Surprisingly, third-party EBV-specific T cells can eliminate EBV-associated posttransplantation lymphoma despite likely immune recognition and rejection of foreign HLA by the patient immune system (31). 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 peptideHLA 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-A2positive 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. Cytotoxic T cells (CTLs) selectively kill target cells that express defined peptides in complex with MHC class I molecules on the cell surface. Most tumor-associated antigens (TAA) are wild-type self-proteins, and T cells that recognize peptides from these antigens with high affinity are deleted during thymic development. Thus, the utility of T cells for detection of self-peptides presented on self-HLA is limited by tolerance. This may be one reason why the number of epitopes identified from TAA after PAT-1251 Hydrochloride 2 decades of intense research amounts to less than 600 (1). The ability to rapidly identify PLCB4 new CTL epitopes would likely facilitate the development of effective immunotherapeutic strategies against cancer. MHC molecules can be isolated from cells and the associated peptides eluted for identification by MS. Ultimately, this approach may provide a description of the entire MHC-bound peptide repertoire: the PAT-1251 Hydrochloride immunopeptidome (2,3). This is, however, a daunting task, and it is unclear whether current MS-based protocols already provide the required sensitivity. Indeed, although 100,000750,000 peptideMHC class I complexes are expressed for each allelic product on the cell surface (for HLA-A and HLA-B loci) (3,4), the largest HLA ligandome identified to date contains 14,065 peptides (5). In contrast, the predicted number of different HLA class I ligands would be 352,000 using the well-renowned computer algorithm NetMHCpan, considering that on average, 4.4% of all nonamers bind HLA class I (6) and that a cell contains at least 8 106distinct nonamers (7). Thus, there is a PAT-1251 Hydrochloride very large gap between the number of confirmed and predicted HLA ligands. In the present study, we investigated if the breadth of the self-immunopeptidome and its utility for T-cell targeting could be evaluated by a combination of three elements. First, because self-tolerance is HLA-restricted, we used T cells from HLA-A*02:01negative (HLA-A2neg) donors as tools to detect self-peptides associated with allogeneic HLA-A2. Second, to induce reactivity against the entire repertoire of epitopes from self-antigens of interest, autologous dendritic cells (DCs) cotransfected with full-length transcripts encoding the target proteins and HLA-A2 were cocultured with the T cells. In this setting, DCs present a multitude of naturally processed self-peptides from predefined self-proteins on the foreign HLA molecule to a T-cell repertoire that has not been affected by tolerance toward this antigenMHC combination. Third, to detect antigen-reactive T cells directly, we used color-coded peptideHLA-A*02:01 multimers with content of a large panel of peptides from the target proteins, predicted to bind HLA-A2 by a computer algorithm. Strikingly, the CTL specificities generated by the approach covered the large majority of epitopes that were predicted by the computer algorithm. Cells reactive with epitopes from the leukemia-associated differentiation antigens myeloperoxidase (MPO) and CD20 were highly peptide- and HLA-specific and responded strongly to target cells endogenously expressing the cognate antigens. These data demonstrate that a wealth of self-epitopes is.