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• W2019510500 abstract "Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease associated with lymphoproliferative neoplasms and characterized by antibodies against plakins and desmoglein 3 (Dsg3). Anti-Dsg3 antibodies have a primary role in blister formation in PNP. In this study, we used phage display to clone monoclonal anti-Dsg3 antibodies from a PNP patient to further characterize their pathogenicity. We isolated 20 unique Dsg3-reactive mAbs, which we classified into four groups according to the heavy-chain complementarity-determining region 3 (CDR3) region. Genetic analyses demonstrated that three antibody groups used the VH1-46 gene (18 clones) and one group used the VH1-02 gene (2 clones). The results of an in vitro keratinocyte dissociation assay and a human skin organ culture injection assay showed that three antibodies displayed pathogenic activity in blister formation with different potencies. Epitope mapping using domain-swapped Dsg3/Dsg2 showed that these pathogenic mAbs bound Ca2+-dependent conformational epitopes in the middle portion of the extracellular region of Dsg3 (EC2 and EC3 domains), in contrast to most previously characterized pathogenic pemphigus vulgaris antibodies, which bound to the EC1 domain of Dsg3. These mAbs reflect the unique polyclonal nature of anti-Dsg3 antibodies in PNP and represent an important tool for detailing the pathophysiological mechanisms of blister formation in PNP. Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease associated with lymphoproliferative neoplasms and characterized by antibodies against plakins and desmoglein 3 (Dsg3). Anti-Dsg3 antibodies have a primary role in blister formation in PNP. In this study, we used phage display to clone monoclonal anti-Dsg3 antibodies from a PNP patient to further characterize their pathogenicity. We isolated 20 unique Dsg3-reactive mAbs, which we classified into four groups according to the heavy-chain complementarity-determining region 3 (CDR3) region. Genetic analyses demonstrated that three antibody groups used the VH1-46 gene (18 clones) and one group used the VH1-02 gene (2 clones). The results of an in vitro keratinocyte dissociation assay and a human skin organ culture injection assay showed that three antibodies displayed pathogenic activity in blister formation with different potencies. Epitope mapping using domain-swapped Dsg3/Dsg2 showed that these pathogenic mAbs bound Ca2+-dependent conformational epitopes in the middle portion of the extracellular region of Dsg3 (EC2 and EC3 domains), in contrast to most previously characterized pathogenic pemphigus vulgaris antibodies, which bound to the EC1 domain of Dsg3. These mAbs reflect the unique polyclonal nature of anti-Dsg3 antibodies in PNP and represent an important tool for detailing the pathophysiological mechanisms of blister formation in PNP. complementarity-determining region 3 desmoglein 3 exfoliative toxin A pemphigus foliaceus paraneoplastic pemphigus pemphigus vulgaris single-chain variable fragment Pemphigus is a group of autoantibody-mediated blistering diseases of the skin and mucous membranes. Classic pemphigus includes two major subtypes: pemphigus vulgaris (PV) and pemphigus foliaceus (PF) (Stanley and Amagai, 2006Stanley J.R. Amagai M. Pemphigus, bullous impetigo, and the staphylococcal scalded-skin syndrome.N Engl J Med. 2006; 355: 1800-1810Crossref PubMed Scopus (350) Google Scholar). Autoimmune targets for these diseases are desmoglein (Dsg) 3 and Dsg 1, respectively. Dsg3 and Dsg1 are members of the cadherin family of cell–cell adhesion molecules localized in desmosomes (Amagai et al., 1991Amagai M. Klaus-Kovtun V. Stanley J.R. Autoantibodies against a novel epithelial cadherin in pemphigus vulgaris, a disease of cell adhesion.Cell. 1991; 67: 869-877Abstract Full Text PDF PubMed Scopus (870) Google Scholar; Stanley, 1993Stanley J.R. Cell adhesion molecules as targets of autoantibodies in pemphigus and pemphigoid, bullous diseases due to defective epidermal cell adhesion.Adv Immunol. 1993; 53: 291-325Crossref PubMed Scopus (224) Google Scholar; Amagai, 1996Amagai M. Pemphigus: autoimmunity to epidermal cell adhesion molecules.Adv Dermatol. 1996; 11: 319-352PubMed Google Scholar). Paraneoplastic pemphigus (PNP) is a unique subtype of pemphigus associated with underlying neoplasms (Anhalt et al., 1990Anhalt G.J. Kim S.C. Stanley J.R. et al.Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.N Engl J Med. 1990; 323: 1729-1735Crossref PubMed Scopus (896) Google Scholar; Mehregan et al., 1993Mehregan D.R. Oursler J.R. Leiferman K.M. et al.Paraneoplastic pemphigus: a subset of patients with pemphigus and neoplasia.J Cutan Pathol. 