Christoph M. Hammers, Jing Chen, Chenyan Lin, Stephen Kacir, Don L

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Persistence of Anti-Desmoglein 3 IgG+ B-Cell Clones in Pemphigus Patients over Years Christoph M. Hammers, Jing Chen, Chenyan Lin, Stephen Kacir, Don L. Siegel, Aimee S. Payne, John R. Stanley Journal of Investigative Dermatology Volume 135, Issue 3, Pages 742-749 (March 2015) DOI: 10.1038/jid.2014.291 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Antibody phage display (APD) libraries: timeline of construction and panning against desmoglein 3 (Dsg3). (a, b) Serum anti-Dsg3 ELISA index values are indicated by boxes. Above the horizontal dotted line are positive values. Red boxes indicate blood used for APD library construction. Periods of complete clinical remission off therapy (CROT) are shaded in turquoise. (c) Enrichment for Dsg3-binding was measured by phage ELISA of the libraries amplified after sequential pannings (further explained in Materials and Methods). Shown is the ratio of the optical density (OD) value from rounds P2–P4 relative to the OD of the unpanned P0 library (with a value of 1), expressed as arbitrary units (a.u.). Journal of Investigative Dermatology 2015 135, 742-749DOI: (10.1038/jid.2014.291) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Desmoglein 3-specific IgG+ B-cell clonal lineages in two pemphigus patients. For patients (a) PV3 and (b) PV1 each clonal lineage is indicated by a circle and Roman numeral, grouped in gray-shaded vertical rectangles by time. Clones identified by PCR are outlined in black; others were determined by antibody phage display (APD). VH/VL-genes used for the VH/VL-chains are shown to the left of each clone. For each clone isolated by APD, VH-chains varying by somatic mutations (SMs) are indicated to the right of the circle (with the Arabic number corresponding to the Roman numeral of each clone and small letters indicating distinct SM patterns; e.g., 2a/2b indicates VH-chains with the same VH-CDR3, differing by SM throughout the rest of the VH-chain). The number of actual mutations (excluding VH-CDR3- and IgG heavy-chain framework 4-regions) compared with germline is indicated in parenthesis. ND: not detected by APD or PCR. Journal of Investigative Dermatology 2015 135, 742-749DOI: (10.1038/jid.2014.291) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 PCR strategy to identify patient-specific VH-complementarity-determining regions 3 (CDR3s) found by antibody phage display (APD) at one time point but not at another. (a) PCR template with forward primer from either CDR1 or CDR2 (F1, F2) and reverse primer from CDR3 (R). (b) PV3 clone III, isolated by APD in 2006, was not found by APD in PV3a (2012), but was identified by PCR (lane T(PV3a)). (c) PV1 clone II was found by APD in 2002, but only by PCR at later times (lanes T1(PV1a) and T2(PV1b)). Deduced amino-acid (aa) sequences from sequencing of PCR products shown below the gels (retrieved), compared with that of the corresponding APD-isolated clone (expected). Presumed somatic mutations (or PCR errors) are in red, VH-CDR2/3 regions in bold font, and primer-covered regions are underlined. Std, 200 bp standard; nC1, negative control (water); nC2, negative control (VH-complementary DNA from an unrelated PV patient); pC1, positive control (monoclonal phagemid DNA of clone); pC2, positive control (polyclonal plasmid DNA from the APD library from which the clone of interest was identified by panning). Journal of Investigative Dermatology 2015 135, 742-749DOI: (10.1038/jid.2014.291) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions