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Journal of Immunological Methods
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Research paper
A novel assay for detecting virus-specific antibodies triggering activation ofFcγ receptors
Eugenia Corrales-Aguilar a,1, Mirko Trilling a,2, Henrike Reinhard a, Eva Mercé-Maldonado a,Marek Widera a, Heiner Schaal a, Albert Zimmermann a, Ofer Mandelboim b, Hartmut Hengel a,⁎a Institute for Virology, Heinrich-Heine-University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germanyb The Lautenberg Center for General and Tumor Immunology, Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel
a r t i c l e i n f o
Abbreviations: ADCC, antibody-dependent cellulEpstein–Barr virus; ELISA, enzyme-linked immunosorreceptor; Fab-g, IgG Fab fragment; Fc-g, IgG Fc fragmmouse antibody; HCMV, human cytomegalovirus; HSVhpi, hours post infection; IVIG, intravenous immunogvirus; RSV, respiratory syncytial virus; HIV, human imm⁎ Corresponding author at: Institute for Virology, Hein
Article history:Received 10 June 2012Received in revised form 12 August 2012Accepted 19 September 2012Available online 27 September 2012
IgG responses are crucial in antiviral defence and instrumental for the serodiagnosis ofinfections. Fcγ receptors (FcγRs), which recognize the Fc-part of IgG, differ regarding their IgGbinding affinity, IgG subclass preference, cellular expression profile and pathogen eliminationmechanisms elicited upon activation. Assessing their activation in vitro is of fundamentalimportance, but technically difficult. Therefore, a novel assay for measuring antiviral IgGantibodies triggering activation of individual host Fcγ receptors was established. The assaycomprises the co-cultivation of virus-infected target cells with immune IgG antibodies andmouse BW5147 hybridoma cells stably expressing chimeric FcγR–CD3ζ chain moleculesconsisting of the extracellular domain of human FcγRIIIA, FcγRIIA or FcγRI, respectively, fusedto the transmembrane and intracellular domains of the mouse CD3ζ chain. Triggering of thechimeric FcγR receptors by immune complexes formed on the surface of IgG-opsonizedvirus-infected target cells resulted in FcγR activation leading to IL-2 secretion by BW5147 cells,which was quantified as a surrogate marker in an ELISA. Target cells infected with varioushuman pathogenic viruses including herpes simplex virus type 1 (HSV-1), Epstein–Barr virus(EBV), human cytomegalovirus (HCMV), measles virus (MV), and respiratory syncytial virus(RSV) or displaying human immunodeficiency virus-1 (HIV-1) gp120 evoke dose-dependentIgG responses demonstrating the universal applicability of the assay. Taken together, a newreliable and simple tool for measuring antibodies triggering activation of Fcγ receptors wasestablished. This assay will be instrumental for defining novel correlates of IgG immunity andthe design of new therapeutic IgGs.
Immune control and elimination of pathogens require closecollaboration between the humoral and cellular components ofthe immune system. Specific receptors recognizing the Fc-partof IgGs (FcγRs) as a ligand are expressed on the surface of awide range of immune cells. Upon IgG binding, FcγRs trigger amultitude of effector mechanisms such as antibody dependentcell cytotoxicity (ADCC), phagocytosis, endocytosis of immunecomplexes, cytokine production, antibody production andfacilitation of antigen presentation (Ravetch and Bolland,2001), thereby linking both branches of immunity. The family
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of FcγRs is comprised of different types, i.e. FcγRI (CD64),FcγRII (CD32) and FcγRIII (CD16) differing in cell distribution,affinities for IgG isotypes and cellular effector functions elicitedupon activation (Gessner et al., 1998;Nimmerjahn andRavetch,2006). FcγRI is constitutively found on monocytes andmacrophages, but cytokine-induced expression also occurs inneutrophil and eosinophil granulocytes and subpopulations ofdendritic cells. FcγRI binds monomeric IgG with high affinity.FcγRII has a low affinity for monomeric IgG but responds toaggregated IgG. FcγRII exists in three isoforms transducingactivating (FcγRIIA, two isoforms with histidine or arginineat position 131) or inhibitory signals (FcγRIIB). FcγRIIIA isexpressed on macrophages and NK cells (Nimmerjahn andRavetch, 2007) and binds monomeric IgG with low affinity,but shows low-to-medium affinity for immune complexes.FcγR-mediated responses are essential for the adaptiveimmune response against viruses and define progression ofdisease (McCullough et al., 1988; Baum et al., 1996; Huberet al., 2001; Chu et al., 2008; Lambotte et al., 2009). Animportant example highlighting the relevance of FcγR functionfor antiviral immunity is represented by findings of Hessell etal. showing that mutations (L234A, L235A; LALA antibody) inthe lower hinge region of a SHIV-reactive monoclonal IgGantibody (called b12), which abrogate recognition by FcγRs,eliminate the protective properties of the b12 antibody in vivo(Hessell et al., 2007).
Both structural and non-structural viral proteins induceantigen-specific IgG responses (Parren and Burton, 2001).Detection of virus-specific IgG is essential for diagnostic purposesinmany clinical settings. The presence of immune IgG is detectedroutinely by prototypic in vitro assays comprising ELISA(Enzyme-linked immunosorbent assay), cell-based immunoflu-orescence assays, immunoblots, hemagglutination inhibition andvirus neutralization tests. However, only the latter methodprovides direct information on an antiviral effector function.
Merely a fraction of virus-specific IgG exerts direct antiviralactivity by i) neutralization of the infectivity of virions throughinhibition of binding to entry receptors or impeding virus fusionwith the host cell membrane, ii) complement activation oriii) FcγR triggering (Hangartner et al., 2006). Several epitopesexposed on viral or cellular surfaces are non-neutralizing whentargeted by IgG. Therefore, subfractions of IgG can elicit furtherimmune functions through activation of complement, increas-ing phagocytosis (opsonization) and eliciting ADCC. These otherantiviral functions of antibodieswere confirmed by the failure ofadoptively transferred neutralizing antibodies (Abs) to protectfrom virus diseases are known to be sensitive to antibodyimmune control (Farrell and Shellam, 1991; Klenovsek et al.,2007). This suggests that non-neutralizing antibodies signifi-cantly contribute to protection against disease or its progressionby triggering other antiviral mechanisms (Wright et al., 2009).
FcγR-mediated responses including ADCC and cytokinerelease are thought to be essential components of the host'simmune response to pathogens and particularly viruses (Shoreet al., 1976; de Noronha et al., 1977; Hessell et al., 2007). ADCCresults in lysis of virus-infected cells when immune IgGactivates FcγR bearing cytotoxic effector cells. Several FcγRscontribute to classical ADCC responses, but FcγRIIIA+ NK cellshave been demonstrated to elicit this process most effectively(Cooper et al., 2001). Several methods tomeasure ADCC in vitrohave been developed (Lichtenfels et al., 1994; Clémenceau et al.,
2006; Gómez-Román et al., 2006; Kantakamalakul et al., 2006;Parekh et al., 2012), but many assays share the disadvantagethat they rely on inhomogeneous effector cells, which varyregarding type and density of FcγR expression. In addition,these effector cells are difficult to prepare and maintain inculture and thus become limiting in their availability. Thesedifficulties could be overcome when using a clonal effector cellpopulation expressing one defined FcγR. We have establisheda set of novel reporter cells for measuring the capacity ofvirus-specific IgG to trigger defined types of FcγRs. The assayscomprise the co-cultivation of virus-infected target cells withtransfectants stably expressing a chimeric FcγR in the presenceof poly- or monoclonal immune IgG. The FcγR chimeras arebearing the extracellular domain of defined FcγRs, i.e. humanFcγRIIIA, FcγRIIA and FcγRI, which are fused to the transmem-brane and intracellular domains of the mouse CD3-ζ chain.Upon activation of the chimeric FcγRs, mouse IL-2 is secretedand can be easily measured by ELISA. Thus, the assay systemquantifies the capacity of virus-immune IgG to trigger FcγRs in areceptor type-specific manner upon recognition of naturallyformed epitopes displayed on the surface of infected target cells.
