The Impact of Toll-Like Receptor Signaling On B Cells Responding to Blood Group A Carbohydrates.

Abstract

[Aim] Bacterial infection following ABO-incompatible organ transplantation has been reported to be associated with refractory antibody-mediated rejection targeting blood type antigens. In this study, we investigated the influence of toll-like receptor(TLR) signaling, through the binding of bacterial-derived ligands, on B cell activation in response to blood group carbohydrate-antigens. [Methods] We have previously demonstrated that B cells responding to blood group A-antigens differentiate into CD11+CD5+B-1a cells, which can be blocked by calcineurin inhibitor(CsA) treatment, in mice, resembling humans. Resting B cells isolated from the splenocytes of Balb/c mice were treated with anti-IgM F(ab), an analog of TI-2 Ags, in the presence or absence of lipopolysaccharide(LPS)(10μg/ml) and CsA in vitro. After 3 days in culture, the phenotypic alteration of proliferating B-cells was analyzed by flow cytometry. Balb/c mice were also immunized with human blood type A-erythrocytes, with or without LPS administration(1 or 10μg/mouse). [Results] CD19+B cells stimulated with anti-IgM F(ab) proliferated vigorously. The proliferating B cells differentiated into CD5+B-1a cells in the absence of LPS, but in the presence of LPS, they differentiated into CD5-B-1b cells. A clinically relevant concentration of CsA(100ng/mL) completely blocked B-1a differentiation, but did not affect B-1b cell differentiation. Consistent with our previous findings, B-cells with anti-A receptors showed an sIgM+CD5+B-1a phenotype in mice immunized with A-erythrocytes. However, those cells showed an sIgM+CD5dim/-B-1b phenotype in mice immunized with A-erythrocytes together with LPS. Anti-A antibody production was augmented by LPS dose-dependently. CsA-treatment significantly inhibited anti-A Ab production in response to immunization with A-erythrocytes, though not in mice treated with LPS. [Conclusions] To our knowledge, these data provide the first evidence that B-cells responding to blood type A carbohydrate, in the presence of TLR signaling, show the sIgM+CD5dim/-B-1b phenotype, and lose susceptibility to inhibition by CsA. Blocking TLR signaling in B-cells with anti-A receptors may be a novel strategy to regulate Ab-mediated rejection associated with bacterial infection, following transplantation.