Alexandra E Hill ¹ Eric Taeyoung Son ¹ Moumita Paul ¹ Chuanmin Wang ¹ Shivanjali Ratnaseelan ¹ Martina Denkova ¹ Pouya Faridi ² Asolina Braun ² Anthony Wayne Purcell ² Nicole Andrea Mifsud ² Alexandra Francesca Sharland ^1*

• ¹ The University of Sydney, Darlington, Australia
• ² Monash University, Melbourne, Victoria, Australia

Mass Spectrometry allied with in-vivo generation of activated alloreactive T cell populations and tetramer screening facilitates the identification of endogenous peptides that are directly recognised in complex with allogeneic Major Histocompatibility class I (MHC I) molecules by alloreactive CD8+ T cells. We had previously used this approach for the discovery of immunogenic self-peptides presented by the allomorph H-2Kb (Kb). In this study, we identified 22 highly immunogenic self-peptides presented by H-2Kd (Kd). Peptide abundance across skin, spleen and liver samples (estimated as the product of the spectral intensity obtained for these samples) was the principal factor influencing recognition of peptide-Kd epitopes. Predicted binding affinity (BA score) and overall peptide hydrophobicity were also independently correlated with immunogenicity, while there was no significant correlation between the IEDB immunogenicity score and the proportion of T cells recognising a given epitope. Eight peptide-Kd epitopes were selected for inclusion in a tetramer panel to detect directly alloreactive CD8+ T cells. This panel bound over 30% of activated alloreactive CD8+ T cells after a prime-boost against Kd . Moreover, the panel identified alloreactive CD8+ T cells within the graft infiltrate, spleen and draining lymph node during rejection of a Kd -bearing heart graft. In conclusion, small animal studies have demonstrated the feasibility of high-throughput approaches for the discovery of pMHC epitopes recognised by directly alloreactive T cells. Translating this approach to the human setting is achievable and will yield both critical insights into the fundamental basis of alloreactivity and powerful tools for immune monitoring in transplantation.