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Journal of Virology, August 2008, p. 8030-8037, Vol. 82, No. 16
0022-538X/08/$08.00+0     doi:10.1128/JVI.00870-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Influenza Virus Infection Augments NK Cell Inhibition through Reorganization of Major Histocompatibility Complex Class I Proteins

Hagit Achdout, Irit Manaster, and Ofer Mandelboim^*

The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel

Received 24 April 2008/ Accepted 20 May 2008

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The killing by natural killer (NK) cells is regulated by inhibitory, costimulatory, and activating receptors. The inhibitory receptors recognize mainly major histocompatibility complex (MHC) class I molecules, while the activating NK receptors recognize stress-induced ligands and viral products. Thus, changes in the expression of the various inhibitory and activating ligands will determine whether target cells will be killed or protected. Here, we demonstrate that after influenza virus infection the binding of the two NK inhibitory receptors, KIR2DL1 and the LIR1, to the infected cells is specifically increased. The increased binding occurs shortly after the influenza virus infection, prior to the increased recognition of the infected cells by the NK activating receptor, NKp46. We also elucidate the mechanism responsible for this effect and demonstrate that, after influenza virus infection, MHC class I proteins redistribute on the cell surface and accumulate in the lipid raft microdomains. Such redistribution allows better recognition by the NK inhibitory receptors and consequently increases resistance to NK cell attack. In contrast, T-cell activity was not influenced by the redistribution of MHC class I proteins. Thus, we present here a novel mechanism, developed by the influenza virus, of inhibition of NK cell cytotoxicity, through the reorganization of MHC class I proteins on the cell surface.

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Natural killer (NK) cells, which belong to the innate immune system, can kill tumor cells and pathogen-infected cells, without prior antigen stimulation (6, 10, 53). The NK cell-mediated killing is controlled by inhibitory, costimulatory, and activating receptors (6, 10, 36, 41). In humans, NK inhibitory receptors are subdivided into three families: killer cell immunoglobulin (Ig)-like receptors (KIR), leukocyte Ig-like receptor (LIR), and C-type lectin receptors. The ligands for most of the NK inhibitory receptors are the major histocompatibility complex (MHC) class I molecules (7, 25, 26, 35, 37). In addition, other non-MHC receptors, such as CEACAM1 (31) and 2B4 (32), also inhibit the killing activity of NK cells.

The direct killing by NK cells is extracted by a few NK activating receptors including CD16, NKG2D (41), CD80 (52), and three natural cytotoxicity receptors (NCRs) (6)—NKp30 (39), NKp44 (9), and NKp46 (6, 36, 40). Thus, killing of target cells is determined by a balance between activating and inhibiting signals, delivered to the NK cell (4). Hence, differences in the expression levels or in the distribution of NK ligands expressed on the target cell surface will have a major effect on NK cell-mediated killing.

The immune system and the invading viruses are in a constant battle. We have demonstrated that the viral hemagglutinin (HA) protein of the influenza virus interacts with both the NKp44 and the NKp46 receptors and that these interactions lead to increased killing of the infected cells (2, 3, 18, 28). We have also shown in vivo that in the absence of NCR1 (the mouse homologue of NKp46), influenza virus infection is lethal (18). The viruses, however, developed a counterattack mechanism and, as demonstrated here, after influenza virus infection the binding of the inhibitory receptors KIR2DL1 and the LIR1 is specifically increased to the infected cells.

We show that the increased binding of these inhibitory receptors leads to the inhibition of NK cell cytotoxicity and that this effect is observed very shortly after influenza virus infection, before the appearance of the HA protein on the cell surface and the consequent killing by NKp46. We also characterize the mechanism that leads to the increased NK inhibition and demonstrate that influenza virus infection results in MHC class I protein accumulation in lipid rafts.

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Cells and transfectants. The cell lines used in the present study were YTS cells, a subline of the YT NK leukemia cell line (54) transfected with either the ecotropic murine retrovirus receptor alone (YTS/eco) or with the ecotropic receptor and KIR2DL1 (YTS/KIR2DL1) (5), the human prostate cancer cell line DU145, and the human Epstein-Barr virus-transformed B-cell line 721.221 (abbreviated as 221). The generation of 221 transfectants expressing HLA-Cw3 (221/Cw3), HLA-Cw4 (221/Cw4), HLA-Cw6 (221/Cw6), HLA-Cw6-green fluorescent protein (GFP) (221/Cw6-GFP), and HLA-A2 proteins was previously described (1, 12, 30). Primary human NK cells were isolated from peripheral blood lymphocytes by using a human NK cell isolation kit and an autoMACS instrument (Miltenyi Biotec, Auburn, CA) (28). T cells were prepared from adult donors expressing HLA-A2 protein as described previously (27).