1993; 20: 203-210Crossref PubMed Scopus (55) Google Scholar). PNP is clinically characterized by the presence of intractable stomatitis and polymorphous skin eruptions. Histological findings include suprabasal acantholysis, keratinocyte necrosis, and vacuolar interface dermatitis (Anhalt et al., 1990Anhalt G.J. Kim S.C. Stanley J.R. et al.Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.N Engl J Med. 1990; 323: 1729-1735Crossref PubMed Scopus (896) Google Scholar). PNP patients are known to possess characteristic autoantibodies against several members of the plakin family (desmoplakins I and II, BPAG1, envoplakin, periplakin, and plectin; Anhalt et al., 1990Anhalt G.J. Kim S.C. Stanley J.R. et al.Paraneoplastic pemphigus. An autoimmune mucocutaneous disease associated with neoplasia.N Engl J Med. 1990; 323: 1729-1735Crossref PubMed Scopus (896) Google Scholar; Oursler et al., 1992Oursler J.R. Labib R.S. Ariss-Abdo L. et al.Human autoantibodies against desmoplakins in paraneoplastic pemphigus.J Clin Invest. 1992; 89: 1775-1782Crossref PubMed Scopus (162) Google Scholar; Hashimoto et al., 1995Hashimoto T. Amagai M. Watanabe K. et al.Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis.J Invest Dermatol. 1995; 104: 829-834Crossref PubMed Scopus (167) Google Scholar; Kim et al., 1997Kim S.C. Kwon Y.D. Lee I.J. et al.cDNA cloning of the 210-kDa paraneoplastic pemphigus antigen reveals that envoplakin is a component of the antigen complex.J Invest Dermatol. 1997; 109: 365-369Abstract Full Text PDF PubMed Scopus (95) Google Scholar; Borradori et al., 1998Borradori L. Trueb R.M. Jaunin F. et al.Autoantibodies from a patient with paraneoplastic pemphigus bind periplakin, a novel member of the plakin family.J Invest Dermatol. 1998; 111: 338-340Crossref PubMed Scopus (48) Google Scholar; Kiyokawa et al., 1998Kiyokawa C. Ruhrberg C. Nie Z. et al.Envoplakin and periplakin are components of the paraneoplastic pemphigus antigen complex.J Invest Dermatol. 1998; 111: 1236-1238Crossref PubMed Scopus (85) Google Scholar; Mahoney et al., 1998Mahoney M.G. Aho S. Uitto J. et al.The members of the plakin family of proteins recognized by paraneoplastic pemphigus antibodies include periplakin.J Invest Dermatol. 1998; 111: 308-313Crossref PubMed Scopus (130) Google Scholar; Proby et al., 1999Proby C. Fujii Y. Owaribe K. et al.Human autoantibodies against HD1/plectin in paraneoplastic pemphigus.J Invest Dermatol. 1999; 112: 153-156Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar), as well as autoantibodies against Dsg3, a common feature of PV (Joly et al., 1994Joly P. Thomine E. Gilbert D. et al.Overlapping distribution of autoantibody specificities in paraneoplastic pemphigus and pemphigus vulgaris.J Invest Dermatol. 1994; 103: 65-72Abstract Full Text PDF PubMed Google Scholar). Of these autoantibodies, those against Dsg3 are considered to have a primary pathogenic role in blister formation in PNP patients (Amagai et al., 1998Amagai M. Nishikawa T. Nousari H.C. et al.Antibodies against desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice.J Clin Invest. 1998; 102: 775-782Crossref PubMed Scopus (271) Google Scholar). Plakins are located in the cytoplasm of keratinocytes, and the circulating antibodies cannot access them. In a previous study, the removal of anti-Dsg3 IgG eliminated the pathogenic activity of PNP sera in passive transfer experiments in neonatal mice, and affinity-purified polyclonal anti-Dsg3 antibodies from PNP patient sera induced blister formation in neonatal mice (Amagai et al., 1998Amagai M. Nishikawa T. Nousari H.C. et al.Antibodies against desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice.J Clin Invest. 1998; 102: 775-782Crossref PubMed Scopus (271) Google Scholar). Epitope analysis of PNP patient sera has revealed that Dsg3 epitopes are distributed more broadly on the extracellular domains of Dsg3 than those of PV patients (Futei et al., 2003Futei Y. Amagai M. Hashimoto T. et al.Conformational epitope mapping and IgG subclass distribution of desmoglein 3 in paraneoplastic pemphigus.J Am Acad Dermatol. 2003; 49: 1023-1028Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). This suggests that the autoimmune response against Dsg3 in PNP is more diversified than that in PV. Phage display has been used to isolate anti-Dsg mAbs from PV and PF patients as single-chain variable fragments (scFvs). Pathogenic and nonpathogenic antibodies have been isolated (Payne et al., 2005Payne A.S. Ishii K. Kacir S. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (190) Google Scholar; Ishii et al., 2008Ishii K. Lin C. Siegel D.L. et al.Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.J Invest Dermatol. 2008; 128: 939-948Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar; Yamagami et al., 2010Yamagami J. Payne A.S. Kacir S. et al.Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity.J Clin Invest. 2010; 120: 4111-4117Crossref PubMed Scopus (47) Google Scholar). Characterizations of these antibodies have revealed that an scFv form of anti-Dsg mAb can reproduce Dsg-binding specificities. Moreover, the pathogenic scFvs in these studies were capable of inactivating Dsgs and inducing blister formation. In addition, genetic analysis of the isolated antibodies revealed both heavy-chain and light-chain restriction, where the heavy chain was restricted to the VH1, VH3, and VH4 genes. The antibodies that bound to Dsg3 and/or Dsg1 were derived from different parental B-cell clones. In this study, to better understand the pathophysiological mechanisms in PNP, we constructed a phage antibody library from PNP patient samples and isolated a cohort of anti-Dsg3 mAbs in the form of scFvs. We characterized the pathogenic activity of these isolated antibodies and characterized the corresponding conformational epitopes on Dsg3. In addition, we analyzed gene usage and the sequences of the variable regions and compared them with those of anti-Dsg3 antibodies isolated from PV patients. We isolated pathogenic anti-Dsg3 mAbs from a PNP patient. We found that the mAbs we isolated recognized the EC2 and EC3 domains of Dsg3, different pathogenic epitopes than those that are commonly recognized by anti-Dsg3 mAbs from PV patients. We constructed a phage antibody library from peripheral blood lymphocytes from a PNP patient. The library was screened through multiple rounds of panning on immobilized baculovirus-produced Dsg3 using standard panning methods. First, we isolated 14 unique Dsg3-reactive monoclonal phage antibodies based on heavy- and light-chain nucleotide sequences. These clones were derived from only one heavy-chain gene (VH1-46) and used the same light-chain gene (2c). We then panned the library in the presence of previously isolated clones (PNP-A1 and PNP-B1) by epitope blocking (Ishii et al., 2008Ishii K. Lin C. Siegel D.L. et al.Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.J Invest Dermatol. 2008; 128: 939-948Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and isolated six additional clones encoded by VH1-46 and VH1-02 heavy-chain genes. Pathogenic anti-Dsg3 mAbs isolated from PV patients use the VH1-46, VH1-69, VH3-07, and VH4b genes (Payne et al., 2005Payne A.S. Ishii K. Kacir S. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (190) Google Scholar; Yamagami et al., 2010Yamagami J. Payne A.S. Kacir S. et al.Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity.J Clin Invest. 2010; 120: 4111-4117Crossref PubMed Scopus (47) Google Scholar). Of the 20 isolated clones from the PNP patient, 18 shared VH1-46. Because the H-CDR3 (heavy-chain complementarity-determining region 3) is important for the pathogenicity of PV and PF antibodies (Yamagami et al., 2010Yamagami J. Payne A.S. Kacir S. et al.Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity.J Clin Invest. 2010; 120: 4111-4117Crossref PubMed Scopus (47) Google Scholar), we classified the 20 isolated clones into four subgroups (A, B, C, and D) according to their H-CDR3 sequences. Three groups (A, B, and C) were encoded by VH1-46 and one group (D) was encoded by VH1-02. Table 1 summarizes the genetic and immunological characterization of representative clones from each group. The nomenclature is as follows: PNP-A1=paraneoplastic pemphigus, group A, clone number 1.Table 1Anti-Dsg3 mAbs isolated from the PNP libraryGenetic characterizationImmunological characterizationHeavy chainLight chainELISAs/IPIBIIFEpitopePathogenicity assaysHumanMouseHumanMouseClone nameGroupV geneD geneJ geneCDR3V geneDsg3Dsg1Dsg21Tested by IPIB.Dsg32Tested by ELISA.Dsg12Tested by ELISA.skinskin/tailCalcium dependencyHuman Dsg31Tested by IPIB.In vitro dissociationOrgan culture human skinPNP-A1AVH1-46D6-13/DN1JH4byycvregiaadgmdywgqVL2c+----+-+EC3++ (Focal)PNP-A2AVH1-46D6-13/DN1JH5byycvregiaadgmdywgqVL2c+--NDND+-NDEC3NDNDPNP-B1BVH1-46D6-13/DN1JH4byycardsipmmghdywgqVL2c+--+-+Weak ++EC2++ (Focal)PNP-B2BVH1-46D6-13/DN1JH4byycardsipmmghdywgqVL2c+--NDND+ND+EC2NDNDPNP-C1CVH1-46D6-25JH4byycargsglivpaayfdywgqVL1g+----+-+EC2+++PNP-D1DVH1-02D4JH4byycasgvvvpaaldldredywgqVL2c+------ND--Abbreviations: CDR3, complementarity-determining region 3; Dsg, desmoglein; IIF, indirect immunofluorescence; IPIB, immunoprecipitation/immunoblotting; ND, not done; PNP, paraneoplastic pemphigus.1 Tested by IPIB.2 Tested by ELISA. Open table in