After specifying important co-stimulatory interactions required for the re-stimulation of FVIII-specific memory B cells, we were interested to study the potential impact of different concentrations of FVIII on this process. We tested a range of concentrations between 1 pg mL−1 and 100 μg mL−1 of FVIII (Fig. 3a) [18]. Re-stimulation of memory B cells could be detected at concentrations of FVIII that were as small as 100 pg mL−1 (Fig. 3a)

[18]. Optimal re-stimulation was achieved at concentrations of 3–10 ng mL−1, which correspond to about 3–10% of the physiological plasma concentration (Fig. 3a) [18]. When we further increased the concentration of FVIII, inhibition of memory B-cell re-stimulation was observed. The MLN8237 solubility dmso inhibition started at a concentration of FVIII of 100–300 ng mL−1 with an almost complete inhibition at 1 μg mL−1 FVIII (Fig. 3a) [18]. The dose-response relation for T-cell re-stimulation was very different from the dose-response relation for memory B-cell re-stimulation. Optimal stimulation of FVIII-specific

T cells was observed at concentrations of 10–30 μg mL−1 FVIII (Fig. 3b,c). Inhibition of T-cell stimulation was seen at concentrations of 100 μg mL−1 FVIII. Based on these results, we conclude that the concentration of FVIII required for inhibition of memory B-cell OSI906 re-stimulation and the concentration required for inhibition of T-cell re-stimulation are very different (Fig. 3a–c), which makes it unlikely that the inhibition of memory B-cell re-stimulation is caused by an inhibition of T-cell stimulation. The major T-cell cytokines found in culture supernatants after stimulation of spleen cells with FVIII were IL-10 and IFN-γ (Fig. 3c), which is consistent with findings we reported previously [13,21]. To further support these results,

we analysed the frequency of FVIII-specific T cells by intracellular cytokine staining 3 days after re-stimulation of this website spleen cells. We compared concentrations of 10 ng mL−1, which re-stimulate, and 20 μg mL−1 FVIII which inhibit memory B-cell differentiation and observed a correlation between the frequency of FVIII-specific T cells producing IL-2, IL-10 or IFN-γ and the concentration of FVIII used for the re-stimulation (data not shown). We did not observe any inhibitory effects of 20 μg mL−1 of FVIII on T-cell stimulation despite the fact that this concentration of FVIII completely blocks the re-stimulation of FVIII-specific memory B cells [18]. Infections, particularly infections from the central venous catheter inserted in patients with haemophilia A and FVIII inhibitors during ITI therapy, commonly cause a rise in anti-FVIII antibody titres [22].