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Article ; Online: SUV39H1 Ablation Enhances Long-term CAR T Function in Solid Tumors.

López-Cobo, Sheila / Fuentealba, Jaime R / Gueguen, Paul / Bonté, Pierre-Emmanuel / Tsalkitzi, Kyriaki / Chacón, Irena / Glauzy, Salomé / Bohineust, Armelle / Biquand, Ariane / Silva, Lisseth / Gouveia, Zelia / Goudot, Christel / Perez, Franck / Saitakis, Michael / Amigorena, Sebastian

Cancer discovery

2023 Volume 14, Issue 1, Page(s) 120–141

Abstract: Failure of adoptive T-cell therapies in patients with cancer is linked to limited T-cell expansion and persistence, even in memory-prone 41BB-(BBz)-based chimeric antigen receptor (CAR) T cells. We show here that BBz-CAR T-cell stem/memory ... ...

Abstract	Failure of adoptive T-cell therapies in patients with cancer is linked to limited T-cell expansion and persistence, even in memory-prone 41BB-(BBz)-based chimeric antigen receptor (CAR) T cells. We show here that BBz-CAR T-cell stem/memory differentiation and persistence can be enhanced through epigenetic manipulation of the histone 3 lysine 9 trimethylation (H3K9me3) pathway. Inactivation of the H3K9 trimethyltransferase SUV39H1 enhances BBz-CAR T cell long-term persistence, protecting mice against tumor relapses and rechallenges in lung and disseminated solid tumor models up to several months after CAR T-cell infusion. Single-cell transcriptomic (single-cell RNA sequencing) and chromatin opening (single-cell assay for transposase accessible chromatin) analyses of tumor-infiltrating CAR T cells show early reprogramming into self-renewing, stemlike populations with decreased expression of dysfunction genes in all T-cell subpopulations. Therefore, epigenetic manipulation of H3K9 methylation by SUV39H1 optimizes the long-term functional persistence of BBz-CAR T cells, limiting relapses, and providing protection against tumor rechallenges. Significance: Limited CAR T-cell expansion and persistence hinders therapeutic responses in solid cancer patients. We show that targeting SUV39H1 histone methyltransferase enhances 41BB-based CAR T-cell long-term protection against tumor relapses and rechallenges by increasing stemness/memory differentiation. This opens a safe path to enhancing adoptive cell therapies for solid tumors. See related article by Jain et al., p. 142. This article is featured in Selected Articles from This Issue, p. 5.
MeSH term(s)	Animals ; Humans ; Mice ; Chromatin ; Immunotherapy, Adoptive ; Methyltransferases/genetics ; Methyltransferases/metabolism ; Neoplasms/genetics ; Neoplasms/therapy ; Receptors, Chimeric Antigen ; Recurrence ; Repressor Proteins/genetics ; Repressor Proteins/metabolism
Chemical Substances	Chromatin ; Methyltransferases (EC 2.1.1.-) ; Receptors, Chimeric Antigen ; Repressor Proteins ; SUV39H1 protein, human (EC 2.1.1.) ; Suv39h1 protein, mouse (EC 2.1.1.)
Language	English
Publishing date	2023-11-07
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2625242-9
ISSN	2159-8290 ; 2159-8274
ISSN (online)	2159-8290
ISSN	2159-8274
DOI	10.1158/2159-8290.CD-22-1350
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Article ; Online: An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles.

Campos-Silva, Carmen / Cáceres-Martell, Yaiza / López-Cobo, Sheila / Rodriguez, María Josefa / Jara, Ricardo / Yáñez-Mó, María / Valés-Gómez, Mar

Methods in molecular biology (Clifton, N.J.)

2021 Volume 2265, Page(s) 323–344

Abstract: Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, ...

Abstract	Most human cells release extracellular vesicles (EVs) of different sizes and composition, containing biomolecules characteristic from the originating tissue. In consequence, when EVs derive from a cancer cell, they also contain tumor antigens. Therefore, isolating and characterizing tumor-derived EVs has attracted great interest as an invaluable source of biomarkers, both for diagnosis and stratification of cancer. In this chapter, we describe a method for flow cytometry assessment of melanoma-derived EVs which are firstly captured onto antibody-coated beads recognizing either a common EV marker, namely, a tetraspanin, or a tumor antigen like the stress-related molecules MICA or PDL1. Then, after staining with a fluorophore-conjugated antibody directed against a different protein present on the EV surface, the EV-bead complex can be visualized in a conventional flow cytometer. The technique allows detection of proteins present on EVs isolated from tissue culture supernatants of melanoma cell lines and, more importantly, directly from plasma.
MeSH term(s)	Cell Line, Tumor ; Extracellular Vesicles/metabolism ; Flow Cytometry ; Humans ; Melanoma/metabolism ; Melanoma/pathology ; Melanoma-Specific Antigens/metabolism
Chemical Substances	Melanoma-Specific Antigens
Language	English
Publishing date	2021-03-11
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-1205-7_24
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Article: Glycosyl-Phosphatidyl-Inositol (GPI)-Anchors and Metalloproteases: Their Roles in the Regulation of Exosome Composition and NKG2D-Mediated Immune Recognition.

