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CHARACTERIZATION OF ALLOREACTIVE MEMORY B CELLS.
Dessislava Kopchaliiska, PhD, Andrea A. Zachary, PhD and Mary S. Leffell, PhD. Baltimore MD, USA, Johns Hopkins University School of Medicine, 21205, Medicine.

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The direct identification of Ag-specific cells with MHC tetramers provides a powerful tool for monitoring T cells. We previously reported that MHC tetramers also specifically bind allo-reactive B cells in peripheral blood samples from our patients who were sensitized to HLA-A2 and /or B7. The frequencies of tetramer + (tet+) B cells correlated with serum level of specific antibody, i.e., historic versus current sensitization. We hypothesized that tetramers bind B lymphocyte antigen receptors on circulating memory B cells. The aim of the current study was to further characterize the CD19+tet+ cells.
Methods: PBMC were isolated from patient blood samples and B cells were enriched by magnetic bead depletion of T cells. Three-color staining with HLA-A*0201GAG-PE (Beckman Coulter) and FITC or APC labeled anti-CD19, CD27 or CD38 monoclonal antibodies (BD Biosciences/ Pharmingen) was used for characterization of the tet+ cells. Data were acquired using a FACSCalibur flow cytometer and analyzed with CellQuest Software (Becton Dickinson).
Results: Samples from 3 patients with well defined antibodies to HLA A2 were tested. The mean frequency of CD19+A*0201 tet+cells for these patients was 6.1% of the total CD19+ population, compared to 0.9% of non-sensitized controls. Of the CD19+tet+ cells from sensitized patients, 68.5 ± 13.4% were also positive for CD27, which is considered a marker for memory B cells. The majority of the CD19+tet+ B cells, 77.0 ± 13.5% also expressed CD38, which is considered a marker for plasma cell precursors.
Conclusion: Circulating CD19+ B cells allo-reactive with HLA-A*0201 tetramers have a phenotype consistent with memory B cells and plasma cell precursors.