Identification of HCMV-derived T cell epitopes in seropositive individuals through viral deletion models.

In healthy individuals, immune control of persistent human cytomegalovirus (HCMV) infection is effectively mediated by virus-specific CD4+ and CD8+ T cells. However, identifying the repertoire of T cell specificities for HCMV is hampered by the immense protein coding capacity of this betaherpesvirus. Here, we present a novel approach that employs HCMV deletion mutant viruses lacking HLA class I immunoevasins and allows direct identification of naturally presented HCMV-derived HLA ligands by mass spectrometry. We identified 368 unique HCMV-derived HLA class I ligands representing an unexpectedly broad panel of 123 HCMV antigens. Functional characterization revealed memory T cell responses in seropositive individuals for a substantial proportion (28%) of these novel peptides. Multiple HCMV-directed specificities in the memory T cell pool of single individuals indicate that physiologic anti-HCMV T cell responses are directed against a broad range of antigens. Thus, the unbiased identification of naturally presented viral epitopes enabled a comprehensive and systematic assessment of the physiological repertoire of anti-HCMV T cell specificities in seropositive individuals.

Key Features Relevant to Select Antigens and TCR From the MHC-Mismatched Repertoire to Treat Cancer.

Adoptive transfer of T cells transgenic for tumor-reactive T-cell receptors (TCR) is an attractive immunotherapeutic approach. However, clinical translation is so far limited due to challenges in the identification of suitable target antigens as well as TCRs that are concurrent safe and efficient. Definition of key characteristics relevant for effective and specific tumor rejection is essential to improve current TCR-based adoptive T-cell immunotherapies. We here characterized in-depth two TCRs derived from the human leukocyte antigen (HLA)-mismatched allogeneic repertoire targeting two different myeloperoxidase (MPO)-derived peptides presented by the same HLA-restriction element side by side comprising state of the art biochemical and cellular in vitro, in vivo, and in silico experiments. In vitro experiments reveal comparable functional avidities, off-rates, and cytotoxic activities for both TCRs. However, we observed differences especially with respect to cytokine secretion and cross-reactivity as well as in vivo activity. Biochemical and in silico analyses demonstrate different binding qualities of MPO-peptides to the HLA-complex determining TCR qualities. We conclude from our biochemical and in silico analyses of peptide-HLA-binding that rigid and high-affinity binding of peptides is one of the most important factors for isolation of TCRs with high specificity and tumor rejection capacity from the MHC-mismatched repertoire. Based on our results, we developed a workflow for selection of such TCRs with high potency and safety profile suitable for clinical translation.

The HLA ligandome landscape of chronic myeloid leukemia delineates novel T-cell epitopes for immunotherapy.

Antileukemia immunity plays an important role in disease control and maintenance of tyrosine kinase inhibitor (TKI)-free remission in chronic myeloid leukemia (CML). Thus, antigen-specific immunotherapy holds promise for strengthening immune control in CML but requires the identification of CML-associated targets. In this study, we used a mass spectrometry-based approach to identify naturally presented HLA class I- and class II-restricted peptides in primary CML samples. Comparative HLA ligandome profiling using a comprehensive dataset of different hematological benign specimens and samples from CML patients in deep molecular remission delineated a panel of novel frequently presented CML-exclusive peptides. These nonmutated target antigens are of particular relevance because our extensive data-mining approach suggests the absence of naturally presented BCR-ABL- and ABL-BCR-derived HLA-restricted peptides and the lack of frequent tumor-exclusive presentation of known cancer/testis and leukemia-associated antigens. Functional characterization revealed spontaneous T-cell responses against the newly identified CML-associated peptides in CML patient samples and their ability to induce multifunctional and cytotoxic antigen-specific T cells de novo in samples from healthy volunteers and CML patients. Thus, these antigens are prime candidates for T-cell-based immunotherapeutic approaches that may prolong TKI-free survival and even mediate cure of CML patients.

The natural HLA ligandome of glioblastoma stem-like cells: antigen discovery for T cell-based immunotherapy.

