NKG2D-CAR memory T cells target pediatric T-cell acute lymphoblastic leukemia in vitro and in vivo but fail to eliminate leukemia initiating cells.

Introduction: Refractory/relapsed pediatric acute leukemia are still clinically challenging and new therapeutic strategies are needed. Interactions between Natural Killer Group 2D (NKG2D) receptor, expressed in cytotoxic immune cells, and its ligands (NKG2DL), which are upregulated in leukemic blasts, are important for anti-leukemia immunosurveillance. Nevertheless, leukemia cells may develop immunoescape strategies as NKG2DL shedding and/or downregulation. Methods: In this report, we analyzed the anti-leukemia activity of NKG2D chimeric antigen receptor (CAR) redirected memory (CD45RA-) T cells in vitro and in a murine model of T-cell acute lymphoblastic leukemia (T-ALL). We also explored in vitro how soluble NKG2DL (sNKG2DL) affected NKG2D-CAR T cells' cytotoxicity and the impact of NKG2D-CAR T cells on Jurkat cells gene expression and in vivo functionality. Results: In vitro, we found NKG2D-CAR T cells targeted leukemia cells and showed resistance to the immunosuppressive effects exerted by sNKG2DL. In vivo, NKG2D-CAR T cells controlled T cell leukemia burden and increased survival of the treated mice but failed to cure the animals. After CAR T cell treatment, Jurkat cells upregulated genes related to proliferation, survival and stemness, and in vivo, they exhibited functional properties of leukemia initiating cells. Discussion: The data here presented suggest, that, in combination with other therapeutic approaches, NKG2D-CAR T cells could be a novel treatment for pediatric T-ALL.

NKG2D Ligands in Liquid Biopsy: The Importance of Soluble and Vesicle-Bound Proteins for Immune Modulation.

The identification of biomarkers allowing diagnostics, prognostics and patient classification is still a challenge in oncological research for patient management. Improvements in patient survival achieved with immunotherapies substantiate that biomarker studies rely not only on cellular pathways contributing to the pathology, but also on the immune competence of the patient. If these immune molecules can be studied in a non-invasive manner, the benefit for patients and clinicians is obvious. The immune receptor Natural Killer Group 2 Member D (NKG2D) represents one of the main systems involved in direct recognition of tumor cells by effector lymphocytes (T and Natural Killer cells), and in immune evasion. The biology of NKG2D and its ligands comprises a complex network of cellular pathways leading to the expression of these tumor-associated ligands on the cell surface or to their release either as soluble proteins, or in extracellular vesicles that potently inhibit NKG2D-mediated responses. Increased levels of NKG2D-ligands in patient serum correlate with tumor progression and poor prognosis; however, most studies did not test the biochemical form of these molecules. Here we review the biology of the NKG2D receptor and ligands, their role in cancer and in patient response to immunotherapies, as well as the changes provoked in this system by non-immune cancer therapies. Further, we discuss the use of NKG2D-L in liquid biopsy, including methods to analyse vesicle-associated proteins. We propose that the evaluation in cancer patients of the whole NKG2D system can provide crucial information about patient immune competence and risk of tumor progression.

ALK-Fusion Transcripts Can Be Detected in Extracellular Vesicles (EVs) from Nonsmall Cell Lung Cancer Cell Lines and Patient Plasma: Toward EV-Based Noninvasive Testing.

BACKGROUND: ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection. METHODS: EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation. RESULTS: Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions. CONCLUSIONS: ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.

A simple immunoassay for extracellular vesicle liquid biopsy in microliters of non-processed plasma.

