Altered Nkp46 Recognition and Elimination of Influenza B Viruses

viruses

Article Altered NKp46 Recognition and Elimination of Inﬂuenza B Viruses

Alexandra Duev-Cohen 1, Batya Isaacson 1, Orit Berhani 1, Yoav Charpak-Amikam 1 , Nehemya Friedman 2, Yaron Drori 2, Michal Mandelboim 2 and Ofer Mandelboim 1,*

1 The Concern Foundation Laboratories at the Lautenberg Center for Immunology and Cancer Research, The Hebrew University Hadassah Medical School, Jerusalem 9112001, Israel; [email protected] (A.D.-C.); [email protected] (B.I.); [email protected] (O.B.); [email protected] (Y.C.-A.) 2 Central Virology Laboratory, Ministry of Health, Public Health Services, Chaim Sheba Medical Center, Tel Hashomer, Ramat-Gan 5265601, Israel; [email protected] (N.F.); [email protected] (Y.D.); [email protected] (M.M.) * Correspondence: [email protected]

Abstract: Every year, millions of people worldwide are infected with influenza, causing enormous health and economic problems. The most common type of influenza is influenza A. It is known that Natural Killer (NK) cells play an important role in controlling influenza A infection, mostly through the recognition of the viral protein hemagglutinin (HA) by the activating receptor, NKp46. In contrast, little is known regarding NK cell recognition of influenza B viruses, even though they are responsible for a third of all pediatric influenza deaths and are therefore included in the seasonal vaccine each year. Here we show that NKp46 also recognizes influenza B viruses. We show that NKp46 binds the HA protein of influenza B in a sialic acid-dependent manner, and identified the glycosylated residue in NKp46, which is critical for this interaction. We discovered that this interaction has a

binding affinity approximately seven times lower than NKp46 binding of influenza A’s HA. Finally, we demonstrated, using mice deficient for the mouse orthologue of NKp46, named NCR1, that

Citation: Duev-Cohen, A.; Isaacson, NKp46 is not important for influenza B elimination. These findings enable us to better understand B.; Berhani, O.; Charpak-Amikam, Y.; the interactions between the different influenza viruses and NK cells that are known to be crucial for Friedman, N.; Drori, Y.; Mandelboim, viral elimination. M.; Mandelboim, O. Altered NKp46 Recognition and Elimination of Keywords: NK cells; NKp46; NCR1; influenza B; hemagglutinin Influenza B Viruses. Viruses 2021, 13, 34. https://doi.org/10.3390/v13010034

Academic Editor: Feng Li 1. Introduction Received: 10 November 2020 Natural Killer (NK) cells are lymphocytes that are part of the innate immune system [1]. Accepted: 23 December 2020 They are critical in the defense against virus-infected cells, bacteria, parasites, fungi, and Published: 27 December 2020 malignant cells [2]. NK cell activity is regulated by the balance of signals received from

Publisher’s Note: MDPI stays neu- inhibitory and activating receptors. The Natural Cytotoxicity Receptors (NCRs), NKp30, tral with regard to jurisdictional claims NKp44, and NKp46, are among the main activating receptors expressed by NK cells. in published maps and institutional NKp30 and NKp46 are constitutively expressed, whereas NKp44 is upregulated after NK affiliations. cell activation [3]. NKp46 recognizes the hemagglutinin (HA) protein of various viruses including influenza A (IAV) [4], poxvirus [5], Sendai virus [4], and Newcastle disease virus [6]. It is also known to bind fungal ligands, such as the EPA adhesins on Candida glabrata [7], and an unknown bacterial ligand of Fusobacterium nucleatum [8]. NKp46 Copyright: © 2020 by the authors. Li- has also been shown to bind the endogenous ligands heparan sulfate [9] and complement censee MDPI, Basel, Switzerland. This factor P [10], but it still has additional unknown tumor [11] and cellular ligands [12,13]. article is an open access article distributed Influenza infections can lead to hospitalization and death among high-risk individuals under the terms and conditions of the (for example, elderly and chronically ill patients), thus posing major health and economic Creative Commons Attribution (CC BY) burdens. Part of IAV’s deadly nature is its ability to change at a high frequency the virus’ license (https://creativecommons.org/ membrane proteins, hemagglutinin (HA) and neuraminidase (NA) [14], a phenomenon licenses/by/4.0/).

Viruses 2021, 13, 34. https://doi.org/10.3390/v13010034 https://www.mdpi.com/journal/viruses Viruses 2021, 13, 34 2 of 11

known as antigenic shift. Accordingly, new influenza strains to which the world popu- lation is not immune arise every year. However, the ability of HA to bind sialic acids is conserved throughout the different strains, since this function facilitates influenza binding and infection of human cells [15]. NK cells take advantage of this property to recognize and kill IAV infected cells. The glycosylated threonine 225 residue on NKp46 interacts with HA and thus activates NK cell function to kill the infected cells [16]. The mouse orthologue of NKp46, called NCR1, also recognizes HA via the same mechanism. This has been shown using mice deficient for Ncr1, in which the influenza infection displays increased lethality [17]. Influenza B (IBV) is somewhat less common than IAV and almost exclusively infects humans. The only two known lineages of IBV were discovered in the late 1970s and are named Victoria and Yamagata [18]. Despite the fact that it mutates at a significantly slower rate than IAV [19], IBV is still responsible for a third of all pediatric influenza deaths and is included in the seasonal vaccine each year [18]. Here we show that NKp46 recognizes IBV via binding to HA. We discovered the mechanism of binding and identified the binding site on NKp46 that is crucial for its interaction with IBV’s HA. We found that the affinity of NKp46 to the HA of IBV is significantly weaker than to that of IAV and that NKp46 is not involved in the clearance of IBV in vivo.

