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Susceptibility to influenza virus infection of bronchial biopsies in asthma

  • Ben Nicholas
    Affiliations
    Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Sir Henry Wellcome Laboratories, Southampton General Hospital, Southampton, United Kingdom

    Southampton NIHR Respiratory Biomedical Research Unit and the NIHR Wellcome Trust Clinical Research Facility, Southampton General Hospital, Southampton, United Kingdom
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  • Sarah Dudley
    Affiliations
    Synairgen Research Ltd, Southampton General Hospital, Southampton, United Kingdom
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  • Kamran Tariq
    Affiliations
    Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Sir Henry Wellcome Laboratories, Southampton General Hospital, Southampton, United Kingdom

    Southampton NIHR Respiratory Biomedical Research Unit and the NIHR Wellcome Trust Clinical Research Facility, Southampton General Hospital, Southampton, United Kingdom
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  • Peter Howarth
    Affiliations
    Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Sir Henry Wellcome Laboratories, Southampton General Hospital, Southampton, United Kingdom

    Southampton NIHR Respiratory Biomedical Research Unit and the NIHR Wellcome Trust Clinical Research Facility, Southampton General Hospital, Southampton, United Kingdom
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  • Kerry Lunn
    Affiliations
    Synairgen Research Ltd, Southampton General Hospital, Southampton, United Kingdom
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  • Sandy Pink
    Affiliations
    Southampton NIHR Respiratory Biomedical Research Unit and the NIHR Wellcome Trust Clinical Research Facility, Southampton General Hospital, Southampton, United Kingdom
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  • Peter J. Sterk
    Affiliations
    Department of Respiratory Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands
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  • Ian M. Adcock
    Affiliations
    Faculty of Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom
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  • Phillip Monk
    Affiliations
    Synairgen Research Ltd, Southampton General Hospital, Southampton, United Kingdom
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  • Ratko Djukanović
    Affiliations
    Clinical and Experimental Sciences, University of Southampton Faculty of Medicine, Sir Henry Wellcome Laboratories, Southampton General Hospital, Southampton, United Kingdom