BACKGROUND: Extracellular vesicles (EVs), released by most cell types, provide an excellent source of biomarkers in biological fluids. However, in order to perform validation studies and screenings of patient samples, it is still necessary to develop general techniques permitting rapid handling of small amounts of biological samples from large numbers of donors. RESULTS: Here we describe a method that, using just a few microliters of patient's plasma, identifies tumour markers exposed on EVs. Studying physico-chemical properties of EVs in solution, we demonstrate that they behave as stable colloidal suspensions and therefore, in immunocapture assays, many of them are unable to interact with a stationary functionalised surface. Using flocculation methods, like those used to destabilize colloids, we demonstrate that cationic polymers increase EV ζ-potential, diameter, and sedimentation coefficient and thus, allow a more efficient capture on antibody-coated surfaces by both ELISA and bead-assisted flow cytometry. These findings led to optimization of a protocol in microtiter plates allowing effective immunocapture of EVs, directly in plasma without previous ultracentrifugation or other EV enrichment. The method, easily adaptable to any laboratory, has been validated using plasma from lung cancer patients in which the epithelial cell marker EpCAM has been detected on EVs. CONCLUSIONS: This optimized high throughput, easy to automate, technology allows screening of large numbers of patients to phenotype tumour markers in circulating EVs, breaking barriers for the validation of proposed EV biomarkers and the discovery of new ones.

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.

Single-reaction multi-antigen serological test for comprehensive evaluation of SARS-CoV-2 patients by flow cytometry.

Here, we describe a new, simple, highly multiplexed serological test that generates a more complete picture of seroconversion than single antigen-based assays. Flow cytometry is used to detect multiple Ig isotypes binding to four SARS-CoV-2 antigens: the Spike glycoprotein, its RBD fragment (the main target for neutralizing antibodies), the nucleocapsid protein, and the main cysteine-like protease in a single reaction. Until now, most diagnostic serological tests measured antibodies to only one antigen and in some laboratory-confirmed patients no SARS-CoV-2-specific antibodies could be detected. Our data reveal that while most patients respond against all the viral antigens tested, others show a marked bias to make antibodies against either proteins exposed on the viral particle or those released after cellular infection. With this assay, it was possible to discriminate between patients and healthy controls with 100% confidence. Analysing the response of multiple Ig isotypes to the four antigens in combination may also help to establish a correlation with the severity degree of disease. A more detailed description of the immune responses of different patients to SARS-CoV-2 virus might provide insight into the wide array of clinical presentations of COVID-19.

An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles.

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.

High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry.

Extracellular vesicles (EVs) provide an invaluable tool to analyse physiological processes because they transport, in biological fluids, biomolecules secreted from diverse tissues of an individual. EV biomarker detection requires highly sensitive techniques able to identify individual molecules. However, the lack of widespread, affordable methodologies for high-throughput EV analyses means that studies on biomarkers have not been done in large patient cohorts. To develop tools for EV analysis in biological samples, we evaluated here the critical parameters to optimise an assay based on immunocapture of EVs followed by flow cytometry. We describe a straightforward method for EV detection using general EV markers like the tetraspanins CD9, CD63 and CD81, that allowed highly sensitive detection of urinary EVs without prior enrichment. In proof-of-concept experiments, an epithelial marker enriched in carcinoma cells, EpCAM, was identified in EVs from cell lines and directly in urine samples. However, whereas EVs isolated from 5-10 ml of urine were required for western blot detection of EpCAM, only 500 µl of urine were sufficient to visualise EpCAM expression by flow cytometry. This method has the potential to allow any laboratory with access to conventional flow cytometry to identify surface markers on EVs, even non-abundant proteins, using minimally processed biological samples.

Exosome beads array for multiplexed phenotyping in cancer.

Exosomes are small extracellular vesicles (EV) released from all cells that differ from others EV in their cellular origin, abundance and biogenesis. These different types of extracellular vesicles are recognized as potential markers of human diseases, including cancer and, in recent years, there has been an important advance in the molecular characterization of exosomes from different types of cancer. In particular, due to their presence and stability in most body fluids and the similarity of their content with tumor cells, exosomes have great potential as non-invasive biomarkers for liquid biopsy. Nevertheless, the use of exosomes for diagnostic purposes has been limited by the lack of reproducible methods. Flow cytometry is a technique well adapted for a reproducible analysis of clinical samples. However, conventional flow cytometers do not allow the detection of particles <300 nm based on forward scattered light (FSC), and therefore do not allow the direct detection of exosomes. To overcome this limitation, the use of microsphere bead-based flow cytometry assays is proposed, which, together with an adequate selection of markers, would contribute to making liquid biopsy based on exosomes a reality. SIGNIFICANCE.

