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Background: Upstream open reading frames (uORFs) represent translational control elements within eukaryotic transcript leader sequences. Recent data showed that uORFs can encode for biologically active proteins and human leukocyte antigen (HLA)-presented peptides in malignant and benign cells suggesting their potential role in cancer cell development and survival. However, the role of uORFs in translational regulation of cancer-associated transcripts as well as in cancer immune surveillance is still incompletely understood. Methods: We examined the translational regulatory effect of 29 uORFs in 13 cancer-associated genes by dual-luciferase assays. Cellular expression and localization of uORF-encoded peptides (uPeptides) were investigated by immunoblotting and immunofluorescence-based microscopy. Furthermore, we utilized mass spectrometry-based immunopeptidome analyses in an extensive dataset of primary malignant and benign tissue samples for the identification of naturally presented uORF-derived HLA-presented peptides screening for more than 2000 uORFs. Results: We provide experimental evidence for similarly effective translational regulation of cancer-associated transcripts through uORFs initiated by either canonical AUG codons or by alternative translation initiation sites (aTISs). We further demonstrate frequent cellular expression and reveal occasional specific cellular localization of uORF-derived peptides, suggesting uPeptide-specific biological implications. Immunopeptidome analyses delineated a set of 125 naturally presented uORF-derived HLA-presented peptides. Comparative immunopeptidome profiling of malignant and benign tissue-derived immunopeptidomes identified several tumor-associated uORF-derived HLA ligands capable to induce multifunctional T cell responses. Conclusion: Our data provide direct evidence for the frequent expression of uPeptides in benign and malignant human tissues, suggesting a potentially widespread function of uPeptides in cancer biology. These findings may inspire novel approaches in direct molecular as well as immunotherapeutic targeting of cancer-associated uORFs and uPeptides. [ABSTRACT FROM AUTHOR]

Recent studies suggest that CLL is an immunogenic disease, which might be effectively targeted by antigen-specific T-cell-based immunotherapy. However, CLL is associated with a profound immune defect, which might represent a critical limitation for mounting clinically effective antitumor immune responses. As several studies have demonstrated that lenalidomide can reinforce effector T-cell responses in CLL, the combination of T-cell-based immunotherapy with the immunomodulatory drug lenalidomide represents a promising approach to overcome the immunosuppressive state in CLL. Antigen-specific immunotherapy also requires the robust presentation of tumor-associated HLA-presented antigens on target cells. We thus performed a longitudinal study of the effect of lenalidomide on the HLA ligandome of primary CLL cells in vitro. We showed that lenalidomide exposure does not affect absolute HLA class I and II surface expression levels on primary CLL cells. Importantly, semi-quantitative mass spectrometric analyses of the HLA peptidome of three CLL patient samples found only minor qualitative and quantitative effects of lenalidomide on HLA class I- and II-restricted peptide presentation. Furthermore, we confirmed stable presentation of previously described CLL-associated antigens under lenalidomide treatment. Strikingly, among the few HLA ligands showing significant modulation under lenalidomide treatment, we identified upregulated IKZF-derived peptides, which may represent a direct reflection of the cereblon-mediated effect of lenalidomide on CLL cells. Since we could not observe any relevant influence of lenalidomide on the established CLL-associated antigen targets of anticancer T-cell responses, this study validates the suitability of lenalidomide for the combination with antigen-specific T-cell-based immunotherapies. [ABSTRACT FROM AUTHOR]

T-cell immunity, mediated by CD4+ and CD8+ T cells, represents a cornerstone in the control of viral infections. Virus-derived T-cell epitopes are represented by human leukocyte antigen (HLA)-presented viral peptides on the surface of virus-infected cells. They are the prerequisite for the recognition of infected cells by T cells. Knowledge of viral T-cell epitopes provides on the one hand a diagnostic tool to decipher protective T-cell immune responses in the human population and on the other hand various prophylactic and therapeutic options including vaccination approaches and the transfer of virus-specific T cells. Such approaches have already been proven to be effective against various viral infections, particularly in immunocompromised patients lacking sufficient humoral, antibody-based immune response. This review provides an overview on the state of the art as well as current studies regarding the identification and characterization of viral T-cell epitopes and approaches of clinical application. In the first chapter in silico prediction tools and direct, mass spectrometry-based identification of viral T-cell epitopes is compared. The second chapter provides an overview of commonly used assays for further characterization of T-cell responses and phenotypes. The final chapter presents an overview of clinical application of viral T-cell epitopes with a focus on human immunodeficiency virus (HIV), human cytomegalovirus (HCMV) and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), being representatives of relevant viruses. [ABSTRACT FROM AUTHOR]

The diversity of human leukocyte antigens (HLAs) enables the presentation of immense repertoires of peptides, including tumor-associated antigens (TAAs). As a surrogate for immunopeptidome diversity, the HLA evolutionary divergence (HED) between individual HLA alleles might directly define the ability to present TAAs, a prerequisite for graft-versus-leukemia effects. We therefore analyzed the impact of HED on survival within a cohort of 171 acute myeloid leukemia (AML) patients after matched donor allogeneic hematopoietic stem cell transplantation (HSCT). Low HED (<25th percentile) of HLA class I (HEDclass I) or HLA-DR antigens (HEDDR) was a strong determinant for adverse overall survival after allogeneic HSCT (OS), with a hazard ratio for death of 1.9 (95% CI 1.2–3.2) and 2.1 (95% CI 1.3–3.4), respectively. Defining a cutoff value for the combined HEDtotal (HEDclass I and HEDDR), the respective 5 year OS was 29.7% and 64.9% in patients with low and high HEDtotal (p < 0.001), respectively. Furthermore, the risk of relapse was significantly higher in patients with low HEDtotal (hazard ratio (HR) 2.2, 95% CI 1.3–3.6) and event-free survival (EFS) was significantly reduced (5 year EFS 25.7% versus 54.4%, p < 0.001). We here introduce HED, a fundamental metric of immunopeptidome diversity, as a novel prognostic factor for AML patients undergoing allogeneic HSCT. [ABSTRACT FROM AUTHOR]

SARS-CoV-2 has spread worldwide, causing millions of deaths and leaving a significant proportion of people with long-term sequelae of COVID-19 ("post-COVID syndrome"). Whereas the precise mechanism of post-COVID syndrome is still unknown, the immune response after the first infection may play a role. Here, we performed a long-term follow-up analysis of 110 COVID-19 convalescents, analyzing the first SARS-CoV-2-directed immune response, vaccination status, long-term symptoms (approximately 2.5 years after first infection), and reinfections. A total of 96% of convalescents were vaccinated at least once against SARS-CoV-2 after their first infection. A reinfection rate of 47% was observed, and lower levels of anti-spike IgG antibodies after the first infection were shown to associate with reinfection. While T-cell responses could not be clearly associated with persistent postinfectious symptoms, convalescents with long-term symptoms showed elevated SARS-CoV-2-specific antibody levels at the first infection. Evaluating the immune response after the first infection might be a useful tool for identifying individuals with increased risk for re-infections and long-term symptoms. [ABSTRACT FROM AUTHOR]

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 proteinMYD88, Leu265Pro (L265P) was identified in up to 90% of different NHL subtype patients. We therefore screened the potential ofMYD88L265P-derived peptides to elicit cytotoxic T cell responses as tumor-specific neoantigens. Based onin silicopredictions, we identified potentialMYD88L265P-containing HLA ligands for several HLA class I restrictions. A set of HLA class IMYD88L265P-derived ligands elicited specific cytotoxic T cell responses for HLA-B*07 and -B*15. These data highlight the potential ofMYD88L265Pmutation-specific peptide-based immunotherapy as a novel personalized treatment approach for patients withMYD88L265P+NHLs that may complement pharmacological approaches targeting oncogenic MyD88 L265P signaling. [ABSTRACT FROM AUTHOR]

