High Throughput Minor Histocompatibility Antigen Discovery by Whole Genome Association Scanning.
Authors: van Bergen Cornelis A.M. , van der Meijden Edith D. , Rutten Caroline E. , van Luxemburg Simone A.P. , Lurvink Elisabeth G.A. , Honders M. Willy , Houwing Jeanine J. , Sampietro M. Lourdes , Willemze Roelof , Falkenburg J.H. Frederik , Griffioen Marieke 
Published: October 2019

Abstract
Abstract 685
Allogeneic stem cell transplantation (allo-SCT) followed by donor lymphocyte infusion (DLI) is used as a curative treatment for patients with malignant diseases. Donor derived T cells mediate graft versus tumor responses by targeting minor histocompatibility antigens (mHags) that are encoded by patient specific single nucleotide polymorphisms (SNPs). Various approaches have been applied for mHag discovery resulting in the characterization of more than 20 mHags. However, restriction to specific HLA types and unfavourable gene expression strongly limits the number of clinically relevant mHags. Recently, it has been demonstrated that whole genome association scanning (WGAs) can be a tool for mHag identification. Here, we present WGAs as a powerful method for high throughput identification of new mHags. From 2 patients that entered complete remission with limited graft versus host disease after allo-SCT and DLI, activated T cells were cloned by flowcytometric cell sorting. After expansion, 232 stably growing T cell clones were obtained. Patient specific recognition in the absence of donor recognition was demonstrated for 78 clones. By using blocking antibodies and a test panel consisting of partially HLA matched EBV-transformed B cell lines (EBV-LCL), we demonstrated that these 78 T cell clones comprised 20 unique mHag reactivities which could be identified to be restricted to HLA-A*02 or B*07. Since WGAs is based on a balanced segregation of test cells in a positive and a negative group, 15 T cell clones were selected recognizing mHags with population frequencies between 20% to 80% for further analysis. To perform WGAs, a test panel was generated containing 80 HLA-A*02 and B*07 positive EBV-LCL for testing of recognition by all selected T cell clones using Interferon-γ Elisa. In parallel, all EBV-LCL were genotyped for 1 million SNPs using bead arrays. All SNP genotype patterns were combined with each individual T cell recognition pattern. The level of matching between both patterns was statistically analyzed using Fisher's exact test, resulting in p-values indicating the significance of association. Significant association (p-value<10-12) between SNP genotypes and a T cell recognition pattern identified a single genomic region for 12 out of 15 T cell clones. In 2 cases no clear discrimination between positive and negative EBV-LCL could be made, suggesting that these T cell clones may not recognize mHags. Incomplete coverage of a genomic region by SNPs on the bead array may explain the lack of association for 1 T cell clone. For 7 T cell clones, significant association was found with array SNPs located in exons of the genes WNK1, SSR1, PRCP, ARHGDIB, PDCD11, EBI3 and APOBEC3B. For 3 other T cell clones, the genes ERAP1, BCAT2 and GEMIN4 were identified based on significant association with SNPs located in non coding regions. Sequence analysis of the coding regions of these genes from patient and donor revealed additional patient specific SNPs that were not included in the bead array. For the remaining associating TTK and ERGIC1 genes, the coding regions were identical between patient and donor, showing that these mHags are not encoded by exon SNPs in the identified TTK and ERGIC1 genes. Differential mHag expression may be induced in these cases by SNPs in adjacent genes that were not identified by SNPs on the bead array or may be the result of SNPs in non coding regulatory regions or in mRNA splice variants. According to the BioGPS gene expression database, a number of genes as identified by WGAs were predominantly expressed in hematopoietic cells, and may therefore encode relevant targets for T cell therapy. Next, we investigated the amino acid polymorphisms encoded by all identified coding SNPs. Peptides spanning the patient type amino acid polymorphism were submitted to HLA binding prediction algorithms. Candidate peptides were synthesized and T cell recognition was demonstrated at concentrations varying from 0.5 to 5000 nM. Recognition of donor type peptides was absent in all cases, validating the identification of 10 novel mHags. In conclusion, these data demonstrate that activation marker based T cell selection and cloning combined with WGAs results in high throughput discovery of multiple mHags. This strategy therefore allows broad characterization of mHags in donor derived T cell responses and selection of clinically relevant mHags for development of T cell based immunotherapy.

Disclosures:
No relevant conflicts of interest to declare.