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

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.

Impaired NK cell recognition of vemurafenib-treated melanoma cells is overcome by simultaneous application of histone deacetylase inhibitors.

Therapy of metastatic melanoma advanced recently with the clinical implementation of signalling pathway inhibitors, such as vemurafenib, specifically targeting mutant BRAFV600E. In general, patients experience remarkable clinical responses under BRAF inhibitor (BRAFi) treatment but eventually progress within 6-8 months due to resistance development. Responding metastases show an increased immune cell infiltrate, including also NK cells, that, however, is no longer detectable in BRAFi-resistant lesions, suggesting NK cell activity should be exploited to prevent disease progression. Here, we examined the effects of BRAFi on the expression of ligands targeting activating NK cells receptors immediately after treatment onset, prior to resistance development. We demonstrate that BRAFV600E mutant melanoma cells cultured in the presence of vemurafenib, strongly decreased surface expression of ligands for NK activating receptors including the NKG2D-ligand, MICA, and the DNAM-1 ligand, CD155, and became significantly less susceptible to NK cell attack. NKG2D-ligand protein downregulation was due to a significant decrease in mRNA levels, already detectable 24 h after drug treatment. Interestingly, vemurafenib-induced MICA downregulation could be counteracted by treatment of melanoma cells with the histone deacetylase (HDAC) inhibitor (HDACi) sodium butyrate, that also upregulated the DNAM1-ligand, Nectin-2. HDACi treatment enhanced surface expression of NKG2D-ligands in the presence of BRAFi, accompanied by recovery of NK cell recognition, but only upon simultaneous drug application. These results suggest that co-administration of BRAFi and HDAC inhibitors as well as having direct effects on melanoma cell survival, could also synergise to improve NK cell recognition and avoid tumour immune evasion.

La expresión del complejo de síntesis de coenzima Q es regulada durante el envejecimiento / Regulation of the expression of coenzyme Q-synthesis complex during ageing

Introducción. El coenzima Q es un componente esencial para la actividad de la cadena de transporte de electrones mitocondrial. En su síntesis están implicados, al menos, 10 proteínas diferentes que conforman un complejo. Nuestro objetivo ha sido el de determinar la evolución de la expresión de los diferentes genes implicados en la síntesis de coenzima Q durante el envejecimiento en ratones. Material y métodos. El ARN mensajero (ARNm) de diferentes órganos (cerebro, hígado, riñón y músculo) de ratones jóvenes (8 meses), maduros (18 meses) y viejos (24 meses) fue extraído utilizando Trizol y analizado por PCR a tiempo real (qPCR) utilizando sondas específicas para los diferentes genes COQ que codifican para los miembros del complejo de síntesis del coenzima Q. Resultados. El hígado fue el órgano que presentó mayores cambios en cuanto a la expresión de ARNm respecto a la edad, afectando tanto a la amplitud de las variaciones como en la significatividad del cambio. En la mayoría de los genes, los niveles de ARNm fueron mayores en los animales maduros que en los jóvenes. Cuando comparamos los niveles de ARNm de los animales jóvenes y viejos solo se encontraron pequeñas reducciones de expresión. El riñón presentó un patrón similar al hígado en cuanto a la evolución de la expresión, aunque con menores incrementos en los animales maduros que los observados en el hígado. Cerebro y músculo esquelético presentaron las menores variaciones de expresión, siendo el músculo el que menores cambios presentó, aunque se observó un patrón similar al encontrado en hígado y riñón, con ligeros incrementos en animales maduros. Discusión. Nuestros resultados indican que la edad es un factor importante a tener en cuenta en el análisis de la expresión de los genes COQ. Además, la expresión de estos genes depende del órgano estudiado. Teniendo en cuenta la importancia del coenzima Q en el metabolismo celular y en el envejecimiento, es obligado un mayor estudio de la regulación génica de su maquinaria de síntesis (AU)