The course of chronic lymphocytic leukemia (CLL), inducing an immunosuppressed state that also affects T cells as central components of adaptive immunity, predisposes patients to develop second malignancies with skin cancer being the most common. Recently, we found that prevalence of memory T cells with specificity for CLL-associated antigens defined by mass spectrometry-based immunopeptidome analysis correlated with a significant survival benefit. Here, we analyzed our CLL patient cohort for second skin (pre)malignancies and found a significantly lower incidence of skin cancer in the patients showing immune responses to CLL-associated antigens. Surprisingly, CLL-associated antigen-specific immune responses did not associate with clinical characteristics including leukocyte, neutrophil, and thrombocyte count, hemoglobin, immunoglobulin levels, or CD8+ and CD4+ T-cell immune status. Our data indicate that the CLL-specific immune signature of a given patient, defined by antigen-specific T-cell responses, might represent an independent marker to identify CLL patients susceptible for the development of skin malignancies. [ABSTRACT FROM AUTHOR]

With the routine use of effective severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, the number of life-threatening coronavirus disease 2019 (COVID-19) courses have largely been reduced. However, multiple COVID-19 convalescents, even after asymptomatic to moderate disease, suffer from post-COVID syndrome, with relevant limitations in daily life. The pathophysiologic mechanisms of post-COVID syndrome are still elusive, with dysregulation of the immune system suggested as a central mechanism. Here, we assessed COVID-19 post-infectious symptoms (5–6 months after PCR-confirmed acute infection) together with the humoral immune response against SARS-CoV-2 in non-hospitalized COVID-19 convalescents, early (5–6 weeks) and late (5–6 months) after their first positive SARS-CoV-2 PCR result. Convalescents reporting several post-infectious symptoms (>3) showed higher anti-spike and anti-nucleocapsid antibody levels 5–6 weeks after PCR-confirmed infection with the latter remained increased 5–6 months after positive PCR. Likewise, a higher post-infectious symptom score was associated with increased antibody levels. Of note, convalescents displaying neuro-psychiatric symptoms such as restlessness, palpitations, irritability, and headache, as well as general symptoms such as fatigue/reduced power had higher SARS-CoV-2-specific antibody levels compared with asymptomatic cases. The increased humoral immune response in convalescents with post-COVID syndrome might be useful for the detection of individuals with an increased risk for post-COVID syndrome. [ABSTRACT FROM AUTHOR]

Background The rapid emergence of the Omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the World Health Organization. Subsequently, Omicron evolved into distinct sublineages (eg, BA.1 and BA.2), which currently represent the majority of global infections. Initial studies of the neutralizing response toward BA.1 in convalescent and vaccinated individuals showed a substantial reduction. Methods We assessed antibody (immunoglobulin G [IgG]) binding, ACE2 (angiotensin-converting enzyme 2) binding inhibition, and IgG binding dynamics for the Omicron BA.1 and BA.2 variants compared to a panel of VOCs/variants of interest, in a large cohort (N = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. Results While Omicron was capable of efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to wild type. Whereas BA.1 exhibited less IgG binding compared to BA.2, BA.2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to Omicron only improved after administration of a third dose. Conclusions Omicron BA.1 and BA.2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind to Omicron. The extent of the mutations within both variants prevents a strong inhibitory binding response. As a result, both Omicron variants are able to evade control by preexisting antibodies. [ABSTRACT FROM AUTHOR]