a new tab Abbreviations: CDR3, complementarity-determining region 3; Dsg, desmoglein; IIF, indirect immunofluorescence; IPIB, immunoprecipitation/immunoblotting; ND, not done; PNP, paraneoplastic pemphigus. As expected, all clones bound to human Dsg3 ELISA. Immunofluorescence analysis of normal human skin showed that representative clones from the three groups encoding VH1-46 (PNP-A1, PNP-B1, and PNP-C1) were bound to the surfaces of keratinocytes. A representative clone from the VH1-02 group (PNP-D1) did not bind to cell surfaces (Figure 1), although the antibody bound to recombinant Dsg3 ELISA and was detected by immunoprecipitation/immunoblotting (data not shown). We tested reactivity in mice by indirect immunofluorescence and ELISA. Only PNP-B1 showed weak cell surface staining of mice keratinocytes by indirect immunofluorescence and bound to mouse Dsg3 ELISA. None of the antibodies bound to mouse Dsg1 ELISA (Table 1). Because three antibodies did not crossreact to mice, we could not use mice to test in vivo pathogenicity. As most pathogenic autoantibodies from PV and PF bind to calcium-sensitive epitopes (Amagai et al., 1995Amagai M. Ishii K. Hashimoto T. et al.Conformational epitopes of pemphigus antigens (Dsg1 and Dsg3) are calcium dependent and glycosylation independent.J Invest Dermatol. 1995; 105: 243-247Crossref PubMed Scopus (87) Google Scholar; Payne et al., 2005Payne A.S. Ishii K. Kacir S. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (190) Google Scholar; Ishii et al., 2008Ishii K. Lin C. Siegel D.L. et al.Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.J Invest Dermatol. 2008; 128: 939-948Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), we analyzed whether the anti-Dsg3 mAbs from the PNP patient would bind calcium-sensitive epitopes. The cell surface binding of keratinocytes by PNP-A1, PNP-B1, and PNP-C1 was abrogated when the human skin was preincubated with EDTA (Figure 1f). This suggests that the clones bound to calcium-dependent conformational epitopes. We further characterized the Dsg3 epitopes recognized by the isolated antibodies by immunoprecipitation/immunoblotting using a series of Dsg3/Dsg2 domain-swapped molecules produced using a baculovirus expression system (Ohyama et al., 2012Ohyama B. Nishifuji K. Chan P. et al.Epitope spreading is rarely found in pemphigus vulgaris by large scale longitudinal study using desmoglein 2-based swapped molecules.J Invest Dermatol. 2012Abstract Full Text Full Text PDF Scopus (59) Google Scholar). PNP-A1 bound to the EC3 domain of Dsg3, whereas PNP-B1 and PNP-C1 bound to the EC2 domain (Figure 2b). We also characterized the epitopes recognized by the polyclonal Dsg3 antibodies from the PNP patient. The epitopes were broad, and covered EC1 to EC4 (Figure 2b). This contrasts with classic PV patients, in whom the dominant epitopes recognized by Dsg3 antibodies are EC1 and EC2 (Figure 2a and b). The isolated mAbs were tested for their ability to inhibit the binding of polyclonal antibodies in the PNP patient's serum to human Dsg3 ELISA. Each scFv competed with the PNP patient's serum in the ELISA plate in serial dilutions. PNP-A1, PNP-B1, PNP-C1, and PNP-D1 inhibited at most only 15% (± 2.4), 11% (±3.2), 9% (±4.3), and 13% (±5.2), respectively (Figure 2c), of the binding of the patient's serum to Dsg3. We further prepared a mixture of equal volumes of the four isolated scFvs and then competed this mixture in serial dilutions with the patient's serum on the ELISA plate. The mixture inhibited binding of polyclonal antibodies in the patient's serum by only 16% (±1.3). This suggests that the isolated mAbs represent only a subset of the polyclonal anti-Dsg3 antibodies in the PNP patient's serum and that the majority of polyclonal anti-Dsg3 antibodies may recognize different epitopes. To examine the pathogenicity of the mAbs, we performed an in vitro dissociation assay using normal human epidermal keratinocytes (Ishii et al., 2005Ishii K. Harada R. Matsuo I. et al.In vitro keratinocyte dissociation assay for evaluation of the pathogenicity of anti-desmoglein 3 IgG autoantibodies in pemphigus vulgaris.J Invest Dermatol. 