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Bullous pemphigoid (BP), the most common autoimmune blistering disease, is induced by autoantibodies to type XVII collagen (COL17). Previous studies demonstrated that COL17 harbors several epitopes targeted by autoreactive T and B cells and that the target epitopes change sequentially during the disease course. To elucidate the details of the humoral immune response to COL17, we used an active BP mouse model in which BP is induced by the adoptive transfer of spleen cells from wild-type mice immunized with human COL17-expressing skin grafting to immunodeficient COL17-humanized (Rag-2 -/- , mouse Col17 -/- , human COL17 + ) mice. By immunoblot analysis, antibodies to the NC16A domain and other extracellular domains (ECDs) of COL17 were detected earlier than antibodies to intracellular domains (ICDs) in the active BP model. Time course analysis by enzyme-linked immunosorbent assay demonstrated a delayed peak of antibodies to ICD epitopes in active BP model. The blockade of CD40-CD40 ligand interaction soon after the adoptive transfer suppressed the production of antibodies to the non-collagenous 16A (NC16A) domain but not to an ICD epitope, suggesting the sequential activation from T and B cells against the ECD epitopes including the NC16A domain to those against ICD epitopes in vivo . Both wild-type mice immunized with a fragment of the NC16A domain and the recipients of those spleen cells produced IgG antibodies to ICD and ECD epitopes, showing intramolecular epitope spreading from the NC16A domain to other epitopes of COL17. Furthermore, we found that a portion of the active BP model mice show intermolecular epitope spreading from human COL17 to murine BP230. The appearance of antibodies to ICD epitopes of COL17 or of antibodies to murine BP230 did not correlate with the skin changes in the mice, suggesting that those antibodies have low pathogenicity. These results suggest that the immune response to the ECD epitopes of COL17, especially to the NC1 6A domain, triggers intramolecular, and intermolecular epitope spreading to ICD epitopes of COL17 and to murine BP230. These novel findings provide insight into the mechanism of epitope spreading in organ-specific, antibody-mediated autoimmune disorders.

Immunoglobulins (Igs) consist of two antigen-binding regions (Fab) and one constant region (Fc). Protein A and protein G are bacterial proteins used for the purification of IgG by virtue of their high affinities for the Fc fragment. Rheumatoid factors are autoantibodies against IgG Fc fragments, which are present in the body under physiological conditions. Little is known about the influence of Fc-binding proteins on the pathogenicity of antibody-induced autoimmune diseases. Pemphigoid diseases are a group of autoimmune subepidermal blistering disorders that includes bullous pemphigoid and mucous membrane pemphigoid. IgGs targeting the non-collagenous NC16A domain of the 180-kDa bullous pemphigoid antigen (BP180) are known to induce skin fragility in mice and the depletion of BP180 in keratinocytes. In this study, mAb against NC16A in combination with Fc-binding proteins was found to enhance BP180 depletion. Although mAb against the C-terminus of BP180 does not show pathogenicity in vivo or in vitro, mAb treatment with Fc-binding proteins clearly induced skin fragility in mice and BP180 depletion in keratinocytes. Anti-BP180 mAbs and Fc-binding proteins were colocalized in the cytoplasm and at the basement membrane zone. Cell adhesion strengths were decreased in parallel with BP180 amounts. Clinically, bullous pemphigoid patients had higher rheumatoid factor titers than controls. Anti-BP180 mAb in combination with high-titer rheumatoid factor serum was found to enhance BP180 depletion. Furthermore, saliva from mucous membrane pemphigoid patients contained larger quantities of bacteria and Fc-binding proteins than controls. Our results suggest that Fc-binding proteins (rheumatoid factor or protein G) may enhance the pathogenicity of autoantibodies in pemphigoid diseases. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd., (Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Background: Mucous membrane pemphigoid (MMP) is a rare chronic autoimmune subepithelial blistering disorder, targeting multiple basement membrane zone (BMZ) proteins including collagen XVII (COL17). Circulating autoantibodies of MMP are often undetected due to their lower titers. The oral mucosa is a valuable substrate for the detection of autoantibodies in MMP patients. However, obtaining normal human oral mucosa is more difficult than obtaining normal human skin. We established immortalized normal human oral mucosal keratinocytes (OMKs) and performed immunoblotting using immortalized OMK lysate for detecting autoantigens in MMP., Methods: Immortalized OMKs were generated from primary OMKs using E6/E7 proteins of HPV. We compared the protein expression levels of major BMZ proteins between primary OMKs and immortalized OMKs. We performed immunoblotting to detect autoantigens using cell lysates from immortalized OMKs in 30 MMP patients., Results: There were no significant differences between primary OMKs and immortalized OMKs in terms of protein expression levels of