Fig 1.
IgG1 and IgG4 PV mAbs target epitopes within the same Dsg3 extracellular domains.
(A) F706 and F779 mAbs bind desmoglein 3 (Dsg3) but not Dsg1. Subclass-switched anti-human Dsg3 F706 and F779 IgG1 and IgG4 were tested by ELISA for binding to Dsg3 and Dsg1. Negative and positive controls were provided by the manufacturer. F71 IgG1 and b12 IgG4 antibodies served as isotype controls. (B) Deduced epitopes of IgG1 and IgG4 variants of PV mAbs show that mAb pairs recognize epitopes within the same Dsg3 extracellular domains.
Fig 2.
Immunoreactivity of anti-Dsg3 PV mAbs by ELISA and IIF.
(A) Serial dilutions of recombinant human IgG1 (♦) or IgG4 (□) variants of mAbs (F706 against Dsg3 in upper left, F779 against Dsg3 in upper right, Px43 against Dsg3 in lower left and Px43 against Dsg1 in lower right) were incubated with Dsg3- coated ELISA plates. Bound antibody was detected using HRP conjugated mouse anti-human (H+L) Ab. Results are the mean of duplicate wells and are representative of 2–3 independent experiments. The relative affinity of each mAb was calculated by Lineweaver-Burk plot. (B) IgG1 and IgG4 variants of F706 and F779 mAbs show comparable immunoreactivity to the Dsg3+ human oropharynx tumor cell line SCC-47. F71 IgG1 and b12 IgG4 served as isotype controls. Other negative controls included HRP substrate OPD added alone or with secondary reagents only (secondary (2nd) biotinylated anti-human Fc Ab (Biotin Ab) or 2nd Ab with HRP-conjugated streptavidin Ab (Bio + SA Abs) in absence of tested PV mAbs. OPD: o-phenylenediamine dihydrochloride. (C) Px43 IgG1 and IgG4 demonstrate different relative affinities by ELISA but comparable affinity by IIF binding of human skin. D: dermal part of skin. Results are representative of 1–2 experiments.
Fig 3.
IgG1 and IgG4 variants of anti-Dsg3 PV mAbs are pathogenic and cause suprabasal blisters in human skin.
(A) 25 μg anti-Dsg3 PV IgG1 or IgG4 was injected ex vivo into human skin sections and incubated for 18 hours prior to harvest for histologic analysis. IgG1 and IgG4 of three anti-Dsg3 PV mAbs, F706, F779 and Px43 induced suprabasal blisters in human skin. PBS/ETA served as negative control. Scale bar, 100 microns. (B) The extent of histologic blistering caused by IgG1 and IgG4 mAb variants is comparable. (C) DIF staining of human skin with PV isotype mAbs illustrates cell surface binding in skin epidermis following injection of mAbs. D: dermal part of skin. Scale bar, 20 microns.
Fig 4.
Pathogenic F706 and F779 IgG1 and IgG4 cause loss of cell surface Dsg3.
Primary human keratinocytes were incubated for 8 hours with 50 μg/mL F706/F779 IgG1 or IgG4. Cells were prepared for IF staining using anti-human IgG or anti-Dsg3 (5G11) primary antibodies. Px44 negative and Px43 positive control antibodies were previously characterized [22]. Scale bar, 20 microns. Results are representative of two independent experiments.