SNP-Based Genome-Wide Identification of Hematopoiesis-Restricted Minor Histocompatibility Antigens
Authors: Hombrink Pleun , Hadrup Sine R , Bakker Arne , Falkenburg J.H. Frederik , Von Dem Borne Peter A. , Schumacher Ton N , Heemskerk Mirjam H.M. 
Published: October 2019

Abstract
T-cell recognition of minor histocompatibility antigens (mHag) plays an important role in the graft-versus-tumor (GVT) effect of allogeneic stem cell transplantation (allo-SCT). However, mHag recognition is also associated with graft-versus-host disease (GVHD). It is assumed that the selective infusion of T cells reactive with hematopoiesis-restricted mHag may help to separate the GVT and GVHD effects of allo-SCT. However, the number of attractive mHag identified to date remains limited. In this study we aimed to determine whether it is feasible to perform genome-wide identification of hematopoiesis-restricted minor histocompatibility antigens. Successful development of such a technology could allow the rapid identification of sets of hematopoiesis-restricted minor histocompatibility antigens required to make antigen-selective adoptive T cell therapy a realistic option. To this purpose we performed microarray analyses of hematopoietic progenitor and non-hematopoietic restricted cell types and merged our data with a microarray database to select approximately 80 genes with a hematopoiesis-restricted gene expression pattern. Subsequently, 322 single nucleotide polymorphism (SNPs) were identified in this set of genes and HLA-A2 binding peptides were predicted in both the normal and alternative reading frames using four different HLA-binding algorithms. The resulting set of 1300 putative HLA-A2 ligands was synthesized and tested for binding affinity using a MHC-peptide exchange technology based on UV-sensitive conditional ligands. Binding affinity was determined based on the ability of the peptides to induce MHC-complex recovery after photo cleavage of the conditional ligand. Based on these affinity measurements 400 high- to medium affinity HLA-A2 binding peptides were selected and used to generate tetramers by MHC-ligand exchange. To enable screening with this vast number of tetramers we developed a combinatorial coding method that allows the detection of up to 25 different T-cell populations in one sample. Furthermore, to overcome the problem of limited availability of patient PBMC and low-frequent T cell responses we performed an in vitro T-cell enrichment and expansion step. In this method tetramer positive lymphocytes are isolated with magnetic cell separation columns and cultured in presence of cytokines, feeders and anti-CD3/28 coated beads. Using this technological platform, we have begun to analyze hematopoiesis-restricted mHag responses in transplant recipients with different hematologic malignancies. This screen confirmed the existence of a known hematopoiesis-restricted mHag response and suggested the presence of a number of novel hematopoiesis-restricted mHag responses. Our data indicate that the technology developed within this project is likely to be of value to the identification of collections of novel minor histocompatibility antigens with potential clinical value.