Exposure to Melan-A/MART-126-35 tumor epitope specific CD8+T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS)

Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA- dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing

Screening of Human Tumor Antigens for CD4+ T Cell Epitopes by Combination of HLA-Transgenic Mice, Recombinant Adenovirus and Antigen Peptide Libraries

BACKGROUND: As tumor antigen-specific CD4+ T cells can mediate strong therapeutic anti-tumor responses in melanoma patients we set out to establish a comprehensive screening strategy for the identification of tumor-specific CD4+ T cell epitopes suitable for detection, isolation and expansion of tumor-reactive T cells from patients. METHODS AND FINDINGS: To scan the human melanoma differentiation antigens TRP-1 and TRP-2 for HLA-DRB1*0301-restricted CD4+ T cell epitopes we applied the following methodology: Splenocytes of HLA-DRB1*0301-transgenic mice immunized with recombinant adenovirus encoding TRP-1 (Ad5.TRP-1) or TRP-2 (Ad5.TRP-2) were tested for their T cell reactivity against combinatorial TRP-1- and TRP-2-specific peptide libraries. CD4+ T cell epitopes thus identified were validated in the human system by stimulation of peripheral blood mononuclear cells (PBMC) from healthy donors and melanoma patients. Using this strategy we observed that recombinant Ad5 induced strong CD4+ T cell responses against the heterologous tumor antigens. In Ad5.TRP-2-immunized mice CD4+ T cell reactivity was detected against the known HLA-DRB1*0301-restricted TRP-2(60-74) epitope and against the new epitope TRP-2(149-163). Importantly, human T cells specifically recognizing target cells loaded with the TRP-2(149-163)-containing library peptide or infected with Ad5.TRP-2 were obtained from healthy individuals, and short term in vitro stimulation of PBMC revealed the presence of epitope-reactive CD4+ T cells in melanoma patients. Similarly, immunization of mice with Ad5.TRP-1 induced CD4+ T cell responses against TRP-1-derived peptides that turned out to be recognized also by human T cells, resulting in the identification of TRP-1(284-298) as a new HLA-DRB1*0301-restricted CD4+ T cell epitope. CONCLUSIONS: Our screening approach identified new HLA-DRB1*0301-restricted CD4+ T cell epitopes derived from melanoma antigens. This strategy is generally applicable to target antigens of other tumor entities and to different HLA class II molecules even without prior characterization of their peptide binding motives

Immunoassays for scarce tumour-antigens in exosomes: Detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma

Abstract Background Tumour-derived exosomes can be released to serum and provide information on the features of the malignancy, however, in order to perform systematic studies in biological samples, faster diagnostic techniques are needed, especially for detection of low abundance proteins. Most human cancer cells are positive for at least one ligand for the activating immune receptor NKG2D and the presence in plasma of NKG2D-ligands can be associated with prognosis. Methods Using MICA as example of a tumour-derived antigen, endogenously expressed in metastatic melanoma and recruited to exosomes, we have developed two immunocapture-based assays for detection of different epitopes in nanovesicles. Although both techniques, enzyme-linked immunosorbent assay (ELISA) and Lateral flow immunoassays (LFIA) have the same theoretical basis, that is, using capture and detection antibodies for a colorimetric read-out, analysis of exosome-bound proteins poses methodological problems that do not occur when these techniques are used for detection of soluble molecules, due to the presence of multiple epitopes on the vesicle. Results Here we demonstrate that, in ELISA, the signal obtained was directly proportional to the amount of epitopes per exosome. In LFIA, the amount of detection antibody immobilized in Au-nanoparticles needs to be low for efficient detection, otherwise steric hindrance results in lower signal. We describe the conditions for detection of MICA in exosomes and prove, for the first time using both techniques, the co-existence in one vesicle of exosomal markers (the tetraspanins CD9, CD63 and CD81) and an endogenously expressed tumour-derived antigen. The study also reveals that scarce proteins can be used as targets for detection antibody in LFIA with a better result than very abundant proteins and that the conditions can be optimized for detection of the protein in plasma. Conclusions These results open the possibility of analyzing biological samples for the presence of tumour-derived exosomes using high throughput techniques.This work has been supported by Grants from Madrid Regional Government [IMMUNOTHERCAN-CM (S2010/BMD-2326)] and the Spanish Ministry of Economy [SAF2015-69169-R (MINEICO/FEDER) and MAT2017-84959-C2-1-R.; the Network of Excellence for Research in Exosomes, Rediex (MINEICO/FEDER); the Consejería de Economía y Empleo del Principado de Asturias (Plan de Ciencia, Tecnología e Innovación 2013-2017), under the Grant GRUPIN14-022. SL-C was a recipient of a FPU fellowship (MECD), and a travel fellowship from the Spanish Group of Extracellular Vesicles (GEIVEX); CC-S was a recipient of a master’s fellowship from “Postgrado de la Fundación Ramón Areces-UAM

