Results

NK cells express sialic acid side chains specific for influenza viral entry

Influenza viruses bind to the terminal sialic acid (SA) of membrane glycoproteins and glycolipids of susceptible cells. They enter into host cells through the interaction of their HA protein with -2,3 and/or -2,6–linked SA. In addition to the lung epithelial cells, multiple cell types including macrophages¹⁵ and dendritic cells^{16, 17} have shown to be targets of influenza virus; and in particular, macrophages are known to express these specific SA side chains. However, the presence of these SAs in immune effectors, such as NK cells, has not been explored. Towards this, we first analyzed the expression of -2,3 and -2,6 SA in NK cells. Results presented in Figure 1a demonstrate the presence of significant levels of -2,3 and -2,6 SA in spleen-derived NK cells. Influenza virus targets the upper respiratory system; therefore, we also analyzed the expression of these SA in the lung-resident NK cells. As shown in Figure 1b, the lung NK cells expressed ample and moderate levels of -2,3 and -2,6 SA, respectively. Interleukin (IL) 2-activated NK cells also expressed comparable levels of -2,3 and -2,6 SA (Figure 1c). This suggests that both primary and IL2-activated NK cells can be targeted by influenza virus.

Figure 1.

Natural killer (NK) cells can be infected by PR8. -2,3 and -2,6 sialic acid (SA) are expressed on primary splenic NK cells (a), primary lung NK cells (b) or interleukin (IL) 2-activated splenic NK cells (c). Total splenocytes, lung-derived lymphocytes or IL2-activated NK cells were stained with anti-CD3, anti-NK1.1 antibodies and lectins SNA-FITC or MAA-FITC that are specific for SA side chains. Gated NK cells (CD3^-NK1.1⁺) were analyzed for the expression of -2,3 and -2,6 SA and presented in (a), (b) and (c). Percent SA positive CD3^-NK1.1⁺ and the mean fluorescence index (MFI) are shown. Negative controls that were used to calculate the percent SA positivity are shown only with the MFI. (d) Intracellular entry of PR8 virus in NK cells. IL2-activated splenic NK cells were mock- or infected with multiplicity of infection 1 (1 MOI) PR8 for 1 h, washed and 24 h later were stained with anti-NCR1, anti-lysosomal-associated membrane protein-1 (LAMP1) and anti-M2 antibodies. Individual or merged images of all three are shown. Data presented in a–d are representatives of three to five experiments each. Representative images of more than 50 individually analyzed cells are shown in (d).

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Detection of PR8 infection in NK cells

To assess the successful entry of influenza virus into NK cells, we analyzed the infected cells for the presence of PR8-derived M2 protein through confocal microscopy. IL2-activated NK cells were cultured in chamber slides, exposed to PR8 virus at a multiplicity of infection 1 for 1 h, washed, cultured for 12 h and stained with anti-NCR1, anti-lysosomal-associated membrane protein-1 (LAMP1) and anti-M2 antibodies. As it is known to be uniquely expressed in NK cells, NCR1 staining was used throughout this study. Figure 2 shows the expected peripheral membrane staining of NCR1 receptor. After attaching to the SA-containing glycoproteins, influenza viruses use clathrin-mediated endocytosis as a major mode of cellular entry.¹⁸ These clathrin-coated vesicles differ from lysosomes, endosomes and cytotoxic secretory vesicles that are positive for LAMP1.¹⁹ Figure 1d shows that virus infected NK cells possess small and unique punctated vesicular structures that are M2 positive. This did not overlap with LAMP1 staining patterns. These distinct structures are similar to those described earlier for intracellular influenza viruses that are localized inside clathrin-coated endocytic vesicles.¹⁸ Our results strongly suggest that PR8 virus can successfully enter NK cells.

Figure 2.

PR8 can infect natural killer (NK) cells in vivo. Lungs sections from the PR8 infected mice were stained for NK cells (anti-NCR1) and virus (anti-M2). Lung sections from days 4, 7 and 10 of post infection along with mock-infected controls are shown. Data presented are representatives of more than 20 individual image panels obtained. A group of three mice were analyzed for each time point.