2. Materials and methods
2.1. Cell lines, viruses and infection conditions
Human MRC-5 lung fibroblasts (ATCC CCL-171), humanHEp-2 (ATCC CCL-23), human HEK 293T (ATCC CRL-11268),African green monkey Vero cells (ATCC CCL-81), TZM-bl cells(Wei et al., 2002) and CD20 transfected 293T cells (a kind giftfrom Irvin S. Y. Chen, University of California) (Morizonoet al., 2010) were maintained in D-MEM (Invitrogen Corp,Life Technologies Corp., Darmstadt, Germany) supplementedwith 10% (v/v) heat-inactivated fetal calf serum (FCS),penicillin (100 U/ml), streptomycin (100 μg/ml) and gluta-mine (2 mM). EBV-transformed marmoset B95-8 cells (ATCCCRL-1612), human U937 cells (ATCC CRL-1593.2), mouseBW5147 thymoma cells (ATCC TIB-47™) and transfectantsthereof were maintained in RPMI 1640 medium containing10% (v/v) FCS, penicillin, streptomycin, glutamine, and sodiumpyruvate (1 mM). BW:FcγR-ζ transfectants were selectedin RPMI medium containing 3 mg/ml G418 (Sigma-Aldrich,Seelze, Germany) or 50 μg/ml Zeocin™ (Invitrogen Corp, LifeTechnologies Corp., Darmstadt, Germany).
The following viruses were used: HCMV strains AD169(Hengel et al., 1995) and HB5 (Borst et al., 1999), mousecytomegalovirus strainMW97.01 (Wagner et al., 1999), HSV-1F(Dingwell et al., 1994), RSV-A strain Long (M.A. Bioproducts,Boston, USA. No. 30–875J) and the MV strain Edmonston-Enders (CSL Behring, Hattersheim amMain, Germany. No. OKEL04). RSV-A clinical isolate was kindly provided by O. Adams,Heinrich-Heine-University Düsseldorf, Germany. Infection ofadherent cells with HCMV, HSV-1F, MV and RSV was enhancedby centrifugation at 800×g for 30 min. If not indicatedotherwise, cells were infected with 2–3 PFU/cell.
2.2. mAbs, human immunoglobulin preparations, human serumsamples and blood donors for effector NK cells
The humanized RSV-specific IgG1 mAb palivizumab(Synagis®,MedImmune, Inc, Gaithersburg,MD, USA) recognizes
Bold: 5′ sequence of FcγR; italics and bold: zeta chain; underlined:restriction enzyme cutting sequence.
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the fusion protein F of RSV (The IMpact-RSV StudyGroup, 1998).The humanized anti-CD20 mAb rituximab (Mabthera®, Roche)was purchased as IgG1, IgG2, IgG3, IgG4 isotypes and IgA fromInvivoGen, Toulouse, France. The humanized anti-HER2 IgG1mAb trastuzumab (Herceptin®) was from Genentech, Inc., SanFrancisco, USA. The humanized b12 and LALA antibodies againstHIV-1 env-coded gp120 were kindly provided by Dr. DennisBurton (The Scripps Research Institute, La Jolla, California, USA).The IVIG preparation Cytotect® (Snydman et al., 1987; Nigro etal., 2005; Hoetzenecker et al., 2007) (batch no. A158024, BiotestPharma GmbH, Dreieich, Germany) containing ELISA-reactivehuman IgG against HCMV, HSV, RSV, EBV andMVwas used as asource of polyclonal human virus-immune IgG. Cytotect® ismanufactured from plasma of healthy volunteer donors (4,500–5,000 donors per batch) from Germany, Austria, Belgium andUSA who are selected for high anti-HCMV titres by ELISA-basedtesting.
For serum preparation and effector cell isolation bloodwas drawn from healthy volunteers after written informedconsent. The experiments were approved by the EthicsCommittee of the University Hospital Düsseldorf (no. 3410)in accordance with the Declaration of Helsinki. As a source ofnon-immune polyclonal IgG, a serum pool of seven MV-ELISAseronegative donors, a serum pool of two HSV/HCMV ELISAseronegative donors and one serum of an EBV-seronegativedonor were used. For assays against HIV-1 gp120, a pool offive different HIV-1 seropositive donors with a viral loadbeneath the detection limit and five seronegative donors asa negative control were used. Purified IgG and IgM fromhuman serum and IgG-depleted human serum as well aspurified human IgG subclasses were purchased from Sigma-Aldrich (Seelze, Germany). For detection of human IgG byflow cytometry, Fc-specific goat anti human IgG-FITC waspurchased from Sigma-Aldrich, Seelze, Germany.
For crosslinking experiments, GAM IgG (Sigma-Aldrich,Seelze, Germany) was used as a secondary reagent afterdecoration of cells with mouse mAbs specific for humanCD16-A/B (SantaCruzBiotechnology, Inc,Heidelberg, Germany),human CD32 (Santa Cruz Biotechnology, Inc, Heidelberg,Germany), human CD64 (Ancell Corporation, Minnesota, USA)and anti-human CD99 (Becton Dickinson, New Jersey, USA).
To obtain Fab-γ and Fc-γ fragments from IVIG, Cytotect®was cleaved using the ImmunoPure® Fab Preparation Kit (PierceProtein Biology, Illinois, USA) according to the manufacturer'sinstructions.
2.3. IgG and IgM ELISAs
Detection of global amounts of virus-specific IgG and IgMwas conducted using ELISA tests from Dade Behring (batchno. 36074 [HSV IgG], 36468 [HCMV IgG], 36294 [MV IgG],36364 [MV IgM], 36331 [EBV]), from Novagnost TM (batchno. RSV-G022 [RSV]) and from LIAISON DiaSorin (batch no.050045/1 [CMV-IgG]).
2.4. Cloning of FcγR-ζ constructs
Cloning of the cDNAs encoding the human FcγRs FcγRIIIA(higher affinity variant with a valine at position 158 (Bowlesand Weiner, 2005)), FcγRIIA (with histidine at position 131)and FcγRI have been described previously (Allen and Seed,
1989; Stengelin et al., 1988). The extracellular portion ofFcγRs was amplified by PCR using a pair of primers, whichcomprise a SpeI restriction site at the 5′-end and the first ninenucleotides of mouse CD3ζ chain transmembrane portion atthe 3′-end (Table 1). The mouse CD3ζ-chain was amplified byPCR using primers, which include the last nine nucleotides ofthe extracellular portion of each FcγR on the 5′-end and theprimer 5′-AAG GCG GCC GCC TTA GCG AGG GGG CCA GGGTCT G-3′ including a NotI restriction site (underlined) at the5′-end. The two amplified fragments were combined anddouble template PCRs were performed with the 5′ SpeI primerand the 3′ NotI primer for the generation of the respectivechimeric FcγR-ζ constructs. The FcγR-ζ constructs were clonedinto pcDNA3.1 expression vector (Invitrogen Corp., California,USA) or into a pcDNA3.1 modified with a Zeocin™ resistancecassette replacing the Geneticin resistance. Constructs wereverified by restriction digest and DNA sequencing (data notshown). The BW:FcγRIIIA-ζ and BW:CD99-ζ transfectants havebeen previously described (Mandelboim et al., 2001, 1999).