López-Cobo, Sheila / Campos-Silva, Carmen / Valés-Gómez, Mar

Frontiers in cell and developmental biology

2016 Volume 4, Page(s) 97

Abstract: Communication within the immune system depends on the release of factors that can travel and transmit information at points distant from the cell that produced them. In general, immune cells use two key strategies that can occur either at the plasma ... ...

Abstract	Communication within the immune system depends on the release of factors that can travel and transmit information at points distant from the cell that produced them. In general, immune cells use two key strategies that can occur either at the plasma membrane or in intracellular compartments to produce such factors, vesicle release and proteolytic cleavage. Release of soluble factors in exosomes, a subset of vesicles that originate from intracellular compartments, depends generally on biochemical and lipid environment features. This physical environment allows proteins to be recruited to membrane microdomains that will be later endocytosed and further released to the extracellular milieu. Cholesterol and sphingolipid rich domains (also known as lipid rafts or detergent-resistant membranes, DRMs) often contribute to exosomes and these membrane regions are rich in proteins modified with Glycosyl-Phosphatidyl-Inositol (GPI) and lipids. For this reason, many palmitoylated and GPI-anchored proteins are preferentially recruited to exosomes. In this review, we analyse the biochemical features involved in the release of NKG2D-ligands as an example of functionally related gene families encoding both transmembrane and GPI-anchored proteins that can be released either by proteolysis or in exosomes, and modulate the intensity of the immune response. The immune receptor NKG2D is present in all human Natural Killer and T cells and plays an important role in the first barrier of defense against tumor and infection. However, tumor cells can evade the immune system by releasing NKG2D-ligands to induce down-regulation of the receptor. Some NKG2D-ligands can be recruited to exosomes and potently modulate receptor expression and immune function, while others are more susceptible to metalloprotease cleavage and are shed as soluble molecules. Strikingly, metalloprotease inhibition is sufficient to drive the accumulation in exosomes of ligands otherwise released by metalloprotease cleavage. In consequence, NKG2D-ligands appear as different entities in different cells, depending on cellular metabolism and biochemical structure, which mediate different intensities of immune modulation. We discuss whether similar mechanisms, depending on an interplay between metalloprotease cleavage and exosome release, could be a more general feature regulating the composition of exosomes released from human cells.
Language	English
Publishing date	2016-09-12
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2016.00097
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Article ; Online: Natural killer (NK) cell-derived extracellular-vesicle shuttled microRNAs control T cell responses.

Dosil, Sara G / Lopez-Cobo, Sheila / Rodriguez-Galan, Ana / Fernandez-Delgado, Irene / Ramirez-Huesca, Marta / Milan-Rois, Paula / Castellanos, Milagros / Somoza, Alvaro / Gómez, Manuel José / Reyburn, Hugh T / Vales-Gomez, Mar / Sánchez Madrid, Francisco / Fernandez-Messina, Lola

eLife

2022 Volume 11

Abstract: Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and ... ...

Abstract	Natural killer (NK) cells recognize and kill target cells undergoing different types of stress. NK cells are also capable of modulating immune responses. In particular, they regulate T cell functions. Small RNA next-generation sequencing of resting and activated human NK cells and their secreted extracellular vesicles (EVs) led to the identification of a specific repertoire of NK-EV-associated microRNAs and their post-transcriptional modifications signature. Several microRNAs of NK-EVs, namely miR-10b-5p, miR-92a-3p, and miR-155-5p, specifically target molecules involved in Th1 responses. NK-EVs promote the downregulation of
MeSH term(s)	Animals ; Extracellular Vesicles/metabolism ; Humans ; Killer Cells, Natural/metabolism ; Mice ; MicroRNAs/genetics ; MicroRNAs/metabolism ; RNA, Messenger/metabolism ; T-Lymphocytes/metabolism
Chemical Substances	MicroRNAs ; RNA, Messenger
Language	English
Publishing date	2022-07-29
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2687154-3
ISSN	2050-084X ; 2050-084X
ISSN (online)	2050-084X
ISSN	2050-084X
DOI	10.7554/eLife.76319
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Article ; Online: CyTOF analysis identifies unusual immune cells in urine of BCG-treated bladder cancer patients.

Castellano, Eva / Samba, Célia / Esteso, Gloria / Simpson, Laura / Vendrame, Elena / García-Cuesta, Eva M / López-Cobo, Sheila / Álvarez-Maestro, Mario / Linares, Ana / Leibar, Asier / Ranganath, Thanmayi / Reyburn, Hugh T / Martínez-Piñeiro, Luis / Blish, Catherine / Valés-Gómez, Mar

Frontiers in immunology

2022 Volume 13, Page(s) 970931

Abstract: High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical ... ...

Abstract	High grade non-muscle-invasive bladder tumours are treated with transurethral resection followed by recurrent intravesical instillations of Bacillus Calmette Guérin (BCG). Although most bladder cancer patients respond well to BCG, there is no clinical parameter predictive of treatment response, and when treatment fails, the prognosis is very poor. Further, a high percentage of NMIBC patients treated with BCG suffer unwanted effects that force them to stop treatment. Thus, early identification of patients in which BCG treatment will fail is really important. Here, to identify early stage non-invasive biomarkers of non-responder patients and patients at risk of abandoning the treatment, we longitudinally analysed the phenotype of cells released into the urine of bladder cancer patients 3-7 days after BCG instillations. Mass cytometry (CyTOF) analyses revealed a large proportion of granulocytes and monocytes, mostly expressing activation markers. A novel population of CD15
MeSH term(s)	Administration, Intravesical ; BCG Vaccine/therapeutic use ; Humans ; Pilot Projects ; Prognosis ; Urinary Bladder Neoplasms/drug therapy ; Urinary Bladder Neoplasms/pathology
Chemical Substances	BCG Vaccine
Language	English
Publishing date	2022-09-15
Publishing country	Switzerland
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2606827-8
ISSN	1664-3224 ; 1664-3224
ISSN (online)	1664-3224
ISSN	1664-3224
DOI	10.3389/fimmu.2022.970931
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Article: Impaired NK cell recognition of vemurafenib-treated melanoma cells is overcome by simultaneous application of histone deacetylase inhibitors.

López-Cobo, Sheila / Pieper, Natalia / Campos-Silva, Carmen / García-Cuesta, Eva M / Reyburn, Hugh T / Paschen, Annette / Valés-Gómez, Mar

Oncoimmunology

2017 Volume 7, Issue 2, Page(s) e1392426

Abstract: Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant ... ...

Abstract	Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant BRAF
Language	English
Publishing date	2017-11-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2645309-5
ISSN	2162-402X ; 2162-4011
ISSN (online)	2162-402X
ISSN	2162-4011
DOI	10.1080/2162402X.2017.1392426
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Article ; Online: Ionomycin Treatment Renders NK Cells Hyporesponsive.

Romera-Cárdenas, Gema / Thomas, L Michael / Lopez-Cobo, Sheila / García-Cuesta, Eva M / Long, Eric O / Reyburn, Hugh T

PloS one

2016 Volume 11, Issue 3, Page(s) e0150998

Abstract: Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and ... ...

Abstract	Natural killer cells are cytotoxic lymphocytes important in immune responses to cancer and multiple pathogens. However, chronic activation of NK cells can induce a hyporesponsive state. The molecular basis of the mechanisms underlying the generation and maintenance of this hyporesponsive condition are unknown, thus an easy and reproducible mechanism able to induce hyporesponsiveness on human NK cells would be very useful to gain understanding of this process. Human NK cells treated with ionomycin lose their ability to degranulate and secrete IFN-γ in response to a variety of stimuli, but IL-2 stimulation can compensate these defects. Apart from reductions in the expression of CD11a/CD18, no great changes were observed in the activating and inhibitory receptors expressed by these NK cells, however their transcriptional signature is different to that described for other hyporesponsive lymphocytes.
MeSH term(s)	Cell Degranulation/drug effects ; Cell Line ; Flow Cytometry ; Humans ; Interferon-gamma/biosynthesis ; Interleukin-2/pharmacology ; Ionomycin/pharmacology ; Killer Cells, Natural/drug effects ; Killer Cells, Natural/immunology ; Transcription, Genetic/drug effects
Chemical Substances	Interleukin-2 ; Ionomycin (56092-81-0) ; Interferon-gamma (82115-62-6)
Language	English
Publishing date	2016
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Intramural ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0150998
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Article ; Online: Immunoassays for scarce tumour-antigens in exosomes: detection of the human NKG2D-Ligand, MICA, in tetraspanin-containing nanovesicles from melanoma.

López-Cobo, Sheila / Campos-Silva, Carmen / Moyano, Amanda / Oliveira-Rodríguez, Myriam / Paschen, Annette / Yáñez-Mó, María / Blanco-López, María Carmen / Valés-Gómez, Mar

Journal of nanobiotechnology

2018 Volume 16, Issue 1, Page(s) 47

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 ... ...

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.
MeSH term(s)	Antigens, Neoplasm/blood ; Cell Line, Tumor ; Exosomes/chemistry ; Histocompatibility Antigens Class I/blood ; Humans ; Immunoassay/methods ; Melanoma/blood ; NK Cell Lectin-Like Receptor Subfamily K ; Nanoparticles/chemistry ; Tetraspanins/blood
Chemical Substances	Antigens, Neoplasm ; Histocompatibility Antigens Class I ; KLRK1 protein, human ; MHC class I-related chain A ; NK Cell Lectin-Like Receptor Subfamily K ; Tetraspanins
Language	English
Publishing date	2018-05-02
Publishing country	England
Document type	Journal Article
ISSN	1477-3155
ISSN (online)	1477-3155
DOI	10.1186/s12951-018-0372-z
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Article ; Online: Transfer of the human NKG2D ligands UL16 binding proteins (ULBP) 1-3 is related to lytic granule release and leads to ligand retransfer and killing of ULBP-recipient natural killer cells.

López-Cobo, Sheila / Romera-Cárdenas, Gema / García-Cuesta, Eva M / Reyburn, Hugh T / Valés-Gómez, Mar

Immunology

2015 Volume 146, Issue 1, Page(s) 70–80

Abstract: After immune interactions, membrane fragments can be transferred between cells. This fast transfer of molecules is transient and shows selectivity for certain proteins; however, the constraints underlying acquisition of a protein are unknown. To ... ...

Abstract	After immune interactions, membrane fragments can be transferred between cells. This fast transfer of molecules is transient and shows selectivity for certain proteins; however, the constraints underlying acquisition of a protein are unknown. To characterize the mechanism and functional consequences of this process in natural killer (NK) cells, we have compared the transfer of different NKG2D ligands. We show that human NKG2D ligands can be acquired by NK cells with different efficiencies. The main findings are that NKG2D ligand transfer is related to immune activation and receptor-ligand interaction and that NK cells acquire these proteins during interactions with target cells that lead to degranulation. Our results further demonstrate that NK cells that have acquired NKG2D ligands can stimulate activation of autologous NK cells. Surprisingly, NK cells can also re-transfer the acquired molecule to autologous effector cells during this immune recognition that leads to their death. These data demonstrate that transfer of molecules occurs as a consequence of immune recognition and imply that this process might play a role in homeostatic tuning-down of the immune response or be used as marker of interaction.
MeSH term(s)	Animals ; CHO Cells ; Cell Degranulation/immunology ; Cell Line ; Cricetinae ; Cricetulus ; Cytotoxicity, Immunologic/immunology ; GPI-Linked Proteins/metabolism ; Glycosylphosphatidylinositols ; Histocompatibility Antigens Class I/immunology ; Humans ; Intercellular Signaling Peptides and Proteins/metabolism ; Intracellular Signaling Peptides and Proteins/metabolism ; Killer Cells, Natural/immunology ; Protein Transport ; Receptors, Natural Killer Cell/immunology
Chemical Substances	GPI-Linked Proteins ; Glycosylphosphatidylinositols ; Histocompatibility Antigens Class I ; Intercellular Signaling Peptides and Proteins ; Intracellular Signaling Peptides and Proteins ; Receptors, Natural Killer Cell ; ULBP1 protein, human ; ULBP2 protein, human ; ULBP3 protein, human
Language	English
Publishing date	2015-09
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	80124-0
ISSN	1365-2567 ; 0019-2805 ; 0953-4954
ISSN (online)	1365-2567
ISSN	0019-2805 ; 0953-4954
DOI	10.1111/imm.12482
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Article: BCG Therapy of Bladder Cancer Stimulates a Prolonged Release of the Chemoattractant CXCL10 (IP10) in Patient Urine.

Ashiru, Omodele / Esteso, Gloria / García-Cuesta, Eva M / Castellano, Eva / Samba, Celia / Escudero-López, Eva / López-Cobo, Sheila / Álvarez-Maestro, Mario / Linares, Ana / Ho, Mei M / Leibar, Asier / Martínez-Piñeiro, Luis / Valés-Gómez, Mar

Cancers

2019 Volume 11, Issue 7

Abstract: Background: ...

Abstract	Background:
Language	English
Publishing date	2019-07-04
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers11070940
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