Phenotypic characterization of immune cell populations in homeostatic and FVIII-challenged severe HA mice

Development of FVIII-neutralizing antibodies (inhibitors) is one of the main complications occurring in 30% of severe hemophilia A (HA) patients undergoing replacement therapy. Both lack of central tolerance, due to the congenital absence of FVIII, and the presence of danger signals are factors involved in inhibitors development, which is definitely related to the immune cells activation. Since recent studies have been proposing new extra-coagulative roles for FVIII in the immuno-hematological biology and have shown the presence of increased levels of several inflammatory cytokines in untreated HA patients, we sought to investigate the effect of FVIII absence on the immune cell populations before and after challenge with a specific (FVIII) or general (OVA) immunological challenge. We also sought to investigate the capability of naïve CD4 T cells to differentiate into T regulatory cells. We employed a mouse model of severe HA and we did not observe any significative change in number and percentage of primary immune populations (e.g. T and B cells) and in the quantitative humoral response against FVIII or OVA when compare to control age- and treatment-matched animals. On the other hand, HA mice challenged with both antigens showed higher conversion of naïve to memory T cells. Interestingly, these differences were detected only when the immunological challenge included an adjuvant while it was absent when FVIII was injected intravenously without any supplemental stimulus, as it happens in the normal therapeutic regimen. Overall, the results obtained in this thesis suggest that immunogenic versus tolerogenic response to FVIII in HA patients could depend on the reciprocal interactions of extrinsic and intrinsic factors among which the pro-inflammatory microenvironment determined by the recurrent microbleedings should be taken in consideration.