2.5. Generation of stable FcγR-ζ BW5147 transfectants andsurface detection of chimeric receptors
TCRαβγζ negative BW5147 thymoma cells were trans-fected with above mentioned constructs using Superfect(Qiagen GmbH, Hilden, Germany) or AMAXA Nucleofection KitV (Lonza Group, Cologne, Germany) following manufacturer'sinstructions. FcγR surface expression was assayed by FACS(FACS CantoII, Becton Dickinson, USA) using mouse anti-human CD16-FITC (Miltenyi Biotec GmbH, Bergisch-Gladbach,Germany), mouse anti-human CD32-FITC (BD Pharmingen™,Erembodegem, Belgium) or mouse anti-human CD64-FITC (BDPharmingen™, Erembodegem, Belgium). To test binding ofmonomeric IgG on the surface of transfectants, human Fcγ-FITC (Rockland Immunochemicals, Pennsylvania, USA) wasused. Analysis was performed using Flowjo Software (Tree StarInc, Oregon, USA).
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2.6. IgG-dependent activation of the BW:FcγR-ζ reporter cells
Assessment of IgG-dependent activation of the BW:FcγR-ζcells was done by incubating mock and virus-infected cells(targets)with two-fold serial dilutions of human sera (or IVIG) inD-MEM10% (v/v) FCS for 30 min at 37 °C in an atmosphere of 5%CO2. The range of total IgG concentrations used for opsonizationwas used as indicated (3.5–0.0035 mg/ml). To remove non-immune IgG, cells were washed three times with D-MEMcontaining 10% (v/v) FCS before co-cultivation with BW:FcγR-ζ reporter cells for 16 h in RPMI 10% (v/v) FCS medium.If not indicated otherwise, experiments were performedin triplicate and the ratio between effector (BW:FcγR-ζtransfectant) and virus-infected target cells was 20/1. Afterco-cultivation for 16 h at 37 °C in a 5% CO2 atmosphere,supernatants were diluted 1/2 in ELISA sample buffer (PBS with10% [v/v] FCS and0.1% [v/v] Tween-20) andmIL-2wasmeasuredby ELISA using the capture Ab JES6-1A12 and the biotinylateddetection Ab JES6-5H4 (BD Pharmingen™, Belgium). Thedetection limit was ~3 pg/ml of IL-2 (data not shown).
When applied to suspension cells, the BW:FcγR-ζ assay wasperformed in V-bottom cell culture wells. 1×104 EBV-infectedB95-8 cellswere added to prediluted sera in RPMI 10% (v/v) FCS.Incubation of cells for 30 min at 37 °C in an atmosphere of 5%CO2 was followed by three rounds of washing by centrifugationto remove unbound IgG. Co-cultivation and IL-2 detection wasperformed as described above. For determination of BW:FcγR-ζcells activation depending on the effector to target (E/T) ratio,mock and HSV-1 infected Vero cells were resuspended, countedand added to prediluted sera in RPMI 10% (v/v) FCS in aV-bottom cell culture well plate. Incubation of cells for 30 minat 37 °C in an atmosphere of 5% CO2 was followed by threerounds of washing by centrifugation to remove unbound IgG.Co-cultivation with variable amounts of BW:FcγR-ζ cells wasdone and IL-2 detection was performed as described above.
To assess activation of the BW:FcγR-ζ cells by IgG aloneor by immune complexes, purified commercially availablehuman IgG or purified HCMV and MCMV virion preparations,respectively, were coated on plates using coating buffer (0.1 MNa2HPO4; pH 9.0).
For assessment of stability in functional response, a batchof BW:FcγRIIIA-ζ cells was passaged 15 times during severalmonths. Different passages were maintained frozen in liquidnitrogen before simultaneous thawing and testing.
For evaluation of antibody binding and activation of BW:FcγRIIIA-ζ cells by HIV-1 env-coded gp120, 4×104 TZM-bl cellswere transiently transfected with SVcrev (Schaal et al., 1993;Krummheuer et al., 2001) and SV-env/GAR (Caputi et al., 2004;Kammler et al., 2001) plasmids using polyethylenimine (Sigma-Aldrich). 48 h later, transfection was monitored by syncytiaformation. BW:FcγRIIIA-ζ activation assay was performed asdescribed above using a pool of five different HIV-1 seropositivedonors and a pool of five seronegative donors as a negativecontrol or two previously described humanized monoclonalantibodies, b12 and LALA.
2.7. IgG subclass binding to FcγR-ζ receptors
Wildtype full length FcγRs were amplified from cDNA ofhuman U937 (ATCC CRL-1593.2) cells using a pair of primers,which include SpeI restriction sites on the 5′-end and one
that includes a NotI restriction sites on the 3′-end (seeTable 1) and verified by DNA sequencing (data not shown).HEK-293T cells were transfected using SuperFect (Qiagen,Germany) with pcDNA3.1 plasmids expressing wildtype fulllength FcγRs or FcγR-ζ constructs and detached from theplate after 48 h using Accutase (Millipore, Life Sciences,Darmstadt, Germany). Resuspended HEK-293T cells wereincubated with 10 μg/ml of trastuzumab (Herceptin®IgG1),palivizumab (Synagis® IgG1), IVIG (polyclonal IgG), humanIgG1, IgG2, IgG3 or IgG4, resp., (Sigma-Aldrich, Germany) for1 h at 4 °C. Cells were washed and stained with anti-humanIgG Fab2-PE (Sigma-Aldrich, Germany). After washing, cellswere counterstained with anti-FcγR-FITC. Ratio of IgG bindingto FcγR wt vs. FcγR-ζ molecules of double stained cells (IgG+
and FcγR+) was calculated.For subclass IgG dependent activation of the BW:FcγR-ζ
cells, CD20 transfected 293T cells were incubated with dif-ferent concentrations of the anti-hCD20 IgG isotype collection(InvivoGen, France). To remove non-bound IgG, cells werewashed three times with D-MEM containing 10% (v/v) FCSbefore co-cultivation with BW:FcγR-ζ reporter cells for 16 h inRPMI 10% (v/v) FCS medium. After co-cultivation for 16 h at37 °C in a 5% CO2 atmosphere, mIL-2 was measured by ELISA.
2.8. CD107a NK cell degranulation assay
NK cells were purified from PBMCs by Lymphoprep(Axis-Shield, Oslo, Norway) differential centrifugation, followedby magnetic sorting through a MACS NK cell isolation kit(Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells werepreactivated overnight with 100 IU/ml of human recombinantIL-2 (PromoKine, PromoCell, Heidelberg, Germany). Goat anti-mouse Fab2 fragment (Sigma-Aldrich, USA) was coated at10 μg/ml on plastic overnight at 4 °C. After washing, gradedconcentrations of mouse anti-human CD16 (BD Pharmingen™,Belgium) were added and the plate was washed after 30 min ofincubation at 37 °C. 1×105 NK cells were added to each welland the CD107a assay was performed as described previously(Alter et al., 2004).
