Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris

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Abstract

Background: Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-desmoglein 3 (Dsg3) IgG autoantibodies; however, the Dsg3-specific B cells that produce anti-Dsg3 IgG are not well characterized. Objectives: Our aims were to develop a flow cytometric method for the isolation of Dsg3-specific B cells from the peripheral blood of patients with active PV and to identify the variable regions within their heavy- and light-chain immunoglobulin genes. Methods: Dsg3-specific B cells were isolated as CD3-IgD-PI-Dsg3E-tag+ cells using recombinant human Dsg3 with an E-tag (rDsg3-E-His) as a probe. Heavy- and light-chain cDNA was produced by PCR from single B cells; these were used to characterize the usage and CDR3 sequence in the variable region of each gene. Results: Staining conditions were optimized using mouse hybridoma cells against human Dsg3 and peripheral blood mononuclear cells (PBMCs) from a PV patient. Individual Dsg3-specific B cells were isolated by FACS from four PV patients at a frequency of 1-18 per 105 PBMCs. CDR3 sequences and identical gene usage in the variable region were identified in several B cells from the same PV patients. Common gene usage was also found among several PV patients. Conclusions: These results suggested clonal expansion of autoreactive B cells and restricted gene usage for autoreactive B cells against Dsg3. Our method for the isolation of Dsg3-specific B cells will allow the systematic analysis of immunoglobulin gene usage in PV patients, which may elucidate the mechanism of immunopathogenesis.

Original languageEnglish
Pages (from-to)98-107
Number of pages10
JournalJournal of Dermatological Science
Volume52
Issue number2
DOIs
Publication statusPublished - 2008 Nov

Fingerprint

Desmoglein 3
Pemphigus
Flow cytometry
Medical Genetics
Flow Cytometry
Blood
B-Lymphocytes
Cells
Genes
Immunoglobulin Genes
Blood Cells
Immunoglobulin G
Immunoglobulin Light Chains
Immunoglobulin D
Immunoglobulin Heavy Chains
Hybridomas
Autoantibodies
Autoimmune Diseases
Immunoglobulins

Keywords

  • Antigen-specific
  • Autoimmune disease
  • B cell
  • Pemphigus vulgaris

ASJC Scopus subject areas

  • Dermatology

Cite this

@article{68671af5b11d43d5a2cdd008c72d9933,
title = "Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris",
abstract = "Background: Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-desmoglein 3 (Dsg3) IgG autoantibodies; however, the Dsg3-specific B cells that produce anti-Dsg3 IgG are not well characterized. Objectives: Our aims were to develop a flow cytometric method for the isolation of Dsg3-specific B cells from the peripheral blood of patients with active PV and to identify the variable regions within their heavy- and light-chain immunoglobulin genes. Methods: Dsg3-specific B cells were isolated as CD3-IgD-PI-Dsg3E-tag+ cells using recombinant human Dsg3 with an E-tag (rDsg3-E-His) as a probe. Heavy- and light-chain cDNA was produced by PCR from single B cells; these were used to characterize the usage and CDR3 sequence in the variable region of each gene. Results: Staining conditions were optimized using mouse hybridoma cells against human Dsg3 and peripheral blood mononuclear cells (PBMCs) from a PV patient. Individual Dsg3-specific B cells were isolated by FACS from four PV patients at a frequency of 1-18 per 105 PBMCs. CDR3 sequences and identical gene usage in the variable region were identified in several B cells from the same PV patients. Common gene usage was also found among several PV patients. Conclusions: These results suggested clonal expansion of autoreactive B cells and restricted gene usage for autoreactive B cells against Dsg3. Our method for the isolation of Dsg3-specific B cells will allow the systematic analysis of immunoglobulin gene usage in PV patients, which may elucidate the mechanism of immunopathogenesis.",
keywords = "Antigen-specific, Autoimmune disease, B cell, Pemphigus vulgaris",
author = "Jun Yamagami and Hayato Takahashi and Takayuki Ota and Masayuki Amagai",
year = "2008",
month = "11",
doi = "10.1016/j.jdermsci.2008.05.002",
language = "English",
volume = "52",
pages = "98--107",
journal = "Journal of Dermatological Science",
issn = "0923-1811",
publisher = "Elsevier Ireland Ltd",
number = "2",