the BMZ proteins, including COL17, laminin 332, integrin α6/β4, collagen VII, and collagen IV. Cell lysates of immortalized OMKs effectively identified MMP autoantigens in 60% (18/30) of MMP sera. We found an interesting case of MMP whose autoantibodies preferentially reacted to the 120-kD protein that is an ectodomain of COL17., Conclusion: We demonstrated that a cell lysate of immortalized OMKs is a reliable substrate for the detection of MMP autoantigens. This newly developed immunoblotting analysis method promises to contribute to the diagnosis of MMP., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The basement membrane zone (BMZ) is framed by hemidesmosomes and extracellular matrix (ECM) including collagen IV (COL4). Hemidesmosomes are multiprotein complexes that include collagen XVII (COL17). BMZ proteins can be targeted in autoimmune subepidermal blistering diseases, e.g., pemphigoid targeting COL17. The blistering mechanisms in pemphigoid have not been fully elucidated, especially in mucous membrane pemphigoid (MMP), which mainly affects the mucosa. In this study, we showed that oral lesions in pemphigoid may be attributed to the inhibition of protein-protein interactions by autoantibodies. Using immunoprecipitation, we revealed that COL17 directly binds to COL4 in normal human keratinocytes and normal human oral keratinocytes. In particular, the C-terminus of COL17 is binding site to COL4 in oral keratinocytes. The precise COL4-binding region on COL17 was determined by protein-protein binding assay to be from amino acid Gly 1175 to Asp 1340 on the C-terminus. MMP-IgG or mAb recognizing the C-terminus hindered the interaction of COL17 with COL4 in oral keratinocytes. Furthermore, keratinocyte adhesion strength to COL4-coated plates was significantly reduced by the treatment of mAb against the C-terminus. In addition, the inflammatory infiltrates around perilesions were significantly less in MMP compared to BP. These results indicate that pemphigoid IgG targeting the C-terminus plays a pathogenic role in blister formation in the oral mucosa to inhibit protein interactions with less inflammation.

Background: Regulatory T (Treg) cells play a crucial role in peripheral immune tolerance in multiple organs, including the skin. Thus far, the effect of peripheral immune tolerance failure on autoantibody-related autoimmune reactions to the skin is unclear., Objective: We sought to elucidate the target autoantigens in the skin under the condition of Treg cell dysfunction caused by forkhead box P3 (Foxp3) gene mutations in scurfy mice and patients with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome., Methods: Sera and skin from scurfy mice and sera from patients with IPEX syndrome were analyzed to detect target autoantigens by using immunofluorescence studies, ELISAs, and immunoblotting. The pathogenicity of scurfy IgG was examined by using a passive transfer experiment. CD4 + T cells from scurfy mice were transferred to immunodeficient mice to examine their pathogenicity. Signal transducer and activator of transcription 6 (Stat6) -/- scurfy mice were analyzed to further clarify the molecular pathway of autoantibody production. Follicular helper T-cell counts are measured in Stat6 -/- scurfy mice and scurfy mice., Results: Scurfy mice spontaneously generated IgG autoantibodies to the dermal-epidermal junction, which had been class-switched from IgM within 12 days after birth. The target autoantigens were murine BP230 and type XVII collagen (COL17). The scurfy polyclonal autoantibodies did not induce skin fragility in neonatal mice. Autoantibody production was induced by CD4 + T cells from scurfy mice and was ameliorated by Stat6 gene knockout in association with a decrease of follicular helper T cells. We also identified autoantibodies to COL17 and BP230 in patients with IPEX syndrome and found an association between production of autoantibodies to COL17 and an eczematous skin phenotype., Conclusions: Dysregulation of Treg cells generates autoantibodies to COL17 and BP230 in vivo., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

The basement membrane zone consists of multiple components, including collagen XVII (COL17), which is the target of bullous pemphigoid. To our knowledge, no research has addressed the differences in basement membrane zone components between the skin and oral mucosa; therefore, we investigated the basement membrane zone proteins, with a focus on COL17. The mRNA and protein expression levels of COL17 were significantly higher in oral keratinocytes than in skin keratinocytes. Hemidesmosomal COL17 expression was markedly higher in oral keratinocytes than in skin keratinocytes, and its level was associated with adhesion strength. Oral keratinocytes adhered to the extracellular matrix more tightly than did skin keratinocytes in vitro. Based on these results, we attempt to explain the clinical diversity of bullous pemphigoid. COL17 depletion was more prominent in skin keratinocytes than in oral keratinocytes after treatment with COL17-NC16A mAbs, which have in vivo pathogenicity. COL17 C-terminus mAbs, which are not pathogenic, facilitated COL17 depletion in combination treatment with COL17-NC16A mAbs in both types of keratinocytes. In summary, the