[Aim] Bacterial infections after ABO-incompatible transplantation are associated with refractory antibody (Ab)-mediated rejection (AMR). We demonstrated that bacterial pathogens provoke B cells, responding to human blood group A-antigens(A-Ag), to develop resistance against calcineurin inhibitors (CNI) via TLR-MyD88 pathway. We also investigated the therapeutic effects of TLR inhibitor (TLR-i) on CNI-resistant AMR. [Methods/Results] CD5⁺B-1a cells(B1a) with B cell receptors (BCR) for A-Ag expanded in the peritoneal cavity of Balb/c mice after sensitization with human A-Ag, and was sensitive to cyclosporin (CsA). However, CD5^dimB-1b cells(B1b) with BCR for A-Ag predominantly expanded in Balb/c mice after exposure to A-Ag together with lipopolysaccharide (LPS), and was resistant to CsA. Consistently, when splenic B cells from Balb/c mice were treated in vitro with anti-IgM F(ab')₂, the B cells differentiated into B1a, which was abrogated by CsA. However, anti-IgM F(ab')₂ treatment and LPS induced differentiation of B1b, indicating CsA-resistance. In MyD88-deficient mice, B cells differentiated into B1a regardless of the LPS-treatment, indicating the dependency of LPS-induced B1b differentiation on TLR-MyD88-signaling (-sig). Western blotting analyses revealed that co-stimulation of TLR in addition to BCR by treating with anti-IgM F(ab')2 and LPS enhanced not only the NF-kB(p65) expression, which is a downstream factor of TLR-sig, but also NFATc1 and NF-kB(p100/p52) expressions, which are downstream factors of calcineurin(CN) activities in BCR-sig in stimulated B cells. However, these were never observed in MyD88-deficient mice. Therefore, the inhibitory effect of CsA on BCR-sig is disabled by the activation of TLR-MyD88-sig in B1b. Notably, in the in vitro model, the molecules downstream of BCR-sig were inhibited by TLR-i even in the presence of LPS. Furthermore, TLR-i nullified the resistance to CsA in B1b, resulting in complete inhibition of anti-A Ab even after exposure to A-Ag together with LPS in an in vivo model. [Conclusions] BCR-crosslinking activates CN/NFATc1, suggesting B1a activation due to BCR-sig, which can be inhibited by CsA. However, LPS induces signals in B1b by activating TLR-MyD88-sig (independent of CN activity) and enhances BCR-sig by activating NFATc1 (a downstream factor of CN) indicating that CsA cannot inhibit B1b. Hence, blocking TLR-sig in B cells is possibly a novel way to treat AMR associated with bacterial infection.

CITATION INFORMATION: Sakai H, Tanaka Y, Ohdan H. Cross-Talk Between TLR and BCR Pathways Induces CNI-Resistance in B-Cells Responding to Blood Group A-Antigens: A Novel Paradigm for Treatment with TLR-Inhibitor for CNI-Resistant AMR. Am J Transplant. 2016;16 (suppl 3).

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