Exploring red blood cells as an antigen delivery system to modulate the immune response towards FVIII in hemophilia A

Background: The main complication in hemophilia A treatment is the development of inhibitory antibodies against factor (F)VIII. Immune tolerance induction, the gold standard for eradicating anti-FVIII antibodies, is efficient in only 60% to 80% of cases. This underscores the need for more efficient induction of tolerance in patients with hemophilia A with FVIII inhibitors.

Objectives: In this study, we explored whether red blood cells (RBCs) can be utilized as antigen delivery system to modulate the immune response against FVIII.

Methods: Two promiscuously HLA-DR-presented peptides derived from the A2 and C1 domains of FVIII were fused to the TAT cell-penetrating peptide and incubated with RBCs.

Results: Biotinylated TAT-A2 and TAT-C1 peptides were found to interact with RBCs as shown by flow cytometry and imaging flow cytometry. Moreover, macrophages efficiently phagocytosed TAT-FVIII peptide-treated RBCs. Using mass spectrometry-based immunopeptidomics we established that TAT-FVIII peptides were presented on major histocompatibility complex class II of macrophages that phagocytosed TAT peptide-pulsed RBCs. Specifically, the TAT-A2 peptide exhibited efficient processing and presentation on HLA-DR molecules. Importantly, incubation of TAT-C1 peptide-treated RBCs-loaded macrophages with a FVIII-specific T-cell hybridoma led to a significant increase in IL-2 production, suggesting functional presentation of TAT-C1-derived peptides by macrophages.

Conclusion: Our findings indicate that RBCs can serve as effective vehicle for the delivery of FVIII-derived peptides to antigen-presenting cells. The successful display of T-cell epitopes on antigen-presenting cell using ex vivo-loaded RBC may be potentially utilized to modulate pathogenic immune responses such as observed in a subset of patients with hemophilia A.