Development of a rapid lateral flow immunoassay test for detection of exosomes previously enriched from cell culture medium and body fluids

Exosomes are cell-secreted nanovesicles (40–200 nm) that represent a rich source of novel biomarkers in the diagnosis and prognosis of certain diseases. Despite the increasingly recognized relevance of these vesicles as biomarkers, their detection has been limited due in part to current technical challenges in the rapid isolation and analysis of exosomes. The complexity of the development of analytical platforms relies on the heterogeneous composition of the exosome membrane. One of the most attractive tests is the inmunochromatographic strips, which allow rapid detection by unskilled operators. We have successfully developed a novel lateral flow immunoassay (LFIA) for the detection of exosomes based on the use of tetraspanins as targets. We have applied this platform for the detection of exosomes purified from different sources: cell culture supernatants, human plasma and urine. As proof of concept, we explored the analytical potential of this LFIA platform to accurately quantify exosomes purified from a human metastatic melanoma cell line. The one-step assay can be completed in 15 min, with a limit of detection of 8.54×105 exosomes/µL when a blend of anti-CD9 and anti-CD81 were selected as capture antibodies and anti-CD63 labelled with gold nanoparticles as detection antibody. Based on our results, this platform could be well suited to be used as a rapid exosome quantification tool, with promising diagnostic applications, bearing in mind that the detection of exosomes from different sources may require adaptation of the analytical settings to their specific composition.FICYT; Ministerio de Educación; Ministerio de Economía y Competitividad; Gobierno Regional de Asturias; Gobierno Regional de Madri

SILAC-based quantification reveals modulation of the immunopeptidome in BRAF and MEK inhibitor sensitive and resistant melanoma cells

BackgroundThe immunopeptidome is constantly monitored by T cells to detect foreign or aberrant HLA peptides. It is highly dynamic and reflects the current cellular state, enabling the immune system to recognize abnormal cellular conditions, such as those present in cancer cells. To precisely determine how changes in cellular processes, such as those induced by drug treatment, affect the immunopeptidome, quantitative immunopeptidomics approaches are essential.MethodsTo meet this need, we developed a pulsed SILAC-based method for quantitative immunopeptidomics. Metabolic labeling with lysine, arginine, and leucine enabled isotopic labeling of nearly all HLA peptides across all allotypes (> 90% on average). We established a data analysis workflow that integrates the de novo sequencing-based tool Peptide-PRISM for comprehensive HLA peptide identification with MaxQuant for accurate quantification.ResultsWe employed this strategy to explore the modulation of the immunopeptidome upon MAPK pathway inhibition (MAPKi) and to investigate alterations associated with early cellular responses to inhibitor treatment and acquired resistance to MAPKi. Our analyses demonstrated significant changes in the immunopeptidome early during MAPKi treatment and in the resistant state. Moreover, we identified putative tumor-specific cryptic HLA peptides linked to these processes that might represent exploitable targets for cancer immunotherapy.ConclusionsWe have developed a new mass spectrometric approach that allowed us to investigate the effects of common MAPK inhibitors on the immunopeptidome of melanoma cells. This finally led to the discovery of new potential targets for cancer immunotherapy

MAPK inhibitors dynamically affect melanoma release of immune NKG2D-ligands, as soluble protein and extracellular vesicle-associated