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To characterize the in vivo infection of NK cells, we intranasally inoculated C57BL/6 mice with 5000 PFU of PR8. Lungs along with trachea were removed at different days of post infection, embedded in OCT, sectioned and stained for NCR1 and M2 proteins. Our results presented in Figure 2 demonstrate localization and persistence of M2 protein inside the NK cells up to tenth day of post infection. NK cells were identified with their unique NCR1 staining pattern in the lung sections. NK cells in the lung sections of mock-infected C57BL/6 mice showed no evidence of viral M2 protein staining. In contrast, NK cells in the lung sections of infected mice consistently showed a strongly stained M2 protein. Maximal amount of viral entry inside the NK cells occurred on day 4 of post infection (Figure 2). PR8 viral titer also peaks on day 4 of post infection (data not shown); thus, the level of viral infection in NK cells proportionately correlated with the viral titer. On day 4 of post infection, M2 staining was also evident in the epithelial and other cell types in the lung (Figure 2). It is interesting to note that on days 7 and 10 of post infections, even though the PR8 is cleared in most part of the lung tissues, our data shows persistence of M2 protein in NK cells.

To further confirm the in vivo infection of NK cells, we isolated the lung-resident lymphocytes from mock-infected and day 4 post-infected mice. NK cells were marked by anti-NCR1 antibody, and additionally stained for LAMP1 and viral M2 proteins (Figure 3a). Staining of trachea from day 4 post infection mice with anti-M2 antibody showed the presence of a considerable amount of M2 protein in epithelial cells, indicating the successful infection of these mice (Figure 3b). NCR1⁺ NK cells from the mock-infected lung tissues contained LAMP1-positive cytotoxic granules, but were negative for viral M2 protein. However, NK cells derived from the day 4 post infection were positive for M2 protein confirming our in vitro observations (Figure 3a). These observations demonstrate for the first time that influenza virus can successfully enter NK cells.

Figure 3.

Primary natural killer (NK) cells from infected mice contain PR8 virus. (a) Lymphocytes from the lungs of mock or PR8 infected mice were isolated and stained for NK cells (anti-NCR1), lysosomal/endosomal vesicles (lysosomal-associated membrane protein-1 (LAMP1)) and PR8 virus (anti-M2). (b) Tissue sections containing trachea of infected mice (day 4 of post infection) were used as positive controls for anti-M2 antibody staining. Data presented are representatives of more than 50 individual cells analyzed. A total of three mice were mock or PR8 infected and their NK cells were purified and analyzed on day 4 of post infection.

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PR8 infection does not affect the expression of activating or inhibitory receptors

Natural killer-cell functions are regulated by activating and inhibitory receptors.²⁰ To determine whether PR8 infection affect the surface expression of NK-cell receptors, we examined CD122, CD11b, CD43, CD49b, CD51 and CD69 (developmental and functional markers); NCR1, NKG2D, NKRP1c, CD244 and Ly49D (activation receptors); and NKG2A, Ly49A, Ly49C/I and Ly49G2 (inhibitory receptors) on gated CD3^-NK1.1⁺ cells. Flow cytometric analyses showed that expressions of all these molecules were comparable between PR8-infected and mock-infected NK cells (Figures 4a–c). This result indicated that PR8 infection does not affect the surface expression of these NK receptors.

Figure 4.

The expression of natural killer (NK)-cell receptors are unaffected after PR8 infection. Interleukin (IL) 2-activated NK cells were mock or PR8 infected and stained for activating (a), inhibitory (b) and developmental/functional (c) NK-cell markers. Percent positive cells and the respective MFI are shown in parenthesis. Data presented are representatives of three independent experiments.

