Danilo Ritz

High-sensitivity HLA class I peptidome analysis enables a precise definition of peptide motifs and the identification of peptides from cell lines and patients' sera.

The characterization of peptides bound to human leukocyte antigen (HLA) class I is of fundamental importance for understanding CD8+ T cell‐driven immunological processes and for the development of immunomodulatory therapeutic strategies. However, until now, the mass spectrometric analysis of HLA‐bound peptides has typically required billions of cells, still resulting in relatively few high‐confidence peptide identifications. Capitalizing on the recent developments in mass spectrometry and bioinformatics, we have implemented a methodology for the efficient recovery of acid‐eluted HLA peptides after purification with the pan‐reactive antibody W6/32 and have identified a total of 27 862 unique peptides with high confidence (1% false discovery rate) from five human cancer cell lines. More than 93% of the identified peptides were eight to 11 amino acids in length and contained signatures that were in excellent agreement with published HLA binding motifs. Furthermore, by purifying soluble HLA class I complexes (sHLA) from sera of melanoma patients, up to 972 high‐confidence peptides could be identified, including melanoma‐associated antigens already described in the literature. Knowledge of the HLA class I peptidome should facilitate multiplex tetramer technology‐based characterization of T cells, and allow the development of patient selection, stratification and immunomodulatory therapeutic strategies.

High‐resolution analysis of the murine MHC class II immunopeptidome

The reliable identification of peptides bound to major histocompatibility complex (MHC) class II is fundamental for the study of the host immune response against pathogens and the pathogenesis of autoimmune conditions. Here, we describe an improved methodology combining immuno‐affinity enrichment of MHC class II complexes, optimized elution conditions and quadrupole Orbitrap mass spectrometry‐based characterization of the immunopeptidome. The methodology allowed the identification of over 1000 peptides with 1% false discovery rate from 108 murine A20 lymphoma cells. The study revealed the I‐Ad‐specific motif in high resolution after multisequence alignment. The methodology was generally applied to the purification of MHC class II from cell lines and murine spleens. We identified 2963 peptides from BALB/c and 2712 from C57BL/6 mouse spleens. The identification of peptides bound to MHC class II in vitro and in vivo will facilitate the characterization of T‐cell specificities, as well as the development of biotherapeutics and vaccines.

Mass spectrometric analysis of the HLA class I peptidome of melanoma cell lines as a promising tool for the identification of putative tumor-associated HLA epitopes.

Melanoma is one of the most immunogenic tumors, and extensive lists of potential tumor rejection antigens have been collected during the last decades. By isolating human leukocyte antigen (HLA) class I complexes from five melanoma cell lines (FM-82, FM-93/2, Mel-624, MeWo and SK-Mel-5) and sequencing HLA-eluted peptides by mass spectrometry, we identified over 10,000 unique peptides with high confidence. The majority of the peptides were 8–11 amino acids in length and were predicted to bind to the respective HLA alleles. Over 250 epitopes, corresponding to previously described tumor-associated antigens, were identified, suggesting that HLA peptidome analysis may facilitate the characterization of putative tumor rejection antigens. MeWo and SK-Mel-5 cell lines were further interrogated for neo-epitopes, revealing one peptide from MeWo cells carrying an amino acid mutation. We also observed a remarkable overlap between A*03:01 peptides eluted from Mel-624 cells and A*03:01 peptides recovered from soluble HLA complexes purified from two melanoma patients, shedding light on the similarity of the HLA peptidome in cell lines and in patient-derived material. The reliable characterization of the HLA class I peptidome in melanoma promises to facilitate the identification of tumor rejection antigens and the development of immunotherapeutic strategies.

The MHC Class II Immunopeptidome of Lymph Nodes in Health and in Chemically Induced Colitis

We recently described a mass spectrometry–based methodology that enables the confident identification of hundreds of peptides bound to murine MHC class II (MHCII) molecules. In this article, we describe its application to the characterization of MHCII-bound peptides isolated from lymph nodes (LNs) of C57BL/6 mice. More than 1000 peptides could be identified in individual analyses, allowing a direct comparison of the MHCII peptidome in different types of normal LNs or in animals with colitis. The peptide length distribution and consensus sequences in axillary, brachial, inguinal, and mesenteric LNs were virtually identical, and a substantial portion of identified peptides corresponded to proteins found in all LNs. However, skin-specific proteins Sbsn and Dmkn and intestine-specific proteins Dmbt1, Krt19, and Maoa, among others, were exclusively identified in skin-draining and mesenteric LNs, respectively. Differences in peptide-presentation patterns were also observed when comparing healthy mice and mice with dextran sodium sulfate–induced colitis. Peptides derived from a subset of proteins (including IgE, Bank1, chondroitin sulfate synthase 2, Cmip, and Fth1) were exclusively identified in mice with colitis, revealing changes in the peptidome associated with the inflammatory process, as well as activation and clonal expansion of B cells.

Sarcoma eradication by doxorubicin and targeted TNF relies upon CD8+ T cell recognition of a retroviral antigen

Antibody-cytokine complexes may offer new tools to treat cancer. Here, we show how TNF-linked antibodies, which recognize tumor-selective splice isoforms of fibronectin (F8-TNF), can be exploited to eradicate sarcomas in immunocompetent mice. We treated mice bearing WEHI-164 fibrosarcoma with a combination of F8-TNF and doxorubicin, curing the majority of treated animals (29/37). Notably, cured mice were resistant to rechallenge not only by WEHI-164 cells but also heterologous C51 or CT26 colorectal tumor cells in a CD8+ T-cell-dependent process. Mechanistic analyses revealed that each tumor cell line presented AH1, a common endogenous retroviral peptide. Numbers of AH1-specific CD8+ T cells exhibiting cytotoxic capacity were increased by F8-TNF plus doxorubicin treatment, arguing that cognate CD8+ T cells contributed to tumor eradication. Sequence analysis of T-cell receptors of CD8+ T cells revealed the presence of H-2Ld/AH1-specific T cells and an expansion of sequence diversity in treated mice. Overall, our findings provide evidence that retroviral genes contribute to tumoral immunosurveillance in a process that can be generally boosted by F8-TNF and doxorubicin treatment. Cancer Res; 77(13); 3644-54. ©2017 AACR.