3. Results
3.1. Stable expression of FcγR-ζ chimeras mediates activation ofBW:FcγR-ζ transfectants
Mouse BW5147 thymoma cells lack functional expressionof TCR α, β, γ and ζ chains (Bonifacino et al., 1988; Letourneurand Malissen, 1989; Wegener et al., 1992) but can bereconstituted to secrete IL-2 upon cross-linking of transfectedCD3-ζ chains or chimeras composed of surface receptors andthe ζ-chain (Wegener et al., 1992). BW5147 cells are thereforeinstrumental for the functional analysis of chimeric surfacereceptors, which utilize the intracellular TCR signal transduc-tionmachinery.We stably transfected BW5147 cells, which areknown to lack expression of endogenous Fc receptors (Daëronet al., 1985), with constructs encoding chimeric FcγRs in whichthe extracellular domains of human FcγRIIIA (Valine 158),FcγRIIA (Histidine 131) and FcγRI, respectively, were fused tothe transmembrane and intracellular domains of the CD3-ζchain (schematically depicted in Fig. 1A). Upon binding of IgGto the FcγR, a TCR-like signal is initiated leading to secretion of
Fig. 1. Stable expression of FcγR-ζ constructs and activation of BW:FcγR-ζ transfectants. A) Schematic representation of FcγR-ζ chimeras. Extracellular domains ofhuman FcγR (white) were fused to the transmembrane domain (light grey) and the signaling module of mouse CD3ζ (dark grey). B) Detection of FcγR chimerason BW5147 transfectants by FACS. Black line, bold: anti-FcγR-FITC mAb. Light grey line: control Ab (anti-CD66-FITC). Medium grey line, bold: Fcγ-FITC.C) Crosslinking experiments with mAb directed against the ectodomain of FcγRs prove intact signal transduction. Goat anti-mouse IgG was coated into 96-wellcell culture plates (2 μg/ml). After blocking and washing, mouse mAbs specific for human CD16-A/B, human CD32 and human CD64 were added. As a negativecontrol, anti-human CD99 mAb was used. After removal of unbound antibodies, 200,000 BW:FcγR-ζ transfectants were added per well. mIL-2 secretion wasdetermined after 16 h.
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mouse IL-2 (mIL-2), which can be accurately quantified byELISA. FACS analysis of stable transfectants demonstrated highlevels of surface expression of chimeric receptors and bindingof Fcγ-FITC fragment, indicating that the IgG Fc-bindingdomains remained functionally intact (Fig. 1B). To proveintegrity of the CD3-ζ chain chimeras, mAbs directed againsteach of the FcγR ectodomains were used in cross-linkingexperiments. All transfectants were highly responsive to thecognate α-FcγR Ab but not to incubation with Abs specific forother FcγR molecules (Fig. 1C).
In conclusion, all BW:FcγR-ζ transfectants respondedefficiently when crosslinked by specific mAbs directed tothe extracellular domain of the chimeric FcγR.
3.2. Conditions of IgG binding required for activation of BW:FcγR-ζ transfectants
To establish experimental conditions for Fc-γ-mediated acti-vation of the BW:FcγR-ζ transfectants, grading concentrations
of commercially available purified human IgG, purified humanIgM and IgG-depleted human serum were coated on plasticplates. Human IgG, but not IgM or IgG-depleted serum,activated the human FcγR reporter cells in a dose-dependentmanner (Fig. 2A depicts representative results obtained withBW:FcγRIIIA-ζ transfectants), indicating that the response isstrictly IgG-dependent. The influence of IgG concentration onthe activation of the BW:FcγR-ζ reporter cells was determinedby using variable amounts of IgG. In contrast to the negativecontrols, a clear dose-dependent activation by IgG wasobserved (Fig. 2A). Thus mIL-2 production induced by theactivation of the chimeric receptors correlated over a widerange with the amount of IgG used for FcγR activation. To testactivation by immune complexes, purified HCMV or mousecytomegalovirus (MCMV) virions were panned on plates andopsonized with grading concentrations of IVIG as a source ofHCMV-specific immune IgG. HCMV particles exerted a strongactivation of FcγRIIIA-ζ reporter cells. No response was detect-able with MCMV virions upon incubation with IVIG (Fig. 2B),
Fig. 2. IgG mediated activation of BW:FcγR-ζ transfectants. A) Purified IgG, IgG-free serum or purified IgM were coated to a plate in binding buffer (0.1 MNa2HPO4 pH 9.0) at different concentrations (5 mg/ml to 0.05 mg/ml). After blocking and washing, 1×105 cells per well of the BW:FcγR-ζ transfectants wereadded. Triplicates were measured. B) Virion preparations of HCMV strain HB5 and MCMV strain MW97.01 were coated in binding buffer (0.1 M Na2HPO4 pH 9.0).After blocking and washing, graded dilutions of Cytotect® (5 mg/ml to 0.05 mg/ml) were added to coated virions and incubated 30 min at 37 °C. After removal ofunbound antibodies, 1×105 cells per well of the BW:FcγR-ζ transfectants were added. Triplicates were measured. C) MRC-5 cells were infected with 2 PFU/cell ofHSV-1. 24 hpi, graded dilutions of Cytotect® (2 mg/ml to 0.02 mg/ml) were added and incubated for 30 min at 37 °C. After removal of unbound antibodies,1×105 BW:FcγR-ζ transfectants were added per well. mIL-2 was determined after 16 h by ELISA. Triplicates were measured. A schematic diagram ofIgG-dependent activation of BW:FcγR-ζ transfectants is shown in the graphics below.
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which is devoid of MCMV-reactive IgGs. Thus, BW:FcγRIIIA-ζtransfectants reliably report the presence of immune IgG boundto epitopes displayed on the surface of viral particles.
To assess the applicability for detection of immunecomplexes composed of human IgG and viral cell surfaceantigens (see graphical abstract, lower panel Fig. 2), we testedIgG-mediated activation of BW:FcγRIIIA-ζ transfectants uponco-cultivationwith HSV-1 infected cells opsonizedwith IVIG asa source of HSV-immune IgG. Dose-dependent triggering wasobserved while mock infected cells did not induce receptoractivation (Fig. 2C). The same type of results were obtainedwith BW:FcγRIIA-ζ and BW:FcγRI-ζ transfectants (data notshown). Taken together, BW:FcγR-ζ reporter cells wereactivated by IgGwhen fixed to amatrix, by immune complexescomposed of IgG and viral particles and by immune complexescomposed of IgG and native antigens displayed on the surfaceof infected cells (schematically depicted in Fig. 2A–C, lowerpanel), suggesting a new cell-based test principle for measur-ing IgG-dependent FcγR activation.
To assess stabilization of response, a batch of BW:FcγRIIIA-ζtransfectantswas passaged several times andmaintained frozenin liquid nitrogen. Cells from different passages were simulta-neously thawed and tested against HSV-F infected fibroblastsopsonized with Cytotect®. The transfectants showed a minimal
functional divergence through passages (data not shown).Therefore, BW:FcγR-ζ transfectants responded to an efficientand comparable extent independent of passage number,allowing their reproducible use for measuring IgG-dependentFcγR activation.