2. Materials and Methods 2.1. Cells and Viruses The cell lines used in this study were the human choriocarcinoma cell line JEG-3 and the murine lymphoma cell line EL4 (ATCC). Human peripheral blood lymphocytes were separated from buffy coats using centrifugation on Ficoll gradient (Lymphoprep) followed by NK cells’ isolation using the Easy Sep Negative selection human NK cells enrichment kit (StemCell Technologies) according to manufacturer’s protocol. Mouse NK cells were isolated from the immune competent Ncr1+/gfp and from the NCR1-deﬁcient Ncr1gfp/gfp mice using the Easy Sep Negative selection mouse NK cells’ enrichment kit (StemCell Technologies) 18 h after intraperitoneal injection of 200 µg poly(I):poly(C) (Sigma). The human inﬂuenza viruses A/Brisbane/59/2007 H1N1 and B/Brisbane/60/2008 (Victoria lineage) used in this study were generated as previously described [20].

2.2. Antibodies, Fusion Proteins, and Compounds Monoclonal antibodies (mAbs) used in the present study included anti-HA speciﬁc to A/Brisbane/59/2007 and anti-HA speciﬁc to B/Brisbane/60/2008 that we kindly received from the International Reagent Resource (IRR) that was established by the Center for Disease Control and Prevention (CDC). The hNKp46.02 antibody was previously described [21]. The anti-NKp46 (9E2) was purchased from Biolegend. The generation of the fusion proteins NKp46-Ig, T225A-Ig, and NCR1-Ig was previously described. The purity of the fusion proteins was near 100%. Treatment of NKp46-Ig with Neuraminidase (NA) beads (Sigma, St. Louis, MO, USA) was performed as previously described [22]. For the ELISA experiments, we used the following recombinant HA proteins fused to an HIS-Tag: HA1 (A/Brisbane/59/2007), HA3 (A/Wisconsin/12/2010), and HA Victoria (B/Brisbane/60/2008) that we kindly received from the International Reagent Resource (IRR) that was established by the CDC.

2.3. FACS Staining of Viruses Cells were coated overnight with 20 µL of A/Brisbane/59/2007 H1N1 or B/Brisbane/60/ 2008 at 37 ◦C. The cells were then washed and incubated with the appropriate antibody (0.2 µg/well) or fusion protein (0.5 µg to 5 µg/well). Then, cells were stained and analyzed using ﬂuorescence-activated cell sorting (FACS). All results were analyzed using the FCSExpress 6 software. Viruses 2021, 13, 34 3 of 11

2.4. ELISA The various recombinant HA proteins mentioned above were coated to an ELISA plate (0.1 µg/well). Blocking was performed with BSA 1%. 0.1 µg/well of NKp46-Ig or anti-HIS-Tag (clone AD1.1.10 from Bio-Rad, Kidlington, Oxford, England) were added followed by the relevant biotinylated secondary antibody from Jackson ImmunoResearch. The plate was then incubated with Streptavidin HRP (Jackson ImmunoResearch, West Grove, PA, USA). Binding was measured by using TMB solution and reading of the results was done with an ELISA reader at a wavelength of 650 nm. The binding of NKp46 to the different HA proteins was normalized to the amount of HA that was bound to the well, which we determined using the anti HIS-tag antibody.

2.5. Surface Plasmon Resonance (SPR) We used HIS capture kit (GE Healthcare, Piscataway, NJ, USA)to coat the different recombinant HA proteins to a CM5 chip (GE Healthcare). After coating of the HA, we performed single cycle kinetics, as described before [23], by pushing different concentrations of NKp46-Ig.

2.6. Killing Assay Targets with or without inﬂuenza were incubated overnight with radioactive 35S labeled methionine. In the same time, human activated NK cells were incubated with hNKp46.02 for the internalization assay, as previously described [21]. The next day, the human NK cells or the mouse NK cells were incubated for 5 h at 37 ◦C with the targets and the cytotoxic activity was measured by 35S release from lysed cells, as previously described [24].

2.7. In Vivo Inﬂuenza Infections All animal work was conducted according to relevant national and international guidelines. The work was approved by the Hebrew University Medical School Ethic committee (Ethics number MD-19-15748-3, date of approval 7/7/19). All experiments were performed using 6–8- weeks-old females of C57BL/6 background. The generation of the Ncr1gfp/gfp mice was described before [25]. Mice were intranasally inoculated with 40µL of A/Brisbane/59/2007 H1N1 or B/Brisbane/60/2008 (Victoria lineage). Five days post infection, lungs were harvested and viral load was assessed.

2.8. Statistical Methods GraphPad Prism software version 8.0 was used for all statistical analyses. For statistical significance, two-tailed Student’s t test analysis was used. A statistical test was considered significant (*) when p < 0.05. ANOVA was used to identify significant group differences See figure legend for further detail.