    Southampton NIHR Respiratory Biomedical Research Unit and the NIHR Wellcome Trust Clinical Research Facility, Southampton General Hospital, Southampton, United Kingdom
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  • On behalf of theU-BIOPRED study group
Published:March 01, 2017DOI:https://doi.org/10.1016/j.jaci.2016.12.964
      To the Editor:
      Influenza causes significant morbidity and mortality, especially in patients with chronic lung diseases.
      • Gerke A.K.
      • Yang M.
      • Tang F.
      • Foster E.D.
      • Cavanaugh J.E.
      • Polgreen P.M.
      Association of hospitalizations for asthma with seasonal and pandemic influenza.
      Infection results in inflammatory cell influx and leads to either resolution or increased lung immunopathology and resulting morbidity,
      • La Gruta N.L.
      • Kedzierska K.
      • Stambas J.
      • Doherty P.C.
      A question of self-preservation: immunopathology in influenza virus infection.
      especially in patients with chronic airways diseases where viruses exacerbate inflammation and, subsequently, symptoms. Those with asthma are more susceptible to influenza and are, therefore, the most common population hospitalized, although, interestingly, they are less likely to develop severe disease or die than those without asthma.
      • Van Kerkhove M.D.
      • Vandemaele K.A.
      • Shinde V.
      • Jaramillo-Gutierrez G.
      • Koukounari A.
      • Donnelly C.A.
      • et al.
      WHO Working Group for Risk Factors for Severe H1N1pdm Infection. Risk factors for severe outcomes following 2009 influenza A (H1N1) infection: a global pooled analysis.
      The mechanisms underlying the increased susceptibility to viral infections in those with asthma are poorly understood, but it has been suggested that the skewing toward TH2 immunity results in deficient TH1 antiviral immunity.
      • Message S.D.
      • Laza-Stanca V.
      • Mallia P.
      • Parker H.L.
      • Zhu J.
      • Kebadze T.
      • et al.
      Rhinovirus-induced lower respiratory illness is increased in asthma and related to virus load and Th1/2 cytokine and IL-10 production.
      Understanding of antiviral immunity in asthma is also complicated by the immunosuppressive effects of inhaled corticosteroids (ICSs) or oral corticosteroids, standard treatments in asthma.
      • Myles P.
      • Nguyen-Van-Tam J.S.
      • Semple M.G.
      • Brett S.J.
      • Bannister B.
      • Read R.C.
      • et al.
      Influenza Clinical Information Network FLU-CIN
      Differences between asthmatics and nonasthmatics hospitalised with influenza A infection.
      The effectiveness of ICSs during exacerbations is unclear because doubling their dose at the time of upper respiratory tract infection fails to prevent asthma exacerbations.
      • Harrison T.W.
      • Oborne J.
      • Newton S.
      • Tattersfield A.E.
      Doubling the dose of inhaled corticosteroid to prevent asthma exacerbations: randomised controlled trial.
      Corticosteroids may protect against severe outcomes in those with asthma with influenza infection, whereas systemic corticosteroids in individuals without asthma cause delayed viral clearance.
      • Lee N.
      • Chan P.K.
      • Hui D.S.
      • Rainer T.H.
      • Wong E.
      • Choi K.W.
      • et al.
      Viral loads and duration of viral shedding in adult patients hospitalized with influenza.
      The primary aim of our study was to compare the susceptibility and inflammatory responses to influenza virus infection of ICS-treated patients with asthma and healthy individuals. We hypothesized that these patients with asthma are more susceptible to influenza infection and that their inflammatory responses during infection are elevated, thereby contributing to asthma exacerbations. In view of ethical and safety difficulties of studying influenza infection in vivo, especially in patients with asthma, the airway responses were studied in bronchial biopsies infected ex vivo, using a bronchial biopsy explant model.
      • Nicholas B.
      • Staples K.J.
      • Moese S.
      • Meldrum E.
      • Ward J.
      • Dennison P.
      • et al.
      A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.
      To mimic in vivo conditions of viral exposure, bronchial biopsies from patients with asthma regularly treated with ICSs were exposed to influenza virus in the presence of exogenous ICS, fluticasone propionate (FP), whereas biopsies from healthy subjects were infected in the absence of this corticosteroid.
      Twenty-four hours after ex vivo infection, biopsies were enzymatically dispersed with collagenase, allowing quantification of infected cells and activation markers by multicolor flow cytometry (see details in this article's Online Repository at www.jacionline.org). In these conditions, epithelial cell infection was not different between health and asthma (Fig 1, Ai), whereas viral shedding was significantly reduced in explants from patients with asthma (Fig 1, Aii). T-lymphocyte activation induced by infection (measured by fold-induction of cell surface HLA-DR expression) was suppressed in the biopsies from patients with asthma (Fig 1, Bi); HLA-DR expression on epithelial cells was unchanged (Fig 1, Bii). Secreted mediator responses, including innate defence (IFN-γ, C-X-C motif chemokine 10 [CXCL-10]), chemokines (CXCL-8, monocyte chemoattractant protein 1, macrophage inflammatory protein 1β), and proinflammatory cytokines (IL-1β, IL-6, TNF-α) were also all blunted in those with asthma when compared with healthy controls (Fig 1, C). Type I interferons were not present in sufficient quantity to measure; however, the finding of lower CXCL-10 quantities in asthmatic explants was consistent with deficient innate antiviral defences in asthma.