Viruses and cell infection. The A/Beijing [A/Beijing/262/95-like (H1N1)], A/Sydney [A/Sydney/5/97-like (H3N2)], A/Moscow [A/Moscow/10/99-like (H3N2)], and A/PR/8/34 (H1N1) viruses were generated as previously described (1). Cells were infected as previously described (1).

MAbs, antivirus sera, and fusion proteins. The monoclonal antibodies (MAbs) used in the present study were as follows: W6/32 and B1.23.2 MAbs directed against MHC class I molecules (33); HC10 MAb directed against the free heavy chain of MHC class I protein; 135.7 MAb directed against HN from SV (3, 28); and H17-L2 MAb directed against HA type 1 (28) and anti-CD71 MAb (Santa Cruz Biotechnology, Inc). Cholera toxin subunit B (CT-B), recombinant horseradish peroxidase (HRP) conjugate (Sigma, St. Louis, MO) was used in the dot blot. Human sera were obtained as previously described (1). The negative control proteins used in the present study were CD99-Ig or CD7-Ig. The production of CD99-Ig, CD7-Ig, NKp46-Ig, LIR1-Ig, and KIR2DL1-Ig fusion proteins by COS-7 cells and the purification on a protein G column were performed as previously described (29).

Flow cytometry and lipid rafts disruption. Cells were stained with either MAbs or Ig fusion proteins as previously described (1). For blocking experiments, cells were first incubated with various MAbs (the final concentrations are indicated in the figure legends) for 1 h on ice, washed, and stained with the appropriate Ig fusion protein. For lipid raft disruption, cells were treated with mevastatin (an inhibitor of sphingolipid and cholesterol synthesis) as previously described (19). Briefly, 10⁶ cells were incubated overnight at 37°C and 5% CO₂ with 5 µg of mevastatin (Sigma-Aldrich). As a control, cells were incubated in equal volumes of dimethyl sulfoxide (DMSO).

Cells were stained with trypan blue and propidium iodide (Biological Industries, Beit Haemek, Israel) to verify viability after influenza virus infection and mevastatin or DMSO treatments.

Cytotoxicity assay. The cytotoxic activity of primary NK cells, cytotoxic-T-lymphocyte (CTL), YTS/eco, and YTS/KIR2DL1 cells against the various target cells was assessed in 5-h ³⁵S release assays as previously described (30). In all assays, spontaneous release was <20% of maximal release.

Biochemical isolation of lipid rafts. Lipid rafts were isolated by 70% Nycodenz gradient equilibrium centrifugation after lysis with the nonionic detergent Triton X-100 (0.2% for the 221 cells) in TNE buffer (150 mM NaCl, 25 mM Tris [pH 7.5], 5 mM EDTA, aprotinin [Sigma-Aldrich] at 1:100, and 1 mM phenylmethylsulfonyl fluoride) for 30 min on ice as previously described (43). A total of 1.5 x 10⁷ cells were pelleted and lysed with 500 µl of flotation lysis buffer. Lysates were centrifuged at low speed, and supernatants were collected and centrifuged again. All subsequent steps were performed on ice. Lysates were adjusted to 35% Nycodenz by adding an equal volume of ice-cold 70% Nycodenz dissolved in TNE. Portions (900 µl) of the samples were loaded at the bottom of TLS-55 tubes (Beckman Instruments, Palo Alto, CA). An 8 to 25% Nycodenz linear step gradient in TNE was then overlaid on the lysate (200 µl each of 25, 22.5, 20, 18, 15, 12, and 8% Nycodenz). The tubes were centrifuged at 50,000 rpm for 3 h at 4°C in a TLS-55 rotor (g_av = 260,000 x g). Twelve fractions, each containing 180 µl, were collected from the top of the tube. Fractions 2 to 6 correspond to the raft-rich microdomains, as detected by the HRP-labeled CT-B staining. In the subsequent experiments, all 12 fractions were separated on a sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis gel detected by electroplating with anti-CD71 or HC10 MAbs. GM1 was detected with CT-B recombinant HRP conjugate by dot blotting.

