Tracking Factor VIII-Specific Memory B Cells in Hemophilia A Patients with Factor VIII Inhibitors.

The development of neutralizing anti-FVIII antibodies (FVIII inhibitors) in about 30% of patients with severe hemophilia A is the most serious complication in the treatment of hemophilia patients with FVIII products. Little information is available on the immunological mechanisms that regulate the development and maintenance of FVIII inhibitors.

Memory B cells are a central component of humoral immunity. They drive the rapid anamnestic antibody response that occurs after re-exposure to antigen and seem to be important for replenishing the pool of long-lived plasma cells to maintain long-term antibody levels in the absence of antigen. Nothing is known about the dynamics of FVIII-specific memory B cells in patients with hemophilia A who develop FVIII inhibitors. Recently, Crotty et al. (J Immunol Methods, 2004) described an in vitro assay to quantify antigen-specific memory B cells in human blood. This assay utilizes a 6-day polyclonal stimulation of peripheral blood mononuclear cells (PBMC) followed by an antigen-specific ELISPOT for the detection of memory B cells that have differentiated into antibody-secreting plasma cells in vitro. We adapted this assay to human FVIII and used it to track FVIII-specific memory B cells in the blood of hemophilia A patients with and without FVIII inhibitors. Human serum albumin was used as a negative control and tetanus toxin as a positive control. The numbers of FVIII-specific, tetanus toxin-specific (positive control) and albumin-specific (negative control) memory B cells were calculated as percentage of total IgG memory B cells.

So far, we have analyzed 14 patients with hemophilia A (age: 6–65 years). 8 were positive and 6 were negative for FVIII inhibitors. For comparison, we have analyzed 20 healthy individuals (age: 19–48 years). 2 out of 8 patients with inhibitors had detectable FVIII-specific memory B cells in their peripheral blood cells. However, none of the patients without inhibitors and none of the healthy individuals had any detectable FVIII-specific memory B cells in their circulation. The detection limit for FVIII-specific memory B cells in patients with inhibitors was about 0.2 % (percent of total IgG memory B cells). Current activities focus on further advancing the method with the aim to improve the detection limit for the detection of FVIII-specific memory B cells. All samples analyzed (including patients and healthy individuals) were negative for human serum albumin-specific memory B cells (negative control). Tetanus toxin-specific memory B cells (positive control) were found in both patients and healthy blood donors. The percentage of tetanus toxin-specific memory B cells in individuals who were vaccinated with tetanus toxoid was in the range of 0.25 – 0.58 % (percent of total IgG memory B cells).

We conclude that the method described is suitable to track FVIII-specific memory B cells in the circulation. We are currently asking the question whether the presence of FVIII-specific memory B cells in the circulation correlates with the persistence of FVIII inhibitors. Furthermore, we will monitor patients with inhibitors during ITI therapy in order to find out whether the disappearance of FVIII-specific memory B cells in the circulation could be an early predictor of a successful ITI outcome.