}

TY - JOUR

T1 - Genetic characterization of human Dsg3-specific B cells isolated by flow cytometry from the peripheral blood of patients with pemphigus vulgaris

AU - Yamagami, Jun

AU - Takahashi, Hayato

AU - Ota, Takayuki

AU - Amagai, Masayuki

PY - 2008/11

Y1 - 2008/11

N2 - Background: Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-desmoglein 3 (Dsg3) IgG autoantibodies; however, the Dsg3-specific B cells that produce anti-Dsg3 IgG are not well characterized. Objectives: Our aims were to develop a flow cytometric method for the isolation of Dsg3-specific B cells from the peripheral blood of patients with active PV and to identify the variable regions within their heavy- and light-chain immunoglobulin genes. Methods: Dsg3-specific B cells were isolated as CD3-IgD-PI-Dsg3E-tag+ cells using recombinant human Dsg3 with an E-tag (rDsg3-E-His) as a probe. Heavy- and light-chain cDNA was produced by PCR from single B cells; these were used to characterize the usage and CDR3 sequence in the variable region of each gene. Results: Staining conditions were optimized using mouse hybridoma cells against human Dsg3 and peripheral blood mononuclear cells (PBMCs) from a PV patient. Individual Dsg3-specific B cells were isolated by FACS from four PV patients at a frequency of 1-18 per 105 PBMCs. CDR3 sequences and identical gene usage in the variable region were identified in several B cells from the same PV patients. Common gene usage was also found among several PV patients. Conclusions: These results suggested clonal expansion of autoreactive B cells and restricted gene usage for autoreactive B cells against Dsg3. Our method for the isolation of Dsg3-specific B cells will allow the systematic analysis of immunoglobulin gene usage in PV patients, which may elucidate the mechanism of immunopathogenesis.

AB - Background: Pemphigus vulgaris (PV) is an autoimmune bullous disease caused by anti-desmoglein 3 (Dsg3) IgG autoantibodies; however, the Dsg3-specific B cells that produce anti-Dsg3 IgG are not well characterized. Objectives: Our aims were to develop a flow cytometric method for the isolation of Dsg3-specific B cells from the peripheral blood of patients with active PV and to identify the variable regions within their heavy- and light-chain immunoglobulin genes. Methods: Dsg3-specific B cells were isolated as CD3-IgD-PI-Dsg3E-tag+ cells using recombinant human Dsg3 with an E-tag (rDsg3-E-His) as a probe. Heavy- and light-chain cDNA was produced by PCR from single B cells; these were used to characterize the usage and CDR3 sequence in the variable region of each gene. Results: Staining conditions were optimized using mouse hybridoma cells against human Dsg3 and peripheral blood mononuclear cells (PBMCs) from a PV patient. Individual Dsg3-specific B cells were isolated by FACS from four PV patients at a frequency of 1-18 per 105 PBMCs. CDR3 sequences and identical gene usage in the variable region were identified in several B cells from the same PV patients. Common gene usage was also found among several PV patients. Conclusions: These results suggested clonal expansion of autoreactive B cells and restricted gene usage for autoreactive B cells against Dsg3. Our method for the isolation of Dsg3-specific B cells will allow the systematic analysis of immunoglobulin gene usage in PV patients, which may elucidate the mechanism of immunopathogenesis.

KW - Antigen-specific

KW - Autoimmune disease

KW - B cell

KW - Pemphigus vulgaris

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U2 - 10.1016/j.jdermsci.2008.05.002

DO - 10.1016/j.jdermsci.2008.05.002

M3 - Article

VL - 52

SP - 98

EP - 107

JO - Journal of Dermatological Science

JF - Journal of Dermatological Science

SN - 0923-1811

IS - 2

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