2005; 124: 939-946Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The normal human epidermal keratinocytes were incubated with the antibodies (1μgml–1) in the presence of 0.5μgml–1 exfoliative toxin A (ETA), which inhibits Dsg1 (Amagai et al., 2000Amagai M. Matsuyoshi N. Wang Z.H. et al.Toxin in bullous impetigo and staphylococcal scalded-skin syndrome targets desmoglein 1.Nat Med. 2000; 6: 1275-1277Crossref PubMed Scopus (384) Google Scholar; Hanakawa et al., 2004Hanakawa Y. Schechter N.M. Lin C. et al.Enzymatic and molecular characteristics of the efficiency and specificity of exfoliative toxin cleavage of desmoglein 1.J Biol Chem. 2004; 279: 5268-5277Crossref PubMed Scopus (56) Google Scholar; Nishifuji et al., 2010Nishifuji K. Shimizu A. Ishiko A. et al.Removal of amino-terminal extracellular domains of desmoglein 1 by staphylococcal exfoliative toxin is sufficient to initiate epidermal blister formation.J Dermatol Sci. 2010; 59: 184-191Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Gentle pipetting will result in fragmentation of the epidermal cell sheet if the antibody is pathogenic. AK23 (IgG form) was used as a positive control (Figure 3). AK23 is a well-characterized mouse bivalent anti-Dsg3 mAb that crossreacts with human Dsg3 (Tsunoda et al., 2003Tsunoda K. Ota T. Aoki M. et al.Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.J Immunol. 2003; 170: 2170-2178Crossref PubMed Scopus (261) Google Scholar). ETA alone was used as a negative control (Figure 3). The dissociation scores for PNP-A1, PNP-B1, and PNP-C1 were 10 (±0.09), 9 (±0.9), and 32 (±10), respectively (n=3). We also prepared mixtures combining two scFvs (PNP-A1+PNP-B1, PNP-B1+PNP-C1 and PNP-A1+PNP-C1) and all the three scFvs, of which the final concentration of the total scFvs was 1μgml–1. The dissociation scores of PNP-A1+PNP-B1, PNP-B1+PNP-C1, and PNP-B1+PNP-C1 were 10 (±1), 10 (±2), and 25 (±3), respectively. The dissociation score of the mixture of the three scFvs was 30 (±6). None of the combined scFvs showed higher dissociation indexes than the single scFvs, indicating that there is no apparent synergism among the scFvs examined in this study. Two scFvs failed to crossreact with mice, and thus neonatal mice could not be used in an in vivo assay. Thus, we assessed pathogenicity using human skin tissues. The scFvs were injected into the dermis of normal human skin in organ culture. The antibodies were injected with a minimal amount of ETA to inactivate Dsg1. An scFv isolated from a PV patient ((D31)2/28; Payne et al., 2005Payne A.S. Ishii K. Kacir S. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (190) Google Scholar) was used as a positive control. (D31)2/28 crossreacts with both Dsg3 and Dsg1, and thus ETA is not required to inactivate Dsg1. Direct immunofluorescence analysis of the skin organ cultures showed binding of PNP-A1, PNP-B1, and PNP-C1 to the surfaces of keratinocytes (data not shown). Hematoxylin and eosin staining showed that PNP-C1 caused typical suprabasal acantholysis, indicating that the antibody is pathogenic (Figure 4). PNP-C1 showed pathogenic activity in a dose-dependent manner when 50, 100, or 150μl scFv (1μgμl–1) was injected. PNP-A1 and PNP-B1 only produced focal acantholysis when 100 or 150μl scFv (1μgμl–1) was injected. We quantitatively evaluated acantholysis by comparing the width of the blister with the width of the whole skin specimen, a measure we called the organ culture acantholysis index (Figure 4). PNP-A1, PNP-B1, and PNP-C1 scored 2.2 (±3.1), 5.4 (±0.9), and 14.1 (±0.5), respectively, when 150μl scFv (1μgμl–1) was injected. The findings of the in vitro dissociation assay and organ culture assay together suggest that PNP-A1, PNP-B1, and PNP-C1 have the ability to induce loss of cell adhesion. Moreover, the lower number of fragments in the dissociation assay and the focal acantholysis in the skin organ culture suggest that PNP-A1 and PNP-B1 are weaker than PNP-C1. PNP-D1 caused neither fragmentation of cell sheets nor blistering in organ culture (data not shown), which suggests that it is not pathogenic. Phage display has previously been used to isolate mAbs from PV (Payne et al., 2005Payne A.S. Ishii K. Kacir S. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (190) Google Scholar) and PF patients (Ishii et al., 2008Ishii K. Lin C. Siegel D.L. et al.Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.J Invest Dermatol. 2008; 128: 939-948Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar; Yamagami et al., 2010Yamagami J. Payne A.S. Kacir S. et al.Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity.J Clin Invest. 