Treatment of patients suffering from hemophilia A involves replacement therapy requiring frequent infusions of exogenous coagulation Factor VIII (FVIII). In approximately 25-33% of patients, the formation of inhibitors, antibodies to the infused FVIII occurs. FVIII inhibitor formation remains one of the most serious complications of FVIII therapy, often rendering further replacement therapy ineffective, making clinical management extremely difficult. Despite extensive investigation, an absolute mechanism for inhibitor formation including identification of defined risk factors has not been elucidated. Investigation into possible parameters influencing FVIII inhibitor formation such as, severity of hemophilia, age, age at first factor replcement, cumulative FVIII exposure, dose, inhibitor testing intervals, causative hemophilia mutation, race, ethnicity, HLA genotype, blood transfusion prior to factor replacement, unrelated immune system activation, type of product including viral inactivation method, other medications and inhibitor assay method has produced little definitive correlation. The immune response in inhibitor patients stems from cooperation between specifically activated B and T lymphocytes. Antibodies made by patient B lymphocytes have been extensively characterized with regard to isotype and FVIII epitopes. IgG antibodies of subclasses IgG4 and IgG1 are most highly represented with their predominant epitopes on the FVIII molecule mapping to the A2 and C2 domains. Although information gathered on the B cell response has been critical to partial development of a mechanism for FVIII inhibitor formation, the complete story must include characterization of the T cell contribution. Elucidation of the interaction between FVIII specific B and T lymphocytes that leads to inhibitor formation in the defined patient population may lead to development of targeted therapies or specific in vitro diagnostic tests to aid in the treatment of inhibitor patients. In addition, elucidation of T cell mechanisms in the formation of FVIII inhibitors may lead to advances in current FVIII tolerization protocols.
The aim of the current project is to identify T cell epitopes on the FVIII molecule and to use peptides composed of epitope sequences to stimulate cultured lymphocytes from inhibitor patients and identify the responsible T cell receptors. The proposed technology used for the current project involves culturing of antigen presenting cell lines (obtained from ATCC or from patients) in the presence of FVIII, allowing for its uptake, processing, and presentation of peptides on the cell surface in the context of Class II MHC molecules. The peptides (ranging in size from 8 to 16 amino acids) will then be eluted, isolated by reverse phase HPLC and identified by direct peptide sequencing. Peptide synthesis procedures will be used to produce sufficient quantities of epitope peptides to be used for stimulation of cultured peripheral blood lymphocytes from inhibitor patients. The novel technique of T cell spectratyping will be used to characterize the repertoire of T cell receptors that have expanded in response to peptide stimulation. This project will be done with the expert technical assistance of Laura Wood, M.S. and in collaboration with Basil Golding, M.D., Dorothy Scott, M.D., and Orit Schrf, Ph.D. Patient material will be provided by Joan Gill, M.D. of the Great Lakes Hemophilia Foundation and the Blood Center of Southeastern Wisconsin.
