Abstract
Allogeneic Hematopoietic Cell Transplantation (HCT) can cure hematologic malignancies through beneficial graft-v-leukemia (GVL) allo-immune responses, but is limited by graft-versus-host disease (GVHD). Previous studies demonstrate allogeneic antibodies (Ab) develop against minor histocompatibility antigens (mHA) encoded on the Y-chromosome (H-Y antigens) after sex-mismatch HCT in association with chronic GVHD and persistent disease remission. We hypothesize that novel mHA can be serologically identified as targets of allo-Ab responses that develop post-transplant. For this study, plasma was collected from five AML patients one year post-transplant, pre-transplant, and from their donors. The technology used to identify allo-Ab responses was human protein microarrays. ProtoArray™ (Invitrogen) displays 5,000 full-length human proteins with N-terminal GST epitopes expressed in baculovirus and affinity purified under native conditions maintaining their cellular enzymatic activities/native conformations. Technical replicates of 10 plasma measured at 1:50, 1:150, 1:500, 1:1500 and 1:4500 provided correlation coefficients of R2=0.94–0.97 confirming technical feasibility. In order to identify targets of allo-Ab, pre-transplant fluorescent signal intensities were subtracted from their one-year plasma results and also screened for the absence in donor sera for all 5000 antigens. While Ab responses were unchanged for 4600 (92%) antigens, new allo-Ab responses targeted 60–75 antigens with fluorescent differences ranging 0.5 to 3 logs. Over 90% of allo-Ab targets have known non-synonymous SNP which when disparate in donor and recipient may elicit alloimmunity. From the index patient, Nucleolar and Spindle Associated Protein 1 (NuSAP1) and Chromatin Assembly Factor 1b (CHAF1b) were two predominant proteins recognized after HCT. These allo-Abs were absent pre and 2 months post-HCT, developed in association with cGVHD at 12 months, and persisted through 18 months. On comparing all the five AML patients using ProtoArrays, we identified two patients of five developed all-ab responses to CHAF1b and NuSAP1. CHAF1b and NuSAP1 were expressed in insect cells and in E.coli, and were validated using ELISA and Western Blotting. We hypothesize alloimmunity in the index patient resulted from disparity between the donor and the recipient. We sequenced all exons for CHAF1b and NuSAP1 but failed to identify disparate non-synonymous coding SNPs between the donor and the index patient. A second hypothesis for immunogeneicity assumes CHAF1b and NuSAP1 are aberrantly expressed as tumor antigens stimulating donor allo immunity after transplantation. We determined if the antibody correlated with disease type. Eight-seven allo-transplant patients after one year post-transplant were screened by ELISA for antibody responses against CHAF1b and NuSAP1. Antibodies against NuSAP1 and CHAF1b are significantly recognized in patients with acute myelogenous leukemia (n=27) as compared to ALL, CLL, CML, MCL, MM and NHL, with a significance of p=0.00028 for CHAF1 and p=0.0011 for NuSAP1 using Wilcox test. Analysis of published AML expression profiles demonstrate CHAF1b and NuSAP1 were mainly present in the bone marrow CD34+ cells and leukemic cell lines and B lymphoblasts as compared to other tissues/cells. We sorted different cell populations and confirmed by RT-PCR results that CHAF1b and NuSAP1 were highly expressed in bone marrow CD34+Thy1+ cells suggesting antibodies specifically target bone marrow stem cells after HCT.
Arabidopsis Protein Microarrays for the High-Throughput Identification of Protein-Protein Interactions