OC 37.3 - Mice possess a more limited natural anti-FVIII antibody repertoire than humans that is generated primarily by marginal zone B cells

Background: 30% of severe hemophilia A patients develop neutralizing anti-FVIII antibodies. The significance of natural non-neutralizing antibodies (NNAs) to FVIII remains unclear.

Aims: To characterize the natural anti-FVIII antibody repertoire in mice and humans.

Methods: Natural anti-FVIII NNAs were quantified in naïve wild-type and FVIII-/- C57Bl6 mice aged 6-20 weeks and in healthy human donors. Murine B cells were harvested and cultured ex vivo in lipopolysaccharide to measure IgM production. Marginal zone B cells (MZBs) and follicular B cells (FOBs) were sorted based on CD21 and CD23 expression. MZBs were depleted via intraperitoneal injections of anti-CD49 and anti-CD11a antibodies. Anti-FVIII antibodies were detected by ELISA and quantified against purified immunoglobulin standards. Inhibitory activity was measured via Bethesda assay.

Results: Non-neutralizing anti-FVIII IgM, but not IgG nor IgA, was detectable in naïve wild-type and FVIII-/- mice and increased with age (p < 0.001). Anti-FVIII IgM was produced by splenic B cells isolated from naïve FVIII-/- mice when stimulated with lipopolysaccharide ex vivo and increased with age (p < 0.05). Total IgM production remained consistent across ages. Anti-FVIII IgM was shown to bind FVIII in vivo, as intravenous administration of FVIII reduced plasma anti-FVIII IgM levels by 44% after 24 hours (p < 0.01). Following depletion of MZBs in naïve FVIII-/- mice, anti-FVIII IgM was significantly reduced compared to mice receiving isotype control antibodies (p < 0.001). When sorted and cultured with lipopolysaccharide, MZBs produced almost all anti-FVIII IgM compared to FOBs (p < 0.01). In human donors, FVIII-specific IgM was detected at the highest concentration, followed by IgG then IgA. No inhibitory antibodies were detected.

Conclusion(s): FVIII-specific NNAs are present in both mice and humans. MZBs are the predominant source of natural anti-FVIII IgM in mice. Anti-FVIII IgM levels rise with age and fall following exogenous FVIII administration, suggesting MZB-derived IgM may be involved in the immune surveillance of FVIII.