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PR8 infection impairs the cytotoxic potential of NK cells

To determine the effects of PR8 infection on NK-cell functions, we first tested the cytolytic potential of mock- and PR8-infected NK cells against EL4^H60 YAC-1 and RMA/S target cells. EL4^H60 is a cell line stably expressing H60, which is a ligand for NKG2D. This cell line was established through transfection of EL4 cells that lack any known NKG2D ligands with a H60-encoding cDNA.²¹ As seen in Figure 5a, PR8 infected NK cells showed reduced cytotoxicity against EL4^H60 compared with that of mock-infected. YAC-1 is a tumor cell line that naturally expresses H60. The killing of this target by infected NK cells was also down regulated (Figure 5b). Cells that lack or have reduced expression of self–major histocompatibility complex (MHC) I molecules are also susceptible to NK-mediated cytotoxicity. PR8-infected NK cells showed reduced cytotoxicity against RMA/S cells, which express lower levels of MHC-I (Figure 5c). Taken together, these data show that PR8 infection of NK cells could negatively modulate their cytotoxic potentials.

Figure 5.

Natural cytotoxicity of natural killer (NK) cells are reduced after PR8 infection. Ability of NK cell to mediate cytotoxicity against EL4^H60 (a, 'induced-self'), YAC-1 (b, 'induced-self and allo') and RMA/S (c, 'missing-self') was analyzed. Interleukin (IL) 2-activated NK cells were mock or PR8 infected (multiplicity of infection 1 (1 MOI)) for 1 h, washed and 24 h later harvested and used in ⁵¹Chromium (⁵¹Cr)-release assay at indicated E:T Ratio. Data presented are average mean values of three independent experiments with s.d.

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PR8 infection of NK cells impairs anti-viral cytokine and chemokine generation

The lung-resident NK cells generate multiple anti-viral cytokines and pro-inflammatory chemokines during influenza infection. To determine the effect of PR8 on NK cells, we next analyzed the generation of IFN-, GM-CSF, MIP-1, MIP-1 and RANTES after receptor-mediated activations. We stimulated PR8- and mock-infected NK cells with mitogenic anti-NCR1, -NKG2D, -Ly49D, -NKPR1c and -CD244 antibodies. Secreted cytokines and chemokines were measured by multiplex assays. As seen in Figure 6, mock-infected NK cells produced a large amount of IFN- and GM-CSF when stimulated with plate-bound antibodies. However, PR8-infected NK cells were severely impaired to produce these cytokines. Further, generation of chemokines MIP-1, MIP-1 and RANTES from infected NK cells were also significantly reduced by PR8-infection (Figure 6). Although significant reductions were seen, the effect on the generation of GM-CSF and RANTES were less compared with others, indicating a possible differential regulation by the PR8 virus. Substantial reduction in cytokine and chemokines could be because of the inabilities of PR8-infected NK cells to produce cytokines or because of a virus-induced defect in the ability of infected NK cells to secrete. To distinguish between these two possibilities, we next analyzed the levels of intracellular IFN- in response to anti-NKG2D antibody. Consistent with the above findings, percentages of IFN--positive NK cells were reduced after PR8 infection (Figure 7). Collectively, these results demonstrate that a direct infection of NK cells by PR8 can impair their ability to generate cytokines and chemokines.

Figure 6.

Cytokine and chemokine generation from major activation receptors of natural killer (NK) cells are significantly reduced after PR8 infection. Interleukin (IL) 2-activated NK cells were mock or PR8 infected (multiplicity of infection 1 (1 MOI)) for 1 h, washed and 24 h later harvested and activated by immobilized plate-bound anti-NCR1, anti-NKG2D, anti-Ly49D, anti-Nkpr1c and anti-CD244 antibodies. Culture supernatants were tested for indicated cytokines and chemokines in a multiplex assay. Data presented are average mean values of three independent experiments with s.d.

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Figure 7.

Level of intracellular IFN- severely reduced in PR8 infected natural killer (NK) cells. Interleukin (IL) 2-activated NK cells were mock-infected (middle panel) or with multiplicity of infection 1 (1 MOI) of PR8 (right panel) for 1 h and 24 h later harvested and activated by immobilized plate-bound anti-NKG2D for 2 h followed by adding Brefeldin-A and incubating for additional 8 h. Mock-infected, non-activated NK cells were used to determine the background levels of intracellular IFN- (left panel). Activated NK cells were harvested and stained for intracellular IFN-. Cells shown are gated for CD3^-NK1.1⁺ NK population. Percentages of CD3^-NK1.1⁺ NK cells were shown for intracellular IFN- positivity. Data presented are representatives of three independent experiments.