      Figure thumbnail gr1
      Fig 1Comparison of bronchial biopsies from healthy subjects and subjects with asthma following influenza virus exposure. A, Effects on (i) epithelial cell infection and (ii) viral shedding. B, Fold change in MHC class II cell surface expression on (i) T lymphocytes and (ii) epithelial cells. C, Mediator secretion from infected biopsies. N = 10 per group compared using Mann-Whitney test. MCP, Monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NP, Influenza A virus nucleoprotein. *P < .05, **P < .005, and ***P < .001.
      Because subjects with asthma in our study were on regular ICSs, the effects on influenza susceptibility of which are unknown, we also sought to determine whether the differences seen in the primary comparator groups reflected the effects of FP or disease. Ex vivo treatment of steroid-naive bronchial explants from healthy participants with FP increased the percentage of virally infected epithelial cells (Fig 2, Ai) without (in contrast to asthmatic explants) affecting viral shedding (Fig 2, Aii) or activation of T lymphocytes (Fig 2, Bi), but epithelial cell surface induction of HLA-DR was suppressed (Fig 2, Bii). We have previously observed elevation of this panel of mediators with influenza infection in healthy subjects, with the exception of IL-8.
      • Nicholas B.
      • Staples K.J.
      • Moese S.
      • Meldrum E.
      • Ward J.
      • Dennison P.
      • et al.
      A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.
      As expected, FP treatment significantly inhibited the secretion of these mediators (Fig 2, C).
      Figure thumbnail gr2
      Fig 2A, Effect of addition of exogenous corticosteroid to bronchial explants from healthy subjects on (i) epithelial cell infection and (ii) viral shedding. B, Fold change in MHC class II cell surface expression following infection on the cell surface of (i) T lymphocytes and (ii) epithelial cells. C, Mediator secretion from infected biopsies. N = 10 per group. Data were compared using Wilcoxon matched pairs signed rank test. MCP, Monocyte chemoattractant protein; MIP, macrophage inflammatory protein; NP, Influenza A virus nucleoprotein. *P < .05, **P < .005, and ***P < .001.
      This study points to important differences between asthma and health in respect of influenza virus handling. However, our hypothesis that the elevated morbidity and mortality caused by influenza infection in people with asthma is associated with increased susceptibility to infection was not fully supported because the initial infection rate was no different between asthma and health (judged by similar proportions of infected epithelial cells). Nevertheless, the blunted inflammatory, including innate immune, responses and T-cell activation (judged by lesser induction of cell surface HLA-DR expression) to infection in ICS-treated patients with asthma do argue in favor of deficient antiinfluenza immunity in these patients. Although the reduced viral shedding in asthmatic tissues (when compared with healthy tissues) could be viewed as a positive phenomenon that limits virus spread, alternatively, it could account for prolonged viral retention, with consequential prolonged recovery and increased risk of viremia. Prolonged viral shedding appears to be a consistent problem associated with systemic corticosteroid therapy in patients hospitalized with influenza, in contrast to antiviral agents that enhance virus clearance.
      • Lee N.
      • Chan P.K.
      • Hui D.S.
      • Rainer T.H.
      • Wong E.
      • Choi K.W.
      • et al.
      Viral loads and duration of viral shedding in adult patients hospitalized with influenza.
      For ethical and safety reasons, it was impossible to wash out the effects of regular treatment with ICS on the asthmatic explant responses to infection. Accepting that the effects of corticosteroids likely differ between healthy and asthmatic tissue, we thought it useful to study the impact of FP treatment on explants from healthy subjects. In contrast to asthma, this showed that FP increased epithelial infection rates, while viral shedding and T-cell activation were unaffected. Similar to asthma, mediator secretion was suppressed. We also found that MHC class II (HLA-DR) induction in healthy airway tissue epithelial cells was suppressed by ex vivo FP treatment. We have previously observed influenza infection-mediated elevation in HLA-DR on the surface of primary bronchial epithelial cells,
      • Wilkinson T.M.
      • Li C.K.
      • Chui C.S.
      • Huang A.K.
      • Perkins M.
      • Liebner J.C.
      • et al.
      an effect replicated in bronchial epithelial cells of our explants, probably occurring in both models as a result of infection-induced secretion of IFN-γ, and suggestive that respiratory epithelial cells potentiate cytotoxic T-cell activity. Corticosteroid suppression of this effect may reflect suppression of innate antiviral mediators including IFN-γ, and could have implications for clearance of virally infected cells from the lungs.
      In summary, the present study shows blunted responses of ICS-treated patients with mild/moderate asthma to influenza virus infection but is unable to differentiate between the impact of corticosteroid and disease itself. The lack of effect of corticosteroids in explants from healthy participants suggests that reduced viral shedding and defective T-cell activation observed in patients with asthma may be independent of corticosteroid treatment. Further study is needed to elucidate the underlying mechanisms.