3.3. Validation of the FcγR-activation assay
3.3.1. The Fc and the Fab part of immune IgG are required forFcγR-ζ activation
To prove the dependence on the Fc-γdomain of immune IgGfor the activation of FcγR-ζ receptors, co-cultivation of reportercells with HSV-1-infected fibroblasts was performed uponopsonization with complete IgG (derived from Cytotect® IVIG)vs. papain cleavage products thereof, i.e. Fab-γ and Fc-γfragments. As expected, the BW:FcγR transfectants were solelyactivated by intact immune IgG while neither Fab fragmentsobtained from Cytotect® IVIG nor soluble Fc-γ fragments alonewere sufficient to elicit activation of BW:FcγRIIIA-ζ cells(Fig. 3A). Fab-γ fragments were expected to fail due to theabsence of the Fc-γ part, whereas Fc-γ fragments did notpossess the ability to bind the antigen and thus were removedduring thewashing procedures. Likewise, FcγRIIA-ζ and FcγRI-ζ
Fig. 3. Validation of FcγR activation assay. A) Fc of IgG is required for activation for the BW:FcγR-ζ assay. IgG fractions after cleavage with papain were analyzedfor activation of the BW:FcγRIIIA-ζ effector cells co-cultivated with HSV-1 strain F infected MRC-5 cells. Cytotect® (as a source of purified IgG, input) anduncleaved IgG output were able to activate BW:FcγRIIIA-ζ cells. Fab fragment and Fc-γ fragment resulting from papain digestion were not able to elicit aresponse. B) Viral infectious dose and c) effector to target ratio influence IgG-mediated FcγR-ζ activation. MRC-5 cells were infected with different PFU/ml ofHSV-1. 24 hpi, 30 μg/ml or 15 μg/ml Cytotect® were added and incubated for 30 min at 37 °C. After removal of unbound antibodies, 1×105 BW:FcγR-ζtransfectants were added per well. mIL-2 was determined after 16 h by ELISA. Triplicates were measured. (C) Vero and mock cells were infected with 2 PFU/cellof HSV-1. 24 hpi, cells were resuspended using trypsin, opsonized using 0.5 mg/ml IVIG and incubated for 30 min at 37 °C. After removal of unbound antibodiesby washing and centrifugation, different amounts of effector BW:FcγR-ζ transfectants were added per well. mIL-2 was determined after 16 h by ELISA. Triplicateswere measured.
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receptors also required intact immune IgG to respond tovirus-infected cells (data not shown).
3.3.2. Viral infectious dose and effector to target ratio influenceIgG-mediated FcγR-ζ activation
As shown in Fig. 2A, the activation of the BW:FcγR-ζreporter cells, varied with the amount of used IgG. However,as a consequence of epitope recognition in this settingstimulating IgGs were not forming immune complexes. Toemulate this physiological situation, graded concentrations ofimmune IgG (Cytotect®) were used upon infection of targetcells with HSV at three different multiplicities of infections(% of cells infected). As shown in Fig. 3B, a concentration-dependent activation of the reporter cells was observed withthe different amounts of IgG bound to the target cells, and theused infectious dose had also impact on the extent of theresponse (Fig. 3B). Another factor influencing the magnitudeof the mIL-2 response by BW:FcγR-ζ reporter cells and thus
the detection limit and the sensitivity of the assay theeffector-to-target cell ratio was applied. As demonstrated inFig. 3C, an 12-fold excess of responder cells was requiredto detect HSV-immune IgG in this setting of 16 h of co-incubation, the detection of which was continuously in-creased over a wide range of added BW:FcγRIIIA-ζ respondercells. Taken together, the amount of immune IgG used foropsonization of target cells and the BW:FcγR-ζ reporter-to-target cell ratio were found to be key determinants of thisassay.
3.3.3. Antigen density measured by monoclonal and polyclonalIgG correlates with BW:FcγRIIIA-ζ transfectant activation
An important factor influencing the magnitude of themIL-2 response by BW:FcγR-ζ reporter cells and thus thedetection limit and sensitivity of the assay was supposed tobe the density of the opsonized antigen and the number ofdisplayed epitopes on the target cells. To test whether there
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exists a direct correlation between antigen display on thesurface of infected cells, opsonizing IgG and activation ofreporter cells, Hep-2 cellswere infectedwith RSV and opsonizedusing graded concentrations of a polyclonal antibody prepara-tion, Cytotect®, and a humanizedmonoclonal IgG1 palivizumabSynagis® directed against the F protein of RSV, respectively.BW:FcγRIIIA-ζ responder cells were used to assess the degree ofactivation mediated by antigen bound antibodies. In parallel,antigen density on RSV-infected cells was measured by FACS(Fig. 4A). The BW:FcγRIIIA-ζ transfectants reacted in a dosedependent manner revealing a congruent response curve withopsonized RSV surface antigens as detected by FACS. Theseresults demonstrated a very low detection limit of the BW:FcγRIIIA-ζ based experimental setting comparable with antigendetection by flow cytometry.
To establish a correlation between expression levels of aviral surface glycoprotein and BW:FcγRIIIA-ζ responses variousamounts of HIV-1 gp120 were expressed by transientlytransfecting graded quantities of a HIV-1 env-encoding plas-mid. Activation of BW:FcγRIIIA-ζ responder cells was observed
Fig. 4. Antigen density correlates with IgG-mediated BW:FcγRIIIA-ζ transfectant actwith different concentrations (2 mg/ml to 2.6 E−05 mg/ml) of Cytotect® as ppreparation was performed at 96 hpi. 1×105 BW:FcγR-ζ transfectants were addedmean fluorescence analysis of antigen density using the same dilutions of opsonanti-human IgG-Fc-FITC antibody. (B) 1×104 TZM-bl cells were transiently transfectfor HIV-1 env expression. BW:FcγRIIIA-ζ activation assay was performed 48 h afterhumanized monoclonal antibodies b12 and LALA. 1×105 BW:FcγR-ζ transfectantswere measured.
when a pool of five HIV-1 seropositive donors (Fig. 4B, leftpanel) but not when a pool of HIV-1 seronegative donors (datanot shown)was used. Likewise, BW:FcγRIIIA-ζ responseswereinduced by a previously described gp120-specific humanizedmonoclonal antibody, b12, (Fig. 4B), while a b12mutatedmAb,LALA, which is known to lack FcγR activating capabilities, failedto elicit IL-2 secretion in BW:FcγRIIIA-ζ transfectants (Fig. 4B,right panel). Importantly, BW:FcγRIIIA-ζ responses induced inthe presence of polyclonal or b12 antibodies were dependenton the IgG concentration used for opsonization as well as theamount of gp120 protein expressed as controlled by syncytiaformation (data not shown). Taken together, antigen densityinfluences the detection limit and the sensitivity of the BW:FcγR-ζ assay.