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Increased binding of KIR2DL1-Ig and LIR1-Ig to the DU145 cell line infected with various strains of human influenza virus. We have previously shown that after infection with influenza virus, the binding of KIR2DL1-Ig and LIR1-Ig to cells expressing the appropriate MHC class I ligands is specifically increased (1). To test whether similar results would be obtained in cells that express endogenous MHC class I proteins, we initially determined the HLA haplotype of DU145 cell line as HLA-A*03, HLA-A*33, HLA-B*50, HLA-B*57, and HLA-Cw*06. The DU145 cell line was infected with various strains of human influenza viruses, and the binding of KIR2DL1-Ig and LIR1-Ig was analyzed. We observed a 3- to 13-fold increase in the binding of KIR2DL1-Ig and a 7- to 25-fold increase in the binding of LIR1-Ig to DU145 cells infected with A/Moscow (H3N2), A/Beijing (H1N1), or A/Sydney virus (H3N2) (Fig. 1A). No change in the binding of the control protein (CD99-Ig) to the infected DU145 cells was observed (Fig. 1A), and no major changes were observed in the expression levels of MHC class molecules (the changes were <5% between influenza virus-infected versus uninfected cells (detected by W6/32 MAb) (Fig. 1B)). Infection of DU145 cells was detected by antivirus serum obtained from individuals vaccinated against all three strains of influenza virus (Fig. 1C).

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FIG. 1. The increased binding of KIR2DL1-Ig and LIR1-Ig to influenza virus-infected DU145 cells is specific. (A) MFI values of KIR2DL1-Ig, LIR1-Ig, and negative control protein (CD99-Ig) binding to the DU145 cell line. Cells were infected with different strains of the indicated human influenza viruses. (B) MFI values of the staining of infected and uninfected DU145 cell line with saturated concentration of W6/32 MAb and negative control antibody. (C) MFI values of infected and uninfected DU145 cells stained with anti-influenza human serum and control serum. For all of the experiments performed in panels A to C, one representative experiment is shown out of six performed. (D to F) The increased binding of inhibitory receptors after influenza virus infection is due to interaction with MHC class I proteins. 221/Cw4 and 221/Cw6 cells infected or not with A/Sydney were incubated in the presence or absence of B1.23.2 for KIR2DL1-Ig blocking and W6/32 MAbs for LIR1-Ig blocking. The final MAb concentrations were 100 and 10 µg/ml for blocking the binding of KIR2DL1-Ig and LIR1-Ig, respectively. The black and red histograms, respectively, represent staining of uninfected or virus-infected cells with KIR2DL1-Ig or LIR1-Ig proteins. The blue and green histograms, respectively, represent the staining of uninfected or infected cells with KIR2DL1-Ig or LIR1-Ig after preincubation with the indicated MAb. The figure shows the results of one representative experiment out of three performed.

To further confirm that the increased binding of KIR2DL1-Ig and LIR1-Ig to infected cells depends on the expression of the appropriate MHC class I molecule, we tested the binding of these proteins to cells expressing a single MHC class I protein. There was no binding of KIR2DL1-Ig to the parental 221 cells or to the 221/Cw3 cells (which is not the ligand for KIR2DL1) whether infected or not (data not shown). Indeed, and in agreement with our previous results (1) (Fig. 1A), we observed an increased binding of KIR2DL1-Ig to infected 221/Cw4 and 221/Cw6 cells (Fig. 1D and E) and increased binding of LIR1-Ig protein to infected 221/Cw6 cells (Fig. 1F). We show the LIR1-Ig binding only to 221/Cw6 cells because we could not detect binding of LIR1-Ig to 221/Cw4 (data not shown). The increased binding was due to enhanced recognition of MHC class I proteins, since preincubation of infected or uninfected 221/Cw4 cells and 221/Cw6 cells with B1.23.2 or with W6/32 MAbs abolished the KIR2DL1-Ig or LIR1-Ig binding, respectively (Fig. 1D to F).