3. Results 3.1. NKp46 Binds IBV To test whether NK cells recognize IBV, we incubated Jeg3 cells with IAV or IBV. Jeg3 cells express very few NK activating ligands and are therefore not killed well by human NK cells, making them ideal candidates for testing the interaction between NK cells and the abovementioned viruses [26]. For IAV we used the H1N1 virus A/Brisbane/59/2007 viral strain and for IBV we used the B/Brisbane/60/2008 viral strain which belongs to the Victoria lineage. Using anti-HA antibodies speciﬁc to either IAV or IBV, we veriﬁed that both viruses bind Jeg3 cells (Figure1a). Next, we generated a fusion protein composed of the extracellular portion of human NKp46 fused to the Fc portion of human IgG1 (named NKp46-Ig) and stained the Jeg3 cells in the presence or absence of the viruses. As we previously reported [4], increased NKp46-Ig staining was observed against Jeg3 cells in the presence of IAV (Figure1b,c data in red). Similarly, we observed increased binding of NKp46-Ig to IBV coated cells (Figure1b,d data in blue ). Collectively, we showed that Viruses 2021, 13, x FOR PEER REVIEW 4 of 12

human IgG1 (named NKp46-Ig) and stained the Jeg3 cells in the presence or absence of Viruses 2021, 13, 34 the viruses. As we previously reported [4], increased NKp46-Ig staining was observed4 of 11 against Jeg3 cells in the presence of IAV (Figure 1b,c data in red). Similarly, we observed increased binding of NKp46-Ig to IBV coated cells (Figure 1b,d data in blue ). Collectively, weNKp46 showed recognizes that NKp46 not only recognizes IAV (as not previously only IAV shown, (as previously [4]), but alsoshown, IBV. [4]), but also IBV.

Figure 1. NKp46 binds IAV and IBV. Jeg3 cells were incubated or not (black histogram) with A/Brisbane/59/2007 H1N1 Figure 1. NKp46 binds IAV and IBV. Jeg3 cells were incubated or not (black histogram) with A/Brisbane/59/2007 H1N1 (IAV) or B/Brisbane/60/2008 (IBV). (a) FACS staining of IAV (red histogram) and IBV (blue histogram) with antibodies (IAV) or B/Brisbane/60/2008 (IBV). (a) FACS staining of IAV (red histogram) and IBV (blue histogram) with antibodies against the hemagglutinin of IAV and IBV, respectively.respectively. ((bb)) TheThe cellscells incubatedincubated withwith thethe indicatedindicated virusesviruses werewere stainedstained with NKp46-Ig fusion protein. The fille filledd histograms and gray, empty histograms represent the staining with secondary antibodies only in the absence or presencepresence of influenza,influenza, respectively. Figure showsshows one representative experiment out of three performed. (c,d) MFI quantification quantification of three independent experiments including data presented in (b). FACS FACS staining with NKp46-Ig against IAV ((cc)) andand IBVIBV ((d)) was normalized to the staining of the cells without influenza. influenza. Values are shown as mean ± SEM.SEM. * * pp << 0.05 0.05 by by two-tailed two-tailed paired paired Student’s Student’s tt test.test.

3.2. NKp46 Has Lower Affinity to the HA of IBV than IAV 3.2. NKp46 Has Lower Affinity to the HA of IBV than IAV We previously demonstrated that the viral ligand of IAV recognized by NKp46 is the HA proteinWe previously [4], so we demonstrated inferred that that NKp46 the viral binds ligand IBV ofin IAV a similar recognized manner. by NKp46Initially, is thewe examinedHA protein binding [4], so weof inferredNKp46 thatto the NKp46 virally binds specific IBV in HAs a similar by manner.ELISA assays Initially, using we recombinantexamined binding HIS-tagged of NKp46 HA to proteins the virally obtained specific from HAs the by ELISAInternational assays usingReagent recombinant Resource (IRR).HIS-tagged ELISA HA plates proteins were obtained coated with from the Internationaldifferent HAs Reagent and then Resource incubated (IRR). with ELISA the NKp46-Igplates were fusion coated protein. with the As different a negative HAs control and thenwe used incubated the HA with of the the H3N2 NKp46-Ig virus fusion from 2010,protein. which As is a negativeknown not control to bind we NKp46 used the[27]. HA As can of the be H3N2seen in virus Figure from 2a, NKp46 2010, which did not is bindknown the not HA to bind of the NKp46 H3N2 [27 ].virus As can (middle be seen inbar, Figure black2a, do NKp46ts) as did expected. not bind theHowever, HA of interestingly,the H3N2 virus NKp46 (middle bound bar, blackboth to dots) the as HA expected. of IBV (blue However, dots) interestingly, and to the HA NKp46 of IAV bound (red dots).both to To the further HA of IBVinvestigate (blue dots) the andNKp46 to the bind HAing, ofIAV we (redmeasured dots). Tothe further binding investigate affinities the of NKp46 to binding, the virally we measured specific HAs. the bindingWe used affinities surface ofplasmon NKp46 resonance to the virally (SPR) specific where HAs. we We used surface plasmon resonance (SPR) where we coated a CM5 chip with anti-HIS antibodies and then bound the HA recombinant proteins of IAV H1N1 (Figure2b) or IBV Victoria (Figure2c). Using single cycle kinetics, we then injected NKp46-Ig in increasing