2010; 120: 4111-4117Crossref PubMed Scopus (47) Google Scholar). In this study, we constructed a phage library from a PNP patient and succeeded in isolating a cohort of anti-Dsg3 mAbs. We proved that three anti-Dsg3 mAbs (PNP-A1, PNP-B1, and PNP-C1) possess the ability, as scFvs, to induce blister formation in the skin. The ability of the isolated antibodies to induce loss of cell adhesion was weaker than that of the positive controls, as assessed by the number of fragments in a dissociation assay and the extent of acantholysis in an organ culture injection assay. The differences in the abilities of the scFvs isolated from the PNP patient in this study and previously isolated antibodies from PV patients to induce loss of cell adhesion may be explained by the epitopes of the antibodies. Whereas PNP-B1 and PNP-C1 bound to the EC2 domain of Dsg3, PNP-A1 bound to EC3. This result contrasts with the previous finding that pathogenic mAbs from PV patients bound to the EC1 region of Dsg3, which is predicted to form the intercellular trans-adhesive interface (Tsunoda et al., 2003Tsunoda K. Ota T. Aoki M. et al.Induction of pemphigus phenotype by a mouse monoclonal antibody against the amino-terminal adhesive interface of desmoglein 3.J Immunol. 2003; 170: 2170-2178Crossref PubMed Scopus (261) Google Scholar; Payne et al., 2005Payne A.S. Ishii K. Kacir S. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (190) Google Scholar). This is the region containing the dominant epitopes in human PV. In a previous study, mouse mAbs were isolated from PV model mice generated by transfer of naive Dsg3-/- splenocytes (Kawasaki et al., 2006Kawasaki H. Tsunoda K. Hata T. et al.Synergistic pathogenic effects of combined mouse monoclonal anti-desmoglein 3 IgG antibodies on pemphigus vulgaris blister formation.J Invest Dermatol. 2006; 126: 2621-2630Crossref PubMed Scopus (43) Google Scholar). Antibodies recognizing the middle portion of Dsg3 proved to be weakly pathogenic. Taken together with our data, these findings suggest that anti-Dsg IgG antibodies that recognize the middle and C-terminal extracellular domains of Dsg3 may also contribute to blister formation. Moreover, our findings indicate that antibodies against the EC2 and EC3 domains of Dsg3 are involved in blister formation. It was previously reported that a combination of weakly pathogenic anti-Dsg3 IgG autoantibodies had synergistic pathogenic effects on blister formation (Kawasaki et al., 2006Kawasaki H. Tsunoda K. Hata T. et al.Synergistic pathogenic effects of combined mouse monoclonal anti-desmoglein 3 IgG antibodies on pemphigus vulgaris blister formation.J Invest Dermatol. 2006; 126: 2621-2630Crossref PubMed Scopus (43) Google Scholar). When the pathophysiology of PNP is considered, the severe phenotype may be caused by the synergistic effects of low-potency pathogenic anti-Dsg3 antibodies that recognize various epitopes. However, the combination of the three isolated mAbs did not show any apparent synergistic effect in dissociation assay (Figure 3). Other anti-Dsg3 mAbs may be required to show synergistic effect on keratinocyte dissociation. It will be interesting to study whether pathogenic antibodies from PNP and PV patients differ in their activation of signal transduction pathways. In addition, it may be interesting to study whether the binding of PNP antibodies to Dsg3 for long periods induces secondary inflammation in the skin, a phenomenon that may be observed in PNP. We analyzed the gene usage of the isolated Dsg3 mAbs. Three groups of mAbs shared the VH1-46 locus, and one group was derived from VH1-02. Some mAbs isolated from PV patients by phage display use the same VH1-46 gene (Payne et al., 2007Payne A.S. Siegel D.L. Stanley J.R. Targeting pemphigus autoantibodies through their heavy-chain variable region genes.J Invest Dermatol. 2007; 127: 1681-1691Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar; Yamagami et al., 2010Yamagami J. Payne A.S. Kacir S. et al.Homologous regions of autoantibody heavy chain complementarity-determining region 3 (H-CDR3) in patients with pemphigus cause pathogenicity.J Clin Invest. 2010; 120: 4111-4117Crossref PubMed Scopus (47) Google Scholar). In one study using the heterohybridoma technique, anti-Dsg3 and anti-Dsg1 antibodies from PV patients did not use VH1-46 (Qian et al., 2007Qian Y. Diaz L.A. Ye J. et al.Dissecting the anti-desmoglein autoreactive B cell repertoire in pemphigus vulgaris patients.J Immunol. 2007; 178: 5982-5990Crossref PubMed Scopus (23) Google Scholar). In another study whose aim was to clone B cells isolated by flow cytometry from peripheral blood of PV patients, some Dsg3-specific B cells used VH1-46 (Yamagami et al., 2008Yamagami J. Takahashi H. Ota T. et al.Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris.J Dermatol Sci. 2008; 52: 98-107Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). This suggests that Dsg3-specific B cells from PNP and PV patients may come from restricted parental B cells. In conclusion, we isolated a cohort of Dsg3 mAbs from a PNP patient, three of which had the ability to induce blister formation. The pathogenic mAbs recognized the middle portion of Dsg3 (the EC2 and EC3 domains), in contrast to the pathogenic PV antibodies. These findings reflect the unique polyclonal nature of anti-Dsg3 IgG antibodies in PNP, and indicate key differences between PNP and PV in terms of the pathophysiological mechanisms of blister formation and autoimmune responses. The antibodies we have isolated will be useful for elucidating the molecular mechanisms of blister formation in PNP and the adhesive function of Dsgs. We used peripheral blood from a 76-year-old male patient with active PNP to construct the PNP library (Figure 5). The patient was diagnosed with B-cell lymphoma through microscopic examination of a lymph node biopsy. This study was approved by the institution review board of Keio University and was conducted according to the Declaration of Helsinki Principles. All samples were used with informed consent. Using previously described methods (Barbas, 2001Barbas C.F. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar), we constructed separate IgG-κ and IgG-λ phage libraries from 1 × 107 mononuclear cells isolated from 50ml peripheral blood collected from the PNP patient. Reverse transcriptase–PCR was used to amplify the Ig variable regions of the heavy (VH) and light chains (VL). The gene fragments were then cloned into the phagemid vector pComb3X (Scripps Institute, La Jolla, CA). The phagemid library was electroporated into the XL-1 Blue strain of E. coli (Stratagene, La Jolla, CA) through superinfection by the helper phage VCSM13 (Stratagene). In this system, filamentous phage particles express scFv antibodies (with a C-terminal (His)6 tag and an HA tag) fused to the pIII bacteriophage coat protein. Recombinant phages were purified from culture supernatants by polyethylene glycol precipitation and resuspended in phosphate-buffered saline (pH 7.4) containing 1% BSA and 1mM CaCl2. The library comprised more than 2 × 108 independent transformants, as determined by titering on XL1-Blue E. coli after transformation. To validate library diversity, we analyzed the sequences of 11 phage clones from the unpanned library. The results showed marked heterogeneity in VH and VL gene usage. ELISA plates coated with recombinant Dsg3 (MBL, Nagoya, Japan) were used to isolate phage clones as described previously (Payne et al., 2005Payne A.S. Ishii K. Kacir S. et al.Genetic and functional characterization of human pemphigus vulgaris monoclonal autoantibodies isolated by phage display.J Clin Invest. 2005; 115: 888-899Crossref PubMed Scopus (190) Google Scholar). Briefly, four wells were incubated with blocking buffer (50mM Tris (pH 7.5), 150mM NaCl, 1mM CaCl2, and 3% skim milk) at room temperature for 1hour. The phage library was diluted into blocking buffer and then incubated in Dsg3-coated wells for 2hours at room temperature. After five washes with Tris-buffered saline/Ca containing 0.1% Tween-20, adherent phages were eluted with 76mM citric acid (pH 2.0) and then neutralized with 2M unbuffered Tris. The eluted phages were amplified in XL1-Blue E. coli and rescued by superinfection with VCSM13. Phages were harvested from bacterial culture supernatants and then repanned four more times. Individual phage clones were isolated at each round of panning and then analyzed for binding to Dsg3 by ELISA using a horseradish peroxidase–conjugated anti-M13 antibody (GE Healthcare Bio-Sciences, Uppsala, Sweden). For epitope-blocked panning, the phage library was first mixed with the purified recombinant scFv clones PNP-A1 and PNP-B1, and then incubated on immobilized Dsg3 for 2hours at room temperature. Recombinant phagemids were purified using a plasmid preparation system (Qiagen Sciences, Germantown, MD), and the VH and VL inserts were sequenced using pComb3X-specific primers as described previously (Barbas, 2001Barbas C.F. Phage Display: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2001Google Scholar). The nucleotide sequences were compared with germline sequences in the V Base sequence directory (http://vbase.mrc-cpe.cam.ac.uk/) to determine their germline gene origins and interrelatedness. The Top10 F′ nonsuppressor strain of E. coli (Invitrogen, Carlsbad, CA) was infected with an individual phage clone, and soluble scFvs were purified from the bacterial periplasmic space using FastBreak (Promega, Madison, WI) and TALON Metal Affinity Resin (Clontech Laboratories, Mountain View, CA). The purity of the scFv preparation was evaluated by SDS-PAGE, followed by Coomassie staining, which showed a single predominant band. The reactivity of scFvs against human Dsg3 was measured by Dsg3 ELISA using a horseradish peroxidase–conjugated anti-HA mAb (3F10; dilution, 1:1,000; Roche Diagnostics, Mannheim, Germany). Immunofluorescence for scFvs was performed on human skin and