Alloreactive cytotoxic T cells provide means to decipher the immunopeptidome and reveal a plethora of tumor-associated self-epitopes

Significance T cells recognize fragments of cellular peptides when presented at the cell surface by HLA molecules. Knowledge of which peptides derived from cellular proteins that are available at the cell surface for T-cell recognition is central to our understanding of T-cell tolerance and immunity. Here, we used alloreactive T cells as tools for detection of self-peptides bound to foreign HLA-A2. Our results indicate that the self-immunopeptidome is far more diverse than previously estimated. Furthermore, our data demonstrate that such self-peptides represent highly attractive targets for T-cell–based cancer immunotherapy. HLA molecules presenting peptides derived from tumor-associated self-antigens (self-TAA) are attractive targets for T-cell–based immunotherapy of cancer. However, detection of such epitopes is hampered by self-tolerance and limitations in the sensitivity of mass spectrometry. Here, we used T cells from HLA-A2–negative donors as tools to detect HLA-A2–bound peptides from two leukemia-associated differentiation antigens; CD20 and the previously undescribed cancer target myeloperoxidase. A high-throughput platform for epitope discovery was designed using dendritic cells cotransfected with full-length transcripts of self-TAA and HLA-A2 to allow presentation of all naturally processed peptides from a predefined self-protein on foreign HLA. Antigen-reactive T cells were directly detected using panels of color-coded peptide–HLA multimers containing epitopes predicted by a computer algorithm. Strikingly, cytotoxic T cells were generated against 37 out of 50 peptides predicted to bind HLA-A2. Among these, 36 epitopes were previously undescribed. The allorestricted T cells were exquisitely peptide- and HLA-specific and responded strongly to HLA-A2–positive leukemic cells with endogenous expression of CD20 or myeloperoxidase. These results indicate that the repertoire of self-peptides presented on HLA class I has been underestimated and that a wealth of self-TAA can be targeted by T cells when using nontolerized T-cell repertoires.