greater amount of COL17 in oral keratinocytes than in skin keratinocytes is associated with the higher strength of oral keratinocyte hemidesmosomal adhesion at the basement membrane zone. Our results may explain why bullous pemphigoid blistering tends to be more prevalent in the skin than in the oral mucosa., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease that clinically demonstrates tense blisters with widespread erythema, histologically demonstrates subepidermal blistering and immunologically demonstrates the presence of circulating autoantibodies against hemidesmosomal molecules. Complement activation has long been regarded as necessary for the generation of the BP. However, certain evidence has recently come to support non-complemental blistering mechanisms. The story of BP blistering mechanisms is a complicated one. This review mainly focuses on a specific blistering mechanism that highlights the role of complements in BP blistering., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Transmembrane collagen XVII (COL17) is a hemidesmosomal component of basal keratinocytes that can be targeted by autoantibodies in autoimmune blistering disorders, including linear IgA dermatosis (LAD). COL17 can be physiologically cleaved within the juxtamembranous extracellular NC16A domain, and LAD autoantibodies preferentially react with the processed ectodomains, indicating that the processing induces neoepitopes. However, the details of how neoepitopes develop have not been elucidated. In this study, we show that C-terminal processing of COL17 also plays a role in inducing neoepitopes for LAD autoantibodies. First, the mAb hC17-ect15 targeting the 15th collagenous domain of COL17 was produced, which showed characteristics similar to LAD autoantibodies. The mAbs preferentially reacted with C-terminally deleted (up to 682 amino acids) recombinant COL17, suggesting that C-terminal processing shows neoepitopes on the 15th collagenous domain. The LAD autoantibodies also react with C-terminal deleted COL17. Therefore, neoepitopes for LAD autoantibodies also develop after C-terminal processing. Finally, the passive transfer of the mAb hC17-ect15 into human COL17-expressing transgenic mice failed to induce blistering disease, suggesting that neoepitope-targeting antibodies are not always pathogenic. In summary, this study shows that C-terminal processing induces dynamic structural changes and neoepitopes for LAD autoantibodies on COL17., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Type XVII collagen (COL17) is a transmembrane protein located at the epidermal basement membrane zone. COL17 deficiency results in premature hair aging phenotypes and in junctional epidermolysis bullosa. Here, we show that COL17 plays a central role in regulating interfollicular epidermis (IFE) proliferation. Loss of COL17 leads to transient IFE hypertrophy in neonatal mice owing to aberrant Wnt signaling. The replenishment of COL17 in the neonatal epidermis of COL17-null mice reverses the proliferative IFE phenotype and the altered Wnt signaling. Physical aging abolishes membranous COL17 in IFE basal cells because of inactive atypical protein kinase C signaling and also induces epidermal hyperproliferation. The overexpression of human COL17 in aged mouse epidermis suppresses IFE hypertrophy. These findings demonstrate that COL17 governs IFE proliferation of neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin.

Macropinocytosis is an endocytic pathway that is involved in the nonselective fluid uptake of extracellular fluid. Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies to type XVII collagen (COL17), which is a component of hemidesmosome. When keratinocytes are treated with BP-IgG, COL17 internalizes into cells by way of the macropinocytosis. We investigated the mechanism of COL17 macropinocytosis using DJM-1 cells, a cutaneous squamous cell carcinoma cell line. First, non-hemidesmosomal COL17 was preferentially depleted by stimulation with the BP-IgG in the DJM-1 cells. To investigate the signaling involved in COL17-macropinocytosis, the inhibition of small GTPase family members Rac1 and Cdc42 was found to strongly repress COL17 internalization; in addition, the Rho inhibitor also partially blocked that internalization, suggesting these small GTPases are involved in signaling to mediate COL17-macropinocytosis. Western blotting using Phostag-SDS-PAGE demonstrated high levels of COL17 phosphorylation in DJM-1 cells under steady-state condition. Treatment with BP-IgG increased the intracellular calcium level within a minute, and induced the overabundant phosphorylation of COL17. The overabundant phosphorylation of COL17 was suppressed by a protein kinase C (PKC) inhibitor. In addition, PKC inhibitor repressed COL17 endocytosis using cell culture and organ culture systems. Finally, the depletion of COL17 was not observed in the HEK293 cells transfected COL17 without intracellular domain. These results suggest that COL17 internalization induced by BP-IgG may be mediated by a PKC pathway. In summary, BP-IgG initially binds to COL17 distributed on the plasma membrane, and COL17 may be internalized by means of a macropinocytic pathway related to the phosphorylation of the intracellular domain by PKC.

