC-I molecules on Listeria-infected and IFN-γ-treated CC531 cells. Overnight incubation of BWZ-Ly49s5 reporter cells with Listeria-infected (black bars) or IFN-γ-treated CC531 (dark grey bars) cells induced increased reporter responses (β-galactosidase activity) as compared with uninfected cells (white bars). As controls, unstimulated (grey bar) and PMA/ionomycin (grey dot bar) activated reporter cells are shown. Addition of 10 μL/well of mAb 8G10 supernatant completely blocked the reporter response. Mean standardized OD 595±SEM is shown. Data represent three independent experiments. Statistical analyses were performed with Student's t-test.

Figure 4.

BWN-Ly49s5 reporter cells recognize nonclassical MHC-I molecules on Listeria-infected and IFN-γ-treated CC531 cells. (A) BWN-Ly49s5 reporter cells coincubated with R2 cells (negative control), YB2/0 cells (positive control) or activated with PMA/ionomycin are shown. (B) Overnight coincubation with Listeria-infected or IFN-γ-stimulated CC531 cells resulted in increased reporter responses (GFP expression) as compared with uninfected cells. The reporter responses were completely blocked by mAb 8G10, but not mAb OX18. (C) The mean percentages±SEM of GFP positive reporter cells after treatment as described above are shown (data from three experiments). A weak, but not statistically significant, increase after coincubation with infected and IFN-γ stimulated cells was observed, p<0.08 and p<0.15, respectively, (Student's t-test, paired test).

Listeria-infected CC531 cells induce IFN-γ production by both NK and NKT cells in vitro

IFN-γ plays a central role for both the innate and the adaptive immune responses that control Listeria infection. IFN-γ can be secreted by NK, NKT and T cells 20. In order to investigate whether spleen cells produce IFN-γ in response to Listeria infection in epithelial cells, splenic lymphocytes of the RT1u haplotype were incubated with either uninfected or infected CC531 cells for 18 h. Both NK and NKT cells produced IFN-γ in response to Listeria-infected cells (Fig. 5). IFN-γ responses were not confined to the Ly49+ NK subset since both Ly49+ and Ly49 subsets responded equally well to the infected cells (Fig. 5B and D). This response likely required direct cell contact as no IFN-γ production was observed when spleen cells were incubated with supernatants from Listeria-infected cells alone (Fig. 5A and C).

Figure 5.

NK and NKT cells produce IFN-γ in response to Listeria-infected CC531 cells. Freshly isolated spleen cells of the RT1u haplotype were incubated for 18 h with either Listeria-infected, supernatant from infected (LM-sup.) or uninfected CC531 cells. NK and NKT cells were analyzed for IFN-γ production by intracellular FACS analysis. (A) Unstimulated, IL-12 activated and cells cultured in LM-sup. are shown. (B) Coincubation of spleen cells with infected CC531 cells induced high amounts of IFN-γ in NK (NKR-P1A+ CD3) and NKT (NKR-P1A+ CD3+) cells. IFN-γ production by Ly49s3 and Ly49i5/s5 NK cells is highlighted. (C and D) Graphs showing the mean percentage±SEM of IFN-γ-producing spleen NK cells after treatment as described above.

Discussion

Most previous studies on experimental in vitro infection with Listeria have employed macrophages. It is well known how Listeria reproduces intracellularly in macrophages and may spread directly from cell to cell 4, 21, 22, a mechanism that facilitates bacterial evasion from immune recognition during systemic infections. The first encounter with Listeria, however, is usually in the gut, where the bacterium enters intestinal epithelial cells and likely spreads to neighbouring cells 4.

We have here employed a chemically induced colon adenocarcinoma cell line from WAG rats (CC531) to establish an in vitro Listeria infection model for epithelial derived cells. The expression of nonclassical RT1u molecules on these cells 23, 24 is particularly pertinent to NK-cell studies, since we have previously characterized NK cell receptors, both inhibitory and activating, that can specifically recognize these ligand(s) 13. The aims of these studies were (i) to establish conditions for Listeria infection of CC531 cells, (ii) to investigate how infection affects MHC class I expression on these cells, (iii) to investigate whether altered MHC class I expression might be sensed by NK-cell receptors (iv) and finally, to investigate putative effector mechanisms in activated NK cells.