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Viruses 2021, 13, 34 dots). To further investigate the NKp46 binding, we measured the binding affinities of 5 of 11 NKp46 to the virally specific HAs. We used surface plasmon resonance (SPR) where we coated a CM5 chip with anti-HIS antibodies and then bound the HA recombinant proteins of IAV H1N1 (Figure 2b) or IBV Victoria (Figure 2c). Using single cycle kinetics, concentrationswe then and injected measured NKp46-Ig the in dissociation increasing concentrations constant, and KD. measured The the afﬁnity dissociation of NKp46 to IBV constant, KD. The affinity of NKp46 to IBV was seven times less than the affinity to IAV was seven times(Figure less 2c). than the afﬁnity to IAV (Figure2c).

Figure 2. NKp46 binds the hemagglutinin protein of IAV better than IBV. (a) Plates were coated with the indicated Commented [MP5]: Figure was changed recombinantFigure HIS-tag 2. NKp46labeled hemagglutinin binds the proteins, hemagglutinin incubated or protein not with ofNKp46-Ig IAV better and assayed than for IBV. binding (a) Plates using were coated with ELISA. Thethe results indicated present recombinantthe NKp46-Ig binding HIS-tag relative labeled to the amount hemagglutinin of HA coated on proteins, the plate (determined incubated using or an not with NKp46-Ig anti HIS-tag antibody). An ANOVA test was performed in order to evaluate significance. Figure shows cumulative results ofand three assayed independent for experiments. binding (b using,c) SPR single ELISA. cycle The kinetics results of NKp46 present vs. the recombinant the NKp46-Ig hemagglutinin binding relative to the proteinsamount of IAV (b) ofand HA IBV ( coatedc). CM5 chip on was the coated plate with (determined anti-HIS-tag antibodies using an and anti then HIS-tagbound with antibody). the recombinant An ANOVA test was HAs expressing HIS-tag. NKp46-Ig was injected with the highest concentration of 6.6 μM in the left panel and 48.2 μM in the rightperformed panel. in order to evaluate significance. Figure shows cumulative results of three independent experiments. (b,c) SPR single cycle kinetics of NKp46 vs. the recombinant hemagglutinin proteins 3.3. Threonine 225 Is Important for the Binding of NKp46 to IBV of IAV (b) and IBV (c). CM5 chip was coated with anti-HIS-tag antibodies and then bound with Since NKp46 binds the HA of IBV with lower affinity than IAV, we next tested the recombinantwhether HAs the expressing recognition HIS-tag.of IBV by NKp46 NKp46-Ig is different was injectedfrom that withof IAV. the We highest previously concentration of 6.6 µM in the leftshowed panel that andthe binding 48.2 µ ofM NKp46 in the to right IAV is panel. dependent on sialic acids present on NKp46 [22]. To test whether the binding of NKp46 to IBV is also dependent on sialic acids we treated NKp46-Ig with neuraminidase, an enzyme known to cleave sialic acid residues 3.3. Threonine[28]. 225 We Is incubated Important the NKp46-Ig for the Bindingfusion protein of NKp46 with neuraminidase to IBV beads (NA beads) Since NKp46for 2 h at binds 37 °C. theThen, HA we ofstained IBV Jeg3 with cells lower in the presence affinity or than absence IAV, of IBV we using next the tested whether treated NKp46-Ig. A significant decrease in the binding of NKp46-Ig was observed the recognitionfollowing of IBV treatment by NKp46 of the fusion is different proteins fromwith NA that (Figure of IAV. 3a), Weindicating previously that the showed that the binding ofbinding NKp46 of NKp46 to IAVto IBV is is dependentsialic acid-dependent. on sialic acids present on NKp46 [22]. To test whether the bindingThe specific of NKp46 glycosylated to IBV residue is also critical dependent for the binding on sialic of NKp46 acids to weIAV treated was NKp46-Ig previously identified to be threonine 225 (Thr 225) [16]. To test whether threonine 225 is with neuraminidase, an enzyme known to cleave sialic acid residues [28]. We incubated the NKp46-Ig fusion protein with neuraminidase beads (NA beads) for 2 h at 37 ◦C. Then, we stained Jeg3 cells in the presence or absence of IBV using the treated NKp46-Ig. A significant decrease in the binding of NKp46-Ig was observed following treatment of the fusion proteins with NA (Figure3a), indicating that the binding of NKp46 to IBV is sialic acid-dependent. The specific glycosylated residue critical for the binding of NKp46 to IAV was previously identified to be threonine 225 (Thr 225) [16]. To test whether threonine 225 is also involved in the recognition of the IBV HA, we generated a fusion protein of NKp46 in which we mutated Thr 225 into alanine (T225A-Ig). We then stained Jeg3 cells in the presence of IBV with NKp46-Ig and T225A-Ig. We observed a significant decrease in the binding to IBV when we used the mutant T225A-Ig as compared to NKp46-Ig (Figure3b). These findings indicate that sialylation and Thr 225 play an important role in NKp46 recognition of IBV, but other unknown elements in NKp46 exist that are essential for NKp46 recognition of IBV. Viruses 2021, 13, x FOR PEER REVIEW 6 of 12

presence of IBV with NKp46-Ig and T225A-Ig. We observed a significant decrease in the binding to IBV when we used the mutant T225A-Ig as compared to NKp46-Ig (Figure 3b). These findings indicate that sialylation and Thr 225 play an important role in NKp46 recognition of IBV, but other unknown elements in NKp46 exist that are essential for Viruses 2021, 13, 34 NKp46 recognition of IBV. 6 of 11

