Suppression of inhibitor formation against FVIII in C57BL6/129 mice with hemophilia A by oral administration of a 1:1 mixture of bioencapsulated CTB-C2 and CTB-HC FVIII antigens. (A) Time line of oral antigen administration and intravenous treatment with BDD-FVIII. Number in circle indicates time-point for tail bleed. (B) Inhibitor titers (in BU per milliliter) after 4 weekly IV injections of FVIII in non-fed animals (“no plant”) or mice fed with WT or FVIII containing plant material. IgG1 (C), IgG2a (D), IgG2b (E) titers against FVIII for the same experimental groups. (B-E) Data are shown for individual mice and as averages ± standard error of the mean (SEM). (F) After the blood draw, mice were killed and spleens collected. Splenocyte cultures for individual mice (n = 3-5 per group) were stimulated in vitro with 10 μg/mL BDD-FVIII for 48 hours. Subsequently, cells were harvested and subjected to quantitative reverse-transcription-PCR analysis. “Fold increase” is change in RNA transcripts of FVIII vs mock-stimulated cultures. The dotted horizontal line indicates the minimally required increase of 2.5-fold for a statistically significant difference. (G) Splenocytes derived from the same experimental mice were subjected to enzyme-linked immunospot analysis for frequency of IL-10 secreting cell population. All data are shown for individual mice and as averages ± SEM. Unpaired 2-tailed Student t tests were used to calculate P values (**P < .01).