040 Single B-cell receptor profiling indicates selective self-reactivity in pemphigus vulgaris

Pemphigus vulgaris (PV) is a potentially fatal blistering disease caused by autoantibodies to desmoglein 3 (Dsg3). Several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and myasthenia gravis, have defective central and/or peripheral B cell tolerance checkpoints, leading to an accumulation of polyreactive B cells that is thought to provide the first hit toward the development of autoimmunity. We investigated whether PV patients have intact tolerance checkpoints to self-antigens other than Dsg3, which would differentiate the mechanism of autoimmunity in PV from those of other autoimmune diseases. Using FACS, we isolated 326 new emigrant and 326 mature naïve B cells from 5 PV patients, as well as corresponding B cells from 3 healthy donors. We performed single cell RT-PCR to amplify each B cell receptor (BCR) and produced them as soluble monoclonal antibodies, which were subsequently screened by ELISA for binding to a panel of classic polyreactivity antigens (dsDNA, insulin, and LPS). In the new emigrant B cell subset, the frequencies of polyreactive BCRs among PV patients varied, with some PV patients having significantly higher rates compared to healthy controls. In the mature naïve B cell subset, the frequencies of polyreactive BCRs among PV patients were similar to those among healthy donors. Polyreactive BCRs did not bind Dsg3, and conversely, anti-Dsg3 BCRs were not polyreactive. These data indicate that the peripheral checkpoint maintains normal B cell tolerance to non-Dsg3 self antigens regardless of whether the central checkpoint is defective, and that polyreactive B cells in PV patients do not significantly contribute to the anti-Dsg3 B cell pool. Collectively, our data suggest that autoimmunity in PV is caused by a selective break in B cell tolerance to Dsg3 in the setting of an otherwise intact peripheral B cell tolerance checkpoint.