3.3.4. CD16-mediated triggering of BW:FcγRIIIA-ζ transfectantsand primary human NK cells is comparable
FcγRIII (CD16) constitutes an activation receptor ex-pressed on a majority of human NK cells (Mandelboim et al.,1999; Cooper et al., 2001). To compare the CD16-mediated
ivation. A) Hep-2 cells were infected with a RSV clinical isolate. Opsonizationolyclonal antibody preparation and palivizumab Synagis® as monoclonalper well. mIL-2 was determined after 16 h by ELISA in triplicates. In parallel,izing antibody preparations was detected by FACS using a secondary goated with different amounts (90 ng–10 ng) of SVcrev and SV-env/GAR plasmidtransfection using a pool of five different HIV-1 seropositive donors and thewere added per well. mIL-2 was determined after 16 h by ELISA. Triplicates
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responsiveness of BW:FcγRIIIA-ζ transfectants versus primaryhuman NK cells, we monitored mIL-2 secretion from BW:FcγRIIIA-ζ versus CD107a mobilization on NK cells, anestablished test for NK cell degranulation (Alter et al., 2004).Effector responses upon CD16 cross-linking by grading con-centrations of a CD16-specific mAb were measured after 4 hof incubation. BW:FcγRIIIA-ζ and primary human NK cellsreacted in a congruent dose response ratio and revealed similarAb concentrations required for triggering of half maximaleffector response (Fig. 5) highlighting a high sensitivity of theBW:FcγRIIIA-ζ assay and a comparable to response to primaryhuman NK cells.
3.3.5. Binding to IgG subclasses and their activation ofBW:FcγR-ζ transfectants
To ensure that chimeric FcγR-ζ molecules exhibit IgGbinding characteristics as wildtype (wt) FcγRs, we performedtransient transfection experiments with plasmids encodingeither chimeric FcγR-ζ's or wt-FcγR's into HEK-293T cells andsubsequent cytofluorometric analysis using human polyclonalIgG preparation (Cytotect® IVIG), purified individual humanIgG subclasses (IgG1–IgG4) or therapeutic humanized mAbstrastuzumab (Herceptin®), palivizumab (Synagis®). The datarevealed largely congruent IgG binding capacities of wt FcγRsand the corresponding CD3-ζ chain chimeras (Fig. 6A). Inagreement with previously published data (Bruhns et al.,2009), the hierarchy of IgG subclasses monomers binding wasmaintained between the wt and FcγR-ζ expressing constructs(data not shown). Namely, FcγRIIIA bound mostly IgG3 andIgG1, FcγRIIA slightly bound IgG3 and FcγRI strongly bound toall IgG subclasses.
Binding of human IgG to FcγRs is adapted to certainsubclasses (Bruhns et al., 2009). To assess whether the BW:FcγR-ζ-based assay reflects the preferential triggering ofCD16 by defined IgG subclasses, CD20 transfected 293T cells(Morizono et al., 2010) were used as antigenic targets usingan anti-hCD20 IgG isotype collection consisting of thevariable region of rituximab and the constant region of allfour IgG subclasses. The IgA constant region was used a as a
Fig. 5. CD16-mediated triggering of BW:FcγRIIIA-ζ transfectants and primaryhuman NK cells is comparably efficient. After coating of GAM F(ab)2fragment, mouse anti human CD16 was added at different concentrationsfor 30 min at 37 °C. After washing, 1×105 MACS-purified polyclonal NK cellsor BW:FcγRIIIA-ζ transfectants per well were added. CD107a degranulationand mIL-2 secretion, respectively, were measured after 4 h of co-cultivationby FACS and ELISA, respectively.
negative control. Upon opsonization of target cells withgrading concentrations of antibody, BW:FcγRIIIA-ζ cellsresponded to all IgG subclasses but not to bound IgA, albeitwith strikingly different efficiency (IgG1>IgG3>IgG4>IgG2)(Fig. 6B). According to the binding data of immune complexesobserved by Bruhns et al. (2009), we concluded that activationof the BW:FcγRIIIA-ζ reporter cells depends on the subclass ofantigen-bound IgG.
3.4. Activation of chimeric FcγRIIA-ζ and FcγRI-ζ receptors byimmune IgG1
To establish a proof of principle for immune IgG-mediatedactivation of chimeric FcγRIIA-ζ and FcγRI-ζ receptors and tocompare the responsiveness of the respective BW:FcγR-ζreporter cells to BW:FcγRIIIA-ζ, a neutralizing humanizedtherapeutic IgG1 mAb, palivizumab (Synagis®) specific forthe glycoprotein F of RSV (The IMpact-RSV Study Group,1998) was used. HEp-2 cells were infected with an RSVclinical isolate before incubated with increasing concentra-tions of antibody. All FcγR-ζ transfectants responded to a lowconcentration of 0.1 μg/ml palivizumab bound to RSV-infected cells, but not mock-infected cells (data for mocknot shown). However, the responsiveness of BW:FcγR-ζdiffer among the 3 individual FcγRs (Fig. 7). Interestingly,the observed detection limit of approximately 0.1 μg/mlpalivizumab is at least 100 times lower as the concentrationof the mAb found in the serum of patients who profited fromits therapeutic effects at 37–72 μg/ml (The IMpact-RSV StudyGroup, 1998). Altogether, as measured by the responses ofthe individual BW:FcγR-ζ transfectants, the activation ofFcγRs by palivizumab-opsonized target cells did not directlycorrelate with the affinities of the FcγRs for immune-complexed IgG1 (Bruhns et al., 2009) specifically concerningthe activation of BW:FcγRI-ζ transfectants. Nevertheless,palivizumab bound to the RSV-F glycoprotein on the surfaceof infected cells forms immune complexes which are potentinducers of all human FcγR types, suggesting that thisantibody may exert additional protective effector functionsbesides virion neutralization.
3.5. Broad applicability of the assays to detect virus-specific IgG
The data presented so far provided a proof of principlethat FcγR-ζ transfectants respond dose-dependently toimmune IgG when bound to epitopes exposed on HSV- andRSV-infected cells (Figs. 3A, C, D and 7), tumour cells (Fig. 6B)as well as purified HCMV virions (Fig. 2B). To assess furtherparameters of the BW5147 FcγR-ζ transfectant-based assayfor the detection of virus-specific IgG, we applied polyclonalIgG including Cytotect®, an IVIG preparation composed ofthe sera of several thousands of healthy HCMV-immunedonors, as a source for immune IgG against a variety ofviruses. Prior to this experiment, we confirmed the exquisiteantigen specificity of the responses of all the BW5147 FcγR-ζchain transfectants by comparison of pooled human sera,which are non-reactive in established virus-specific IgGELISAs for HSV, HCMV and Measles virus (‘non-immune’ inFig. 8) with those, which are reactive in the virus-specific IgGELISA assays (‘immune’ in Fig. 8). When graded concentrationsof Cytotect® were applied for opsonization of virus-infected
Fig. 6. Validation of FcγR activation assay: (A) Chimeric and wt receptors bind IgG to a similar extent. HEK 293T cells were transfected using SuperFect with FcγRwt or FcγR-ζ constructs and detached after 48 h using Accutase (Millipore). Resuspended HEK 293T cells were incubated with 10 μg/ml of trastuzumab (Her.)Herceptin® (humanized IgG1), palivizumab (Syn.) Synagis® (humanized IgG1), IVIG (Cytotect® polyclonal IgG), IgG1, IgG2, IgG3 or IgG4 for 1 h at 4°C. Cellswere washed and stained with anti-human IgG Fab2-PE. After washing, cells were stained with anti-FcγR-FITC. The ratio of IgG binding from FcγR wt vs. FcγR-ζconstructs of double stained cells (IgG+ and FcγR+) is indicated. (B) IgG subclasses dependent activation of BW:FcγR-ζ transfectants. CD20 transfected 293T cellswere used as antigenic targets using different concentration of an anti-hCD20 isotype collection consisting of the variable region of rituximab and the constantregion of all 4 IgG subclasses. IgA constant region was used a as a negative control. Antibodies were added and incubated for 30 min at 37 °C. After removal ofunbound antibodies, 1×105 BW:FcγR-ζ transfectants were added per well. mIL-2 was determined after 16 h by ELISA. Triplicates were measured.
