If not otherwise indicated, whole splenocytes were used as APCs in most experiments.
To evaluate the composition of APCs, splenocytes were stained for CD3e PerCP-eFluor 710 , CD19 FITC , CD11c APC , CD11b PE-Cy7 and HLA-DR BV605 and analyzed using a BD FACS Aria III and FlowJo software . Nonspecific binding through Fc gamma receptors was blocked by a mixture of anti-CD16 and anti-CD32 antibodies .Splenocytes were prepared as described before.
The FVIII peptide repertoire presented by APCs was monitored by analyzing the peptide specificities of subsequently activated FVIII specific CD4+ T cell clones. In detail, APCs from naïve HLA-DRB1*1501 mice were incubated with human recombinant FVIII, thrombin activated FVIII or FVIII in complex with human purified plasma-derived VWF. Equal units of human FVIII and human VWF were co-incubated in serum-free medium at room temperature for one hour to facilitate complex building.
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