Simple Summary: Immunosurveillance of cancer is mediated by T cell-based recognition of tumor-associated antigens, i.e., short peptides that are presented on the surface of cells on human leukocyte antigen (HLA) molecules. This encourages the analysis of the entirety of HLA-presented peptides, the so-called immunopeptidome, of malignant and benign cells, in order to identify novel therapeutic targets presented exclusively on malignant cells. In the present study, we aim to investigate the role of previously described immunopeptidome-defined antigen presentation in chronic lymphocytic leukemia (CLL) patients for clinical characteristics and disease outcome. We observed that higher yields of presented total and CLL-exclusive peptides were associated with a more favorable disease course, suggesting efficient immunosurveillance in a subgroup of patients and the possibility of further investigating T cell-based therapeutic approaches for CLL. Chronic lymphocytic leukemia (CLL) is characterized by recurrent relapses and resistance to treatment, even with novel therapeutic approaches. Despite being considered as a disease with low mutational burden and thus poor immunogenic, CLL seems to retain the ability of eliciting specific T cell activation. Accordingly, we recently found non-mutated tumor-associated antigens to play a central role in CLL immunosurveillance. Here, we investigated the association of total and CLL-exclusive HLA class I and HLA class II peptide presentation in the mass spectrometry-defined immunopeptidome of leukemic cells with clinical features and disease outcome of 57 CLL patients. Patients whose CLL cells present a more diverse immunopeptidome experienced fewer relapses. During the follow-up phase of up to 10 years, patients with an HLA class I-restricted presentation of high numbers of total and CLL-exclusive peptides on their malignant cells showed a more favorable disease course with a prolonged progression-free survival (PFS). Overall, our results suggest the existence of an efficient T cell-based immunosurveillance mediated by CLL-associated tumor antigens, supporting ongoing efforts in developing T cell-based immunotherapeutic strategies for CLL. [ABSTRACT FROM AUTHOR]

• Association of different COVID-19 symptoms with SARS-CoV-2-directed immune responses. • Antibody levels are increased with severe fever, headache, and loss of smell and taste. • Decreased T cell responses are associated with severe cough or loss of smell and taste. Besides SARS-CoV-2-directed humoral immune responses, T cell responses are indispensable for effective antiviral immunity. Recent data have shown a correlation between COVID-19 symptoms and humoral immune response, but so far, little is known about the association of SARS-CoV-2-directed T cell responses and disease severity. Herein, we evaluated the prevalence of different clinical COVID-19 symptoms in relation to SARS-CoV-2-directed humoral and cellular immune responses. The severity of eight different symptoms during acute infection were assessed using questionnaires from 193 convalescent individuals and were evaluated in relation to SARS-CoV-2 antibody levels and intensity of SARS-CoV-2-specific T cell responses 2–8 weeks after positive polymerase chain reaction. Although increased IgG serum levels could be associated with severity of most symptoms, no difference in T cell response intensity between different symptom severities was observed for the majority of COVID-19 symptoms. However, when analyzing loss of smell or taste and cough, awareness of more severe symptoms was associated with reduced T cell response intensities. These data suggest that rapid virus clearance mediated by SARS-CoV-2-specific T cells prevents severe symptoms of COVID-19. [ABSTRACT FROM AUTHOR]

Simple Summary: The immunopeptidome represents the entirety of peptides that are presented on the surface of cells on human leukocyte antigen (HLA) molecules and are recognized by the T-cell receptors of CD4+ and CD8+ T-cells. Malignant cells present tumor-associated antigens essential for tumor immune surveillance, which can be targeted by T-cell-based immunotherapy approaches. For ovarian carcinomas, various tumor-associated antigens, such as Mucin-16 and Mesothelin, have been described. The aim of our study is to analyze immunopeptidome-defined tumor antigen presentation in ovarian carcinoma patients in relation to clinical characteristics and disease outcomes to define potential biomarkers. Our work demonstrates the central role of HLA-DR-restricted peptide presentation of the tumor antigen Mesothelin and of CD4+ T-cell responses for tumor immune surveillance, and underlines Mesothelin as a prime target antigen for novel immunotherapeutic approaches for ovarian carcinoma patients. T-cell recognition of HLA-presented antigens is central for the immunological surveillance of malignant disease and key for the development of novel T-cell-based immunotherapy approaches. In recent years, large-scale immunopeptidome studies identified naturally presented tumor-associated antigens for several malignancies. Regarding ovarian carcinoma (OvCa), Mucin-16 (MUC16) and Mesothelin (MSLN) were recently described as the top HLA class I- and HLA class II-presented tumor antigens, respectively. Here, we investigate the role and impact of immunopeptidome-presented tumor antigens on the clinical outcomes of 39 OvCa patients with a follow-up time of up to 50 months after surgery. Patients with a HLA-restricted presentation of high numbers of different MSLN-derived peptides on their tumors exhibited significantly prolonged progression-free (PFS) and overall survival (OS), whereas the presentation of MUC16-derived HLA class I-restricted peptides had no impact. Furthermore, a high HLA-DRB gene expression was associated with increased PFS and OS. In line, in silico prediction revealed that MSLN-derived HLA class II-presented peptides are predominantly presented on HLA-DR allotypes. In conclusion, the correlation of MSLN tumor antigen presentation and HLA-DRB gene expression with prolonged survival indicates a central role of CD4+ T-cell responses for tumor immune surveillance in OvCa, and highlights the importance of immunopeptidome-guided tumor antigen discovery. [ABSTRACT FROM AUTHOR]

