High Throughput Screening for Antibody Responses Against H-Y Antigens and Their X-Variants in Allogeneic Hematopoeietic Stem Cell Transplantation,

Abstract 4097

Allogeneic hematopoietic stem cell transplantation (alloSCT) is an effective treatment for patients with hematologic malignancies. At the Leiden University Medical Center, T-cells are depleted from the stem cell graft to reduce the incidence and severity of graft-versus-host disease (GvHD). Donor T-cells are readministered later as donor lymphocyte infusions (DLI), to preserve graft-versus-leukemia (GvL) reactivity via recognition of polymorphic peptides on recipient malignant cells.

Various studies have provided evidence that not only T-cells but also antibodies play a role in the development of clinical responses (GvL and GvHD) after alloSCT. Only a limited number of polymorphic (allo) and non-polymorphic (auto) antigens, however, have been identified as targets for antibodies with potential biological relevance.

We developed a high throughput Luminex bead assay for simultaneous measurement of antibodies against multiple antigens in a small sample volume. Antibody responses against antigens encoded by the Y-chromosome (H-Y antigens), which share 90–99% homology with their X-variants, have previously been reported to correlate with chronic GvHD and (maintenance of) disease remission in sex-mismatched alloSCT. We produced 3 full length H-Y antigens (RPS4Y, ZFY and DBY) as well as their respective X-variants (RPS4X, ZFX and DBX) in E.Coli as overlapping recombinant protein fragments. The protein fragments were coupled to individual Luminex bead IDs and 400 serum/plasma samples from 54 patients with chronic myeloid leukemia collected before and during treatment with alloSCT and DLI were screened for specific antibody binding to the antigen-coupled beads.

Pre-existing antibodies against H-Y antigens were measured in 2/18 female and 3/36 male patients. In all of these responses ZFY was targeted, except for one female who had detectable antibodies against DBY. In 4 male patients, anti-H-Y responses were induced selectively after alloSCT (1 patient) or after subsequent DLI (3 patients). Treatment-induced antibodies against H-Y antigens were not observed in any of the female patients. Three of the 4 male patients with treatment-induced antibodies against H-Y antigens were transplanted with a female donor, out of a total of 11 female-to-male transplantations. In these patients, ZFY was targeted alone or in combination with antibodies against RPS4Y or DBY. In 1 out of 25 male-to-male transplantations, antibodies against DBY were induced after treatment with DLI.

Notably, not only H-Y antigens were targets for antibodies but also their respective X-encoded homologues. Pre-existing antibodies against RPS4X, ZFX or DBX were detected in 5 patients. Of these, 1 female and 2 males also displayed antibodies against ZFY. In 2 other male recipients pre-existing antibodies against only RPS4X were measured. Furthermore, antibodies against X-variants were frequently detected selectively after alloSCT and DLI. Treatment-induced antibodies against RPS4X, ZFX or DBX were found in 3, 5 and 4 patients, respectively, and often co-developed with antibodies against the corresponding H-Y homologue. These co-developing antibodies were predominantly found in male recipients transplanted with a female donor after administration of DLI.

In conclusion, we demonstrate that 3 ubiquitously expressed intracellular H-Y antigens are frequent targets for antibodies in alloSCT recipients. Treatment-induced H-Y reactive antibodies were measured most in male patients transplanted with female donors compared to other sex-matched and –mismatched settings. Antibodies against H-Y antigens often showed cross-reactivity against the respective X-encoded homologue. Our data therefore suggest that antibodies specific for intracellular H-Y and X antigens develop as a consequence of substantial lysis of patient cells during clinical responses induced after DLI.