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target cells, triggering of all BW5147 FcγR-ζ transfectants wasobserved, while control BW5147 CD99-ζ cells did not respond.While all viruses included in the test panel elicited responses,the efficiency of FcγR triggering was strikingly different(Fig. 9). In general, the activation of BW:FcγRIIIA-ζ cells wasmore prominent than BW:FcγRIIA-ζ, while despite of the highaffinity of monomeric IgG to FcγRI (Bruhns et al., 2009) BW:FcγRI-ζ transfectants were the least stimulated. Furthermore,substantial variations among viruses were observed sinceFcγRIIA-ζ and FcγRI-ζ were clearly less activated uponco-cultivationwith HCMV- and EBV-infected cells as comparedto HSV and both paramyxoviruses MV and RSV. Notably, theIgG concentration, which sufficed to elicit a robust FcγRactivation were by far the lowest in the case of HSV-1compared to the other viruses of the panel (Fig. 9 — pleasenote the different IgG concentrations used). In conclusion, theFcγR-ζ-based assays are broadly applicable to various viruses.The titer of virus-specific IgG present within a large pool of
human sera that is able to elicit FcγR responses depends on theparticular virus and the individual FcγR type, and no correla-tion of FcγR titers with the concentration of neutralizingantibodies was observed (data not shown).
4. Discussion
We have developed a new assay designed to detect andquantify virus-immune IgG, which is able to trigger FcγRsupon opsonization of infected target cells. Taking advantageof a comprehensive set of FcγR-ζ chimeric receptors, thebinding of poly- or monoclonal IgG to virus-infected targetcells was translated into IL-2 secretion by stably transfectedBW5147 cells. The simultaneous accurate determination ofIgG specificity on the one hand and FcγR-effector capabilitieson the other hand within the global IgG response will allow abetter definition of correlates of antiviral immunity. Beyondthat, the new assay principle has a broad applicability to
Fig. 8. Antigen specificity of FcγR-ζ responses triggered by immune IgG. MRC-5 cellswith HSV (2 PFU/cell) for 24 h or Measles virus (2 PFU/cell) for 72 h. Afterwards, cesera were compared with ELISA non-reactive (non-immune) sera at 2 mg/ml of IgGratio of 20/1 and cultures were incubated for 16 h. mIL-2 was determined by ELISA
Fig. 7. The humanized antibody palivizumab Synagis® directed againstthe RSV-F protein activates efficiently the BW:FcγR-ζ effector cells: BW:FcγRIIA-ζ and BW:FcγRI-ζ react to IgG in a dose dependent manner. Hep-2cells were infected with a RSV clinical isolate. Opsonization with differentconcentrations (10 μg/ml to 0.1 μg/ml) of palivizumab Synagis® 72 hpi wasperformed. 1×105 BW:FcγR-ζ transfectants were added per well. mIL-2 wasdetermined after 16 h by ELISA. Triplicates were measured.
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immunology including areas where characterization ofpathogenic or protective IgG responses (e.g. autoimmunity,vaccinology) is desirable. Last but not the least, it could guidemolecular designing and functional optimization of thera-peutic IgGs.
4.1. Setup of the assay system
Despite the fact that IgG binding to FcγRs is decisive duringthe induction and the effector phase of immune responses,surprisingly little convenient methodology exists to measurethese immune responses in vitro. This limitation can beattributed to the lack of simple, reliable and standardizedassays, which might contribute to the relative neglect for FcγRtriggering Abs. ADCC measured in vitro represents a surrogatefor FcγRIII-mediated IgG responses. However, the use ofprimary heterogeneous effector cell populations like PBMC orisolated and in vitro propagated NK cell populations oftengenerates problems in ADCC assays due to the considerablevariability in FcγR and NK cell marker expression as wellas a fluctuating activation status of primary effector cells.
were infected with HCMV HB5 (2 PFU/cell) for 72 h, Vero cells were infectedlls were opsonized with IgG of pooled human sera. ELISA-reactive (immune)concentration. After washing, BW:FcγR-ζ effector cells were added at an E/T. Triplicates were measured. * indicates ODb0.05.
Fig. 9. Detection of virus-immune IgG triggering host FcγRs in a large human IVIG IgG preparation. MRC-5 cells were infected with HCMV AD169 (2 PFU/cell) for72 h, Vero cells were infected with HSV (2 PFU/cell) for 24 h and measles virus (2 PFU/cell) for 72 h, Hep-2 cells were infected with RSV (2 PFU/cell) for 72 h,and the EBV-infected B95.8 cell line were opsonized with IVIG Cytotect® at the indicated IgG concentrations. After removal of unbound IgG, BW:FcγR-ζ effectorcells were added at a E/T ratio of 20/1 and cultures were incubated for 16 h. Amount of mIL-2 were determined by ELISA. Note that the same dilutions of IVIGwere used for all viruses except HSV-1. n.t: not tested. Triplicates were measured.
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This imponderability makes the interpretation of test resultsdifficult. As a consequence, the assessment of immune IgGtitres using ADCC assays is neither reliable nor sensitive andfor these reasons unsuitable for routine diagnostics. Our testsystem has several advantages over the traditional ADCCassays: i) a homogeneous effector cell population expressingonly one defined FcγR omitting the need of cell preparationfrom cell donors; ii) a high intra- and interassay reproducibilitybased on a constant and unlimited effector cell population andavailable immune IgG standards; iii) a comprehensive panel ofFcγR-bearing responder cells allowing to differentiate IgG–FcγR responses; iv) low detection limits; and v) an excellentsensitivity of the test generating quantifiable data. In practicalterms, the BW:FcγR-ζ effector cells are relatively easy tomaintain and the assay procedure does not require radioactiveisotopes.
4.2. Viral antigens activating FcγRs
Detection of virus-immune Ab in classical neutralizationassays is based on very few antigenic determinants present onthe virion surface, which are involved in critical steps of virusattachment and target cell entry. In contrast, the bulk ofvirus-immune IgG generated in response to infection isdirected to non-neutralizing epitopes formed by structuralsurface proteins, non-structural viral polypeptides and internalproteins of the virion, which are all lacking neutralizingactivity. These IgG specificities comprise the bulk of biophysicalbinding Ab reacting in ELISA tests. The targets of the IgGdetected in the BW:FcγR-ζ assays represent a discrete class ofvirus antigens that are characterized by their disposition on thesurface of the infected host cell and thus include both structuraland non-structural transmembrane proteins, depending on the
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protein coding content of a given virus. However, the new testprinciple records those IgGs that become able to activate adefined FcγR when bound to a viral determinant in its nativeconformation displayed on the surface of an infected cell.Several features will enhance or restrict this ability, includingthe subclass of the bound IgG molecule, the number and thedensity of the epitopes on the target cell surface, the fluidity ofthe target cell membrane, which controls IgG dislocation uponFcγR ligation, and conformational rearrangements of theformed immune complex (Woof and Burton, 2004).
