Immunopeptidomic identification of SLA-derived HLA class II ligands recognized by human T cells, using a strategy adapted for xenotransplantation
Ken Kawada1, Kenta Iwasaki2, Susumu Tomono3, Yuko Miwa 2, Masato Shizuku1, Satoshi Ashimine1, Kohei Ishiyam1, Sachiko Akashi-Takamura3, Ekser Burcin1, Takaaki Kobayashi4.
1Department of Renal Transplant Surgery, Aichi Medical University School of Medicine, Nagakute, Japan; 2Department of Kidney Diseases and Transplant Immunology, Aichi Medical University School of Medicine, Nagakute, Japan; 3Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute, Japan; 4Division of Abdominal Transplant Surgery, Stritch School of Medicine, Loyola University, Maywood, IL, United States
Background: Recent advances in genetic engineering have drastically extended xenograft survival in NHPs, facilitating the advancement of xenotransplantation toward clinical application. However, long-term graft acceptance remains a major barrier in humans, and the genetic targets required to mitigate human anti-xenogeneic immune responses have not been fully defined. This study aimed to identify peptide epitopes derived from porcine antigens that are presented by human dendritic cells via HLA class II, as potential targets for future genetic modifications to reduce T-cell mediated rejection.
Methods: CD14⁺ monocytes isolated from human peripheral blood were cultured with irradiated porcine liver-derived endothelial cells lacking GGTA1, CMAH, and β4GalNT2 for four days with IL-4 and GM-CSF, followed by two additional days with IL-1β and TGFα to induce differentiation into dendritic cells. After cell lysis, HLA class II-bound peptides were immunoprecipitated using anti-HLA-DR antibodies and analyzed by nanoLC-qTOF/MS/MS. Peptide sequences were matched to the Immune Epitope Database for immunogenicity prediction. Selected peptides were synthesized and evaluated by ELISPOT assays using PBMCs from healthy donors.
Results: Peptides derived from swine leukocyte antigen (SLA) class I and II molecules, along with other porcine proteins, were consistently identified in all three donors. Several predicted immunogenic peptides elicited cytokine production in ELISPOT assays, indicating T-cell recognition of these epitopes.
Conclusion: Donor-specific anti-HLA antibodies (DSAs), including those cross-reactive to SLA, are associated with xenograft rejection, yet reliable immunological markers for recipient stratification are lacking. In allotransplantation, the presence of shared T-cell epitopes has been associated with de novo DSA formation. Our findings demonstrate that SLA-derived peptides can be presented by human HLA class II molecules and are recognized by human T cells, suggesting their relevance in the adaptive immune response to xenografts. These epitopes represent potential targets for donor genome editing to enhance long-term xenograft survival in clinical settings.