Risk factors initiating tolerance breakdown towards coagulation factor VIII during immune tolerance induction therapy

Hemophilia A (HA) is a hereditary bleeding disorder caused by a deficiency in coagulation factor VIII (FVIII). Despite FVIII replacement therapy, a significant proportion of patients develop neutralizing FVIII-specific antibodies (inhibitors), rendering the treatment ineffective. To address this, a high-dose regimen known as immune tolerance induction (ITI) has been developed, aiming to re-establish peripheral FVIII tolerance and eradicate these inhibitors. In a recent study, our group identified FVIII-specific regulatory T cells (Tregs) as key players in ITI success. Here, we investigate how immunological challenges, specifically <em>Staphylococcus aureus (USA300 LAC)</em> infection, can impact the stability of peripherally induced tolerance. <br> Using in vivo and in vitro models, we demonstrate that <em>Staphylococcus aureus</em> infection disrupts ITI by reactivating FVIII-specific B cells through direct stimulation of TLR2 and subsequent activation of the mTOR/STAT3 pathways, which are downregulated in tolerized B cells when compared to prophylactically activated cells. Infection induced a spike in FVIII-specific inhibitor production by directly stimulating B cells and by promoting the differentiation of Tregs and naïve T cells into follicular helper T cells, thereby enhancing the humoral response. Although FVIII-specific Tregs expand <em>in vivo</em> during infection, they acquire pro-inflammatory features, including increased proliferation and expression of transcription factors such as T-bet. Functional suppression assays <em>in vitro</em> reveal that these bacteria-exposed Tregs are dysfunctional, exhibiting reduced suppressive capacity and phenotypic instability driven by TLR2 signaling. <br> Importantly, PD-1 stimulation mitigates infection-induced B cell activation by inhibiting the mTOR pathway and the STAT3 pathway, possibly through PTEN activation, identifying PD-1 signaling as an attractive candidate for therapy to prevent <em>S. aureus</em>-mediated tolerance modulation. This may apply to other bacterial infections potently activating the mTOR/STAT3 pathway. <br> Collectively, the data elucidates mechanisms by which bacterial infections destabilize immune tolerance and identify candidate biomarkers and therapeutic targets to predict and prevent ITI failure.