CD40-CD40L interactions are a key event in T-cell-dependent humoral immune responses [30]. The

results from studies on the significance of these interactions for the differentiation of memory B cells into ASC, however, are in conflict. Several reports suggest that CD40 signalling is important for the terminal differentiation of B cells and for antibody secretion [31–34]. Other reports show that CD40 signalling prevents the terminal differentiation of B cells [35–39]. Our results indicate that the re-stimulation of FVIII-specific memory B cells and their subsequent differentiation into anti-FVIII ASC requires CD40-CD40L interaction. The blockade of these interactions prevented the formation of anti-FVIII ASC Crenolanib in vitro and reduced it significantly in vivo [17]. We believe that the blockade of CD40-CD40L interactions in our system downregulates T-cell activation and, more importantly, blocks the interaction between activated T cells and memory B cells. Based on the successful use of high-dose FVIII for the induction of immune tolerance in patients with haemophilia A [1], we speculated on the issue of whether the re-stimulation of FVIII-specific memory B cells was affected in any significant manner by high concentrations of FVIII. Our results demonstrate that concentrations of FVIII buy Napabucasin that are below the physiological plasma concentration

of 100 ng mL−1 (1 U mL−1) re-stimulate FVIII-specific memory B cells and induce their differentiation into ASC in vitro, whereas concentrations that are above the physiological plasma concentration inhibit this process. These results support the idea that the inhibition or eradication of FVIII-specific memory B cells might be an early event in the downregulation of established Chloroambucil anti-FVIII antibody responses in patients. The eradication of memory B cells would prevent their differentiation into ASC and, moreover, may lead to a deficiency of effective antigen-presenting cells

required for the re-stimulation of FVIII-specific T cells. The induction of regulatory T cells rather than effector T cells could be the consequence of this deficiency. Currently, it is not clear, however, whether high-dose FVIII ITI therapy in patients would lead to local FVIII concentrations that are comparable with the concentrations that we used in our in vitro experiments. Further studies are necessary to investigate this hypothesis. Toll-like receptors recognize invading pathogens such as viruses and bacteria and serve as an important link between innate and adaptive immunity [40,41]. Given the importance of TLR for the regulation of adaptive immune responses, we speculated as to how the triggering of TLR would influence the regulation of FVIII-specific memory B cells.