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Mutations in PR8-NS1 augment the functional impairment of NK cells

PR8 infection did not alter the expression levels of activating receptors of NK cells. Hence, it is possible that the PR8 virus may target signaling pathways in NK cells. Our earlier work in NK cells demonstrated that phosphotidyl inositol 3-kinase subunit p85 (PI3K-p85) has a critical role in their cytokine/chemokine generation and cytotoxicity.²² Further, we and others have shown that the PR8-derived NS1 has the ability to directly interact with PI3K-p85 subunit.^{23, 24, 25} As PR8 can enter NK cells, it could allow the viral NS1 to bind and alter the functions of PI3K-p85 with a direct impact on the effector functions. On the basis of these observations, we hypothesized that abolishing the binding ability of NS1 to PI3K-p85 could result in the loss of PR8's ability to manipulate NK-cell functions. Toward this, we generated an NS1-mutated PR8 virus (PR8-NS1-SH2/SH3-mt). NS1 contains two polyproline motifs (164–167: PSLP and 212–216: PPLTP) that are required to bind to the p85 subunit of PI3K. The NS1 protein also contains a consensus SH2-binding site, which is similar to the previously described YXXM motif (89–93: YLTDM). The five prolines and the tyrosine were swapped for alanines or phenylalanine to generate a mutated NS1 that failed to interact with PI3K-p85 (Figure 8a).^{24, 25}

Figure 8.

Infection with PR8-NS1-SH2/SH3-mt virus augments the functional impairment of natural killer (NK) cells. (a) NS1-mutated PR8 virus (PR8-NS1-SH2/SH3-mt) was generated by reverse genetics. Three putative motifs in NS1 protein that have the potential to interact with PI3K-p85 are altered in the PR8-NS1-SH2/SH3-mt virus. A tyrosine phenylalanine in a consensus SH2 binding motif (89–93: YLTDM); prolines alanines in two polypropyline motifs (164–167: PSLP and 212–216: PPLTP) were made. (b) The ability of NK cell to mediate cytotoxicity against EL4^H60 after PR8-NS1-SH2/SH3-mt virus infection. Interleukin (IL) 2-activated NK cells were mock-, multiplicity of infection 1 (1 MOI) of PR8 or PR8-NS1-SH2/SH3-mt virus infected for 24 h, harvested and used in ⁵¹Chromium (⁵¹Cr)-release assay at indicated E:T Ratio. (c) Impairment in the NCR1 and NKG2D-mediated IFN- generation after PR8 or PR8-NS1-SH2/SH3-mt virus infection. (d) Infection-induced cell death after PR8 or PR8-NS1-SH2/SH3-mt virus infection. IL2-activated splenic NK cells were mock, PR8 or PR8-NS1-SH2/SH3-mt virus infected for 1 h, washed and 24 h later tested with Annexin V and 7AAD. Data presented in b–d are average mean values or representatives of three to five independent experiments.

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Using this PR8-NS1-SH2/SH3-mt and the wild type PR8 viruses, we infected the IL2-activated NK cells. NK cells were infected with multiplicity of infection 1 of PR8 or PR8-NS1-SH2/SH3-mt for 1 h, washed and 24 h later expression patterns of activating (NCR1, NKG2D, CD244 and Ly49D), inhibitory (NKG2A, Ly49A, Ly49C/I and Ly49G2) and developmental markers (CD122, CD11b, CD43, CD49b, CD51 and CD69) were analyzed. Owing to PR8-NS1-SH2/SH3-mt infection none of these receptors were modulated (data not shown). Next, we quantified the levels of functional impairment of the PR8-NS1-SH2/SH3-mt-infected NK cells. The ability of NK cells to mediate cytotoxicity against tumor cells was quantified. Contrary to our expectations, infection of NK cells with NS1 mutant PR8 virus further reduced NK cytotoxicity compared with those infected with PR8 (Figure 8b). Similar to cytotoxicity, generation of IFN- in response to anti-NCR1 or anti-NKG2D was further reduced (Figure 8c).

