Global Assessment of B Cell Alloimmunity Using 5000 Human Protein Microarrays.

Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but its full potential is limited by graft-versus-host disease (GVHD). Recent studies demonstrate allogeneic antibodies develop against mHA encoded on the Y-chromosome, called H-Y antigens develop after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize novel mHA can be serologically identified as targets of allogeneic antibody responses that develop after transplant and were absent pretransplant. Over 70,000 nonsynonymous single nucleotide polymorphisms (nsSNPs) encode polymorphic amino acid residues in human proteins that are potential mHA, and their analysis requires a novel high-throughput approach. ProtoArray™ (Invitrogen) displays five thousand full-length human proteins expressed as N-terminal GST-fusion proteins in a baculovirus system that are affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. These human antigens are printed in duplicate on nitrocellulose-coated glass slides. In order to determine if targets of allogeneic antibodies can be detected using microarray technology we tested three cGVHD patients for de novo Ab development after HCT using ProtoArrays™.

Plasma from three male patients with AML was collected 1) prior to myeloablative HCT 2) from each HLA-identical sibling donor, and 3) one year after HCT. All three had developed extensive chronic GVHD. Plasma was diluted 1:150 and incubated on two microarrays providing replicate results. After washing, each microarray was incubated with anti-human IgG conjugated to Alexa Fluor 647 dye and detected. Negative controls include buffer, BSA, and GST, and their maximum fluorescent signals with these samples were 5,631 units with mean SD of 546. In contrast, influenza A protein is a positive control with fluorescent signal ranging 30,000–45,000 units. Correlation coefficients (R² values) between duplicate slides ranged from 0.85 to 0.91.

Data analysis was performed using Invitrogen’s Prospector Analyzer Software. Fluorescent intensity is measured for each “spot” and individual antigen results are reported as both flourescent signal intensity and a Z-score which is a measure of the intensity of a given signal relative to all of the other human protein targets reported in units of standard deviation. In order to identify human protein targets of allogeneic antibodies, each target antigen’s pretransplant Z-score was subtracted from his respective one year post-transplant Z-score yielding the “ΔZ” for that antigen. Using a conservative ΔZ of 0.1, these three patients developed new antibody responses for 67, 66, and 74 human proteins. Ninety-two percent of these proteins have known nsSNPs. No single protein was recognized by “new” antibodies in all three proteins, however polymorphic proteins Growth Arrest Specific-7 (GAS7), laminin A/C, and ribosomal protein S19 (RPS19) were recognized by two of the three patients after HCT. Results from plasma samples collected 3 and 6 months after HCT demonstrate the progression of alloimmune immune response with few new antibody responses at 3 months.

Conclusion: Protein microarrays are an innovative, powerful tool for high-throughput global assessment of B cell alloimmunity after HCT. Microarray technology provides sufficient reproducibility for candidate mHA discovery. These novel mHA require validation by large clinically characterized patient samples.