Epitope-Dependent Pathogenicity of Antibodies Targeting a Major Bullous Pemphigoid Autoantigen Collagen XVII/BP180 Mayumi Wada, Wataru Nishie, Hideyuki.

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Epitope-Dependent Pathogenicity of Antibodies Targeting a Major Bullous Pemphigoid Autoantigen Collagen XVII/BP180 Mayumi Wada, Wataru Nishie, Hideyuki Ujiie, Kentaro Izumi, Hiroaki Iwata, Ken Natsuga, Hideki Nakamura, Yoshimasa Kitagawa, Hiroshi Shimizu Journal of Investigative Dermatology Volume 136, Issue 5, Pages 938-946 (May 2016) DOI: 10.1016/j.jid.2015.11.030 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Recombinant COL17 proteins and epitopes of monoclonal antibodies (mAbs). (a) Constructs of recombinant proteins. mAb C17-C1 reacts within amino acid Gly1316 to Gly1342, and mAb TS39-3 reacts within amino acid Asp522 to Gln545. DDDDK means a Flag-tag. (b) mAb C17-C1 reacts with both 180-kDa full-length and COL3, but not with NC6, COL11, or COL15 recombinant proteins, suggesting that the mAb targets the end-most C-terminal region of COL17. (c) mAb C17-C1 reacts with C-22K but not with C-16K (arrow). Journal of Investigative Dermatology 2016 136, 938-946DOI: (10.1016/j.jid.2015.11.030) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Indirect immunofluorescence studies of monoclonal antibody (mAb) C17-C1. (a) Indirect immunofluorescence using normal human skin as a substrate and 1:1,600 diluted mAb C17-C1 (concentration of original mAb: 1 mg/ml). Bar = 100 μm. (b) mAb C17-C1 does not react to COL17-lacking skin from a non-Herlitz junctional epidermolysis bullosa patient. Stars indicate blisters. Bar = 100 μm. (c) Indirect immunofluorescence using 1 M NaCl-split skin shows that mAb C17-C1 strongly binds to epidermal side. Separation within dermal-epidermal junction is indicated by stars. Bar = 100 μm. (d) Indirect immunofluorescence using normal human skin and secondary Abs targeting a different subclass of IgG reveals mAb C17-C1 to be IgG1. d, dermis; e, epidermis. Bar = 100 μm. Journal of Investigative Dermatology 2016 136, 938-946DOI: (10.1016/j.jid.2015.11.030) Copyright © 2016 The Authors Terms and Conditions

Figure 3 Monoclonal antibody (mAb) TS39-3, but not mAb C17-C1, induces skin detachment in COL17-humanized mice. Forty-eight hours after mAb injection, mAb TS39-3 targeting the NC16A domain of COL17 induces skin detachment (arrow). In contrast, mAb C17-C1 against the C-terminal region fails to induce skin fragility. Hematoxylin and eosin (H&E) staining reveals detachment of dorsal skin only in mice injected with mAb TS39-3 (star). Direct immunofluorescence shows linear deposition of IgG but not C3 at the dermal-epidermal junction in a similar manner for both mAb C17-C1 and TS39-3 injected mice skin and tongue mucosa (arrowheads). Bar = 100 μm. Journal of Investigative Dermatology 2016 136, 938-946DOI: (10.1016/j.jid.2015.11.030) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Monoclonal antibody (mAb) C17-C1 does not reduce COL17 expression. mAb TS39-3 reduces expression of COL17 in treated normal human epidermal keratinocytes (arrows), whereas no effects are observed in cells treated with mAb C17-C1 and control mouse IgG1. Depletion of COL17 was detectable starting 3 hours after incubation of mAb TS39-3. Journal of Investigative Dermatology 2016 136, 938-946DOI: (10.1016/j.jid.2015.11.030) Copyright © 2016 The Authors Terms and Conditions

Figure 5 Monoclonal antibody (mAb) C17-C1 does not induce internalization of immune complexes (ICs). (a) Approximately 20–30 minutes after mAb treatment, ICs of COL17 are internalized into the cytoplasm of normal human epidermal keratinocytes treated with mAb TS39-3. In contrast, internalization of ICs is not observed in cells treated with mAb C17-C1. ICs internalized into the cytoplasm are indicated by arrows. Scale bar: 10 μm. (b) Two hours after treatment of normal human epidermal keratinocytes by Alexa488-conjugated mAb TS39-3 together with CF555-conjugated mAb C17-C1. Note that mAb C17-C1 (green) is internalized into the cytoplasm with mAb TS39-3 (red) after binding with the cell surface of NHEKs. Bar = 10 μm. (c) mAb TS39-3 and C17-C1 strongly deposit at the dermal-epidermal junction, and ICs induced by mAb TS39-3 are internalized into the cytoplasm of basal keratinocytes in organ-cultured normal human skin (arrow). Note that ICs induced by mAb C17-C1 are not internalized. Bar = 10 μm. Journal of Investigative Dermatology 2016 136, 938-946DOI: (10.1016/j.jid.2015.11.030) Copyright © 2016 The Authors Terms and Conditions

Figure 6 Macropinocytosis-mediated endocytosis of immune complexes (ICs) by monoclonal antibody (mAb) TS39-3. (a) N-ethylmaleimide inhibits ICs internalization of ICs. (b–d) Sucrose, nystatin, and genistein fail to inhibit internalization. (e) 5-(N-ethyl-N-isopropyl) amiloride (EIPA) blocks COL17 internalization, suggesting that mAb TS39-3-induced internalization of ICs is mainly mediated by macropinocytosis. Bar = 10 μm. Journal of Investigative Dermatology 2016 136, 938-946DOI: (10.1016/j.jid.2015.11.030) Copyright © 2016 The Authors Terms and Conditions