4.3. Comparing IgG-mediated FcγR responses to viruses
Using a very large and representative human IgG pool(Cytotect®), we were able to observe a notably differentmagnitude of BW:FcγR-ζ responses directed to specific viruses,among which the herpes viruses HCMV and EBV induced theweakest reactivities (Fig. 9). This is unexpected considering thevery large coding capacity (EBV 184 kbp, HCMV 235 kbp) andproteome of the herpes viruses encompassing up to 60transmembrane glycoproteins many of which have a potentialcell surface disposition. In contrast, MV and RSV representserologically monotypic viruses expressing only very fewantigenic transmembrane glycoproteins, i.e. HA and F or G, Fand SH, respectively, which reach a high density on the cellsurface (Forthal et al., 1993, 1994; Osiowy et al., 1994; Caneet al., 1996; Wu et al., 2005). With regard to HCMV, severalfeatures of the virus are likely to dampen the FcγR activatingefficiency of the IgG response: i) the expression of virus-encoded FcγRs binding to the Fc domain of HCMV-immune IgGthus preventing host FcγR activation (Sprague et al., 2008;Lilley et al., 2001; Budt et al., 2004; Atalay et al., 2002) and(Corrales-Aguilar, E.; Merce-Maldonado, E. and Hengel, H.unpublished observation); ii) retrieval of highly immunogenicneutralizing glycoproteins like gB from the cell surfaceresulting in very low surface densities (Radsak et al., 1996)and iii) a considerable sequence variability of certain non-essential transmembrane glycoprotein-encoding genes encodingpotential IgG targets on infected cells (Dolan et al., 2004). Thesefeatures further imply that the antigen display between HCMVparticles and infected target cells substantially differs. Inter-estingly, HSV-1, which also belongs to the herpes virus familyand possesses virally encoded FcγRs (Johnson and Feenstra,1987; Dubin et al., 1991), induced the strongest reactivity inthe assay. This can be explained by the fact that anti-HSV IgGtitre in IVIG determined in ELISA assays exceeds considerablythe IgG against other herpes viruses. Consequently, IgG re-sponses to virus-infected cells segregate and could representindependent correlates of immunity.
4.4. FcγR activating IgG — a correlate of viral immunity?
Many clinical observations as well as experimental resultsin B cell deficient mice have established a crucial role ofimmune IgG in preventing virus infection and the resolutionof disease (Sanna and Burton, 2000; Parren and Burton,2001). Neutralization of virus infectivity by Ab is an effectiveway to blunt infection in vitro, attributing many examples ofvaccine-mediated protection to neutralizing IgG. Also, IgGrecognizing surface antigens is predisposed to mediate directantiviral effects when interacting with FcγR-bearing immune
effector cells, when activating complement or by modulationof viral replication in infected cells (Burton, 2002). A numberof persisting viruses replicate in the presence of neutralizingIgG (Jonjić et al., 1994; Polić et al., 1998), supporting the ideathat neutralizing antibodies may not generally be suitable asan adequate correlate of humoral immunity and effective as atherapeutic tool. Recently, Hessell et al. identified FcγR butnot complement binding IgG to be crucial in IgG-mediatedprotection against SHIV in macaques (Hessell et al., 2007).Here we show that by a systematic exchange of the heavychain determining the IgG subclass, dramatic consequencescan be observed for the ability of immune complexes toactivate FcγRIII (Fig. 6B). This finding is clearly addressingthe need to differentiate the effector profiles of IgGs despitesharing preserved antigen specificity. The application of thenovel FcγR-specific methodology should provide furtherinsight into the subcomposition of virus-specific IgG anddefine protective Ab subfractions in response to infection andvaccination (Pulendran and Ahmed, 2011).
4.5. Future applications
When adapted to Fc receptors specific for other Igsubclasses like IgA or IgM, the test should readily beextendable to the detection of immune IgA or IgM responses.Besides the versatile application as a new immunoassay toolfor medical virology, parasitology or bacteriology, the assayprinciple described here can be used in many areas ofimmune diagnostics and development. Ample of evidencewas collected over the last few years demonstrating that theengagement of defined FcγRs is of extreme importance forthe therapeutic effects of tumor-specific IgG (Clynes et al.,2000; Nimmerjahn and Ravetch, 2005; Lowe et al., 2007),anti-inflammatory effects of intravenous IgG (Kaneko et al.,2006; Park-Min et al., 2007) and IgG-mediated autoimmunediseases. This knowledge has greatly stimulated approachesto engineer IgG as a therapeutic tool for immune interven-tion. The new assay system described here could be helpful toimprove the effectiveness of such IgGs by designing optimalFc-γ–FcγR interactions.
5. Conclusions
We established a set of novel reporter cells for measuringthe capacity of virus-specific IgG to trigger defined types ofFcγRs. The assay system quantifies the capacity of virus-immune IgG to trigger FcγRs in a receptor type-specificmannerupon recognition of naturally formed epitopes displayed on thesurface of infected target cells. Substantial differences be-tween viruses opsonized with a large pool of immunoglobulinpreparation to activate FcγRs was observed. Furthermore,individual types of FcγRs were triggered to a different extent,revealing FcγRIIIA responses to be most prominent. Targetcells infected with various human pathogenic viruses evokeddose-dependent IgG responses demonstrating its universalapplication. Taken together, a new reliable and simple tool formeasuring antibodies triggering activation of the host Fcγreceptors was generated. This assay could be extremely usefulfor defining a new IgG antiviral subfraction and will aid in thedevelopment of new therapeutic IgGs.
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Acknowledgments
We thank D. Johnson (Oregon Health and SciencesUniversity, Portland, OR), O. Adams (Institute for Virology,Heinrich-Heine-University Düsseldorf) for providing virusesand sera, B. Seed (Massachusetts General Hospital, Boston,MA) for cDNA constructs of human CD16, CD32 and CD64,I. S. Y. Chen (University of California) for CD20 expressing cells,G. Ebers (Biotest, Dreieich, Germany) for Cytotect® and Ann J.Hessell and Richard Burton for b12 and LALA antibodies (TheScripps Research Institute, La Jolla, California, USA). TZM-blcells were obtained through the NIH AIDS Research andReference Reagent Program, Division of AIDS, NIAID, NIHfrom Dr. John C. Kappes, Dr. Xiaoyun Wu and Tranzyme Inc.The expert technical assistance of A. Voges and M. Thieme isgratefully acknowledged.
Role of funding source
This work was supported by funds of the DeutscheForschungsgemeinschaft (He 2526/6-2, GK1045) and theEuropean Commission (QLRT-2001-01112, MRTN-CT-2005-019248). ECA was supported by the German AcademicExchange Service (DAAD). The funders had no role in studydesign, data collection and analysis, decision to publish, orpreparation of the manuscript.
Conflict of interests
The authors have declared that no competing interestsexist.
Authors' contributions
ECA, MT, HR, EMM, MW, HS, and HH conceived anddesigned the experiments. ECA, MT, HR, EMM, and MWperformed the experiments. ECA, MT, and HH analyzed thedata. AZ and OM contributed reagents/materials/analysistools. ECA, MT, and HH wrote the paper.
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