FVIII Specific Memory B Lymphocytes in Hemophilia A with Inhibitors Treated by Immune Tolerance Induction.

Hemophilia A (HA) is a life-threatening hemorrhagic bleeding disorder caused by deficiency of Factor VIII (FVIII). Twenty to thirty percent of HA patients treated by FVIII develop inhibitors. The presence of these inhibitors in high titer (>5 Bethesda Units (BU)/ml) requires modification of the treatment regimen: use of products that by-pass the action of FVIII for haemorrhagic accidents and treatment protocols by immune tolerance induction (ITI) to eradicate inhibitors. The aim of our study was to enumerate and characterize FVIII-specific memory B-cells in peripheral blood by using polyclonal activation of enriched B-cells and an ELISpot assay in hemophilia patients with inhibitors.

Two groups of patients were prospectively included. First group included six severe HA patients treated by ITI; three with inhibitors (mean 3.04 ± 0.67 BU/ml; mean 14.6±4.2 years old) while the three others had a past of inhibitors, successfully treated by ITI (<0.6BU/ml; mean 18.6 ±6.3 years old). Second group were six severe HA who never developed inhibitors (mean 19.5±1.7 years old). Circulating FVIII specific memory B-cells were enumerated in each cohort and 13 controls using the ELISpot system as previously described. The results were expressed as the number of FVIII specific Secreting Cells (SCs) (IgG, IgA or IgM)/10⁶ B-cells.

In the first group,circulating FVIII-specific IgM-SCs were detected in 5 patients (1.07-45/10⁶ B-cells), FVIII-specific IgA-SCs in 6 patients ( 2.9-7.5/10⁶ B-cells).Only in patients with detectable inhibitors, FVIII-specific IgG-SCs (4-5.2/10⁶ B-cells) were detected. In the second group FVIII-specific IgM-SCs (between 5 and 52 per 10⁶ B-cells) were found, one patient had FVIII-specific IgA-SCs (2/10⁶ B-cells).

We were able to detect FVIII specific memory B-cells. Before the treatment by ITI, the FVIII-specific immune memory seems to be characterized by the presence of circulating FVIII-specific memory B-cells belonging to the three isotype classes whereas FVIII-specific IgG-memory B-cells could not be found after a successful treatment. These results suggest an evolution of the pool of FVIII-specific circulating memory B-cells during the period of ITI. It would be interesting to study the FVIII-specific memory B-cells at the different periods of ITI (before, during and at the end of the protocol).The significance of FVIII specific memory B-cells in HA patients without inhibitor deserve to be study.

No relevant conflicts of interest to declare.