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

Abstract

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 leukemiaassociated 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.C ytotoxic 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 2 decades of intense research amounts to less than 600 (1). The ability to rapidly identify 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 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 100,000-750,000 peptide-MHC 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 × 10 6 distinct nonamers (7). Thus, there is a very large gap between the numb...

Role of the MHC restriction during maturation of antigen‐specific human T cells in the thymus

“…In particular, around half of MelA-, PGT-and NS3-A2 specific alloreactive cell lines efficiently recognized A2 + target cells unloaded with relevant exogenous peptide, reflecting their ability to interact with other naturally expressed p-A2 complexes. These observations are in agreement with murine and human studies showing that the alloreactive TCR repertoire encompasses a continuum of peptide-specific to peptidedegenerate receptors [35][36][37], and that negative thymic selection probably leads to depletion of peptide-promiscuous T-cell subsets restricted by thymic MHC alleles [38,39]. Although the average functional avidities of tetramer-specific cell lines derived from A2…”

How an alloreactive T-cell receptor achieves peptide and MHC specificity

“…The surprising specificity of many alloreactive TCRs and structural studies of alloreactive complexes have challenged these theories (3,12,13). Highly specific alloreactive T cells and TCRs are being explored for targeting what otherwise would be poorly immunogenic shared tumor antigens and have been adopted for treatment of posttransplantationassociated lymphoproliferative disease (4,6,7).…”

“…Central tolerance mechanisms exist to avoid self-reactivity, hindering the identification of highly avid T cells specific for shared (nonmutated) tumor antigens. The phenomenon of specificity in alloreactivity has thus been exploited to identify T cells capable of recognizing shared tumor antigens with high potency, using antigen-specific T cells from mismatched donors to circumvent tolerance (4,5). In a related fashion, antigen-specific alloreactive T cells have been used to target conditions such as posttransplantation Epstein-Barr virus-associated lymphoma (6,7).…”

Analysis of Major Histocompatibility Complex (MHC) Immunopeptidomes Using Mass Spectrometry*

“…for vaccinia virus, HIV, hepatitis C virus, human papillomavirus, and human respiratory syncytial virus) per experiment using optimal in vitro model systems (27, 72, 132, 147, 149 -151). Interestingly, the development of a highthroughput cytotoxic T cell-based platform for epitope discovery was recently reported (152). The results generated by this technology indicated that the repertoire of self-HLA peptides has been clearly underestimated by MS studies.…”