Metastatic melanoma presents, in many cases, oncogenic mutations in BRAF, a MAPK involved in proliferation of tumour cells. BRAF inhibitors, used as therapy in patients with these mutations, often lead to tumour resistance and, thus, the use of MEK inhibitors was introduced in clinics. BRAFi/MEKi, a combination that has modestly increased overall survival in patients, has been proven to differentially affect immune ligands, such as NKG2D-ligands, in drug-sensitive vs. drug-resistant cells. However, the fact that NKG2D-ligands can be released as soluble molecules or in extracellular vesicles represents an additional level of complexity that has not been explored. Here we demonstrate that inhibition of MAPK using MEKi, and the combination of BRAFi with MEKi in vitro, modulates NKG2D-ligands in BRAF-mutant and WT melanoma cells, together with other NK activating ligands. These observations reinforce a role of the immune system in the generation of resistance to directed therapies and support the potential benefit of MAPK inhibition in combination with immunotherapies. Both soluble and EV-associated NKG2D-ligands, generally decreased in BRAF-mutant melanoma cell supernatants after MAPKi in vitro, replicating cell surface expression. Because potential NKG2D-ligand fluctuation during MAPKi treatment could have different consequences for the immune response, a pilot study to measure NKG2D-ligand variation in plasma or serum from metastatic melanoma patients, at different time points during MAPKi treatment, was performed. Not all NKG2D-ligands were equally detected. Further, EV detection did not parallel soluble protein. Altogether, our data confirm the heterogeneity between melanoma lesions, and suggest testing several NKG2D-ligands and other melanoma antigens in serum, both as soluble or vesicle-released proteins, to help classifying immune competence of patients

Persister state-directed transitioning and vulnerability in melanoma

©2022. This manuscript version is made available under the CC-BY 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/ This document is the Published Manuscript version of a Published Work that appeared in final form in Nature Communications. To access the final edited and published work see https://doi.org/10.1038/s41467-022-30641-9Item en revisión. Pendiente de cumplimentar metadatos.Melanoma is a highly plastic tumor characterized by dynamic interconversion of different cell identities depending on the biological context. Melanoma cells with high expression of the H3K4 demethylase KDM5B (JARID1B) rest in a slow-cycling, yet reversible persister state. Over time, KDM5Bhigh cells can promote rapid tumor repopulation with equilibrated KDM5B expression heterogeneity. The cellular identity of KDM5Bhigh persister cells has not been studied so far, missing an important cell state-directed treatment opportunity in melanoma. Here, we have established a doxycycline-titratable system for genetic induction of permanent intratumor expression of KDM5B and screened for chemical agents that phenocopy this effect. Transcriptional profiling and cell functional assays confirmed that the dihydropyridine 2-phenoxyethyl 4-(2-fluorophenyl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexa-hydro-quinoline-3-carboxylate (termed Cpd1) supports high KDM5B expression and directs melanoma cells towards differentiation along the melanocytic lineage and to cell cycle-arrest. The high KDM5B state additionally prevents cell proliferation through negative regulation of cytokinetic abscission. Moreover, treatment with Cpd1 promoted the expression of the melanocyte-specific tyrosinase gene specifically sensitizing melanoma cells for the tyrosinase-processed antifolate prodrug 3-O-(3,4,5-trimethoxybenzoyl)-(-)-epicatechin (TMECG). In summary, our study provides proof-of-concept for a dual hit strategy in melanoma, in which persister state-directed transitioning limits tumor plasticity and primes melanoma cells towards lineage-specific eliminatio

Clinical and genetic characteristics of BAP1-mutated non-uveal and uveal melanoma

BackgroundScreening for gene mutations has become routine clinical practice across numerous tumor entities, including melanoma. BAP1 gene mutations have been identified in various tumor types and acknowledged as a critical event in metastatic uveal melanoma, but their role in non-uveal melanoma remains inadequately characterized.MethodsA retrospective analysis of all melanomas sequenced in our department from 2014–2022 (n=2650) was conducted to identify BAP1 mutated samples. Assessment of clinical and genetic characteristics was performed as well as correlations with treatment outcome.ResultsBAP1 mutations were identified in 129 cases and distributed across the entire gene without any apparent hot spots. Inactivating BAP1 mutations were more prevalent in uveal (55%) compared to non-uveal (17%) melanomas. Non-uveal BAP1 mutated melanomas frequently exhibited UV-signature mutations and had a significantly higher mutation load than uveal melanomas. GNAQ and GNA11 mutations were common in uveal melanomas, while MAP-Kinase mutations were frequent in non-uveal melanomas with NF1, BRAF V600 and NRAS Q61 mutations occurring in decreasing frequency, consistent with a strong UV association. Survival outcomes did not differ among non-uveal melanoma patients based on whether they received targeted or immune checkpoint therapy, or if their tumors harbored inactivating BAP1 mutations.ConclusionIn contrast to uveal melanomas, where BAP1 mutations serve as a significant prognostic indicator of an unfavorable outcome, BAP1 mutations in non-uveal melanomas are primarily considered passenger mutations and do not appear to be relevant from a prognostic or therapeutic perspective