The fact that B1.23.2 MAb and W6/32 MAb inhibited the binding of KIR2DL1 and LIR1, respectively, was not surprising as the LIR1 and KIR2DL1 receptors recognize different epitopes on HLA-C (8, 11, 16, 23, 30). These different epitopes are recognized by the two antibodies used. The B1.23.2 MAb, which recognizes the region around residue 80, blocked the binding of KIR2DL1 (which interact in the same region), while W6/32 MAb, which interacts with the 3 chain of MHC class I (21, 48), blocked the binding of LIR1 (Fig. 1D to F). Thus, the increased binding of the inhibitory receptors after influenza virus infection is specific, is not confined to a certain epitope on MHC class I proteins, and is not restricted to a particular MHC class I protein.

The increased KIR2DL1 binding after influenza virus infection is functional. The functional consequences of the increased binding of the inhibitory receptors to the infected cells was tested by using the YTS NK cell line, which lacks the activity of both inhibitory receptors and activating NCRs. We used two transfectants of YTS cells: cells expressing the ecotropic murine retrovirus receptor (YTS/eco), which were used as a control, and YTS/eco cells expressing KIR2DL1 (YTS/KIR2DL1) (5). In addition, we used two different 221/Cw6-GFP transfectants that differ in the expression levels of HLA-Cw6-GFP molecules (shown in Fig. 2A and D). The results showed that the level of HLA-Cw6-GFP expression on 221/Cw6 cells correlates with efficient inhibition of killing by YTS/KIR2DL1 (Fig. 2B and E). In agreement with our binding results, increased inhibition of killing by YTS/KIR2DL1 cells was observed when 221/Cw6^low cells were infected with influenza virus (Fig. 2B).

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FIG. 2. The increased binding of KIR2DL1-Ig to infected cells is functional. (A and D) Fluorescence-activated cell sorting (FACS) analysis of various 221/Cw6-GFP transfectants (open histogram). The background (gray histogram) represents the autofluorescence of parental 221 cells. YTS/KIR2DL1 cells (B and E) and YTS/eco cells (C and F) were tested in killing assays against various 221/Cw6-GFP cells either infected or not infected with A/Sydney. The various effector/target (E:T) ratios are indicated in the figure. The figure shows the results of one representative experiment out of four performed.

The control YTS/eco cells efficiently lysed all target cells (Fig. 2C and F). The killing of uninfected 221/Cw6^low cells by YTS/eco was slightly higher than that of the other targets; however, this difference is insignificant since such differences were not observed in other killing assays and were not evident in with the 221/Cw6^high cells (Fig. 2F). YTS/KIR2DL1 cells efficiently lysed 221 and 221/Cw3 cells regardless of whether or not these cells were infected (Fig. 2B and E and data not shown).

Hierarchy in NK inhibition and activation. NKp46 is active in all peripheral blood NK cells. We have previously shown that NKp46 recognizes the HA of influenza virus, and ca. 50% of the NK clones tested exhibited increased killing of influenza virus-infected cells, regardless of whether the target cells express MHC class I proteins (1, 28). In YTS cells, however, the NKp46 is inactive for unknown reasons, and hence efficient inhibition is observed after influenza virus infection (Fig. 2). Thus, what is the physiological importance of the increased class I mediated inhibition after influenza virus infection? We hypothesized that these two opposing mechanisms of influenza virus-mediated inhibition and activation would occur at different time points after infection.

To test this hypothesis, we examined the killing of the DU145 cell line by using bulk peripheral blood NK cells expressing the entire repertoire of inhibitory and activating NK receptors. As can be seen in Fig. 3A, 5 h after influenza virus infection, NK cytotoxicity was inhibited. This inhibition was correlated with increased KIR2DL1-Ig binding (Fig. 3B). In contrast, there was no change in the binding of NKp46-Ig and in the binding of the negative control fusion protein 5 h postinfection (Fig. 3B). Therefore, the increased inhibition occurs shortly after infection. Importantly, there was no elevation in HA (a ligand for the NK activating receptors, NKp46 and the NKp44) (3, 28) expression, on the surface of infected cells, at this time point, 5 h postinfection (Fig. 3C). In marked contrast, increased killing of infected cells was observed 15 h postinfection and was even further enhanced 24 h later (Fig. 3A). The observed increased killing correlated with an increased NKp46 binding and the appearance of HA on the cell surface (Fig. 3B and C). The enhanced killing was observed with bulk NK cells derived from some donors but not in others (data not shown). The fact that in some donors activation could not be observed is not surprising. Since DU145 cells express MHC class I proteins, it is likely that in some donors the appearance of HA on the surfaces of infected cells will tip the balance toward activation, while in other individuals with different combinations of inhibitory receptors it will not.