We found that Listeria infection induced upregulation of both classical and nonclassical MHC class I molecules on this epithelial cell line. These could possibly be sensed by both innate and adaptive immune effector mechanisms. The finding that the activating Ly49s5 receptor responded to increased levels of nonclassical MHC class I molecules was particularly intriguing as the physiologic functions of these activating receptors have been elusive. Specific binding of inhibitory Ly49 receptors to MHC class I ligands has been firmly established and serves as a fail-safe mechanism to prevent killing of normal cells expressing sufficient amounts of “self”-MHC class I molecules. Some have speculated that recognition of “self”-nonclassical MHC class I molecules might trigger weak, but physiologically insignificant NK-cell responses, and that the physiological ligands of activating rodent Ly49 receptors might be pathogen-encoded MHC-like molecules 25. Although these speculations cannot be formally tested in the present experiments, we have not found any MHC-like molecules encoded within the Listerial genome (our unpublished observations).

On the basis of the present data, we therefore hypothesize that Listeria induces an upregulation of “self”-MHC class I molecules on infected cells that contributes to their recognition and destruction by NK cells. This hypothesis requires three elements. First, it requires that NK cells are found in the proximity of infected cells in vivo. Intestinal intraepithelial lymphocytes comprise a subpopulation of NK (NKR-P1A+CD3) cells, but pilot experiments with cocultures of intestinal intraepithelial lymphocytes and Listeria-infected CC531 cells have not yielded evidence for IFN-γ production. It is difficult to draw definitive conclusions from these experiments, however, as the purity of NK cells in these studies was low. Second, this hypothesis necessitates that NK-cell activation is not over-ridden by inhibition. The activating Ly49s5 and inhibitory Ly49i5 receptors, which likely recognize the same MHC class I ligands, are mainly expressed by different subsets of NK cells 13. This finding suggests that Ly49s5-expressing NK cells may be able to respond to the Listeria-infected epithelial cells and macrophages unencumbered by inhibitory effects due to Ly49i5. Finally, this hypothesis requires that the Ly49s5+ NK cells are not hyporesponsive.

The in vitro survival of Listeria-infected CC531 cells is typically 3 days. Coincubation of infected CC531 cells with splenic NK and NKT cells inhibited Listeria growth and increased cell survival (our unpublished observations). We have observed release of IFN-γ from splenic NK cells after coincubation with infected target cells. These data, combined with our previously published in vivo data 12, show that innate immune cells likely play a prominent role in controlling Listeria infection. We could not, however, infer that this response was due to a direct interaction between NK cells and Listeria-infected cells as encounters with other accessory cells could not be excluded.

We speculate that NK-cell cytotoxic effects or NK/NKT cell-derived cytokines may help control Listeria infection in rats. Since IFN-γ release was not confined to the Ly49+ subset, but was a general feature of NK and NKT cells, the ability of the innate immune system to respond promptly to Listeria infections is not necessarily dependent solely on Ly49 receptors 26, 27. As in other potentially life-threatening infections, the innate immune system shows redundancy, and failure to respond by one mechanism (Ly49 responding to altered MHC class I expression) may be compensated by other receptor systems.

Some nonclassical rat MHC class I molecules encoded within the RT1-CE region display features of classical MHC class I molecules. This includes the ability to present peptides and to be upregulated by IFN-γ, but they are usually expressed at five to tenfold lower surface density compared with classical RT1-A-encoded molecules 17. Although clearly functional in allotransplant settings in vivo28, 29, it has been difficult to show cytotoxic responses against RT1-CE-encoded antigens on unmanipulated target cells in vitro, probably due to their low-surface expression. The inflammatory upregulation of RT1-CE encoded molecules on Listeria-infected cells may be a physiologic signal for immune activation through a repertoire of activating Ly49 receptors directed exclusively against class Ib molecules.

In conclusion, our data provide support for the idea that the repertoire of activating Ly49 receptors on NK cells may physiologically recognize nonclassical MHC class I molecules on Listeria-infected target cells, contributing to their destruction. This interpretation is supported by our previously published data, showing that rats depleted of their NK cells and specifically depleted of the NK subset expressing Ly49 receptors are rendered more susceptible to Listeria infection 12. The apparent contradiction between these findings and similar studies in mice, where NK cells have been proposed to inhibit the immune defense against Listeria30, 31, may be explained by species-specific differences in the repertoire of Ly49 receptors. Compared with mice, rats have a larger and more diverse repertoire of activating Ly49 receptors for MHC-I ligands 32 that may provide a more robust mechanism by which infected targets can trigger NK cells. We have previously shown that NK mediated killing of allogeneic cells is dependent on these activating Ly49 receptors and that they recognize ligands encoded within the nonclassical MHC class I region. This study suggests that the same activating Ly49 receptors might be involved in the recognition of Listeria-infected cells. It should be kept in mind, however, that the in vivo setting may be more complex and may require accessory cells to activate NK cells before their recognition and effector functions come into play.