Glioblastoma is the most frequent malignant primary brain tumor. In a hierarchical tumor model, glioblastoma stem-like cells (GSC) play a major role in tumor initiation and maintenance as well as in therapy resistance and recurrence. Thus, targeting this cellular subset may be key to effective immunotherapy. Here, we present a mass spectrometry-based analysis of HLA-presented peptidomes of GSC and glioblastoma patient specimens. Based on the analysis of patient samples (n = 9) and GSC (n = 3), we performed comparative HLA peptidome profiling against a dataset of normal human tissues. Using this immunopeptidome-centric approach we could clearly delineate a subset of naturally presented, GSC-associated HLA ligands, which might serve as highly specific targets for T cell-based immunotherapy. In total, we identified 17 antigens represented by 41 different HLA ligands showing natural and exclusive presentation both on GSC and patient samples. Importantly, in vitro immunogenicity and antigen-specific target cell killing assays suggest these peptides to be epitopes of functional CD8+ T cell responses, thus rendering them prime candidates for antigen-specific immunotherapy of glioblastoma.

The immunopeptidomic landscape of ovarian carcinomas.

Immunotherapies, particularly checkpoint inhibitors, have set off a revolution in cancer therapy by releasing the power of the immune system. However, only little is known about the antigens that are essentially presented on cancer cells, capable of exposing them to immune cells. Large-scale HLA ligandome analysis has enabled us to exhaustively characterize the immunopeptidomic landscape of epithelial ovarian cancers (EOCs). Additional comparative profiling with the immunopeptidome of a variety of benign sources has unveiled a multitude of ovarian cancer antigens (MUC16, MSLN, LGALS1, IDO1, KLK10) to be presented by HLA class I and class II molecules exclusively on ovarian cancer cells. Most strikingly, ligands derived from mucin 16 and mesothelin, a molecular axis of prognostic importance in EOC, are prominent in a majority of patients. Differential gene-expression analysis has allowed us to confirm the relevance of these targets for EOC and further provided important insights into the relationship between gene transcript levels and HLA ligand presentation.

Identification of Immunogenic Epitopes by MS/MS.

The interrogation of cell surface-presented immunogenic epitopes is of great importance to differentiate diseased cells in consequence to malignant transformation or viral infections. On the basis of this knowledge, next-generation immunotherapies against cancers, autoimmunity, or infectious diseases can be developed. The identification of altered peptide repertoires of transformed cells renders mass spectrometry-based analysis indispensable. This is evident considering the low correlation of gene or protein expression alterations, respectively, with changes in the peptide repertoire rendering those analyses less informative. Nevertheless, immunogenicity of peptides appearing to be exclusively found on diseased cells has to be finally proven in T cell-based assays. This review highlights the capabilities and limitations of mass spectrometry in the identification of entire immunopeptidomes, as well as individual potential immunogenic epitopes with a strong focus on cancer. Furthermore, an overview of state-of-the-art immunogenicity screens is presented.

HLA class I-restricted MYD88 L265P-derived peptides as specific targets for lymphoma immunotherapy.

Genome sequencing has uncovered an array of recurring somatic mutations in different non-Hodgkin lymphoma (NHL) subtypes. If affecting protein-coding regions, such mutations may yield mutation-derived peptides that may be presented by HLA class I proteins and recognized by cytotoxic T cells. A recurring somatic and oncogenic driver mutation of the Toll-like receptor adaptor protein MYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential of MYD88L265P-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based on in silico predictions, we identified potential MYD88L265P-containing HLA ligands for several HLA class I restrictions. A set of HLA class I MYD88L265P-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential of MYD88L265P mutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients with MYD88L265P+ NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling.

HLA ligandomics identifies histone deacetylase 1 as target for ovarian cancer immunotherapy.

