A Novel Tissue-Restricted Minor Histocompatibility Antigen Resulting from Differential Expression Due to a Deletion/Insertion Polymorphism in the P2X5 Purinergic Receptor Gene.
Authors: Dolstra Harry , de Rijke Bjorn , van Horssen Agnes , Maas Frans , Otterud Britt , van de Wiel-van Kemenade Elly , De Witte Theo 
Published: April 2020

Abstract
Minor histocompatibility antigens (mHags) that are selectively expressed by hematopoietic cells, including hematopoietic tumor cells, represent attractive targets for T cell-based immunotherapy after MHC-matched allogeneic stem cell transplantation (SCT) to boost the graft-versus-tumor response without causing graft-versus-host disease. We isolated a CD8+ cytotoxic T lymphocyte (CTL) clone from a SCT recipient that recognized a novel HLA-B*0702-restricted mHag with lymphoid lineage-specific expression. In vitro CTL recognition experiments revealed that this mHag was expressed by EBV-transformed B cells, CD40-activated B cells and PHA-stimulated T cells, but not expressed by cells of myeloid origin and non-hematopoietic fibroblasts. Furthermore, testing a panel of HLA-B*0702 transduced cell lines showed that several B lymphoid tumor cell lines express this mHag, which was designated as Lymphoid Restricted Histocompatibility antigen-1 (LRH-1). To define the chromosomal localization of the LRH-1 coding gene, we used genetic linkage analysis using EBV-lymphoblastoid cell lines (LCL) form the Centre d’Etude Polymorphism Humain (CEPH) reference families that have been extensively mapped for thousands of genetic markers. EBV-LCL generated from individuals of six CEPH reference families were retrovirally transduced with HLA-B*0702 and tested for CTL recognition. After determining the LRH-1 segregation pattern, linkage and haplotype analysis revealed that the LRH-1 coding gene maps to a 1.64 Mb region on chromosome 17p13.2 between marker D17S1845 and D17S1584. A database search identified 19 candidate genes that are localized within this region. Real-time RT-PCR analysis revealed that 3 genes showed an expression profile that corresponds to the CTL recognition pattern of which the purinergic receptor gene P2X5 was the most promising candidate. Transfection of P2X5 cDNA cloned from a homozygous antigen-positive individual into 293-HLA-B*0702 cells resulted in efficient recognition by the CTL clone. Testing of different P2X5 transcript variants and deletion constructs revealed that the nucleotide sequence coding the antigenic peptide is located within exon 3 of P2X5 transcript variant 1. In this region, we identified a synthetic nonameric peptide that sensitized antigen-negative donor EBV-LCL to CTL recognition, with half-maximal recognition observed at a peptide concentration of 20 nM. To identify the basis for LRH-1 disparity, exon 3 of the P2X5 alleles of the donor and recipient were sequenced and compared. The antigen-negative donor contained a homozygous C-nucleotide deletion/insertion polymorphism (DIP) resulting in a frame-shift in the protein at the third residue of the epitope. Transfection of the donor P2X5 transcript variant 1 into 293-HLA-B*0702 cells confirmed that the C-nucleotide DIP within the donor allele was associated with resistance to CTL recognition. These results provide the first evidence that mHag can result from differential protein expression as a consequence of a homozygous DIP in a transcript variant of the encoding gene.