Immunopeptidomics of blood coagulation factor IX: a core peptide derived from the protease domain is promiscuously...

Hemophilia B is an X-linked bleeding disorder that results in a qualitative or quantitative factor IX (FIX) deficiency. A subset of hemophilia B patients treated with FIX replacement therapy develop antibodies against FIX. The development of FIX inhibitors can present as allergic or anaphylactic reactions in some patients. It is currently unknown why a subset of hemophilia B patients develop inhibitors. CD4⁺ T cells recognizing FIX-derived peptides on major histocompatibility complex (MHC) class II on antigen-presenting cells are crucial for the development of class-switched, high-affinity anti-FIX antibodies.

In this study, we explored MHC class II presentation of FIX-derived peptides by antigen presenting cells.

We incubated purified FIX with monocyte derived dendritic cells of healthy, human leukocyte antigen-typed donors. MHCII peptide complexes were immunopurified, followed by peptide elution and mass spectrometry-based analysis of eluted peptides.

We analyzed 12 healthy donors; of them, 8 presented FIX-derived peptides. The majority of FIX peptides were derived from the serine protease domain of FIX. With the exception of 1 donor, all of them presented at least one peptide containing the same core sequence, "NVIRIIPHHN" (residues 295-306 of FIX).

Taken together, we identified a promiscuously presented peptide derived from the protease domain of FIX. Based on its frequent presentation in multiple donors, we speculate that this peptide may be recognized by FIX-specific CD4⁺ T cells that develop in hemophilia B patients with inhibitors. Peptides identified in our study can be used for inhibitor risk stratification and development of novel therapies to induce tolerance.