The recent approval of clincially effective immune checkpoint inhibitors illustrates the potential of cancer immunotherapy. A challenging task remains the identification of specific targets guiding immunotherapy. Facilitated by technical advances, the direct identification of physiologically relevant targets is enabled by analyzing the HLA ligandome of cancer cells. Since recent publications demonstrate the immunogenicity of ovarian cancer (OvCa), immunotherapies, including peptide-based cancer vaccines, represent a promising treatment approach. To identify vaccine peptides, we employed a combined strategy of HLA ligandomics in high-grade serous OvCa samples and immunogenicity analysis. Only few proteins were naturally presented as HLA ligands on all samples analyzed, including histone deacetylase (HDAC) 1 and 2. In vitro priming of CD8(+) T cells demonstrated that two HDAC1/2-derived HLA ligands can induce T-cell responses, capable of killing HLA-matched tumor cells. High HDAC1 expression shown by immunohistochemistry in 136 high-grade serous OvCa patients associated with significantly reduced overall survival (OS), whereas patients with high numbers of CD3(+) tumor-infiltrating lymphocytes (TILs) in the tumor epithelium and CD8(+) TILs in the tumor stroma showed improved OS. However, correlating HDAC1 expression with TILs, high levels of TILs abrogated the impact of HDAC1 on OS. This study strengthens the role of HDAC1/2 as an important tumor antigen in OvCa, demonstrating its impact on OS in a large cohort of OvCa patients. We further identified two immunogenic HDAC1-derived peptides, which frequently induce multi-functional T-cell responses in many donors, suitable for future multi-peptide vaccine trials in OvCa patients.

Human CD8(+) T Cells Target Multiple Epitopes in Respiratory Syncytial Virus Polymerase.

Respiratory syncytial virus (RSV) infection is a serious health problem in young children, immunocompromised patients, and the elderly. The development of novel prevention strategies, such as a vaccine to RSV, is a high priority. One strategy is to design a peptide-based vaccine that activates appropriate CD8(+) T-cell responses. However, this approach is limited by the low number of RSV peptide epitopes defined to date that activate CD8(+) T cells. We aimed to identify peptide epitopes that are presented by common human leukocyte antigen types (HLA-A*01, -A*02, and -B*07). We identify one novel HLA-A*02-restricted and two novel HLA-A*01-restricted peptide epitopes from RSV polymerase. Peptide-HLA multimer staining of specific T cells from healthy donor peripheral blood mononuclear cell, the memory phenotype of such peptide-specific T cells ex vivo, and functional IFNγ responses in short-term stimulation assays suggest that these peptides are recognized during RSV infection. Such peptides are candidates for inclusion into a peptide-based RSV vaccine designed to stimulate defined CD8(+) T-cell responses.

Combined Immunoscore of CD103 and CD3 Identifies Long-Term Survivors in High-Grade Serous Ovarian Cancer.

OBJECTIVE: Increased numbers of tumor-infiltrating lymphocytes (TILs) in high-grade serous ovarian cancer (HGSC) are associated with improved clinical outcome. Intraepithelial localization of TILs might be regulated by specific homing receptors, such as CD103, which is widely expressed by intraepithelial lymphocytes. Given the emerging role of CD103 TILs, we aimed to assess their contribution to the prognostic value of immunoscoring in HGSC. METHODS: The density of intratumoral CD3 and CD103 lymphocytes was examined by immunohistochemistry on a tissue microarray of a series of 135 patients with advanced HGSC and correlated with CD4, CD8, CD56, FoxP3, and TCRγ T-cell counts, as well as E-cadherin staining and conventional prognostic parameters and clinical outcome. RESULTS: Both the presence of CD103 cells, as well as high numbers of intraepithelial CD3 lymphocytes (CD3E), showed a significant correlation with overall survival, in the complete series, as well as in patients with optimal debulking and/or platinum sensitivity. Combining CD3 and CD103 counts improved prognostication and identified 3 major subgroups with respect to overall survival. The most pronounced effect was demonstrated for patients with optimally resected and platinum-sensitive tumors. Patients with CD3/CD103 tumors showed a 5-year survival rate at 90%, CD3/CD103 at 63%, and CD3/CD103 at 0% (P < 0.001). CONCLUSIONS: These results suggest that combined assessment of CD103 and CD3 counts improves the prognostic value of TIL counts in HGSC and might identify patients with early relapse or long-term survival based on the type and extent of the immune response.