      Methods

       Influenza virus preparation

      A/H3N2/Wisconsin/67/2005 seed stocks were obtained from the National Institute for Biological Standards and Control, propagated in embryonated specific pathogen free chicken eggs, and, subsequently, purified from egg allantoic fluid by sucrose density gradient ultracentrifugation (Virapur LLC, San Diego, Calif). Stock and conditioned media viral titers were determined by MDCK plaque assay (see details below).

       Participants and sample collection

      Ten healthy participants and 10 subjects with mild/moderate asthma on regular ICSs were recruited as part of the Unbiased BIOmarkers Predictive of REspiratory Disease outcomes project.
      • Shaw D.E.
      • Sousa A.R.
      • Fowler S.J.
      • Fleming L.J.
      • Roberts G.
      • Corfield J.
      • et al.
      U-BIOPRED Study Group
      Clinical and inflammatory characteristics of the European U-BIOPRED adult severe asthma cohort.
      Participants were matched for age and sex, but subjects with asthma had significantly reduced lung function and evidence of increased airway inflammation and atopy (Table E1). Fiberoptic bronchoscopy was performed according to a standard research protocol.
      • Djukanovic R.
      • Wilson J.W.
      • Lai C.K.
      • Holgate S.T.
      • Howarth P.H.
      The safety aspects of fiberoptic bronchoscopy, bronchoalveolar lavage, and endobronchial biopsy in asthma.
      Up to 10 endobronchial biopsies were taken from the subcarinae of large airways.

       Ex vivo infection of bronchial explants

      The protocol for ex vivo infection was that reported recently
      • Nicholas B.
      • Staples K.J.
      • Moese S.
      • Meldrum E.
      • Ward J.
      • Dennison P.
      • et al.
      A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.
      with a minor modification (use of RPMI instead of AIM-V culture medium to increase infection efficiency by reducing the concentration of serum albumin). Following collection during bronchoscopy, explants were rested overnight in pairs in 24-well culture dishes containing 500 μL of RPMI supplemented with glutamine and penicillin/streptomycin in a humidified tissue culture incubator at 37°C, 5% CO2. They were then cultured in RPMI medium supplemented with glutamine alone (RPMI-G) and treated with either 100 nM FP or carrier control (0.1% v/v dimethyl sulfoxide) for 2 hours before adding log7.0 plaque-forming units (pfu) of virus or the equivalent volume of virus diluent (0.4% w/v sucrose in 0.5 mM HEPES buffer, pH 7.4) in the presence of carrier/FP and incubated for 2 hours. Explants were then washed 3 times with basal RPMI medium to remove excess virus and incubated for a further 22 hours in RPMI-G containing carrier/FP, after which conditioned media were centrifuged (400g) to remove cellular material and stored at −80°C. Samples for plaque assay were stored in 40% (w/v) sucrose-50 mM HEPES buffer (pH 7.4).

