Understanding FVIII immunogenicity in severe Haemophilia A (HA)-patients: Intracellular fate of endogenous FVIII variants in IPS derived endothelial cells

In about 20-30% of patients with severe Haemophilia A (HA), treatment with replacement FVIII is complicated due to the development of inhibitory antibodies against the substituted concentrates. The mutation type and position in the protein plays a pivotal role in the risk of inhibitor development. It is believed that F8 nonsense null mutations provoke higher immunogenicity against replacement FVIII by lacking self-FVIII protein that could be presented to the immune system. However, surprisingly, nonsense mutations located in the light chain (A3C1C2 domains; last 1/3 of the protein), have higher risk to develop inhibitors when compared to nonsense mutations located in the heavy chain (A1A2B domains; first 2/3 part of protein). Accordingly, the highest inhibitor risk (70%) appears, for two preterminal stop codons (PTCs) located in the A3 domain. This is against expectation as the protein should be largely available to get presented and recognised as self-protein. Molecular mechanism to explain this phenomenon is still lacking, however it was suggested that a rapid degradation due to the stop mutation could be responsible. To study the most native fate of FVIII mRNA and protein we are using induced pluripotent stem cells (iPSCs) as cellular model. We generated iPSCs from one HApatient with a heavy chain located PTC at position Y431X (P-A2X, high inhibitor), two HA-patients with the light chain located PTC R1941X (P1-A3X & P2-A3X, both associated with high inhibitor), one HApatient with an I22 inversion (P3-I22I, no inhibitor) and one wild type sample (Cm) to differentiate these cells into vascular endothelial cells (vECs). Using overlapping RT-PCR, FVIIIspecific ELISA and domain-specific immunostaining, we studied here the intracellular fate of mutant FVIII mRNA and protein in IPSC differentiated vECs. All three nonsense mutation samples contain full-length F8 mRNA, while I22I F8 mRNA shows the expected break between exons 22–23. A polyclonal antigen assay detected significant amounts of intracellular FVIII protein in all four patient samples. Immunostaining of differentiated vECs using different FVIII domain-specific monoclonal antibodies revealed a mutation-specific effect. The I22 inversion presents intracellular FVIII diffusely distributed throughout the cytoplasm, not co-localizing with vWF. The heavy chain located mutation (P-A2X) shows the same localization for FVIII as the wild type sample, indicating a low rate read through. The light chain located PTC (P1-A3X & P2-A3X) presents in both patients an abnormal intracellular trafficking pattern for FVIII when stained with an antibody targeting the A2 domain, indicating that the high immunogenicity for this specific mutation is not attributable to the absence of protein but to an altered/modified intracellular route.

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