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FIG. 3. Hierarchy in NK inhibition and activation. (A) Killing experiment. DU145 cells uninfected or infected with A/PR/8 for the indicated time points were tested for killing by NK cells obtained from a healthy donor at a 10:1 effector/target ratio. (B) In parallel with the killing assays, FACS experiments were performed to test the binding of KIR2DL1-Ig, NKp46-Ig, and a control protein (CD99-Ig) to the DU145 cells. (C) Binding of anti-HA MAb and a control antibody. Representative data from one out of four experiments are shown.

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FIG. 5. Accumulation of MHC class I molecules in lipid raft microdomains after influenza virus infection. Lipid raft microdomains were identified by the specific raft marker GM1 (the numbers in the figure indicate the fraction number). Detection of GM1 was performed with HRP-labeled CT-B, followed by dot blot analysis on uninfected cells (A) or on influenza virus-infected cells (B). An equal volume (32 µl) obtained from each of the 12 fractions was immunoblotted with anti-CD71 to observe the detergent-soluble nonraft fractions in uninfected cells (C) and in infected cells (D). MHC class I proteins were detected with the anti-free-heavy-chain MHC class I protein HC10 MAb (in uninfected cells [E] or in infected cells [F]). The figure shows the results of a representative experiment out of three performed.

CTL killing is not influenced by influenza virus infection. It would be beneficial for the virus to confer increased NK cell inhibition through the increased binding to the MHC class I proteins. However, it would be a major disadvantage for the virus to cause an increase in the TCR binding to the same MHC class I proteins, since this would result in better killing of the infected cells by CTLs. To test whether increased CTL recognition would also be observed after influenza virus infection, we generated 221 cells expressing HLA-A2 protein (Fig. 4A) and CTL clones that were specific for the HA1-derived peptide (GILGFVFTL) presented on HLA-A2. As noted above, we observed an increased binding of LIR1-Ig to 221/A2 cells after influenza virus infection (Fig. 4B), while there was no change in the HLA-A2 level detected by W6/32 MAb (Fig. 4C).

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FIG. 4. Influenza virus infection does not alter CTL killing. An increase in the binding of LIR1-Ig to 221/A2 A/Sydney infected cells is demonstrated. (A) FACS staining of 221 cells expressing HLA-A2 (221/A2) cells with W6/32 MAb. (B) MFI values of LIR1-Ig and a control fusion protein (CD99-Ig) staining of 221/A2 cells infected or not with A/Sydney. (C) MFI values of W6/32 staining and a control antibody staining of 221/A2 cells infected or not with A/Sydney. For panels A to C, the results of one representative experiment out of five performed are shown. (D) CTL activity against infected 221/A2 cells loaded with the appropriate peptide is not influenced by the infection. CTLs were tested in killing assays against the 221 and 221/A2 cells infected with or without A/Sydney and loaded with 10 µg of specific peptide (see Results for the sequence) or with DMSO. The effector/target ratio was 5:1. The figure shows the results for one representative experiment out of five performed.

To test whether influenza virus infection would result in increased CTL cytotoxicity, we infected 221 and 221/A2 cells with A/Sydney virus that express an HA protein (H3) and that do not contain the GILGFVFTL peptide found in H1 (and was used for the generation of the CTL clones). As expected, no killing was observed when infected or uninfected 221/A2 cells were used in the absence of the specific HA peptide (Fig. 4D). An efficient killing was observed when 221/A2 cells were loaded with the HA peptide. However, no change in the killing was noted when the 221/A2 cells were loaded with the HA peptide and infected with A/Sydney virus. No killing was observed when 221 cells (infected or uninfected, peptide loaded or not) were used (Fig. 4D). Thus, we concluded that, in contrast to NK cell cytotoxicity, CTL killing is not augmented by influenza virus infection.

Accumulation of MHC class I proteins in lipid raft microdomains after infection. Our observations suggested that qualitative and not quantitative changes in the MHC class I proteins were responsible for the increased NK cell inhibition. One possible explanation for the increased binding of the inhibitory receptors might be the synthesis of a new protein, which associates with MHC class I molecules and affects the binding of the inhibitory receptors. However, we observed a rapid (Fig. 3B) increase in the binding and, therefore, this assumption is unlikely. It is more plausible that the increased binding is a result of a reorganization of MHC class I at the cell surface.