neonatal mouse skin and tail. Binding was detected through staining with rat anti-HA mAb (3F10; dilution, 1:100; Roche Diagnostics), followed by an Alexa Flour 488-conjugated anti-rat IgG (dilution, 1:200; Invitrogen). Normal human epidermal keratinocytes (a gift from Ehime University) were grown on collagen-coated 12-well microplates (Iwaki Science Products, Tokyo, Japan) until confluent. Thereafter, 1.2mM CaCl2 and scFvs (1μgml–1) were added, and the plates were incubated for 12hours at 37°C. ETA (0.5μgml–1) was added and incubated for 2hours. The cells were washed twice with phosphate-buffered saline (+), resuspended in 1ml phosphate-buffered saline (+), and pipetted five times using a 1-ml pipette. The cells were fixed by adding formaldehyde (5%). A few drops of 0.02% crystal violet were added to stain the particles, with the plates being incubated overnight to allow the stain to be absorbed. Three pictures were taken and were analyzed manually. The dissociation index was calculated from the number of fragments (N) using the following formula: ((N with mAb–N with ETA only)/(N with AK23–N with ETA only)) × 100. For the combination of scFvs, we prepared mixtures containing equal concentrations of two scFvs (PNP-A1+PNP-B1, PNP-B1+PNP-C1, and PNP-A1+PNP-C1) or all of the three scFvs, whose final concentration of the total scFvs was 1μgml–1. To determine the epitopes of the PNP patient, as well as the scFvs, we used the following baculovirus-produced recombinant human Dsg3/Dsg2 domain-swapped molecules tagged with an E-tag and a histidine tag: Dsg3-EC1 (1-101), Dsg3-EC2 (95-221), Dsg3-EC3 (212-328), Dsg3-EC4 (320-440), and Dsg3-EC5 (440-551; Ohyama et al., 2012Ohyama B. Nishifuji K. Chan P. et al.Epitope spreading is rarely found in pemphigus vulgaris by large scale longitudinal study using desmoglein 2-based swapped molecules.J Invest Dermatol. 2012Abstract Full Text Full Text PDF Scopus (59) Google Scholar). Baculovirus-infected insect cell culture supernatants containing recombinant molecules were incubated with the serum or scFvs for 30minutes, and then immunoprecipitated with protein G Sepharose 4 Fast Flow beads (GE Healthcare Bio-Sciences) for serum or anti-HA agarose (Sigma-Aldrich, St Louis, MO) for scFvs (at 4°C overnight with gentle rotation). After washing with Tris-buffered saline/Ca, immunoprecipitates were resuspended in Laemmli sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). Membranes were probed with a horseradish peroxidase–conjugated anti-E-tag antibody (dilution, 1:5,000; GE Healthcare Bio-Sciences). Anti E-tag antibody (GE Healthcare Bio-Sciences) was used as positive control. Specimens of normal skin that were left over after skin biopsies were obtained from our dermatology outpatient clinic. After fat removal, the specimens were cut into pieces with a diameter of 4mm. Intradermal injection of 100μl purified scFv and low-dose ETA was then performed using an insulin syringe, and the specimens were transferred to Transwells (Corning, NY) containing MCDB medium (a gift from Ehime University) and 1.2mM CaCl2. At 24hours, the skin was harvested for direct immunofluorescence and histology. The organ culture acantholysis index was calculated as (width of blister/total width of specimen) × 100. Inhibition ELISA was performed as described previously (Ishii et al., 2008Ishii K. Lin C. Siegel D.L. et al.Isolation of pathogenic monoclonal anti-desmoglein 1 human antibodies by phage display of pemphigus foliaceus autoantibodies.J Invest Dermatol. 2008; 128: 939-948Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The diluted PNP serum competed with an scFv mixture in a Dsg3 ELISA, and was detected using horseradish peroxidase–conjugated anti-human IgG (MBL). The following calculation was used: % inhibition=(1-[(OD PNP serum with competitors-OD AM3-13)/(OD PNP serum without competitors-OD AM3-13)]) × 100, where AM3-13 is an scFv isolated from a thrompocytopenic purpura patient. We thank Dr Aimee Payne (University of Pennsylvania) for providing the scFv-positive control from a PV patient, Dr Tsuyoshi Hata for valuable advice regarding the dissociation assay, and Drs Bungo Ohyama and Takashi Hashimoto for sharing the Dsg3/Dsg2 domain-swapped molecules. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Health and Labour Sciences Research Grants for Research on Measures for Intractable Diseases from the Ministry of Health, Labor and Welfare of Japan." @default.
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• W2019510500 title "Pathogenic Anti-Desmoglein 3 mAbs Cloned from a Paraneoplastic Pemphigus Patient by Phage Display" @default.
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