One possible explanation for these observations could be infection-induced cell death. Earlier studies have shown that the binding of NS1 to PI3K-p85 redirected signaling events to initiate pro-survival pathways and, thereby, delayed host cell apoptosis.^{24, 25} Thus, the mutated NS1 that is unable to bind to PI3K-p85 could have failed to initiate these pro-survival pathways resulting in increased apoptosis. Therefore, 24 h after infection, we tested NK cells with Annexin V (early phase of apoptosis) and 7AAD (late phase of apoptosis). Our results show that infection with PR8 caused moderate early and late phase of NK cell death (Figure 8d), and infection with PR8-NS1-SH2/SH3-mt virus only slightly augmented this cell death. We conclude that the functional impairment caused by PR8 in NK cells may not depend on the ability of NS1 to manipulate the functions of PI3K-mediated signaling cascades. Thus, our results suggest the existence of additional new mechanisms by which influenza virus can negatively regulate the effector functions of lymphocytes.

Discussion

Influenza A virus is a negative-strand RNA virus that can cause severe disease in humans and animals. Influenza virus primarily infects and replicates in the lung epithelial cells. However, their ability to infect and modulate the functions of effector lymphocytes has not been analyzed. Here, our results show that the PR8 virus can enter and modulate the functions of NK cells.

Influenza enters the host cells by specifically binding to glycoproteins that possess -2,3 and -2,6-linked SAs. Our current results show that NK cells express abundant quantities of these glycosyl side chains facilitating the binding and entry of PR8 virus. In vitro exposure to the virus followed by staining for viral M2 protein showed the presence of PR8 virus inside the NK cells. Further, NK cells that are present in the lung sections or isolated from infected mice confirmed the presence of viral proteins. As viral HA functions as ligand that facilitates the entry into susceptible cells and as an activating ligand for NCR1 receptor, it is possible that PR8 indeed uses NCR1 to enter inside the NK cells. It is also important to note that NCR1 is uniquely expressed on NK cells and not on T, B or NKT cells. Therefore, if NCR1 is used as a conduit for viral entry, NK cells could be the only target for viral manipulation. PR8 entry into NK cells was analyzed using anti-M2 and not anti-HA antibodies. This argues against the possibility that NK cells preferentially internalized NCR1/HA complexes and not the whole PR8 virus. As PR8 inside the NK cells underwent only a limited replication, it appears that the influenza virus does not use NK cells as a typical host cell similar to that of the lung epithelial cells. Limited replication of influenza virus on NK cells is not surprising, as its infection in macrophage and dendritic cell is usually non-productive.^{11, 12} Limited infection of influenza virus in human NK cells was also observed.¹³ If not for replication, does influenza infection affect NK-cell functions? Our results show that PR8 can affect both cytotoxicity and cytokine/chemokine generation of NK cells.

One possible mechanism by which PR8 can manipulate effector functions is to alter the expression of activating receptors in NK cells. NK cells express multiple activation receptors. NCR family contains three human (NKp46, NKp44 and NKp30) members and one murine (NKp46) member.^{26, 27, 28} The recognition of HA⁹ on host cells by NCR1 constitutes a critical step for the activation of NK cells during influenza infection. NKG2D has an important role in the recognition of virus-infected cells through the recognition of 'induced-self' ligands. NKG2D is expressed on all human and murine NK cells and it recognizes MIC-A/B,²⁹ ULBP-1/2/3³⁰ (in human) and H60,^{31, 32} Rae-1////,^{31, 32} Mult-1³³ (in mouse). Ly49D associates with both DAP10 and DAP12^{34, 35} and recognizes H2-D^d as its ligand.³⁶ Nkrp1c is a subunit of NK1.1 complex and it is a unique cell marker expressed on NK and NKT cells that associates with FcR to mediate its signal.³⁷ CD244 is expressed in both human and murine NK cells, and recruits SH2 domain containing SAP to propagate its signals. A direct infection of NK cells by PR8 did not alter the expression levels of any of these activation receptors. Further, our results also indicate that PR8 did not have any effect on inhibitory, developmental or functional status-related receptors on NK cells.