One of the most dramatic events that occurs at the cell surface after influenza virus infection is the formation of lipid rafts (24). Lipid raft microdomains are assumed to function as selective concentration devices for protein-protein complexes providing platforms for signal transduction (45). In addition, the generation of an efficient immune response sometimes depends on the accumulation of the appropriate proteins in the lipid rafts (20, 22).

To test whether the specific increased binding of KIR2DL1-Ig and LIR1-Ig to infected cells was due to accumulation of MHC class I proteins in lipid rafts, we performed flotation assays using influenza virus-infected or uninfected 221/Cw6 cells. In agreement with previous publications demonstrating the formation of lipid rafts after influenza virus infection (20, 22), we noticed an increased raft formation after influenza virus infection (Fig. 5A and B). In the uninfected cells, lipid rafts were detected in fractions 2 and 3 (Fig. 5A), while after infection, increased raft formation was observed in fractions 2 to 7 (Fig. 5B). The nonraft marker CD71 was equally detected both in influenza virus-infected and in uninfected cells in fractions 9 to 12 (Fig. 5C and D). Therefore, increased lipid raft formation was observed in infected cells, and the lipid raft fractions can be clearly distinguished from the nonraft fractions.

Next, we examined whether MHC class I molecules accumulate in lipid rafts after infection. In the uninfected cells, a small amount of MHC class I molecules is present in the raft fractions, while most of the molecules are present in the nonraft fractions. However, after influenza virus infection, there is an accumulation of MHC class I molecules in the lipid raft fractions (Fig. 5E and F).

Disruption of lipid rafts abolishes the increased binding of the NK inhibitory receptors. To confirm that the increased binding of KIR2DL1-Ig and LIR1-Ig to infected cells depends on the accumulation of MHC class I molecules in lipid raft microdomains, we treated the influenza virus-infected and uninfected cells with mevastatin, which disrupts lipid rafts, and tested the binding of KIR2DL1-Ig and LIR1-Ig.

Figure 6A shows the increased binding of LIR1-Ig to 221/Cw3 after influenza virus infection (MFI 36 for uninfected cells compared to a median fluorescence intensity [MFI] of 307 in influenza virus-infected cells). After mevastatin treatment, the binding of LIR1-Ig to 221/Cw3 cells was abolished (MFI = 41). These changes in LIR1-Ig binding were not a result of alterations in HLA-Cw3 expression, since the expression of HLA-Cw3 protein did not vary between the infected and uninfected, mevastanin-treated or untreated cells, as was detected by W6/32 MAb (data not shown). Similar results were obtained with HLA-Cw6 expressing 721.221 cells (data not shown). Similar results were obtained when 221/Cw4 cells were stained with KIR2DL1-Ig (Fig. 6B). There was no change in the binding of CD7-Ig (the control protein) to the cells after influenza virus infection or mevastatin treatment (data not shown).

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FIG. 6. Lipid raft disruption leads to a decrease in LIR1-Ig and KIR2DL1-Ig binding to infected cells. Mevastatin treatment leads to a loss in inhibitory receptor binding. MFI values of LIR1-Ig (A) and KIR2DL1-Ig (B) bound to 221/Cw3 or 221/Cw4 cells, respectively, are presented. Transfected 221 cells were infected or not with strain A/Sydney and treated with mevastatin (+) or with DMSO (–). The figure shows the results of one representative experiment out of four performed.

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NK cytotoxicity is controlled by a delicate balance between inhibitory and activating NK receptors (6, 10, 41, 53). We have previously demonstrated that, upon influenza virus infection, the viral HA protein is recognized by two NK killer receptors, NKp44 and NKp46, and that this recognition leads to enhanced killing by most of the NK clones (2, 3, 18, 28). We show here that a short time after influenza virus infection there is an increased inhibition of NK cell cytotoxicity, whereas NKp46-mediated activation is observed only later. What is the physiological importance of these time differences between inhibition and activation? One possible explanation is that the influenza virus has developed an escape mechanism to avoid NK cell attack and that the NK cells coevolved with the virus to develop a killing mechanism that is based on the recognition of HA by NKp46 and NKp44. We suggest that this coevolution enables both the virus and its host to survive. The influenza virus causes epidemics by the easy transmission of the virus between different individuals. Thus, at early time points the virus should gain power by producing enough viruses that might provide a critical mass for transmission. Therefore, early after infection, the virus developed an inhibitory mechanism that is not based on the synthesis of new proteins, and NK cells are inhibited through the reorganization of MHC class I proteins on infected cells. However, efficient inhibition of NK cell activity will result in the killing of the host. Indeed, we showed that, in the absence of NKp46, influenza virus infection is lethal (18). Therefore, at later time points after infection, HA appears on the surfaces of infected cells, and these infected cells are recognized by NKp46 and NKp44.