Bullous pemphigoid (BP) is a major autoimmune blistering skin disorder, in which a majority of the autoantibodies (autoAbs) target the juxtamembranous extracellular noncollagenous 16A domain (NC16A) domain of hemidesmosomal collagen XVII. BP-autoAbs may target regions of collagen XVII other than the NC16A domain; however, correlations between epitopes of BP-autoAbs and clinical features have not been fully elucidated. To address correlations between the clinical features and specific epitopes of BP-autoAbs, we evaluated the epitope profiles of BP-autoAbs in 121 patients. A total of 87 patients showed a typical inflammatory phenotype with erythema and autoAbs targeting the anti-NC16A domain, whereas 14 patients showed a distinct noninflammatory phenotype, in which autoAbs specifically targeted the midportion of collagen XVII, but not NC16A. Interestingly, this group clinically showed significantly reduced erythema associated with scant lesional infiltration of eosinophils. Surprisingly, 7 of the 14 cases (50.0%) received dipeptidyl peptidase-IV inhibitors for the treatment of diabetes. Dipeptidyl peptidase-IV inhibitors were used in 3 of 76 (3.9%) typical cases of BP with autoAbs targeting NC16A; thus, dipeptidyl peptidase-IV inhibitors are thought to be involved in the development of atypical noninflammatory BP. This study shows that the autoAb profile differentiates between inflammatory and noninflammatory BP, and that noninflammatory BP may be associated with dipeptidyl peptidase-IV inhibitors., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

In bullous pemphigoid, the common autoimmune blistering disorder, IgG autoantibodies target various epitopes on hemidesmosomal transmembrane collagen XVII (COL17)/BP180. Antibodies (Abs) targeting the extracellular noncollagenous 16th A domain of COL17 may be pathogenic; however, the pathogenic roles of Abs targeting non-noncollagenous 16th A regions are poorly understood. In this study using a pathogenic and a nonpathogenic monoclonal antibody (mAb) targeting the noncollagenous 16th A domain (mAb TS39-3) and the C-terminus domain (mAb C17-C1), respectively, we show that endocytosis of immune complexes after binding of Abs to cell surface COL17 is a key phenomenon that induces skin fragility. Passive transfer of IgG1 mouse mAb TS39-3 but not mAb C17-C1 induces dermal-epidermal separation in neonatal human COL17-expressing transgenic mice. Interestingly, mAb C17-C1 strongly binds with the dermal-epidermal junction of the recipient mice skin, suggesting that binding of Abs with COL17 is insufficient to induce skin fragility. In cultured normal human epidermal keratinocytes treated with these mAbs, mAb TS39-3 but not mAb C17-C1 internalizes immune complexes after binding with cell surface COL17 via macropinocytosis, resulting in reduced COL17 expression. This study shows that pathogenicity of Abs targeting COL17 is epitope dependent, which is associated with macropinocytosis-mediated endocytosis of immune complexes and finally results in the depletion of COL17 expression in basal keratinocytes., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Pemphigoid is a common autoimmune blistering disorder in which autoantibodies target transmembrane collagen XVII (COL17), a component of hemidesmosomes in basal keratinocytes. The ectodomain of COL17 can be cleaved from the cell surface within the juxtamembranous extracellular NC16A domain, and, interestingly, certain autoantibodies of pemphigoid patients preferentially react with the shed ectodomain. These findings suggest that COL17 ectodomain shedding generates neoepitopes on the shed form; however, the regulatory mechanism of the shedding in in vivo skin and the pathogenicity of the neoepitope-targeting antibodies still are uncertain. To address these issues, we