The allo-antigen specificity of a T cell driving acute cellular rejection of a kidney allograft 2302494

Abstract Introduction While alloreactive CD8+ T cells are the main drivers of direct acute cellular rejection (ACR), their targets are poorly understood. Old models posit that TCRs on alloreactive T cells bind their targets non-specifically. However, recent data suggest that many alloreactive T cells are allospecific, recognizing unique peptide-allo-HLA complexes. Using scRNA/TCR-seq we showed that kidney biopsies from ACR patients had limited numbers of clonally expanded CD8+ T cells (CD8EXP). Here, our aim was to identify the alloantigens recognized by the CD8EXP TCRs. Methods K562 cells singly expressing donor mismatched class I HLA proteins, HLA-A*32:01 and -B*14:01, were used to stimulate Jurkat cells expressing the CD8EXP TCRs. An A*32:01 responsive TCR was used to screen an A*32:01 yeast display library. Hits from the screen were used in a molecular mimicry search (MimicMaker) of the K562 transcriptome to find potential targets. Hits from the search were compared to peptides identified by immunopeptidomics, tested for their ability to stimulate the patient-derived TCR, and mapped to the donor kidney transcriptome. Results The clone 7 TCR responded to A*32:01 on K562 cells only, indicating HLA restriction. Clone 7 failed to respond when co-cultured with A*32:01+ T2 cells in the presence of rationally designed peptides, confirming allospecificity. Yeast display screening yielded 5 highly stimulatory peptides. A MimicMaker scan of the K562 transcriptome for mimics of these peptides, along with peptidomics, identified 72 candidate targets. From these, only a single peptide strongly stimulated clone 7. This peptide is likely the biologically relevant target of the clone 7 TCR. Its source protein is highly expressed in the donor kidney. Conclusion Our interdisciplinary approach has allowed us to identify a target of a patient-derived allospecific TCR. Broader deployment of this approach is likely to yield further success, paving the way for improving transplant outcomes by linking alloreactive TCRs to their targets. Funding Source R21AI91060, R21AI169863, T32GM145773 Topic Categories Transplantation Immunology (TRAN)