In contrast to the early increased in NK cell inhibition, influenza virus infection did not enhance CTL activity. One possible explanation for this phenomenon is the fact that the NK inhibitory receptors bind to their cognate MHC ligands in a very fast on and off rates, whereas the TCR binds to the same ligands at a higher affinity (51). Thus, clustering of MHC class I proteins into lipid rafts would increase the avidity of the inhibitory receptors toward their ligands and would therefore have a major impact on NK cells but not on CTL activity.

The influenza virus assembles and buds off the host cell, especially from the lipid raft microdomains that are induced after infection (38, 44, 47, 55). Lipid rafts are assumed to function as selective concentration devices for protein-protein complexes, forming platforms for different receptors that are important in signal transduction (46). Indeed, it has been previously reported that after receptor accumulation in lipid raft microdomains there is an increased avidity of their binding to their suitable ligands (14, 15, 22, 34).

Normally, MHC class I proteins are not located in a particular area of the cell surface but are rather spread on the cell surface. Here we demonstrate that after influenza virus infection MHC class I proteins accumulate in the lipid raft microdomains (Fig. 5). We show that this accumulation is responsible for the early increase in NK inhibition, since the increased binding of LIR1-Ig and KIR2DL1-Ig to the infected cells was abolished after raft disruption (Fig. 6). There are several examples of enhanced inhibition of NK killing through the manipulation of MHC class I proteins by viruses. The early human cytomegalovirus (CMV) protein, gpUL40, contains a ligand for HLA-E that can mediate its upregulation and therefore allows inhibition of NK cell killing through the HLA-E recognition by CD94/NKG2A (49, 50). An additional mechanism that is used by both human and mouse CMV is mediated through glycoproteins homologous to MHC class I heavy chain; it is designated UL18 in human CMV (42) and M144 in mouse CMV (17). UL18 is recognized by LIR1, and this interaction permits UL18-mediated inhibition of LIR1-positive NK cells (13). To the best of our knowledge, there is no other example describing increased NK inhibition without changing the levels or nature of MHC class I proteins present on the cell surface. Here we show that shortly after infection, influenza virus induced the reorganization of MHC class I to accumulate in lipid rafts. Such redistribution probably causes complexes of MHC class I proteins, which increase NK inhibition without altering the killing mediated by CTLs.

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A recent publication on dengue virus-mediated NK inhibition suggests that membrane-associated MHC class I reorganization is involved in the augmented inhibition (O. Hershkovitz, A. Zilka, A. Bar-Ilan, S. Abutbul, A. Davidson, M. Mazzon, B. M. Kümmerer, A. Monsoengo, M. Jacobs, and A. Porgador, J. Virol. 82:7666-7676, 2008). Combined with our similar results for the influenza virus, this puts forward a general mechanism implied by certain viruses.

In addition, the generation of 721.221 cells expressing C26 GFP was previously described (C. R. Almeida and D. M. Davis, J. Immunol. 177:6904-6910, 2006).

 
This study was supported by research grants from the ICRF, the BSF, the Israel Science Foundation, and the AICR and by grants from the European Consortium (MRTN-CT-2005 and LSCH-CT-2005-518178) and the Israel Ministry of Health (KKL). O.M. is a Crown Professor of Molecular Immunology.

 
* Corresponding author. Mailing address: The Lautenberg Center for General and Tumor Immunology, The Hebrew University Hadassah Medical School, Jerusalem 91120, Israel. Phone: 972-2-6757515. Fax: 972-2-6424653. E-mail: oferm{at}ekmd.huji.ac.il

Published ahead of print on 4 June 2008.

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Journal of Virology, August 2008, p. 8030-8037, Vol. 82, No. 16
0022-538X/08/$08.00+0     doi:10.1128/JVI.00870-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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