produced rabbit antibodies specifically reacting with N-terminal cleavage sites of the shed COL17 ectodomain. The antibodies showed that certain amounts of the human COL17 ectodomain are cleaved physiologically at Gln(525) in in vivo skin. In contrast, migrating human keratinocytes cleave COL17 at Leu(524) but not at Gln(525). The passive transfer of antibodies reacting with an N-terminal cleavage site of the mouse COL17 ectodomain into neonatal wild-type mice failed to induce blister formation, even though the antibodies bound to the dermal-epidermal junctions, indicating that cleavage site-specific antibodies have reduced or absent pathogenicity for blister formation. This study shows the ectodomain shedding of COL17 to be a physiological event in in vivo human skin that probably generates nonpathologic epitopes on the cleavage sites., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Bullous pemphigoid (BP) is a common autoimmune blistering skin disease in which two hemidesmosomal components--the transmembrane collagen XVII (BP180 or BPAG2) and the plakin family protein BP230 (BPAG1)--are targeted by autoimmunity. Of these, collagen XVII (COL17) is thought to be a major autoantigen, and vital roles of IgG autoantibodies in blister formation have been elucidated. However, BP shows distinct features, including pruritic urticarial erythema and eosinophilic infiltration, which may be independent of IgG-mediated autoimmunity. Recently, it has been revealed that sera from certain patients with BP contain IgE autoantibodies to COL17 and that IgE autoantibodies bind to peri-lesional dermal-epidermal junctions. Mouse models have demonstrated that IgE antibodies to COL17 induce erythema and eosinophilic infiltration in skin. In addition, the successful treatment of severe BP with omalizumab, a humanized monoclonal antibody targeting IgE, has been reported. These findings suggest that both IgG and IgE autoantibodies to COL17 may be involved in the BP pathogenesis. This article summarizes IgE-mediated autoimmunity to COL17 in BP., (Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

In bullous pemphigoid (BP), the most prevalent autoimmune blistering disease, type XVII collagen (COL17) is targeted by circulating autoantibodies. BP is thought to be an autoantibody-mediated complement-fixing blistering disease, and a juxtamembranous noncollagenous 16A (NC16A) domain spanning Glu(490) to Arg(566) was proved to be the main pathogenic region on COL17, although precise pathogenic epitopes within NC16A have not been elucidated. In this study, we showed that injection of rabbit IgG Abs targeting Asp(522) to Gln(545) induced skin fragility associated with in vivo deposition of IgG and complement in neonatal COL17-humanized mice. Notably, immunoadsorption of rabbit anti-NC16A IgG Ab with this epitope (Asp(522) to Gln(545)) or the anti-NC16A IgG administered together with the peptides of this epitope as a decoy ameliorated skin fragility in the injected neonatal COL17-humanized mice compared with the anti-NC16A IgG alone even though all of the mice showed both IgG and complement deposition. These results led us to investigate an additional, complement-independent mechanism of skin fragility in the mice injected with anti-COL17 Abs. The rabbit anti-NC16A IgG depleted the expression of COL17 in cultured normal human keratinocytes, whereas immunoadsorption of the same IgG with this epitope significantly suppressed the depletion effect. Moreover, passive transfer of F(ab')(2) fragments of the human BP or rabbit IgG Abs against COL17 demonstrated skin fragility in neonatal COL17-humanized mice. In summary, this study reveals the importance of Abs directed against distinct epitopes on COL17, which induce skin fragility in complement-dependent as well as complement-independent ways.

