Post Transplant Allogeneic Antibody Responses Form against Annexin 8

Allogeneic hematopoietic cell transplantation can cure hematologic malignancies through graft versus leukemia effects but are limited by graft versus host disease. Antibodies against human Y chromosome antigens are associated with graft versus host disease and we hypothesized that antibodies could conversely be associated with post transplant graft versus leukemia effects. We identified an index male patient with chronic lymphocytic leukemia who received a female PBSC graft following reduced intensity conditioning allogeneic hematopoietic cell transplantation. This F→M had developed allogeneic antibodies against H-Y antigen UTY. We hypothesized allogeneic antibodies develop against autosomal minor histocompatibility antigens and could be identified as targets of allo-antibodies. Protein microarrays (Invitrogen Protoarray) featuring 8000 baculovirus expressed human proteins with N-terminal GST fusions were screened with plasma samples from the index patient and antibodies binding to potential graft versus leukemia antigens were detected with fluorescently tagged secondary antibodies. We compared post-transplant with pre-transplant and donor plasma fluorescent intensities and identified new post transplant antibody responses against annexin 8 which were not present prior to transplant or in the donor. Our finding of new antibody responses against annexin 8 was confirmed by Western blot against E.coli expressed affinity purified recombinant V5-His6 tagged annexin 8.

Annexin 8 is a membrane associated protein postulated to play a role in cellular proliferation. Recent genomic analyses have identified three loci coding for three separate annexin 8 genes clustered on chromosome 10 in a region of high frequency copy number variation (CNV). In addition, the annexin 8 mRNA transcript undergoes alternative splicing with one variant being identified from a choriocarcinoma cell line. Both CNV and alternative splicing may result in immunologic disparities between donor and recipient inducing alloimmunity.. Therefore, we tested these possibilities by synthesizing recombinant V5-His6 annexin 8 proteins corresponding to the three possible isoforms and using them in an ELISA to screen 187 patients with a variety of malignancies for post-allogeneic transplant antibodies. The recombinant proteins included the alternative splice variant from choriocarcinoma which differed from the common variant by the insertion of a 38 amino acid sequence after the second exon and the deletion of two 57 and 32 amino acid sequences from exons 6, 7, and 10 of the common variant, as well as two versions of the common variant which differed by a G to C nonsynonymous single nucleotide polymorphism resulting in a change from glycine to alanine. These two recombinant common variant isoforms correspond to the reported sequences of two of the annexin 8 loci affected by copy number variation. Thus reactions to either, but not both, might indicate disparities in annexin 8 copy number between recipient and donor. Of 187 patients, 22 had significant antibody reactions (defined as greater than 1 standard deviation above the mean in normal controls) to either of the common variant isoforms but only 4 had reactions to both isoforms suggesting that antibodies were differentially recognizing annexin 8 isoforms based on a single nonsynonymous single nucleotide polymorphism. Six of 187 patients had significant antibody reactions against the splice variant isoform alone, suggesting that the alternatively inserted exon may be the antibody target.

Taken together, our findings suggest that annexin 8 may be a target of the post-transplant humoral response due to immune disparities between recipient and donor caused by copy number variation as well as alternative splicing.