Introduction. Coenzyme Q is an essential component in the activity of the mitochondrial electron transport chain. Its synthesis involves, at least, a complex of ten different proteins. In this study, an attempt is made to determine the evolution of the expression of the genes involved in coenzyme Q synthesis during mouse ageing. Material and methods. The messenger RNA (mRNA) of different organs, such as brain, liver, kidney and skeletal muscle from young (8 months), mature (18 months), and old (24 months) mice was extracted by using Trizol and was then analysed by real time PCR (qPCR) using specific primers for all the known components of the coenzyme Q-synthesis complex (COQ genes). Results. Liver showed the highest age-dependent changes in mRNA levels of the different components of Q-synthesis complex, affecting the extent of the variation as well as the significance of the change. In most of the cases, mRNA levels of the different components were higher in mature animals compared to young and old animals. When mRNAs of young and old animals were compared, only minor reductions of mRNA levels were found. Kidney showed a pattern similar to that found in liver as regards the changes in expression, although with lower increases in mature animals than those observed in the liver. Brain and skeletal muscle showed low variations, with muscle being the tissue with less changes, although a pattern similar to that found in liver and kidney was found, with slight increases in mature animals. Discussion. The results of this study indicate that ageing is an important factor affecting COQ gene expression, but its effect depends on the organ, and that mature animals show higher levels of mRNA than young and old animals. Taken into consideration the importance of coenzyme Q in cell metabolism and ageing, a more detailed study is needed to understand the gene regulation of the coenzyme Q-synthesis mechanisms during ageing (AU)

[Regulation of the expression of coenzyme Q-synthesis complex during ageing]. / La expresión del complejo de síntesis de coenzima Q es regulada durante el envejecimiento.

INTRODUCTION: Coenzyme Q is an essential component in the activity of the mitochondrial electron transport chain. Its synthesis involves, at least, a complex of ten different proteins. In this study, an attempt is made to determine the evolution of the expression of the genes involved in coenzyme Q synthesis during mouse ageing. MATERIAL AND METHODS: The messenger RNA (mRNA) of different organs, such as brain, liver, kidney and skeletal muscle from young (8 months), mature (18 months), and old (24 months) mice was extracted by using Trizol and was then analysed by real time PCR (qPCR) using specific primers for all the known components of the coenzyme Q-synthesis complex (COQ genes). RESULTS: Liver showed the highest age-dependent changes in mRNA levels of the different components of Q-synthesis complex, affecting the extent of the variation as well as the significance of the change. In most of the cases, mRNA levels of the different components were higher in mature animals compared to young and old animals. When mRNAs of young and old animals were compared, only minor reductions of mRNA levels were found. Kidney showed a pattern similar to that found in liver as regards the changes in expression, although with lower increases in mature animals than those observed in the liver. Brain and skeletal muscle showed low variations, with muscle being the tissue with less changes, although a pattern similar to that found in liver and kidney was found, with slight increases in mature animals. DISCUSSION: The results of this study indicate that ageing is an important factor affecting COQ gene expression, but its effect depends on the organ, and that mature animals show higher levels of mRNA than young and old animals. Taken into consideration the importance of coenzyme Q in cell metabolism and ageing, a more detailed study is needed to understand the gene regulation of the coenzyme Q-synthesis mechanisms during ageing.

Glycosyl-Phosphatidyl-Inositol (GPI)-Anchors and Metalloproteases: Their Roles in the Regulation of Exosome Composition and NKG2D-Mediated Immune Recognition.

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.