Bullous pemphigoid, the most common autoimmune blistering disease, is induced by autoantibodies against type XVII collagen. Passive transfer of IgG or IgE antibodies against type XVII collagen into animals has revealed not only the pathogenicity of these antibodies but also the subsequent immune responses, including complement activation, mast cell degranulation, and infiltration of neutrophils and/or eosinophils. In vitro studies on ectodomain shedding of type XVII collagen have also provided basic knowledge on the development of bullous pemphigoid. The pathogenic role of autoreactive CD4+ T lymphocytes in the development of the pathogenic autoantibodies to type XVII collagen should also be noted., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Bullous pemphigoid (BP), the most common autoimmune blistering disease, is caused by autoantibodies against type XVII collagen (COL17). We recently demonstrated that CD4+ T cells were crucial for the production of anti-COL17 IgG and for the development of the BP phenotype by using a novel active BP mouse model by adoptively transferring immunized splenocytes into immunodeficient COL17-humanized mice. Noncollagenous 16A (NC16A) domain of COL17 is considered to contain the main pathogenic epitopes of BP, however, the pathogenicity of COL17 NC16A-reactive CD4+ T cells has never been elucidated. To address this issue, we modulated the immune responses against COL17 in active BP model by using anti-CD40 ligand (CD40L) monoclonal antibody MR1, an inhibitor of the CD40-CD40L interaction, in various ways. First, we show the essential role of CD4+ T cells in the model by showing that CD4+ T cells isolated from wild-type mice immunized with human COL17 enabled naïve B cells to produce anti-COL17 NC16A IgG in vivo. Second, we show that the activation of anti-COL17 NC16A IgG-producing B cells via CD40-CD40L interaction was completed within 5 days after the adoptive transfer of immunized splenocytes. Notably, a single administration of MR1 at day 0 was enough to inhibit the production of anti-COL17 NC16A IgG and to diminish skin lesions despite the presence of restored anti-COL17 IgG at the later stage. In contrast, the delayed administration of MR1 failed to inhibit the production of anti-COL17 NC16A IgG and the development of the BP phenotype. These results strongly suggest that COL17 NC16A-reactive CD4+ T cells play a pivotal role in the production of pathogenic autoantibodies and in the development of active disease in experimental BP model., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bullous pemphigoid, the most common autoimmune blistering disease, is induced by autoantibodies against type XVII collagen. Passive transfer of IgG or IgE antibodies against type XVII collagen into animals has revealed not only the pathogenicity of these antibodies but also the subsequent immune responses, including complement activation, mast cell degranulation, and infiltration of neutrophils and/or eosinophils. In vitro studies on ectodomain shedding of type XVII collagen have also provided basic knowledge on the development of bullous pemphigoid. The pathogenic role of autoreactive CD4+ T lymphocytes in the development of the pathogenic autoantibodies to type XVII collagen should also be noted., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Bullous pemphigoid (BP), the most common autoimmune blistering disease, is caused by autoantibodies against type XVII collagen (COL17). To establish an active stable BP animal model that demonstrates the persistent inflammatory skin lesions initiated by the anti-human COL17 Abs, we used COL17-humanized (COL17(m-/-,h+)) mice that we recently produced. First, we generated immunodeficient Rag-2(-/-)/COL17-humanized mice by crossing Rag-2(-/-) mice with COL17-humanized mice. Then, splenocytes from wild-type mice that had been immunized by grafting of human COL17-transgenic mouse skin were transferred into Rag-2(-/-)/COL17-humanized mice. The recipient mice continuously produced anti-human COL17 IgG Abs in vivo and developed blisters and erosions corresponding to clinical, histological, and immunopathological features of BP, although eosinophil infiltration, one of the characteristic histological findings observed in BP patients, was not detected in the recipients. Although the depletion of CD8(+) T cells from the immunized splenocytes was found to produce no effects in the recipients, the depletion of CD4(+) T cells as well as CD45R(+) B cells was found to inhibit the production of anti-human COL17 IgG Abs in the recipients, resulting in no apparent clinical phenotype. Furthermore, we demonstrated that cyclosporin A significantly suppressed the production of anti-human COL17 IgG Abs and prevented the development of the BP phenotype in the treated recipients. Although this model in an immunodeficient mouse does not exactly reproduce the induction mechanism of BP in human patients, this unique experimental system targeting humanized pathogenic Ag allows us to investigate ongoing autoimmune responses to human molecules in experimental animal models.

Activation of the complement cascade via the classical pathway is required for the development of tissue injury in many autoantibody-mediated diseases. It therefore makes sense to block the pathological action of autoantibodies by preventing complement activation through inhibition of autoantibody binding to the corresponding pathogenic autoantigen using targeted Fab antibody fragments. To achieve this, we use bullous pemphigoid (BP) as an example of a typical autoimmune disease. Recombinant Fabs against the non-collagenous 16th-A domain of type XVII collagen, the main pathogenic epitope for autoantibodies in BP, were generated from antibody repertoires of BP patients by phage display. Two Fabs, Fab-B4 and Fab-19, showed marked ability to inhibit the binding of BP autoantibodies and subsequent complement activation in vitro. In the in vivo experiments using type XVII collagen humanized BP model mice, these Fabs protected mice against BP autoantibody-induced blistering disease. Thus, the blocking of pathogenic epitopes using engineered Fabs appears to demonstrate efficacy and may lead to disease-specific treatments for antibody-mediated autoimmune diseases.