SNP-Based Genome-Wide Identification of Hematopoiesis-Restricted Minor Histocompatibility Antigens

• Published in: Blood 2008-11, Vol.112 (11), p.814-814
• Main Authors: Hombrink, Pleun, Hadrup, Sine R, Bakker, Arne, Falkenburg, J.H. Frederik, Von Dem Borne, Peter A., Schumacher, Ton N, Heemskerk, Mirjam H.M.
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood.V112.11.814.814

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