       Flow cytometric analysis of infection and cell activation markers

      Postinfection, tissue pieces were digested in RPMI containing 1 mg/mL collagenase I for 60 minutes with agitation. The dispersed cells were then filtered through 100-μm filters to remove tissue debris and resuspended in 100 μL of fluorescence-activated cell sorting buffer (0.5% BSA, 2 mM EDTA in Dulbecco's PBS) containing 2 mg/mL human IgG for 10 minutes on ice. Cells were then stained for leukocytes (the pan-leukocyte marker CD45 conjugated to PECF594), epithelial cells (the epithelial cell marker CD326 conjugated to PerCPCy5.5), T lymphocytes (the T-lymphocyte specific marker CD3 conjugated to phycoerythrin-Cy7), and MHC class II (HLA-DR conjugated to antigen-presenting cell-Cy7) using mAbs directed against each extracellular marker, or appropriate isotype control antibodies conjugated to the relevant fluorophores. Cells were then fixed and permeabilized using proprietary reagents (BD Fix/perm kit, BD Biosciences, Oxford, United Kingdom [UK]), and cells infected with A/H3N2/Wisconsin/67/2005 virus were detected using fluorescein isothiocyanate–conjugated monoclonal anti-influenza nucleoprotein antibody (AbCAM, Cambridge, UK). Flow cytometry of the stained cells was performed using a FACSAria (BD) with appropriate filters and settings. Appropriate isotype and fluorescence-matched antibodies were added to separate samples to aid gating of cell populations (Table E2). Epithelial cells were identified by following a gating strategy modified from previous reports,
      • Nicholas B.
      • Staples K.J.
      • Moese S.
      • Meldrum E.
      • Ward J.
      • Dennison P.
      • et al.
      A novel lung explant model for the ex vivo study of efficacy and mechanisms of anti-influenza drugs.
      on the basis of size, and then excluding the leukocyte marker CD45, and including positive staining for the epithelial cell marker CD326 (Fig E1, A). Viral infection in epithelial cells was gated against epithelial cells from uninfected tissue stained with the mAb against viral nucleoprotein (Fig E1, B). T lymphocytes were identified from the CD45-positive population by expression of the T-lymphocyte marker CD3. The applied viral concentration was retitrated in these altered conditions using the previously described gating strategy, to ensure that epithelial cell infection versus viral dose was linear 24 hours postinfection (Fig E1, C), resulting in an optimized final dose of log7.0 pfu. For quantification of cell surface HLA-DR ligand expression on T lymphocytes and epithelial cells, specific mean fluorescence intensity (sMFI) was calculated by subtracting the MFI of the cell population stained with isotype antibody from the MFI of the cell population stained with specific antibody (Fig E1, D). Fold-induction of the expression of HLA-DR on the surface of T lymphocytes was used as a marker of T-cell activation.
      • Ko H.S.
      • Fu S.M.
      • Winchester R.J.
      • Yu D.T.
      • Kunkel H.G.
      Ia determinants on stimulated human T lymphocytes: occurrence on mitogen- and antigen-activated T cells.
      Data were analyzed using FACS Diva software v5.0.3 (BD).

       Viral shedding plaque assay

      Cell-free tissue conditioned medium samples containing 40% (w/v) sucrose and 50 mM HEPES buffer (pH 7.4) were stored at −80°C until use. Samples were then thawed and immediately prepared by serial dilution in infection buffer (Dulbecco modified Eagle medium containing l-glutamine, sodium pyruvate, penicillin streptomycin, and nonessential amino acids). Diluted media samples were then applied to 90% confluent monolayer cultures of MDCK cells with agitation for 1 hour at 37°C, 5% CO2. Media were then removed and the cultures overlayed with cellulose/methylcellulose biopolymer (Sigma-Aldrich, Poole, Dorset, UK) prepared in minimal essential medium (MEM), supplemented with sodium bicarbonate, HEPES, BSA, and diethylaminoethyl-dextran hydrochloride. Overlay was also supplemented with TRTPCK trypsin (Worthington Biochemical Corp, Reading, Berks, UK) at a final concentration of 0.25 ng/mL. Cultures were incubated for a further 48 hours and then the overlays removed, and the monolayers stained with crystal violet to visualize the plaques. Plaques were manually counted and the viral titer (pfu/mL) was then adjusted for the dilution factor and expressed as the number of pfu per milliliter.