Monoclonal antibodies to murine thrombospondin-1 and thrombospondin-2 reveal differential expression patterns in cancer and low antigen expression in normal tissues

There is a considerable interest for the discovery and characterization of tumor-associated antigens, which may facilitate antibody-based pharmacodelivery strategies. Thrombospondin-1 and thrombospondin-2 are homologous secreted proteins, which have previously been reported to be overexpressed during remodeling typical for wound healing and tumor progression and to possibly play a functional role in cell proliferation, migration and apoptosis. To our knowledge, a complete immunohistochemical characterization of thrombospondins levels in normal rodent tissues has not been reported so far. Using antibody phage technology, we have generated and characterized monoclonal antibodies specific to murine thrombospondin-1 and thrombospondin-2, two antigens which share 62% aminoacid identity. An immunofluorescence analysis revealed that both antigens are virtually undetectable in normal mouse tissues, except for a weak staining of heart tissue by antibodies specific to thrombospondin-1. The analysis also showed that thrombospondin-1 was strongly expressed in 5/7 human tumors xenografted in nude mice, while it was only barely detectable in 3/8 murine tumors grafted in immunocompetent mice. By contrast, a high-affinity antibody to thrombospondin-2 revealed a much lower level of expression of this antigen in cancer specimens. Our analysis resolves ambiguities related to conflicting reports on thrombosponding expression in health and disease. Based on our findings, thrombospondin-1 (and not thrombospondin-2) may be considered as a target for antibody-based pharmacodelivery strategies, in consideration of its low expression in normal tissues and its upregulation in cancer.

Membranal and Blood-Soluble HLA Class II Peptidome Analyses Using Data-Dependent and Independent Acquisition

The interaction between HLA class II peptide complexes on antigen‐presenting cells and CD4+ T cells is of fundamental importance for anticancer and antipathogen immunity as well as for the maintenance of immunological tolerance. To study CD4+ T cell reactivities, detailed knowledge of the presented peptides is necessary. In recent years, dramatic advances in the characterization of membranal and soluble HLA class I peptidomes could be observed. However, the same is not true for HLA class II peptidomes, where only few studies identify more than hundred peptides. Here we describe a MS‐based workflow for the characterization of membranal and soluble HLA class II DR and DQ peptidomes. Using this workflow, we identify a total of 8595 and 3727 HLA class II peptides from Maver‐1 and DOHH2 cells, respectively. Based on this data, a motif‐based binding predictor is developed and compared to NetMHCIIpan 3.1. We then apply the workflow to human plasma, resulting in the identification of between 34 and 152 HLA‐DR and between 100 and 180 HLA‐DQ peptides, respectively. Finally, we implement a data‐independent acquisition workflow to increase reproducibility and sensitivity of HLA class II peptidome characterizations.

Data Independent Acquisition of HLA Class I Peptidomes on the Q Exactive Mass Spectrometer Platform

The characterization of peptides presented by human leukocyte antigen (HLA) class I molecules is crucial for understanding immune processes, biomarker discovery, and the development of novel immunotherapies or vaccines. Mass spectrometry allows the direct identification of thousands of HLA‐bound peptides from cell lines, blood, or tissue. In recent years, data‐independent acquisition (DIA) mass spectrometry methods have evolved, promising to increase reproducibility and sensitivity over classical data‐dependent acquisition (DDA) workflows. Here, we describe a DIA setup on the Q Exactive mass spectrometer, optimized regarding the unique properties of HLA class I peptides. The methodology enables sensitive and highly reproducible characterization of HLA peptidomes from individual cell lines. From up to 16 DDA analyses of 100 million human cells, more than 10 000 peptides could be confidently identified, serving as basis for the generation of spectral libraries. This knowledge enabled the subsequent interrogation of DIA data, leading to the identification of peptide sets with >90% overlap between replicate samples, a prerequisite for the comparative study of closely related specimens. Furthermore, >3000 peptides could be identified from just one million cells after DIA analysis using a library generated from 300 million cells. The reduction in sample quantity and the high reproducibility of DIA‐based HLA peptidome analysis should facilitate personalized medicine applications.

Purification of soluble HLA class I complexes from human serum or plasma deliver high quality immuno peptidomes required for biomarker discovery

Soluble human leukocyte antigen class I (sHLA)‐peptide complexes have been suggested to play a role in the modulation of immune responses and in immune evasion of cancer cells. The set of peptides eluted from sHLA molecules could serve as biomarker for the monitoring of patients with cancer or other conditions. Here, we describe an improved sHLA peptidomics methodology resulting in the identification of 1816 to 2761 unique peptide sequences from triplicate analyses of serum or plasma taken from three healthy donors. More than 90% of the identified peptides were 8–11mers and 74% of these sequences were predicted to bind to cognate HLA alleles, confirming the quality of the resulting immunopeptidomes. In comparison to the HLA peptidome of cultured cells, the plasma‐derived peptides were predicted to have a higher stability in complex with the cognate HLA molecules and mainly derived from proteins of the plasma membrane or from the extracellular space. The sHLA peptidomes can efficiently be characterized by using the new methodology, thus serving as potential source of biomarkers in various pathological conditions.