Low human leukocyte antigen (HLA)-DR expression might compromise CD4+ T-cell-mediated anti-tumor immunity. Its immunological and clinical significance however remain undefined in non-promyelocytic acute myeloid leukemia (AML). Taking advantage of mass spectrometry-based immunopeptidome analysis of primary AML samples (n = 31), we studied the implications of low HLA-DR expression for antigen presentation and analyzed its association with disease characteristics and survival within a cohort of 399 AML patients. Remarkably, overall HLA-DR/DQ immunopeptidome diversity was preserved in AML with low HLA-DR expression (HLA-DRlow AML) and was associated with a shift in HLA-DR/DQ allotype abundances (HLA-DQ to HLA-DR/DQ ligand ratio 0.36 vs 0.19 in HLA-DRlow and HLA-DRhigh patients, respectively). Consistent with unimpaired antigenicity, survival was similar in HLA-DRlow and HLA-DRhigh patients. Demonstrating for the first time that overall HLA-DR/DQ antigen presentation is preserved in HLA-DRlow AML, our findings provide a rationale for the non-inferior outcome observed in HLA-DRlow AML patients. [ABSTRACT FROM AUTHOR]

Cyclin A1 is a promising antigen for T cell therapy being selectively expressed in high-grade ovarian cancer (OC) and acute myeloid leukemia (AML) stem cells. For adoptive T cell therapy, a single epitope has to be selected, with high affinity to MHC class I and adequate processing and presentation by malignant cells to trigger full activation of specific T cells. In silico prediction with three algorithms indicated 13 peptides of Cyclin A1 9 to 11 amino acids of length to have high affinity to HLA-A*02:01. Ten of them proved to be affine in an HLA stabilization assay using TAP-deficient T2 cells. Their immunogenicity was assessed by repetitive stimulation of CD8+ T cells from two healthy donors with single-peptide-pulsed dendritic cells or monocytes. Intracellular cytokine staining quantified the enrichment of peptide-specific functional T cells. Seven peptides were immunogenic, three of them against both donors. Specific cell lines were cloned and used in killing assays to demonstrate recognition of endogenous Cyclin A1 in the HLA-A*02:01-positive AML cell line THP-1. Immunopeptidome analysis based on direct isolation of HLA-presented peptides by mass spectrometry of primary AML and OC samples identified four naturally presented epitopes of Cyclin A1. The immunopeptidome of HeLa cells transfected with Cyclin A1 and HLA-A*02:01 revealed six Cyclin A1-derived HLA ligands. Epitope p410–420 showed high affinity to HLA-A*02:01 and immunogenicity in both donors. It proved to be naturally presented on primary AML blast and provoked spontaneous functional response of T cells from treatment naïve OC and, therefore, warrants further development for clinical application. [ABSTRACT FROM AUTHOR]

