Factor VIII Is a Potential Autosomal B Cell Minor Histocompatibility Antigen in Chronic Gvhd.

Abstract 1167

Poster Board I-189

Alloantibody responses to HY antigens have been well described in patients with chronic graft versus host disease (cGVHD), but specific B cell reactivity to autosomal minor histocompatibility antigens has not been described. Recently, genetic polymorphisms in hemophiliacs treated with recombinant factor VIII (F8) have been associated with an increased risk of developing F8 antibody inhibitors (Viel KR NEJM 2009), highlighting the possibility that F8 is an alloantigen target in cGVHD. From a patient with cGVHD manifesting with a clinically significant antibody inhibitor to F8, we identified a novel non-synonymous mutation resulting in a glycine (G) to arginine (R) substitution at codon 2076 (G2076R) in the patient/recipient's F8 gene not present in the donor. To visualize the G to R substitution, homology modeling was performed using the SWISS-MODEL server. The G2076R substitution was found to alter the surface of the C1 domain. The homology model is expected to be particularly reliable in this case because the side chain is on the surface, making conformational changes in peptide backbone structure unlikely. Enzyme-Linked Immunosorbent Assays (ELISA) using peptides synthesized with or without the G2076R mutation were employed to measure direct IgG binding. The donor had pre-existing alloantibodies to the mutated peptide, while the recipient, prior to hematopoietic stem cell transplantation (HSCT), did not have a pre-existing allo-IgG response to the mutated peptide. The recipient had no IgG response to native or mutated peptide or whole, unmutated F8 before HSCT or at 1,2,3,6 or 9 months after HSCT, but a specific IgG response to the G2076R containing peptide developed at 11 months post-HSCT just prior to clinically evident coagulopathy. Negative control amino acid substitutions (G2076A) did not result in IgG binding and negative control G2076R peptide (oriented on the ELISA so that R was not available for IgG binding) also did not result in IgG binding. An IgG response to whole, unmutated recombinant F8 protein as measured by ELISA and Western Blot was detected by 11 months post-HSCT. The recipient was treated with a 4 week course of rituximab and corticosteroids and had a complete clinical response with normalization of coagulation parameters. Alloreactivity in the recipient was no longer detectable 6 months later, although IgG responses to unmutated F8 protein remained, suggesting that rituximab resulted in removal of the alloantibody producing B cell. Persistence of IgG reactive to unmutated whole F8 also suggests epitope spreading occurred, resulting in the development of antibodies to other F8 epitopes that contribute to the autoreactivity found in this patient. Thus, we demonstrate ‘proof of principal’ that B cell responses to autosomal minor histocompatibility antigens occur in human cGVHD.