Natural killer cells control influenza infection by destroying the infected cells to release the viral particles that can be neutralized by specific antibodies.³⁸ Mice that lack NK cells succumbed to influenza, demonstrating their critical role in controlling influenza infection.^{3, 4, 39} For that reason, PR8 could have evolved evasion strategies that target the effector functions of NK cells. Our results show that infection of NK cells with PR8 leads to a significant reduction of their cytotoxicity against tumor cells that represent 'induced-self' (EL4^H60 and YAC-1) and 'missing-self' (RMA/S). This indicates that a direct infection by influenza virus can interfere with the ability of NK cells to effectively recognize and kill the host cells that harbor this virus.

Natural killer cells are also a critical source of multiple pro-inflammatory cytokines and chemokines. Our present findings indicate that a direct infection of NK cells by PR8 reduces the generation of IFN-, GM-CSF, MIP-1, MIP-1 and RANTES when stimulated through NCR1, NKG2D, Ly49D, Nkpr1c or CD244. These findings have significant implications on both innate and adaptive immune responses. NK cells can control infection by secreting IFN- that directly inhibit influenza replication resulting in protection.⁴⁰ Therefore, a reduction in the level of IFN- can increase the severity of the disease. NK-derived cytokines and chemokines also regulate B-cell and T-cell functions and for instance, INF- secreted by NK cells is also one of the key regulator of antibody-isotype switching and secretion, controlling the quality of B-cell responses.^{41, 42, 43} During influenza infection, NK cells are required for the generation of influenza virus-specific CTL in vitro and in vivo.⁴⁴ NK cells also participate in adaptive immune responses by clearing influenza-infected cells through antigen-specific antibodies by ADCC.⁴⁵ Thus, this ability of PR8 to manipulate NK-cell functions suggests a new mechanism employed by influenza virus with implications to both the innate and adaptive immune responses.

The infection of NK cells with PR8 did not alter the expression levels of any of the activating or inhibitory receptors. Further, the viral titers from the culture supernatant indicate that this infection is moderate and the NK cells do not support the active replication of PR8. Irrespective of these, critical effector functions mediated by the infected NK cells were significantly compromised. Thus, it is possible that the PR8 virus may target signaling pathways that regulate the cytotoxicity and cytokine/chemokine generation. We and others have shown that the PR8-derived NS1 has the ability to directly interact with and modulate the function of PI3K-p85 subunit whose phosphorylation regulates NK-mediated cytokine generation and cytotoxicity.^{22, 23, 24, 25} Towards this, we used a PR8 virus that expressed a mutated NS1 protein (PR8-NS1-SH2/SH3-mt) that failed to interact with PI3K-p85.^{24, 25} In contrary to our expectations, mutation of NS1 resulted in the additional reduction of NK cell-mediated cytotoxicity and cytokine generation. On the basis of these results, we conclude that PR8 employs new mechanisms that does not involve SH2/SH3 domain of NS1 to target signaling pathways in NK cells.

Viral infections can lead to cell death. However, to achieve a high level of replication, during early phase of infection, influenza virus uses its NS1 protein to delay host cell apoptosis by the activation of PI3K/Akt-dependent pro-survival pathways.²⁴ NS1 protein with mutations in the SH2/SH3-binding motifs failed to interact with SH domains of PI3K-p85 and did not activate the PI3K/Akt pathway.²⁵ Thus, disruption of the NS1 binding to p85 could have induced apoptosis. In our study, infection of NK cells with PR8 or NS1-mutated PR8 caused moderate levels of cell death (23–29%) compared with that of mock-infected (8–14%). However, the moderate level of cell death alone does not explain the severe reductions in the effector functions of NK cells. Taken together, we conclude that influenza virus can down modulate the effector functions of NK cells; however, the molecular mechanism is yet to be understood.

