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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.

SUBMITTER: Hammers CM

PROVIDER: S-EPMC4294994 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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

Hammers Christoph M CM Chen Jing J Lin Chenyan C Kacir Stephen S Siegel Don L DL Payne Aimee S AS Stanley John R JR

The Journal of investigative dermatology 20140714 3

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) a ...[more]

PMID: 25142730

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Project description:Pemphigus foliaceus (PF) is an autoimmune blistering disease caused by autoantibodies (Abs) against desmoglein 1 (Dsg1). PF sera contain polyclonal Abs which are heterogeneous mixture of both pathogenic and non-pathogenic Abs, as shown by isolation of monoclonal Abs (mAbs).To investigate how pathogenic and non-pathogenic anti-Dsg1 Abs contribute to blister formation in PF.Using organ-cultured human skin, we compared the effect of a single pathogenic anti-Dsg1 IgG mAb, a single non-pathogenic anti-Dsg1 IgG mAb, and their mixture on blister formation as analyzed by histology, subcellular localization of IgG deposits and desmosomal proteins by confocal microscopy, and desmosomal structure by electron microscopy. In addition, we measured keratinocyte adhesion by an in vitro dissociation assay.24h after injection, a single pathogenic anti-Dsg1 IgG caused a subcorneal blister with IgG and Dsg1 localized linearly on the cell surface of keratinocytes. A single non-pathogenic anti-Dsg1 IgG bound linearly on the keratinocytes but did not induce blisters. A pathogenic and a non-pathogenic IgG mAb injected together caused an aberrant granular pattern of IgG and Dsg1 in the lower epidermis with blister formation in the superficial epidermis. Electron microscopy demonstrated that the mixture of mAbs shortened desmosomal lengths more than a single mAb in the basal and spinous layers. Furthermore, although Dsg1 clustering required both cross-linking of Dsg1 molecules by the non-pathogenic IgG plus a pathogenic antibody, the latter could be in the form of a monovalent single chain variable fragment, suggesting that loss of trans-interaction of Dsg1 is required for clustering. Finally, a p38MAPK inhibitor blocked Dsg1 clustering. When pathogenic strength was measured by the dissociation assay, a mixture of pathogenic and non-pathogenic IgG mAbs disrupted keratinocyte adhesion more than a single pathogenic mAb. This pathogenic effect was only partially suppressed by the p38MAPK inhibitor.These findings indicate that a polyclonal mixture of anti-Dsg1 IgG antibodies enhances pathogenic activity for blister formation associated with p38MAPK-dependent Dsg1 clustering and that not only pathogenic antibodies but also non-pathogenic antibodies coordinately contribute to blister formation in PF.

Project description:BackgroundA number of autoimmune diseases have been clinically and pathologically characterized. In contrast, target antigens have been identified only in a few cases and, in these few cases, the knowledge of the exact epitopic antigenic sequence is still lacking. Thus the major objective of current work in the autoimmunity field is the identification of the epitopic sequences that are related to autoimmune reactions. Our labs propose that autoantigen peptide epitopes able to evoke humoral (auto)immune response are defined by the sequence similarity to the host proteome. The underlying scientific rationale is that antigen peptides acquire immunoreactivity in the context of their proteomic similarity level. Sequences uniquely owned by a protein will have high potential to evoke an immune reaction, whereas motifs with high proteomic redundancy should be immunogenically silenced by the tolerance phenomenon. The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models. Here the hypothesis has been applied to pemphigus diseases and the corresponding desmoglein autoantigens.MethodsDesmoglein 3 sequence similarity analysis to the human proteome followed by dot-blot/NMR immunoassays were carried out to identify and validate possible epitopic sequences.ResultsComputational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous. The epitopic peptide corresponded to the amino acid REWVKFAKPCRE sequence, was located in the extreme N-terminal region (residues 49 to 60), and had low redundancy to the human proteome. Sequence alignment showed that human desmoglein 1 and 3 share the REW-KFAK-RE sequence as a common motif with 75% residue identity.ConclusionThis study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

Project description:Keratins are crucial for the anchorage of desmosomes. Severe alterations of keratin organization and detachment of filaments from the desmosomal plaque occur in the autoimmune dermatoses pemphigus vulgaris and pemphigus foliaceus (PF), which are mainly caused by autoantibodies against desmoglein (Dsg) 1 and 3. Keratin alterations are a structural hallmark in pemphigus pathogenesis and correlate with loss of intercellular adhesion. However, the significance for autoantibody-induced loss of intercellular adhesion is largely unknown. In wild-type (wt) murine keratinocytes, pemphigus autoantibodies induced keratin filament retraction. Under the same conditions, we used murine keratinocytes lacking all keratin filaments (KtyII k.o.) as a model system to dissect the role of keratins in pemphigus. KtyII k.o. cells show compromised intercellular adhesion without antibody (Ab) treatment, which was not impaired further by pathogenic pemphigus autoantibodies. Nevertheless, direct activation of p38MAPK via anisomycin further decreased intercellular adhesion indicating that cell cohesion was not completely abrogated in the absence of keratins. Direct inhibition of Dsg3, but not of Dsg1, interaction via pathogenic autoantibodies as revealed by atomic force microscopy was detectable in both cell lines demonstrating that keratins are not required for this phenomenon. However, PF-IgG shifted Dsg1-binding events from cell borders toward the free cell surface in wt cells. This led to a distribution pattern of Dsg1-binding events similar to KtyII k.o. cells under resting conditions. In keratin-deficient keratinocytes, PF-IgG impaired Dsg1-binding strength, which was not different from wt cells under resting conditions. In addition, pathogenic autoantibodies were capable of activating p38MAPK in both KtyII wt and k.o. cells, the latter of which already displayed robust p38MAPK activation under resting conditions. Since inhibition of p38MAPK blocked autoantibody-induced loss of intercellular adhesion in wt cells and restored baseline cell cohesion in keratin-deficient cells, we conclude that p38MAPK signaling is (i) critical for regulation of cell adhesion, (ii) regulated by keratins, and (iii) targets both keratin-dependent and -independent mechanisms.

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

Publications

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

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