Materials and methods

Animals

Briefly, 8- to 10-wk-old PVG.7b (RT1c or c; i.e. RT1-Ac -B/Dc -CE/N/Mc (class Ia-class II-class Ib) abbreviated c-c-c), PVG.1U (RT1u-u-u), AO (RT1u-u-u), PVG.1N (RT1n-n-n), PVG.23 (RT1u-av1-av1), PVG.1L (RT1l-l-l) and PVG.1LV1 (RT1l-l-lv1) rats were bred in our animal house or were purchased from Harlan (Bicester, UK). Rats were regularly screened for common pathogens and housed in compliance with guidelines set by the Experimental Animal Board under the Ministry of Agriculture of Norway.

Bacteria and infection

L. monocytogenes (strain L 242/73 type 4b, originally a gift from A. de Klerk, Department of Toxicology, Pathology and Genetics, National Institute of Public Health and the Environment (RIVM), Bilthoven, The Netherlands 33) were grown in TSB broth until mid-log phase as described previously 12. After addition of glycerol (15% v/v), 2 mL aliquots were stored frozen at −70°C until use. Cells were infected at an MOI of 5:1 and incubated at 37°C, 5% CO2 for 1 h, followed by washing with RPMI 1640 medium and resuspension in complete RPMI (cRPMI; RPMI 1640 supplemented with 10% FBS, 5×10–5 M 2-ME, L-glutamine) supplemented with 10 μg/mL gentamycin (Sigma-Aldrich) to kill extracellular bacteria. The infected cells were harvested after 48–72 h. Giemsa-stained (Sigma-Aldrich) cytospins were routinely analyzed to monitor the infection.

Cells and IFN-γ stimulation

The colon carcinoma epithelial cell line (CC531) was a kind gift from Dr. Peter Kuppen, Department of Surgery, Leiden University Medical Center, Leiden, The Netherlands. The CC531 cell line is a dimethylhydrazine-induced adenocarcinoma from the colon of a WAG rat (RT1u haplotype) 23. An in vitro cell line was established by Peter Kuppen 24. It was grown in cRPMI. CC531 cells were stimulated with 100 U/mL rat IFN-γ (a gift from PH van der Meide, University Medical Center Utrecht, Utrecht, The Netherlands). The rat macrophage cell line R2 originates from pleural macrophages induced by a silica injection in the pleural cavity of Wistar rats 34. It expresses classical and nonclassical MHC-I molecules not recognized by mAb 8G10 (our unpublished observations) and does not activate Ly49s5 reporter cells (Fig. 4). YB2/0 cells were also used as target cells for Ly49s5 reporters (expresses classical and nonclassical MHC-I molecules of RT1u haplotype).

FACS analysis and Ab-blocking experiments

CC531 cells were labelled with mAb OX18 (anti-classical MHC-I), OX6 (anti-MHC-II) and 8G10 (anti-nonclassical MHC-I). mAb R73 (anti-TCRα/β) and STOK6 (anti-Ly49s3) were used as isotype-matched controls (Table 1). In MHC class I blocking experiments, 3 μg/well of purified mAb or 10 μL supernatants of OX18, TIB96, STOK6 or 8G10 was added to the target cells 20–30 min prior to the addition of reporter cells.

Table 1. List of used mAb
mAbAntigen(s) Subclass
OX6MHC-IINon polymorphicMouse IgG1
OX18MHC-Ia/bNon polymorphicMouse IgG1
AAS1MHC-Ia/bMHC-I of certain haplotypesRat IgG2a
8G10MHC-IbRT1-CE of u haplotypeRat IgG2b
3.2.3NKR-P1A Mouse IgG1
Wen23NKp46 Mouse IgG1
DAR13Ly49i3, -s3, -i4 and -s4 Mouse IgG1
Fly5Ly49i5 and -s5 Mouse IgG1
STOK6Ly49s3 Rat IgG2b
STOK27NKR-P1B Rat IgG2a
G4.18CD3 Mouse IgG3
R73TCRα/β Mouse IgG1
TIB96Igh-5b (IgD b allotype) Mouse IgG1

Reporter assays

BWZ-Ly49s5 reporter cells were produced by stably transfecting BWZ cells (BW5147 T-cell line with LacZ gene under the control of 3×NFAT-1 promoter) with the activating Ly49s5 receptor and human DAP12. The LacZ gene encodes β-galactosidase. This enzyme generates chlorophenol red when it acts on its substrate chlorophenol red-β-D-galactopyranoside (Sigma-Aldrich), and chlorphenol red can be quantified in a colorimeter 35. Reporter cells were maintained in cRPMI with 1 μg/mL Puromycin and 0.5 mg/mL of Hygromycin B (Invitrogen, Carlsbad, CA, USA). BWZ-Ly49s5 cells were plated at 1×105 cells/well in cRPMI supplemented with 10 ng/mL of phorbol myristic acid (PMA). Uninfected or Listeria-infected CC531 colon carcinoma cells were added at 1×105 cells/well. In parallel, blocking mAb to MHC class I molecules were added. Plates were incubated overnight at 37°C and reporter cell activation (as defined by β-galactosidase production) was evaluated by incubation with 150 μM chlorophenol red-β-D-galactopyranoside in PBS supplemented with 100 mM 2-ME, 9 mM MgCl2 and 0.125% Nonidet P-40. After sufficient colour development, absorbance was measured at 595 nm and corrected for background absorbance at 650 nm.

BWN-Ly49s5 reporter cells were made by stably transfecting BWN.3G cells (BW5147 cells with enhanced green fluorescent protein under the control of 3×NFAT-1 promoter) with a Ly49s5-TCRζ chimeric construct. This construct was made by fusing the extracellular and transmembrane parts of the Ly49s5 receptor with the inverted intracellular part of the CD3ζ-chain. This construct was then subcloned into a pMX vector that coexpresses the rat DAP12 adapter molecule. The BWN-Ly49s5 cells were maintained in cRPMI supplemented with 0.5 mg/mL Hygromycin B and 1 mg/mL G418 sulfate (Invitrogen). Reporter cell responses towards uninfected or Listeria-infected CC531 cells were evaluated by FACS analysis for enhanced green fluorescent protein expression. In Ab-blocking experiments, 3 μg of purified Ab or 10 μL supernatants were added before the addition of effectors cells.

Analysis of IFN-γ production by splenic NK and NKT cells

Spleen cells were separated on Lymphoprep and incubated with either uninfected, Listeria-infected or supernatant from Listeria-infected CC531 cells in the presence of 125 U/mL IL-2 for 18 h. IL-12 (2 ng/mL) stimulation was used as a positive control. In brief, 10 μg/mL Brefeldin A (Sigma-Aldrich) was added and the cells were incubated for an additional 3–4 h. In total, 1×106 spleen cells were labelled with different combinations of conjugated mAb: 3.2.3-FITC (anti-NKR-P1A), PE-conjugated G4.18 (anti-CD3) or 10/78 (anti-NKR-P1A) (both from Pharmingen), DAR13 (anti-Ly49s3, -s4, -i3, -i4) 36 and Fly5 (anti-Ly49i5, -s5) 13 followed by PE or PerCP-conjugated streptavidin. Cells were then permeabilized with 0.5% saponin (Sigma-Aldrich) and intracellularly stained with anti-rat IFN-γ mAb (Pharmingen) according to the standard protocols.

Statistical analysis

Graphics and statistical analysis were performed with the GraphPad Prism software. Data are presented as the mean±SEM. Comparisons between experimental groups were performed with the one-way ANOVA and a Student's t-test. p-Values less than 0.05 were considered statistically significant.

Acknowledgements

The authors thank Stine Martinsen and Ulla Heggelund for excellent technical assistance. This work was supported by grants from the Norwegian Cancer Society, the Research Council of Norway, Anders Jahre's Foundation for the promotion of Science and the University of Oslo. The authors also thank Kurt Wonigeit for kindly providing them with the 8G10 hybridoma.

Conflict of interest: The authors declare no financial or commercial conflict of interest.

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