Methods

Mice, virus and cell lines

The C57BL/6 mice were maintained in pathogen-free conditions at the Biological Resource Center (BRC) of the Medical College of Wisconsin (MCW), Milwaukee, WI, USA. All the animal protocols used were approved by the IACUC, BRC and MCW. Target cells, EL4^H60 RMA/S and YAC-1 and their culture conditions were described.⁴⁶ Madin–Darby Canine Kidney (MDCK) were purchased from ATCC (Rockville, MD, USA) and cultured in RPMI 1640 medium with 10% fetal bovine serum. Mouse adapted human influenza virus A/PR/8/34 (PR8) was used as described.²³ NS1 mutant PR8 virus was described previously.^{24, 25} Viral titers were determined by plaque-forming assays.

PR8 infections

In vivo.C57BL/6 mice were intra nasally challenged with of 5000 PFU of PR8 virus in sterile phosphate-buffered saline in a total volume of 30 l through one nostril. Mock infections were carried out using only sterile phosphate-buffered saline without the virus. After days 4, 7 and 10 of post infection, lungs were collected and used for lymphocytes isolation or tissue mounting. For lymphocyte preparation, lungs was minced finely into pieces, washed, 500 l 10 mg ml^-1 collagenase (C5138–100 mg, Sigma, St Louis, MO, USA) and 500 l 1 mg ml^-1 DNase I, bovine pancreas (D4527-20KU, Sigma, St Louis, MO, USA) were added for digestion 1 h at 37 with gentle vortex (400 r.p.m.). Remaining tissue pellets were transferred into cell strainer and smashed gently with 5-ml syringe plunge and washed. Cell suspensions were incubated with 5 ml of RBC lysis buffer for 5 min and then washed. For generating cryosections, whole lungs were mounted with OCT and used in immunofluorescence analyses.

In vitro.IL-2 activated NK cells were incubated with PR8 virus at a multiplicity of infection 1. After 1 h at 37 °C, cells were washed three times and cultured in RPMI 1640 medium with 10% fetal bovine serum and 1000 U ml^-1 of IL2 for additional 12 or 24 h before it is used for different analyses.

Confocal microscopy

Lymphocytes from digested lung tissues at day 4 of post infection were purified using Ficoll density gradient. Purified lymphocytes were cultured in poly-L-lysine coated 8-wells chamber slides. Cell preparations or tissue sections were fixed with 2% paraformaldehyde. Single cell preparations cultured in chamber slides were additionally permeabilized before staining. Slides were blocked for 1 h with 10% fetal bovine serum and 1% BSA in phosphate-buffered saline followed by goat anti-mouse NCR1 (BD Biosciences, San Jose, CA, USA), anti-M2 and anti-mouse LAMP1-PE antibodies (e-Bioscience, San Diego, CA, USA) for overnight. After three washes with phosphate-buffered saline, cells were incubated with rabbit anti-goat Alexa Flour 488 (Invitrogen, Carlsbad, CA, USA) and goat anti-mouse Alexa Flour 633 for additional 1 h and slides were mounted with VECTASHIELD (Vector Laboratories, Burlingame, CA, USA). Images were obtained using Olympus FluoView FV1000 MPE microscope (Center Valley, PA, USA) that is equipped with multiphoton capabilities (MaiTai DSBB-OL: 710–990 nm and MaiTai DSHP-OL: 690–1040 nm).