Figure 3. The binding of NKp46 to IBV is dependent on the sialic acid present on threonine 225. (a) NKp46-Ig fusion Figure 3. The binding of NKp46 to IBV is dependent on the sialic acid present on threonine 225. (a) NKp46-Ig fusion proteins werewere treatedtreated or or not not with with Neuraminidase Neuraminidase beads beads (NA (NA beads) bead ands) and then then incubated incubated with with Jeg3 Je cellsg3 cells in the in presencethe presence of IBV. of TheIBV. graphsThe graphs summarize summarize average average of three of independent three independent experiments. experiments. The results Thewere results normalized were normalized to staining to ofstaining the cells of with the untreatedcells with NKp46-Ig.untreated NKp46-Ig. Values are shownValues asare mean shown± SEM.as mean * p < ± 0.05SEM. by * two-tailedp < 0.05 by paired two-tailed Student’s pairedt test. Student’s (b) Jeg3 t test. incubated (b) Jeg3 or notincubated with IBV or wasnot with stained IBV with was NKp46-Ig stained with (No IBVNKp46-Ig = black (No histogram; IBV = black +IBV histogram; = blue histogram) +IBV = blue or the histogram) mutant NKp46 or the T225A-Ig mutant (NoNKp46 IBV T225A-Ig = black, filled(No IBV histogram; = black, +IBVfilled = histogram; blue, filled +IBV histogram). = blue, filled The gray, histogram). filled histograms The gray, andfilled gray, histograms empty histograms and gray, representempty histograms the staining represent with secondary the staining antibodies with onlysecondary in the absenceantibodies or presenceonly in ofthe influenza, absence respectively.or presence Figureof influenza, shows onerespectively. representative Figure experiment shows one out representati of three performed.ve experiment (c) MFI out quantification of three performed. of three independent (c) MFI quantification experiments includingof three independent experiments including data presented in (b). FACS staining with T225A-Ig against IBV was normalized to data presented in (b). FACS staining with T225A-Ig against IBV was normalized to the staining of the cells with NKp46-Ig. the staining of the cells with NKp46-Ig. Values are shown as mean ± SEM. * p < 0.05 by two-tailed paired Student’s t test. Values are shown as mean ± SEM. * p < 0.05 by two-tailed paired Student’s t test. 3.4. Human NK Cells Require NKp46 to Efficiently Eliminate IBV 3.4. HumanTo investigate NK Cells the Require functional NKp46 implications to Efficiently of Eliminate NKp46 binding IBV to IBV we used an anti- NKp46To mAb, investigate named the hNKp46.02, functional implicationsthat we previo of NKp46usly generated. binding toThis IBV specific we used antibody, an anti- whenNKp46 incubated mAb, named with cells hNKp46.02, expressing that NKp46, we previously leads to internalization generated. This and specific degradation antibody, of thewhen receptor; incubated therefore, with cells NKp46 expressing is downregulated NKp46, leads from to internalizationthe cell surface and[21]. degradation of the receptor;We incubated therefore, bulk NKp46 primary is downregulated human NK cells from thewith cell an surface isotype [21 control]. or with hNKp46.02We incubated for 16 h bulk at 37 primary °C. As humanwe previously NK cells reported with an isotype [21], such control incubation or with hNKp46.02resulted in 50%for 16 reduction h at 37 ◦C. of As NKp46 we previously surface reportedexpression [21 due], such to incubationNKp46 internalization resulted in 50% (Figure reduction 4a). Theseof NKp46 NK surfacecells were expression subsequently due to incubated NKp46 internalization with 35S-labeled (Figure Jeg34a). cells These in the NK presence cells were or subsequently incubated with 35S-labeled Jeg3 cells in the presence or absence of IBV and a cytotoxicity assay was performed. In the absence of IBV, Jeg3 cells were weakly killed and the hNKp46.02 antibody reduced the killing even further (although, not in a statistically significant manner, Figure4b). When Jeg3 cells were incubated with IBV, increased killing was observed. The increased killing was dependent on NKp46, since killing was reduced Viruses 2021, 13, x FOR PEER REVIEW 7 of 12

absence of IBV and a cytotoxicity assay was performed. In the absence of IBV, Jeg3 cells

Viruses 2021, 13, 34 were weakly killed and the hNKp46.02 antibody reduced the killing even further7 of 11 (although, not in a statistically significant manner, Figure 4b). When Jeg3 cells were incubated with IBV, increased killing was observed. The increased killing was dependent onto baselineNKp46, since levels killing of uninfected was reduced Jeg3 cellsto baseline when levels NK cells of uninfected were treated Jeg3 with cells hNKp46.02 when NK cells(Figure were4b). treated with hNKp46.02 (Figure 4b).

