Factor VIII-Specific Memory B-Cells in Patients with Hemophilia A.
Authors: van Helden Pauline M.W. , Kaijen Paul H.P. , van den Berg H. Marijke , Voorberg Jan 
Published: October 2019

Abstract
The quick anamnestic antibody response seen after recurrent exposure to antigen involves memory B- cells that, helped by T-cells, undergo rigid proliferation and subsequent differentiation into antibody producing cells. The presence of a pool of memory B-cells allows for a rapid response to antigens which quickly eliminates incoming pathogens. In the context of immune responses to therapeutic agents such as blood coagulation factor VIII (FVIII), antigenic re-stimulation of specific memory B-cells is undesirable. In approximately 25% of hemophilia A patients replacement therapy is hampered by inhibitory antibodies that bind to FVIII. Currently, the FVIII-specific memory B-cell compartment in patients with hemophilia A has remained poorly characterized. We have developed a protocol that allows for identification and quantification of circulating memory B-cells in patients with hemophilia A. CD19+ B-cells were sorted on a layer of irradiated EL4B5 thymoma cells expressing CD40L in the presence of the supernatant of mitogen-stimulated T-cells. These experimental conditions, that mimic the interaction of B-cells with an activating helper CD4+ T-cell, induce proliferation of memory B-cells and allow them to differentiate into antibody secreting cells (ASC) in an antigen-independent manner. After 9–10 days of culture, total IgG and FVIII-specific IgG was determined by ELISA and number of ASC was determined by ELISpot. We analyzed blood samples of five multi-transfused patients (>50 FVIII administrations) who never experienced any inhibitor episode, five patients who experienced inhibitory antibodies in the past but were successfully treated with immune tolerance induction and 6 patients with an inhibitor at the time of blood sampling. The ELISA set-up appeared to be more sensitive than ELISpot showing ASC producing anti-FVIII antibodies varying from 0.2–50 ng/ml. In contrast, ELISpot analysis only allowed for detection of B-cell clones producing over ~4 ng/ml of FVIII-specific IgG. Frequencies of FVIII-specific memory B-cells varied from 0–0.027% of total number of circulating peripheral B-cells. The relative amount of circulating memory B-cells did not correspond to inhibitor titers as measured in a Bethesda assay. The highest frequencies were observed in patients suffering from anamnestic response to FVIII suggesting the importance of antigenic stimulation for maintenance of memory B-cell levels. This is further supported by the low frequency that was observed in a high-titer inhibitor patient who had not been treated with FVIII for several months prior to blood sampling. Surprisingly, we detected FVIII-specific memory B-cells in two multi-transfused patients who did not experience any inhibitor episode in the past. These B-cells were present in a low frequency however and developed into ASC producing only limited amounts of anti-FVIII antibodies. These observations suggest that peripheral blood memory B-cells can develop in the absence of clinically relevant inhibitors.