CD4 T cells specific for factor VIII are present at high frequency in healthy donors and comprise naïve and memory cells.

We investigated the frequency and subset origin of circulating factor VIII (FVIII)-specific CD4 T cells in healthy donors. Total CD4 T cells and purified CD4 T-cell subsets were stimulated with FVIII-loaded autologous dendritic cells and challenged for specificity in interferon-γ enzyme-linked immunospots. The number of specific T-cell lines allowed estimation of the frequency of T cells circulating in the blood of the donors. All the 16 healthy donors generated strong in vitro T-cell responses, leading to the generation of 154 FVIII-specific T-cell lines. The mean frequency of FVIII-specific CD4 T cells in healthy donors was similar to that of T cells specific for foreign antigens and greater than that of T cells specific for known immunogenic therapeutic proteins. Normal levels of endogenous FVIII in healthy donors therefore do not prevent a significant escape of FVIII-specific CD4 T cells from negative thymic selection. FVIII-specific T cells mainly originated from both the naïve and central memory cell subsets, but their frequencies remained low as compared with those of cells specific for foreign antigens in immunized donors. The observation of a spontaneous generation of FVIII-specific memory T cells without a global expansion suggests peculiar peripheral tolerance mechanisms to FVIII in healthy donors.