Background: Clear cell renal cell carcinoma (ccRCC) is the dominant subtype of renal cancer. With currently available therapies, cure of advanced and metastatic ccRCC is achieved only in rare cases. Here, we developed a workflow integrating different -omics technologies to identify ccRCC-specific HLA-presented peptides as potential drug targets for ccRCC immunotherapy. Methods: We analyzed HLA-presented peptides by MS-based ligandomics of 55 ccRCC tumors (cohort 1), paired non-tumor renal tissues, and 158 benign tissues from other organs. Pathways enriched in ccRCC compared to its cell type of origin were identified by transcriptome and gene set enrichment analyses in 51 tumor tissues of the same cohort. To retrieve a list of candidate targets with involvement in ccRCC pathogenesis, ccRCC-specific pathway genes were intersected with the source genes of tumor-exclusive peptides. The candidates were validated in an independent cohort from The Cancer Genome Atlas (TCGA KIRC, n = 452). DNA methylation (TCGA KIRC, n = 273), somatic mutations (TCGA KIRC, n = 392), and gene ontology (GO) and correlations with tumor metabolites (cohort 1, n = 30) and immune-oncological markers (cohort 1, n = 37) were analyzed to characterize regulatory and functional involvements. CD8+ T cell priming assays were used to identify immunogenic peptides. The candidate gene EGLN3 was functionally investigated in cell culture. Results: A total of 34,226 HLA class I- and 19,325 class II-presented peptides were identified in ccRCC tissue, of which 443 class I and 203 class II peptides were ccRCC-specific and presented in ≥ 3 tumors. One hundred eighty-five of the 499 corresponding source genes were involved in pathways activated by ccRCC tumors. After validation in the independent cohort from TCGA, 113 final candidate genes remained. Candidates were involved in extracellular matrix organization, hypoxic signaling, immune processes, and others. Nine of the 12 peptides assessed by immunogenicity analysis were able to activate naïve CD8+ T cells, including peptides derived from EGLN3. Functional analysis of EGLN3 revealed possible tumor-promoting functions. Conclusions: Integration of HLA ligandomics, transcriptomics, genetic, and epigenetic data leads to the identification of novel functionally relevant therapeutic targets for ccRCC immunotherapy. Validation of the identified targets is recommended to expand the treatment landscape of ccRCC. [ABSTRACT FROM AUTHOR]

Immunotherapeutic approaches, including allogeneic stem cell transplantation and donor lymphocyte infusion, have significantly improved the prognosis of leukemia patients. Further efforts are now focusing on the development of immunotherapies that are able to target leukemic cells more specifically, comprising monoclonal antibodies, chimeric antigen receptor (CAR) T cells, and dendritic cell- or peptide-based vaccination strategies. One main prerequisite for such antigen-specific approaches is the selection of suitable target structures on leukemic cells. In general, the targets for anti-cancer immunotherapies can be divided into two groups: (1) T-cell epitopes relying on the presentation of peptides via human leukocyte antigen (HLA) molecules and (2) surface structures, which are HLA-independently expressed on cancer cells. This review discusses the most promising tumor antigens as well as the underlying discovery and selection strategies for the development of anti-leukemia immunotherapies. [ABSTRACT FROM AUTHOR]

Direct analysis of HLA-presented antigens by mass spectrometry provides a comprehensive view on the antigenic landscape of different tissues/malignancies and enables the identification of novel, pathophysiologically relevant T-cell epitopes. Here, we present a systematic and comparative study of the HLA class I and II presented, non mutant antigenome of multiple myeloma (MM).Quantification of HLA surface expression revealed elevated HLA molecule counts on malignant plasma cells compared with normal B cells, excluding relevant HLA downregulation in MM. Analyzing the presentation of established myeloma-associated T-cell antigenson the HLA ligandome level, we found a substantial proportion of antigens to be only infrequently presented on primary myelomas or to display suboptimal degrees of myeloma specificity. However, unsupervised analysis of our extensive HLA ligand data set delineated a panel of 58 highly specific myeloma-associated antigens (including multiple myeloma SET domain containing protein) which are characterized by frequent and exclusive presentation on myeloma samples. Functional characterization of these target antigens revealed peptide-specific, preexisting CD81 T-cell responses exclusively in myeloma patients, which is indicative of pathophysiological relevance. Furthermore, in vitro priming experiments revealed that peptide-specific T-cell responses can be induced in response-naive myeloma patients. Together, our results serve to guide antigen selection for T-cell-based immunotherapy of MM. [ABSTRACT FROM AUTHOR]

A correction to this paper has been published: https://doi.org/10.1007/s00262-021-02977-6 [ABSTRACT FROM AUTHOR]