Identification of Y-Chromosomally Encoded Minor Histocompatibility Antigens Using a Reverse Immunology Approach

Introduction: In allogeneic hematopoietic cell transplantation (HCT), minor histocompatibility antigens (mHags) are known to play an important role in generating immune responses leading to graft-versus-leukaemia (GVL) effects and graft-versus-host-disease (GVHD). mHags are results of polymorphisms in the recipients genome, which cause expression of peptides that can be recognised by donor T-cells. Y-chromosomally encoded proteins constitute a constant source of mHags relevant in allogeneic HCTs with female donor and male recipient due to the disparities between these and their homologue X-chromosomally encoded counterparts.

Methods: A panel containing 8-11 mer peptides encompassing multiple putative and known mHags encoded by the Y-chromosome was designed using a bioinformatics predictor of peptide-HLA binding, NetMHCpan. These peptides were synthesized and used to screen for peptide-specific T-cell responses in peripheral blood mononuclear cells (PBMCs) obtained post-HCT from male recipients of female donor grafts. Following in vitro stimulation, PBMCs were analysed with an inteferon-γ ELISpot assay. When a response was found, the T-cells were further analyzed with intracellular cytokine staining (ICS) and flow cytometry to determine whether it was a CD4- or a CD8-response. The optimal epitope and the HLA-restriction was determined by tetramer staining.

Results: In one male recipient of a female donor graft a T-cell response was observed with ELISpot against the peptides YFYYNAFHWAI and RESEEESVSL. ICS and flow cytometry revealed that both were CD8 responses. Both peptides were earlier described mHags restricted by HLA-A*24:02 and HLA-B60, respectively. Tetramer staining confirmed that the optimal epitopes were YYNAFHWAI and RESEEESVSL presented on HLA-A*24:02 and HLA-B*40:01 (a member of the previously designated HLA-B60 specificity), respectively. PBMCs obtained post HCT from five other male recipients of female donor grafts have been analysed for T-cell responses with ELISpot. Responses have been observed and further analysis is ongoing.

Conclusion: Using a HLA-tetramer approach to identify the optimal epitopes of two known mHags encoded by the Y-chromosome as well as the presenting HLA restriction elements at high resolution, we have demonstrated the feasibility of a reverse immunology approach in mHag discovery.