395 in Vitro Expansion of Desmoglein-Specific B Cells of Patients with Pemphigus

The ability to expand B cells in vitro has become a useful tool for studying human immunity. We have developed a culture system to support the expansion of human mature B cells purified from peripheral blood. Murine bone marrow stromal cell line MS-5 was used as feeder cells to support the growth of human B cells. Single B cells expanded by 46,689 ± 4,105 (mean ± s.e.m.) fold after 12 days of culture. The monoclonal IgM and IgG levels in the culture supernatant were 9.3 ± 1.4 μg/mL and 10.5 ± 0.8 μg/mL, respectively, which could be readily screened by ELISA. Pemphigus vulgaris (PV) and foliaceus (PF) are severe blistering diseases of the skin that are caused by autoantibodies directed against desmoglein-1 (DSG1) and DSG3. Utilizing in vitro B cells expansion system made it possible to better understand the frequency of DSG1/ DSG3 specific B cells in PV/PF patients. B cells were isolated from patients with clinically active PV (n=2), PF (n=2) and healthy controls (HC) (n=2). Peripheral blood B cells (10/well) were cultured in 96 well plates for 12 days. Supernatants were tested for the presence of DSG1 or DSG3 antibodies by ELISA. The frequency of wells with ELISA values outside of 3x the interquartile range (IQR) was calculated for each subject. Supernatants from pemphigus patients showed a higher frequency of wells producing IgG anti DSG1 or DSG3 than controls (Mean frequency of wells > 3x IQR: PV/PF = 4. 87%; HC = 1.89%, p = 0.03). Indirect immunofluorescence staining with supernatants of high outlier wells showed intracellular IgG deposits in the epidermis, which are consistent with the pattern seen in the staining with serum of PV/PF patients. In conclusion, our culture system enabled us to identify and expand DSG-specific B cells, and to visualize an increase in frequency of DSG-specific B cells in peripheral blood of patients with active PV/PF.