A274 Aims: Antibodies (Abs) against blood group A-carbohydrate determinant are a major barrier to ABO-incompatible organ transplantation. We have previously demonstrated that the B cells bearing surface IgM (sIgM) recognizing A-antigen (A-Ag) were CD5+, CD11b+, B-1a cells in blood group O human peripheral blood. Recently it has been proven that B-1a cells are specific Ag-selected and this selection can be blocked by Cyclosporine (CsA). But it’s also known that CsA has no effect on anti-A Abs producing cells. Since we previously reported that both of Abs-producing cells and B-1 cells express sIgM B cell receptor, which can bind specific Ag, those cells with anti-A sIgM may be targeted for specific elimination by synthetic A-carbohydrates conjugated with a cytotoxic constituent. The present study proved that mice, resembling to human, had CD5+ B-1a cells with anti-A sIgM and could produce natural anti-A IgM. Thereby we have used mice for investigating the effects of a novel approach consisting of treatment with synthetic A-carbohydrates and CsA on B cells responding to blood group A-Ag. Methods: In vitro, Spl cells from Balb/c mice were cultured with various concentrations of A-BSA, and then cultured with 50 fold diluted rabbit anti-BSA Abs, followed by culture with 25 fold diluted rabbit complement in a 37°C, 5% CO2 cell incubator. All cells were conducted to ELISPOT assay to detect the frequencies of anti-A Abs-producing cells. For in vivo experiment, Balb/c mice were i.p. injected with A-BSA diluted in PBS at a does of 100ug/mouse in day 0 and then injected with 20 fold diluted rabbit anti-BSA Abs diluted in PBS at a dose of 1ml/mouse 6 hours later after A-BSA injection. CsA were administered by daily i.p. injection for 2 weeks from day 1. At day 14, 5×108 A-RBCs were i.p. injected to boost anti-A Abs production. A-determinant binding B cells were detected by staining fluorescein-labeled synthetic blood group A trisaccharide (GalNAcα1-3Fucα1-2Gal) conjugated to bovine serum albumin (BSA) (A-BSA) and anti-mouse IgM. Anti-A IgM serum titer was determined by indirect ELISA. Results: After culture spleen cells with A-BSA/anti-BSA Abs, the frequency of anti-A Abs-producing B cells decreased in parallel to the concentration of A-BSA. The in vivo single injection of A-BSA and anti-BSA Abs in Balb/c mice resulted in specific but temporal reduction of both A-binding B cells percentage and anti-A IgM in their sera. The concomitant treatment with daily injection of CsA completely inhibited reappearance of B cells bearing receptor for A-determinants, there by significantly inhibited production of anti-A Abs even after immunization with human A red blood cells (p<0.01). This prevention of anti-A Abs production did not affect production of total serum IgM and IgG levels, indicating the specificity of this strategy. Conclusions: Specific and permanent depletion of A -reactive B cells needs two steps: First, administration of A-BSA specific targets mature A-reactive B-1 cells (mainly in PerC) and anti-A Abs producing cells in Spl by binding to their BCRs, further administrated anti-BSA Abs recognizes the BSA component in A-BSA molecule, thus induces complement activity to kill these B cells. Second, administration of CsA blocks newly formed A-reactive B-1 cells differentiation from B-0 cells. By such treatment, specific and permanent A-reactive B cell depletion can be achieved.
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