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Shared VH1-46 gene usage by pemphigus vulgaris autoantibodies indicates common humoral immune responses among patients.

Cho MJ, Lo AS, Mao X, Nagler AR, Ellebrecht CT, Mukherjee EM, Hammers CM, Choi EJ, Sharma PM, Uduman M, Li H, Rux AH, Farber SA, Rubin CB, Kleinstein SH, Sachais BS, Posner MR, Cavacini LA, Payne AS - Nat Commun (2014)

Bottom Line: Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs.Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding.Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies (autoAbs) against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies (Abs) from four PV patients and identify pathogenic VH1-46 autoAbs from all four patients. Unexpectedly, VH1-46 autoAbs had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs. Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 Abs require few to no mutations to acquire Dsg3 autoreactivity, which may favour their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

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Anti-Dsg3 mAbs reproduce the PV phenotype. (a)Indirect immunofluorescence staining of human skin with scFv mAbs demonstrates cell surface binding typical of PV. Negative control is an scFv mAb against an irrelevant Ag. Positive control is a previously characterized anti-Dsg3 scFv mAb, (D31)2/2918. Scale bar, 20 μM. (b) VH1-46 mAbs, but not VH5a mAb, induce suprabasal skin blisters in human skin organ culture. Scale bar, 100 μM. Data are representative of 3 independent experiments.
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Figure 1: Anti-Dsg3 mAbs reproduce the PV phenotype. (a)Indirect immunofluorescence staining of human skin with scFv mAbs demonstrates cell surface binding typical of PV. Negative control is an scFv mAb against an irrelevant Ag. Positive control is a previously characterized anti-Dsg3 scFv mAb, (D31)2/2918. Scale bar, 20 μM. (b) VH1-46 mAbs, but not VH5a mAb, induce suprabasal skin blisters in human skin organ culture. Scale bar, 100 μM. Data are representative of 3 independent experiments.

Mentions: Binding of mAbs to the surface of human keratinocytes was confirmed by indirect IF staining of normal human skin sections (Figure 1a), similar to indirect IF patterns typical of PV sera. All five VH1-46 anti-Dsg3 mAbs, in the presence of exfoliative toxin to inactivate compensatory adhesion by Dsg1, induced suprabasal blisters in human skin explants (Figure 1b). The VH5a anti-Dsg3 mAb did not cause epidermal blisters despite detectable binding to the cell surface of human keratinocytes by direct IF analysis.


Shared VH1-46 gene usage by pemphigus vulgaris autoantibodies indicates common humoral immune responses among patients.

Cho MJ, Lo AS, Mao X, Nagler AR, Ellebrecht CT, Mukherjee EM, Hammers CM, Choi EJ, Sharma PM, Uduman M, Li H, Rux AH, Farber SA, Rubin CB, Kleinstein SH, Sachais BS, Posner MR, Cavacini LA, Payne AS - Nat Commun (2014)

Anti-Dsg3 mAbs reproduce the PV phenotype. (a)Indirect immunofluorescence staining of human skin with scFv mAbs demonstrates cell surface binding typical of PV. Negative control is an scFv mAb against an irrelevant Ag. Positive control is a previously characterized anti-Dsg3 scFv mAb, (D31)2/2918. Scale bar, 20 μM. (b) VH1-46 mAbs, but not VH5a mAb, induce suprabasal skin blisters in human skin organ culture. Scale bar, 100 μM. Data are representative of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4120239&req=5

Figure 1: Anti-Dsg3 mAbs reproduce the PV phenotype. (a)Indirect immunofluorescence staining of human skin with scFv mAbs demonstrates cell surface binding typical of PV. Negative control is an scFv mAb against an irrelevant Ag. Positive control is a previously characterized anti-Dsg3 scFv mAb, (D31)2/2918. Scale bar, 20 μM. (b) VH1-46 mAbs, but not VH5a mAb, induce suprabasal skin blisters in human skin organ culture. Scale bar, 100 μM. Data are representative of 3 independent experiments.
Mentions: Binding of mAbs to the surface of human keratinocytes was confirmed by indirect IF staining of normal human skin sections (Figure 1a), similar to indirect IF patterns typical of PV sera. All five VH1-46 anti-Dsg3 mAbs, in the presence of exfoliative toxin to inactivate compensatory adhesion by Dsg1, induced suprabasal blisters in human skin explants (Figure 1b). The VH5a anti-Dsg3 mAb did not cause epidermal blisters despite detectable binding to the cell surface of human keratinocytes by direct IF analysis.

Bottom Line: Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs.Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding.Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies (autoAbs) against desmoglein 3 (Dsg3). Here, we clone anti-Dsg3 antibodies (Abs) from four PV patients and identify pathogenic VH1-46 autoAbs from all four patients. Unexpectedly, VH1-46 autoAbs had relatively few replacement mutations. We reverted antibody somatic mutations to their germline sequences to determine the requirement of mutations for autoreactivity. Three of five VH1-46 germline-reverted Abs maintain Dsg3 binding, compared with zero of five non-VH1-46 germline-reverted Abs. Site-directed mutagenesis of VH1-46 Abs demonstrates that acidic amino-acid residues introduced by somatic mutation or heavy chain VDJ recombination are necessary and sufficient for Dsg3 binding. Our data suggest that VH1-46 autoantibody gene usage is commonly found in PV because VH1-46 Abs require few to no mutations to acquire Dsg3 autoreactivity, which may favour their early selection. Common VH gene usage indicates common humoral immune responses, even among unrelated patients.

Show MeSH
Related in: MedlinePlus