The HLA Ligandome Comprises a Limited Repertoire of O-GlcNAcylated Antigens Preferentially Associated With HLA-B*07:02

Mass-spectrometry based immunopeptidomics has provided unprecedented insights into antigen presentation, not only charting an enormous ligandome of self-antigens, but also cancer neoantigens and peptide antigens harbouring post-translational modifications. Here we concentrate on the latter, focusing on the small subset of HLA Class I peptides (less than 1%) that has been observed to be post-translationally modified (PTM) by a O-linked N-acetylglucosamine (GlcNAc). Just like neoantigens these modified antigens may have specific immunomodulatory functions. Here we compiled from literature, and a new dataset originating from the JY B cell lymphoblastoid cell line, a concise albeit comprehensive list of O-GlcNAcylated HLA class I peptides. This cumulative list of O-GlcNAcylated HLA peptides were derived from normal and cancerous origin, as well as tissue specimen. Remarkably, the overlap in detected O-GlcNAcylated HLA peptides as well as their source proteins is strikingly high. Most of the O-GlcNAcylated HLA peptides originate from nuclear proteins, notably transcription factors. From this list, we extract that O-GlcNAcylated HLA Class I peptides are preferentially presented by the HLA-B*07:02 allele. This allele loads peptides with a Proline residue anchor at position 2, and features a binding groove that can accommodate well the recently proposed consensus sequence for O-GlcNAcylation, P(V/A/T/S)g(S/T), essentially explaining why HLA-B*07:02 is a favoured binding allele. The observations drawn from the compiled list, may assist in the prediction of novel O-GlcNAcylated HLA antigens, which will be best presented by patients harbouring HLA-B*07:02 or related alleles that use Proline as anchoring residue.

Natural and cryptic peptides dominate the immunopeptidome of atypical teratoid rhabdoid tumors

BackgroundAtypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear which epitopes T cells might recognize on AT/RT cells.MethodsHere, we report a comprehensive mass spectrometry (MS)-based analysis of naturally presented human leukocyte antigen (HLA) class I and class II ligands on 23 AT/RTs. MS data were validated by matching with a human proteome dataset and exclusion of peptides that are part of the human benignome. Cryptic peptide ligands were identified using Peptide-PRISM.ResultsComparative HLA ligandome analysis of the HLA ligandome revealed 55 class I and 139 class II tumor-exclusive peptides. No peptide originated from the SMARCB1 region. In addition, 61 HLA class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas, but no concordance was found with extracranial tumors. More than 80% of AT/RT exclusive peptides were able to successfully prime CD8+ T cells, whereas naturally occurring memory responses in AT/RT patients could only be detected for class II epitopes. Interestingly, >50% of AT/RT exclusive class II ligands were also recognized by T cells from glioblastoma patients but not from healthy donors.ConclusionsThese findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA class I and class II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.

Acid Stripping after Infection Improves the Detection of Viral HLA Class I Natural Ligands Identified by Mass Spectrometry

Identification of a natural human leukocyte antigen (HLA) ligandome is a key element to understand the cellular immune response. Advanced high throughput mass spectrometry analyses identify a relevant, but not complete, fraction of the many tens of thousands of self-peptides generated by antigen processing in live cells. In infected cells, in addition to this complex HLA ligandome, a minority of peptides from degradation of the few proteins encoded by the viral genome are also bound to HLA class I molecules. In this study, the standard immunopeptidomics strategy was modified to include the classical acid stripping treatment after virus infection to enrich the HLA ligandome in virus ligands. Complexes of HLA-B*27:05-bound peptide pools were isolated from vaccinia virus (VACV)-infected cells treated with acid stripping after virus infection. The HLA class I ligandome was identified using high throughput mass spectrometry analyses, yielding 37 and 51 natural peptides processed and presented untreated and after acid stripping treatment VACV-infected human cells, respectively. Most of these virus ligands were identified in both conditions, but exclusive VACV ligands detected by mass spectrometry detected on acid stripping treatment doubled the number of those identified in the untreated VACV-infected condition. Theoretical binding affinity prediction of the VACV HLA-B*27:05 ligands and acute antiviral T cell response characterization in the HLA transgenic mice model showed no differences between HLA ligands identified under the two conditions: untreated and under acid stripping condition. These findings indicated that acid stripping treatment could be useful to identify HLA class I ligands from virus-infected cells.

NATURAL AND CRYPTIC PEPTIDES DOMINATE THE IMMUNOPEPTIDOME OF ATYPICAL TERATOID RHABDOID TUMORS

Atypical teratoid/rhabdoid tumors (AT/RT) are highly aggressive CNS-tumors of infancy and early childhood. Hallmark is the surprisingly simple genome with inactivating mutations or deletions in the SMARCB1 gene as the oncogenic driver. Nevertheless, AT/RTs are infiltrated by immune cells and even clonally expanded T cells. However, it is unclear, which epitopes T-cells might recognize on AT/RT cells. Here, we report a comprehensive MS-based analysis of naturally presented HLA-class-I and class-II ligands on 23 AT/RTs. Comparative HLA ligandome analysis of the HLA-ligandome revealed 55 class-I and 139 class-II tumor-exclusive peptides. No peptide originated from the SMARCB1-region. In addition, 61 HLA-class I tumor-exclusive peptide sequences derived from non-canonically translated proteins. Combination of peptides from natural and cryptic class I and class II origin gave optimal representation of tumor cell compartments. Substantial overlap existed with the cryptic immunopeptidome of glioblastomas but no concordance was found with extracranial tumors. More than 80% of AT/RT-exclusive peptides were able to successfully prime CD8+ T-cells, whereas naturally occurring memory responses in AT/RT-patients could only be detected for class-II epitopes. Interestingly, >50% of AT/RT-exclusive class-II ligands were also recognized by T-cells from glioblastoma patients but not from healthy donors. These findings highlight that AT/RTs, potentially paradigmatic for other pediatric tumors with a low mutational load, present a variety of highly immunogenic HLA-class-I and class-II peptides from canonical as well as non-canonical protein sources. Inclusion of such cryptic peptides into therapeutic vaccines would enable an optimized mapping of the tumor cell surface, thereby reducing the likelihood of immune evasion.

IMMU-08. THE ANTIGENIC LANDSCAPE OF GLIOBLASTOMA - REFINING THE TARGETS FOR IMMUNOTHERAPY

We provide a comprehensive analysis of the antigenic landscape of glioblastoma using a multi-omics approach including ligandome mapping of the Human Leukocyte Antigen (HLA) ligandome, next generation sequencing (NGS) as well as an in-depth characterization of tumor-infiltrating lymphocytes (TIL) using mass cytometry and ultra-deep sequencing of the T-cell receptor (TCR). Tumor-exclusive HLA class I and class II ligands (immune precipitation and LC-MS/MS) of 24 isocitrate dehydrogenase 1 wild type glioblastoma samples and 10 autologous primary glioblastoma cell lines were defined in comparison to an HLA ligandome normal tissue reference database (n > 418). We found 11,496 glioblastoma exclusive HLA class I ligands (2,064 shared with cell lines; 3,754 on ≥ 2 glioblastoma samples). On the source protein level, 239 glioblastoma exclusive proteins were identified; among them 54 were also found in cell lines. For HLA class II ligands the analysis revealed 11,870 glioblastoma exclusive peptides (444 shared with cell lines; 3,420 on ≥ 2 glioblastoma samples) and 278 glioblastoma exclusive proteins; among which 18 were present also in cell lines. Moreover, whole-exome sequencing and whole RNA sequencing of 13 tumor samples was performed with the aim to predict neoantigens. On average 5,662 somatic missense effects were identified per patient (min: 4,258; max: 7,479). Candidate peptides are grouped into (i) in silico predicted neoepitopes, (ii) tumor-exclusivity on HLA, (iii) gene expression (e.g. cancer testis antigens). Top-ranking candidates from each group will be tested with regards to their immunogenicity in an autologous setting (TIL, peripheral blood mononuclear cells, patient derived tumor cells). Finally, the peptide and immunogenicity data is correlated with the immune phenotype of the TIL compartment as well as the TCR repertoire of the sample.

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.