The “inflamed self” – perturbations of the murine immunopeptidome during ischaemia-reperfusion injury and transplantation

Introduction: Recognition of allogeneic donor transplantation antigens as “non-self” by recipient T cells drives transplant rejection. Alloreactive CD8+ T cells recognise epitopes composed of allogeneic MHC class I complexed with a range of endogenous self-peptides. Sterile inflammation frequently accompanies transplantation, due to factors including ischemia reperfusion injury (IRI) in solid organ transplantation, or cytotoxic preconditioning for hematopoietic stem cell transplantation. Inflammatory mediators such as interferon gamma (IFN-γ) increase MHC class I expression, and alter antigen-presentation pathways, expanding the peptide repertoire (immunopeptidome) presented by MHC class I. During inflammation, proteins and/or peptides can become post-translationally modified (PTM). Oxidative stress increases usage of non-canonical start codons CUG / UUG and the proteins produced from these unconventional translation pathways can generate novel self-antigens contributing to graft immunogenicity.
Methods: Here we have applied mass spectrometry to characterise the murine immunopeptidome under different inflammatory settings. These include renal IRI in C57BL/6 (H-2b) mice, reduced intensity conditioning (RIC) and GvHD in BALB/c (H-2d) mice and heart
graft rejection in BALB/c to C57BL/6, and C57BL/6 to BALB/c models. The number of unique peptides, cumulative abundance, and frequency of common PTMs including phosphorylation, deamidation, cysteinylation, and acetylation were recorded for H-2Kb, H-2Db, H-2Kd or H-2Ld in target tissues exposed to each perturbation. The immunogenicity of selected peptides was evaluated using tetramer staining of alloreactive CD8 T cells from the heart graft infiltrate.
Results: All perturbations examined altered the steady-state immunopeptidome. In acute kidney injury, the number of unique peptides increased in response to both direct injury (IRI) and remote injury (contralateral). Peptide phosphorylation was prominent among H-2Kb restricted peptides from injured kidneys. Immunopeptidome diversity also increased during heart graft rejection. Increased peptide hydrophobicity is associated with a shift from usage of the constitutive proteasome in peptide processing towards immunoproteasome usage. In the fully allogeneic heart graft model, total peptide hydrophobicity was significantly higher for H-2Kb restricted 8mers from rejecting hearts compared to naïve C57BL/6 hearts. Both conditioning and GvHD alter the immunopeptidome. A large proportion of peptides are present in GvHD and / or conditioned intestine but not present in normal small intestine (Figure 1). 

A selection of abundant tissue-specific &/or perturbation-specific peptides was assessed for recognition by activated alloreactive CD8+ T cells, all 14 tested were immunogenic (Figure 2). 

Conclusions: Changes in peptide processing and in the PTM landscape shape the immunopeptidome during IRI, graft rejection and GvHD. Changes in the number of PTM peptides varied depending upon the condition and presenting MHC I allomorph.
Increased peptide hydrophobicity suggests increased presentation of immunoproteasome-derived peptides. Peri-transplant perturbations likely increase the immunogenicity of the graft or host target tissue by altering the immunopeptidome.