NK-cell preparation and analysis of -2,3 and -2,6-linked SA

Natural killers cells were purified as described.⁴⁶ Single cell suspension of spleens were made and used. IL2-activated splenic NK cells were generated using following the protocol. Briefly, single cell suspensions from the spleen were passed through nylon wool columns to deplete adherent populations consisting of B cells and macrophages. Nylon wool-non-adherent cells were cultured with 1000 U ml^-1 of IL2 (NCI-BRB-Preclinical Repository, Maryland, MD, USA). Purity of the NK cell cultures was checked by flow cytometry and preparations with more than 90% of NK1.1⁺ cells were used. The distribution of -2,3 and -2,6-linked SAs on prepared NK cells were detected by flow cytometry. Primary or IL2-activated NK cells (1 10⁶) were blocked with anti-mouse CD16/32 (1 g per 10⁶ cells) followed by staining with anti-CD3 PB (145-2C11, e-Bioscience), NK1.1-APC (PK136, e-Bioscience) and 100 l of 1:400 dilution of SNA-FITC or 1:800 MAA-FITC (Vector Laboratories, Burlingame, CA, USA) for 30 min at 4 °C. Cells were washed, re-suspended in FACS washing buffer and analyzed by flow cytometry in LSR-II using FACSDiva software (Becton Dickinson, Franklin Lake, NJ, USA).

Flow-cytometric analysis of NK markers after virus infection

The cell preparations were stained with fluorescent-labeled monoclonal antibodies as previously described.⁴⁶ Antibodies for NK1.1 (Nkpr1c) (PK136), CD3 (145-2C11), NKG2D (A10), NKG2A (16a11), CD11b (M1/70), CD43 (1B11), CD49b (DX5), CD51 (RMV-7), CD69 (H1.2F3), CD122 (5H4) and Ly49I (YLI-90) were obtained from e-Bioscience. Antibodies for CD244 (2B4), Ly49A (A1), Ly49D (4E5), Ly49C/I (5E6) and Ly49G2 (4D11) were obtained from BD Biosciences. Antibody for NCR1 (259018) was purchased from R&D System (Minneapolis, MN, USA). Standard flow cytometry analysis was carried out in LSR-II using FACSDiva software.

NK-cell cytotoxicity assay

Natural killer cell-mediated cytotoxicity was quantified using ⁵¹Chromium-labeled target cells, including, EL4^H60 YAC-1 and RMA/S. At 24 h after infection, infected or mock-infected NK cells were mixed with target cells at varied E:T Ratio and incubated at 37 °C for 4 h. Plates were spun down and supernatants were harvested, and the release of ⁵¹Chromium into culture supernatants was measured using the gamma counter. Percent specific lysis was calculated using amounts of absolute, spontaneous and experimental ⁵¹Chromium-release from target cells.⁴⁶

Quantification of cytokines and chemokines

PR8-, PR8-NS1-SH2/SH3-mt- or mock-infected NK cells were activated with titrated concentrations of plate-bound anti-NCR1 (259018), anti-NKG2D (A10), anti-Ly49D (4E5), anti-Nkpr1c (PK136) and anti-CD244 (2B4) monoclonal antibodies for 18 h. The secreted IFN-, GM-CSF, MIP-1, MIP-1 and RANTES in the supernatants were quantified by conventional enzyme linked immunosorbent assay (ELISA) or multiplex assays (Bio-Rad, Richmond, CA, USA). Intracellular staining of IFN- in mock or PR8 infected NK cells was carried out using plate bound anti-NKG2D (5 g ml^-1) activation as previously described.⁴⁷

Apoptosis assay

PR8 or NS1 mutant PR8 virus infected NK cells were stained with Annexin V and 7-ADD using Annexin V-PE apoptosis detection kit (BD Pharmingen, Franklin Lake, NJ, USA). Flow cytometry was carried out in LSR-II by using the FACSDiva software.

Statistics

Statistical analysis was performed by two-tail, unpaired, Student's t-test using Microsoft Excel 2003 software to compare the differences in results obtained using PR8 infected and mock-infected NK cells. P-values of 0.05 were considered significant.

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Acknowledgements

H.G. was supported by MCW-Cancer Center Postdoctoral Fellowship. This work is supported in part by National Institutes of Health (NIH) grants R01 A1064826-01, U19 AI062627-01, NO1-HHSN26600500032C (to SM). We thank laboratory members for critical review of the manuscript. We also thank BRI-Flow core for help with flow cytometry.