Persistence of anti-desmoglein 3 IgG(+) B-cell clones in pemphigus patients over years

Abstract

Pemphigus vulgaris (PV) is a prototypic tissue-specific autoantibody-mediated disease, in which anti-desmoglein 3 (Dsg3) IgG autoantibodies cause life-threatening blistering. We characterized the autoimmune B-cell response over 14 patient years in two patients with active and relapsing disease, then in one of these patients after long-term remission induced by multiple courses of rituximab (anti-CD20 antibody). Characterization of the anti-Dsg3 IgG(+) repertoire by antibody phage display (APD) and PCR indicated that six clonal lines persisted in patient 1 (PV3) over 5.5 years, with only one new clone detected. Six clonal lines persisted in patient 2 (PV1) for 4 years, of which five persisted for another 4.5 years without any new clones detected. However, after long-term clinical and serologic remission, ∼11 years after initial characterization, we could no longer detect any anti-Dsg3 clones in PV1 by APD. Similarly, in another PV patient, ∼4.5 years after a course of rituximab that induced long-term remission, anti-Dsg3 B-cell clones were undetectable. These data suggest that in PV a given set of non-tolerant B-cell lineages causes autoimmune diseases and that new sets do not frequently or continually escape tolerance. Therapy such as rituximab, aimed at eliminating these aberrant sets of lineages, may be effective for disease because new ones are unlikely to develop.

Figure 1. APD libraries: timeline of construction and panning against Dsg3

(a, b) Serum anti-Dsg3 ELISA index values are indicated by boxes. Above 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 methods). Shown is the ratio of the optical density (O.D.) value from rounds P2–P4 relative to the O.D. of the unpanned P0 library (with a value of 1), expressed as arbitrary units (A.U.).

Figure 2. Dsg3-specific IgG⁺ B-cell clonal lineages in patients (a) PV3 and (b) PV1

Each clonal lineage is indicated by circle and Roman numeral, grouped in gray-shaded vertical rectangles by time. Clones identified by PCR are outlined in black, others were determined by APD. VH/VL genes used for the V_H/V_L-chains are shown to the left of each clone. For each clone isolated by APD, V_H-chains varying by somatic mutations (SM) 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 indicate V_H-chains with the same V_H-CDR3, differing by SM throughout the rest of the V_H-chain). Number of actual mutations (excluding V_H-CDR3- and IgG heavy chain framework 4-regions) compared to germline are indicated in parenthesis. ND: not detected by APD or PCR.

Figure 3. PCR-strategy to identify patient-specific V_H-CDR3s found by 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 to that of the corresponding APD-isolated clone (expected). Presumed somatic mutations (or PCR errors) are in red, V_H-CDR2/3 regions in bolded font, primer-covered regions are underlined. Std, 200 bp standard; nC1, negative control (water); nC2, negative control (V_H-cDNA from an unrelated PV patient); pC1, positive control (monoclonal phagemid DNA of clone); pC2, positive control (polyclonal plasmid DNA from APD-library from which clone of interest was identified by panning).