Figure 4. When expression of NKp46 is reduced from the cell surface, NK cells are not able to effectively kill IBV. NK cells Figure 4. When expression of NKp46 is reduced from the cell surface, NK cells are not able to effectively kill IBV. NK cells ◦ a were incubated withwith hNKp46.02 internalizinginternalizing antibodyantibody forfor 1616 hh atat 37 °C.C. ( a) Reduction Reduction of the the expression expression of NKp46 NKp46 receptor receptor from the cell surface was verified veriﬁed by FACS staining. The Medi Medianan Fluorescence Intensity (MFI) of of the the staining staining is is presented. presented. The graphs summarize average of three independentindependent experiments. The results were normalizednormalized to staining of the NK cells incubated with with an an isotype isotype control. control. Values Values are are shown shown as as mean mean ± ±SEM.SEM. *** *** p

3.5. The Mouse Orthologue of NKp46 Recognizes IBV but Does not Mediate Killing by Mouse NK3.5. CellsThe Mouse Orthologue of NKp46 Recognizes IBV but Does not Mediate Killing by Mouse NK Cells We next wanted to determinedetermine whetherwhether the NKp46NKp46 mousemouse orthologue,orthologue, NCR1, also recognizes IBV. For this we incubated IBV with EL4 cells, a murine lymphoma cell line, and verified verified binding using anti-HAs' anti-HAs’ antibodies (Figure (Figure 55a).a). We thenthen usedused aa previouslypreviously generated fusion protein comp composedosed of of NCR1 fused to human IgG1, called NCR1-Ig [25], [25], and saw that NCR1-Ig binds both IBV (Figure 55b,db,d datadata inin blue)blue) andand IAVIAV (Figure(Figure5 b,c5b,c data data inin red). To understandunderstand the the significance significance of theof the binding binding we performed we performed killing killing assays withassays murine with murineNK cells NK extracted cells extracted from the from immune the immune competent, competent, Ncr1+/gfp Ncr1+/gfp (HET), mice (HET), or frommice theor from Ncr1 thedeficient, Ncr1 deficient, Ncr1gfp/gfp Ncr1gfp/gfp (KO), mice (KO), that mice we that previously we previously generated generated [25]. In[25]. the In NCR1 the NCR1 KO KOmouse mouse the the Ncr1 Ncr1 gene gene was was replaced replaced with with a greena green fluorescent fluorescent protein protein (GFP). (GFP). InIn thethe HET immuneimmune competent competent mice mice one one allele allele of of Ncr1 was still present and the other allele was replaced by GFP. GFP. Hence, all NK cells were labeled with GFP. The presence of GFP in the NK cells enables a reliable and clean isolation of murine NK cells. We incubatedincubated thethe NK NK cells cells obtained obtained from from the th twoe two strains strains of mice of mice with with EL4 inEL4 the in pres- the presenceence or absence or absence of IAV of IAV and and IBV. IBV. As expected, As expected, and inand agreement in agreement with with previous previous results results [17], [17],when when we used we Ncr1+/gfpused Ncr1+/gfp (HET) (HET) NK cells NK that cells express that express the NCR1 the receptor, NCR1 wereceptor, observed we increased killing of IAV (Figure5e). This increased killing was dependent on NCR1 because observed increased killing of IAV (Figure 5e). This increased killing was dependent on in its absence no difference was observed when IAV was present or absent (Figure5e). NCR1 because in its absence no difference was observed when IAV was present or absent Moreover, NK cells isolated from HET and KO mice killed uninfected EL4 to a similar (Figure 5e). Moreover, NK cells isolated from HET and KO mice killed uninfected EL4 to extent, showing no general effect of reduced cytotoxic ability. However, when the cells a similar extent, showing no general effect of reduced cytotoxic ability. However, when were incubated with IBV, no significant increased killing was observed in the HET mice NK similarly to the KO mice NK cells (Figure5e), indicating that NCR1 may not play a role in the killing of IBV in mice.

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the cells were incubated with IBV, no significant increased killing was observed in the Viruses 2021, 13, 34 HET mice NK similarly to the KO mice NK cells (Figure 5e), indicating that NCR1 8may of 11 not play a role in the killing of IBV in mice.

Figure 5. NCR1 binds to IBV but does not activate killing by mouse NK cells. (a,b) EL4 cells were incubated or not (black Figure 5. NCR1 binds to IBV but does not activate killing by mouse NK cells. (a,b) EL4 cells were incubated or not (black histogram)histogram) with with IAV or IBV.IBV. ((aa)) FACSFACS staining staining of of IAV IAV (red (red histogram) histogram) and and IBV IBV (blue (blue histogram) histogram) with with antibodies antibodies against against the thehemagglutinin hemagglutinin of IAV of IAV and IBV,and respectively.IBV, respectively. (b) The (b cells) The incubated cells incubated with the with indicated the indi virusescated wereviruses stained were with stained NKp46-Ig with NKp46-Igfusion protein. fusion The protein. filled histogramsThe filled andhistograms gray empty and histogramsgray empty represent histograms the stainingrepresent with the secondarystaining with antibodies secondary only antibodiesin the absence only or in presencethe absence of influenza, or presen respectively.ce of influenza, Figure respectively. shows one Figure representative shows one experiment representative out ofexperiment three performed. out of three(c,d) MFIperformed. quantification (c,d) MFI of quantification three independent of three experiments independent including experiments data presentedincluding data in (b presented). FACS staining in (b). FACS with NCR1-Igstaining withagainst NCR1-Ig IAV (c )against and IBV IAV (d) ( wasc) and normalized IBV (d) was to thenormalized staining ofto thethe cellsstaining without of the influenza. cells without Values influenza. are shown Values as mean are ±shownSEM . as mean ± SEM. * p < 0.05 by two-tailed paired Student’s t test. (e) NK cells from Ncr1+/gfp (HET) and Ncr1gfp/gfp (KO) * p < 0.05 by two-tailed paired Student’s t test. (e) NK cells from Ncr1+/gfp (HET) and Ncr1gfp/gfp (KO) mice were mice were extracted and incubated with EL4 cells in the presence or absence of IAV and IBV. Killing assay was performed extracted and incubated with EL4 cells in the presence or absence of IAV and IBV. Killing assay was performed as mentioned as mentioned above. An ANOVA test was performed in order to evaluate significance. Values are shown as mean ± SEM. ± p *above. p < 0.05. An Figure ANOVA shows test cumulative was performed results in of order three to independent evaluate significance. experiments. Values are shown as mean SEM. * < 0.05. Figure shows cumulative results of three independent experiments. 3.6. NKp46 Is not Involved in the Killing of IBV in Vivo 3.6. NKp46To determine Is Not Involved the physiological in the Killing ofrelevance IBV In Vivo of our findings we injected mice intranasallyTo determine with IAV the or physiological IBV. Five days relevance post infection of our findings we sacrificed we injected the mice mice and intranasally assessed viralwith load IAV in or the IBV. lungs. Five daysAs previously post infection reported we sacrificed [17], when the we mice infected and assessedmice with viral IAV load we observedin the lungs. that As Ncr1gfp/gfp previously reported(KO) exhibits [17], when severely we infected impaired mice withviral IAVclearance, we observed thus indicatingthat Ncr1gfp/gfp that the (KO) elimination exhibits of severely IAV is impaireddependent viral on clearance, NCR1 (Figure thus indicating6a). Importantly, that the whenelimination the mice of IAV were is dependent injected with on NCR1 IBV, (Figureno difference6a). Importantly, in viral whenclearance the mice between were Ncr1gfp/gfpinjected with and IBV, Ncr1+/+ no difference mice inwas viral observed clearance (Figure between 6b). Ncr1gfp/gfp These results and suggest Ncr1+/+ that mice the lowerwas observed affinity binding (Figure 6ofb). NKp46 These to results the HA suggest of IBV that might the not lower be enough affinity bindingto activate of NKp46the NK cellsto the in HAvivo. of IBV might not be enough to activate the NK cells in vivo.

Viruses 2021, 13, x FOR PEER REVIEW 9 of 12 Viruses 2021 , 13, 34 9 of 11

Figure 6. NCR1 is critical for the control of IAV but not IBV infection in vivo. Ncr1+/+ and Ncr1gfp/gfp mice were Figure 6. NCR1 is critical for the control of IAV but not IBV infection in vivo. Ncr1+/+ and Ncr1gfp/gfp mice were intranasallyintranasally infected infected with IAV with (a )IAV or IBV(a) or (b IBV). After (b). After ﬁve five days, days, lungs lungs were were collected collected and and real-timereal-time PCR PCR was was performed. performed. n = n = 5 mice in each5 mice group. in each Values group. are Values shown are asshown mean as ±meanSEM. ± SEM. * p < * 0.05p < 0.05 by by two-tailed two-tailed Student’s Student’s tt test.test.

