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

Department of Dermatology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.

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 10(5) 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.

PMID: 18586466 [PubMed - in process]