Identification of a Novel Immunodominant HLA-B*07: 02-restricted Adenoviral Peptide Epitope and Its Potential in Adoptive Transfer Immunotherapy.

Adenovirus infections of immunocompromised patients, particularly following allogeneic hematopoietic stem cell transplantation, are associated with morbidity and mortality. Immunotherapy by adoptive transfer of hexon-specific and penton-specific T cells has been successfully applied, but many approaches are impeded by the low number of HLA class I-restricted adenoviral peptide epitopes described to date. We use a novel method to identify naturally presented adenoviral peptide epitopes from infected human cells, ectopically expressing defined HLA, using peptide elution and liquid chromatography-mass spectrometry analysis. We show that the previously described HLA-A*01:01-restricted peptide epitope LTDLGQNLLY from hexon protein is naturally presented, and demonstrate the functionality of LTDLGQNLLY-specific T cells. We further identify a novel immunodominant HLA-B*07:02-restricted peptide epitope VPATGRTLVL from protein 13.6 K, and demonstrate the high proliferative, cytotoxic, and IFN-γ-producing capacity of peptide-specific T cells. Lastly, LTDLGQNLLY-specific T cells can be detected ex vivo following adoptive transfer therapy, and LTDLGQNLLY-specific and VPATGRTLVL-specific T cells have memory phenotypes ex vivo. Given their proliferative and cytotoxic capacity, such epitope-specific T cells are promising candidates for adoptive T-cell transfer therapy of adenovirus infection.

A combined approach of human leukocyte antigen ligandomics and immunogenicity analysis to improve peptide-based cancer immunotherapy.

The breakthrough development of immune checkpoint inhibitors as clinically effective novel therapies demonstrates the potential of cancer immunotherapy. The identification of suitable targets for specific immunotherapy, however, remains a challenging task. Most peptides previously used for vaccination in clinical trials were able to elicit strong immunological responses but failed with regard to clinical benefit. This might, at least partly, be caused by an inadequate peptide selection, usually derived from established tumor-associated antigens which are not necessarily presented as human leukocyte antigen (HLA) ligands. Recently, HLA ligandome analysis revealed cancer-associated peptides, which have been used in clinical trials showing encouraging impact on survival. To improve peptide-based cancer immunotherapy, our group established a combined approach of HLA ligandomics and immunogenicity analysis for the identification of vaccine peptides. This approach is based on the identification of naturally presented HLA ligands on tumor samples, the selection of tumor-associated/tumor-specific HLA ligands and their subsequent testing for immunogenicity in vitro. In this review, we want to present our pipeline for the identification of vaccine peptides, focusing on ovarian cancer, and want to discuss differences to other approaches. Furthermore, we want to give a short outlook of a potential multi-peptide vaccination trial using the novel identified peptides.

An impedance-based cytotoxicity assay for real-time and label-free assessment of T-cell-mediated killing of adherent cells.

The in vitro assessment of T-cell-mediated cytotoxicity plays an important and increasingly relevant role both in preclinical target evaluation and during immunomonitoring to accompany clinical trials employing targeted immunotherapies. For a long time, the gold standard for this purpose has been the chromium release assay (CRA). This end point assay, however, shows several disadvantages including the inevitable use of radioactivity. Based on electrical impedance measurements (using the xCELLigence system), we have established a label-free assay, facilitating the real-time monitoring of T-cell-mediated cytotoxicity. The coculture of peptide-specific T-cell lines with peptide-loaded target cells reproducibly led to a decrease in impedance due to induced apoptosis and detachment of target cells. Comparing our results to the standard CRA assay, we could demonstrate that impedance-based measurements show comparable results after short incubation periods (6h) but outperform the CRA both in reproducibility and sensitivity after prolonged incubation (24h), enabling the detection of target cell lysis with an effector to target ratio as low as 0.05:1. The impedance-based assay represents a valuable and highly sensitive tool for label-free real-time high throughput analysis of T-cell-mediated cytotoxicity.