4. Discussion 4. DiscussionInfluenza infections constitute a major health and economic burden [29]. According Influenzato the World infections Health Organization constitute a major(WHO), health up to and650,000 economic people burdendie of influenza [29]. According every to the Worldyear. HealthTo evade Organization recognition by (WHO), the immune up to system, 650,000 IAV people viruses die undergo of influenza rapid every changes, year. To evadein recognition particular in by their the HA immune and NA system, proteins. IAV This viruses high mutation undergo rate rapid is one changes, of the causes in particular for the inability of the adaptive immune system to develop memory against this virus. In this in their HA and NA proteins. This high mutation rate is one of the causes for the inability process however, the ability of HA to bind sialic acid remains unchanged. This property of theis adaptivecrucial forimmune the virus systemto attach toand develop infect the memory cell [30].against One of the this major virus. NK In receptors, this process however,NKp46, the uses ability the ofsialylated HA to bindthreonine sialic 225 acid residue remains to bind unchanged. the HA protein This property of IAV [16]. is crucial for theAccordingly, virus to attach the virus and evolved infect the and cell developed [30]. One mechanisms of the major to evade NK receptors, recognition NKp46, by NK uses the sialylatedcells. The threoninevirus uses 225its NA residue enzyme to bindto clea theve the HA sialic protein acids of present IAV [16 on]. NKp46, Accordingly, thus the viruspreventing evolved and recognition developed of mechanismsthe infected tocell evades [22]. recognitionFurthermore, by recent NK cells. H3N2 The isolates virus uses its NAdisplay enzyme lower to sialic cleave acid the affinity sialic due acids to presenta mutation on in NKp46, the glycosylation thus preventing on the HA recognition protein, of the infectedand, indeed, cells [NKp4622]. Furthermore, is unable to recentrecognize H3N2 these isolates viruses display [27]. It loweris a continuous sialic acid and affinity due tounrelenting a mutation battle in the between glycosylation our NK cells on and the HAthe influenza protein, virus and, family. indeed, NKp46 is unable to Interestingly, it was not known whether NK cells recognize IBV. Influenza B viruses recognize these viruses [27]. It is a continuous and unrelenting battle between our NK cells diverged from influenza A viruses at some point in the distant past and are, therefore, and thenewer influenza viruses virus[18]. family.This virus mutates at a significantly lower rate than IAV and is Interestingly,composed of only it was one notserotype known and whether two lineag NKes, cellsYamagata recognize and Victoria IBV. Influenza [31]. Although B viruses divergednot as from deadly influenza as IAV, AIBV viruses is the reported at some cause point of in death the distantof one-third past of and all infected are, therefore, children newer virusesevery [18]. year, This as virus stated mutates by the at WHO. a significantly IBV is more lower dangerous rate than IAVto children and is composeddue to age- of only one serotypedependent and sialic two acid lineages, composition. Yamagata Sialic andacid Victoriacomposition [31 ].in Although the human not respiratory as deadly tract as IAV, IBV ischanges the reported during cause aging of with death higher of one-third α-2,3-linked of all sialic infected acids’ children expression every in children year, as statedand by the WHO.more α IBV-2,6-linked is more sialic dangerous acids in to adults. children Interestingly, due to age-dependent while IAV HA sialicbinds acidto 2,6-linked composition. Sialicsialic acid acids, composition IBV HA can in thebind human both α-2,3 respiratory and α-2,6-linked tract changes sialic acids. during Therefore, aging children with higher are more susceptible to infection by IBV [32]. α-2,3-linked sialic acids’ expression in children and more α-2,6-linked sialic acids in adults. Here we showed that NKp46 binds to the HA of IBV mainly through sialic acids Interestingly,present on while the threonine IAV HA 225 binds of NKp46. to 2,6-linked This mode sialic of acids,binding IBV is similar HA can to that bind of both IAV α[16].-2,3 and α-2,6-linkedInterestingly, sialic the acids. presence Therefore, of sialic children acids, per are se, more is not susceptiblesufficient for to binding infection to HA by as IBV most [32 ]. HereNK cell we receptors showed express that NKp46 sialic acids, binds but to thewe previously HA of IBV showed mainly that through most NK sialic cell acids presentreceptors on the are threonine not able 225to interact of NKp46. with ThisHA [33]. mode Moreover, of binding when is similarwe stained to thatcells ofin IAVthe [16]. Interestingly,presence of the IBV presence with the ofmutated sialic acids,T225A-Ig per we se, could is not still sufficient observe some for binding to to the HA as most NK cell receptors express sialic acids, but we previously showed that most NK cell receptors are not able to interact with HA [33]. Moreover, when we stained cells in the presence of IBV with the mutated T225A-Ig we could still observe some binding to the influenza virus. Thus, there are elements other than the presence of sialic acids which determine the specificity of NK receptor-HA interactions. Next, we demonstrated that NKp46 has a lower affinity against the HA of IBV as compared to IAV. Nevertheless, the NK cells were still able to kill cells in the presence of Viruses 2021, 13, 34 10 of 11

IBV, and this was dependent on NKp46. Interestingly, when assessing whether mouse NK cells were able to kill cells in the presence of IBV, we observed that, although NCR1 is able to bind IBV, this interaction does not induce killing. In this assay both IAV and IBV were evaluated, and mouse NK cells expressing NCR1 were able to efficiently kill cells in the presence of IAV but not IBV. This observation suggests that NCR1 may not play a role in the clearance of IBV. We confirmed this assessment using an in vivo system where we observed that the presence or absence of NCR1 did not influence viral clearance of IBV. It is possible that the lower affinity of NKp46 to IBV HA is because the HA protein of IBV binds α-2,3 as well as α-2,6-linked sialic acids. The sialic acids present on the threonine 225 of NKp46 are mainly α-2,6-linked sialic acids [34]. Moreover, it has been shown that IBV, in general, has lower receptor binding affinities than influenza A viruses due to critical difference in the sialic acid binding site of HA, namely a Phe-95 versus Tyr-98 [35]. This difference influences the conformation of the protein and might explain the limited host range of influenza B viruses [32].

Author Contributions: Conceptualization, A.D.-C. and O.M.; methodology, A.D.-C.; investigation, A.D.-C.; resources, O.M.; mice work, A.D.-C., B.I. and Y.C.-A.; viruses’ preparation, N.F., Y.D. and M.M.; writing—original draft preparation, A.D.-C.; writing—review and editing, O.B. and O.M.; supervision, O.M.; funding acquisition, O.M. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the European Research Council Marie Currie program Further support came from the GIF foundation, the ICRF professorship grant, The DKFZ-MOST grant, the Israeli Science Foundation (Moked), and The Israel-China ISF grant (all to O.M.). Institutional Review Board Statement: All animal work was conducted according to relevant national and international guidelines. The work was approved by the Hebrew University Medical School Ethic committee (Ethics number MD-19-15748-3, date of approval 7/7/19). The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Institu- tional Review Board of Hadassa medical organization, Israel (protocol code 0030-12HMO, date of approval 30/11/20). Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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