       Measurement of inflammatory mediators

      A bespoke 8-plex immunoassay to quantify a range of cytokines/chemokines (IFN-γ, IL-6, IL-8, CXCL-10/interferon gamma-induced protein 10 (IP-10), monocyte chemoattractant protein 1, macrophage inflammatory protein 1β, TNF-α, and IL-1β) was purchased from Mesoscale Discoveries (Rockville, Md) and performed according to the manufacturers' instructions. Briefly, conditioned media samples were diluted 1:1 in buffer containing carrier protein and applied to microtiter plates precoated with antibodies directed against the 8 mediators to be measured. A dilution series of mediators in the culture medium diluent was also applied to the plate to construct standard curves of mediator concentration. A detection antibody prediluted in blocking buffer was applied, and then the signal analyzed on a SECTOR 2400 MSD plate reader. Data were analyzed using MSD discovery workbench software. Cytokine quantities were interpolated from standard curves generated using mixtures of recombinant proteins. Baseline secretion from noninfected untreated controls was subtracted from infected and/or treated wells. Values were then adjusted for wet tissue weight (mg) to give the concentration of mediator per milligram per milliliter culture medium.
      Figure thumbnail fx1
      Fig E1Leukocytes and epithelial cells dispersed from bronchial biopsies were phenotypically identified by flow cytometry. A, Gating strategy: (i) Size using forward scatter properties, (ii) CD45-PE/CF594 to identify leukocytes, (iii) CD3-PECy7 on the CD45+ cell population to identify T lymphocytes, and (iv) CD326-PerCPCy5.5 on the CD45- population to identify epithelial cells. B, Quantification of epithelial cell influenza infection within bronchial biopsies by flow cytometric detection of influenza viral nucleoprotein (NP-FITC) in infected biopsies compared with mock-infected control explants. C, Viral dose- response curve performed in resected lung tissue explants. D, HLA-DR (MHC class II) expression on the surface of T lymphocytes and epithelial cells (HLA-DR-APCCy7) with isotype control used to subtract from positive stains to calculate the sMFI. APC, Antigen-presenting cell; FITC, fluorescein isothiocyanate; IFV, influenza virus; PE, phycoerythrin.
      Table E1Clinical characteristics of healthy subjects and volunteer donors with moderate asthma of bronchial tissue explants
      ParameterHealthy controlsSubjects with moderate asthmaP value
      N1010
      Age (y)27 (22.5-48.0)35.5 (23.3-48.5).647
      Data were compared using the Mann-Whitney U test.
      Sex (M/F)6/46/41.0
      Data were compared using the chi-square test.
      FEV14.23 (3.03-5.14)3.55 (2.12-4.59).142
      Data were compared using the Mann-Whitney U test.
      FEV1 % predicted109.1 (99.6-112.2)95.9 (63.1-108.0).017
      Data were compared using the Mann-Whitney U test.
      Inhaled steroid dose (μg/d)NA280 (165.4-394.6)<.001
      Data were compared using the Mann-Whitney U test.
      SABA (μg/d)NA140 (89.98-190.00)<.001
      Data were compared using the Mann-Whitney U test.
      Blood eosinophils (%)2.87 (1.69-5.87)3.15 (2.04-4.89).986
      Data were compared using the Mann-Whitney U test.
      Total blood IgE33.3 (7.1-63.5)89.6 (31.0-179.5).023
      Data were compared using the Mann-Whitney U test.
      Atopy (yes)3/108/10.025
      Data were compared using the chi-square test.
      Feno18.25 (11.38-33.38)33.75 (21.50-100.10).027
      Data were compared using the Mann-Whitney U test.
      Sputum eosinophils (%)02.39 (0-3.68).045
      Data were compared using the Mann-Whitney U test.
      Sputum neutrophils (%)11.4 (7.1-15.7)64.5 (21.9-74.2).006
      Data were compared using the Mann-Whitney U test.
      F, Female; Feno, fraction of exhaled nitric oxide; M, male; NA, not applicable/available; SABA, short-acting beta-agonist.
      Data shown are medians (interquartile range).
      Data were compared using the Mann-Whitney U test.
      Data were compared using the chi-square test.
      Table E2List of antibody clones and fluorophores used for detection of cell types and activation markers on cells dispersed from bronchial biopsy explants
      TargetAntibody sourceClone no.IsotypeFluorochromeAntibody concentration (mg/mL)Optimized dose (μL per 100 μL reaction)
      Influenza A virus nucleoproteinAbcam431IgG1FITC0.11
      CD326 (EpCAM)Beckton DickinsonEBA-1IgG1λPerCP-Cy5.50.00210
      CD45InvitrogenH130IgG1PE-CF594NA2
      CD3BDSK7IgG1κPE-Cy7NA5
      HLA-DRBDL243IgG2aκAPC-Cy70.055
      APC, Antigen-presenting cell; EpCAM, epithelial cell adhesion molecule; FITC, fluorescein isothiocyanate; NA, not available; PE, phycoerythrin.

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