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α-Galactosyltransferase-deficient (GalT-/-) pigs lacking Galα1,3Galβ1,4GlcNAc (Gal) epitopes facilitate the longer survival of nonhuman primate recipients. However, the Abs induced by T-cell-independent (TI) non-Gal glycan Ags cause refractory delayed rejection of xenografts, even from GalT-/- pigs. Therefore, therapeutic strategies that inhibit the refractory B cell immune response against TI glycan Ags are essential for the success of xenotransplantation. In our previous study, B cells responding to Gal Ags showed the CD5– B-1b phenotype and resistance to calsineurin inhibitors (CNI) in vivo, and Ag-induced B-1b-cell differentiation in vitro was associated with Toll-like receptor (TLR) signals. Herein, we provide the rationale behind a novel therapeutic approach to inhibit the B-1b-cell-mediated refractory production of Abs against transplant-related TI glycan Ags.We explored the molecular mechanisms behind CNI resistance in B-1b activation in vitro, using cells from MyD88- and TRIF-deficient (downstream factors of TLRs) and wild-type (WT) mice. Using WT and GalT-/- mice, we examined the therapeutic effect of TLR-signaling blockade in inhibiting corresponding anti-glycan Abs in mice immunized with two types of known transplant-related glycan Ags; namely, human blood group A (A-erhythrocytes, A-RBC) and Gal (rabbit-erhythrocytes, rabbit-RBC) Ags, which induce CD5+ B-1a and CD5– B-1b immune responses, respectively.MyD88-dependent TLR stimulation in vitro enhanced downstream factors of the B cell receptor (BCR)-calcineurin pathway, including NFATc1, indicating the molecular mechanism of CNI resistance in B-1b cells. Blockade of the TLR signals attenuated NFATc1 expression in B-1b cells, and dual blockade of BCRs and TLRs by CNI and a TLR-inhibitor (-i) abrogated refractory B-1b-cell differentiation. A-RBC immunization expanded the B-1a cells with BCRs for A Ags and elevated the serum anti-A Ab levels in WT mice. CNI markedly inhibited both events. LPS (a TLR4 agonist) stimulation and A-RBC immunization expanded the B-1b cells with BCRs for A Ags and further increased the serum levels of anti-A Abs, which were not inhibited by CNI. Injection of the TLR-i before A-RBC and LPS administration completely inhibited the elevation of anti-A Ab levels. Rabbit-RBC immunization expanded both the B-1b cells with BCRs for Gal Ags and anti-Gal Ab-producing cells in the spleen, and elevated the serum anti-Gal Ab levels in GalT-/- mice, which showed resistance to CNI. TLR-i and MyD88-i treatment overcame the CNI resistance. Dual blockade of BCRs (by a Bruton’s tyrosine kinase inhibitor) and TLR-MyD88 signals (by a histone deacetylase inhibitor) abrogated the refractory B-1b-cell response against anti-Gal glycan Ags.The dual blockade of BCRs and TLR-MyD88 signals is a novel and effective therapeutic strategy for overcoming refractory transplant-related anti-glycan Ab production for the success of xenotransplantation.