IFNγ-induced PD-L1 expression is JAK2 but not JAK1 dependent and its inhibition enhances NK-cetuximab mediated ADCC of HNSCC cells

Programmed death ligand 1 (PD-L1) is an immunosuppressive molecule expressed by many cancer types, including a large proportion of head and neck cancers (HNC), and ligation of its receptor, programmed death 1 (PD-1), induces exhaustion of effector T cells. It has been shown that interferon gamma (IFNγ) induces PD-L1 expression in many cancer types including glioblastoma, melanoma, lung and kidney cancer. Importantly, the stimuli and mechanism for PD-L1 upregulation in HNC cells are not well characterized. IFNγ signals through Janus Kinase 1/2 (JAK1/2) heterodimer complex and mediates signal transducer and activator of transcription 1 (STAT1) phosphorylation, leading to type I cytokine expression, upregulation of antigen presentation, and tumor cell recognition by cytolytic T lymphocytes (CTL). We investigated basal PD-L1 expression and the mechanism by which IFNγ signaling upregulates PD-L1 in HNC cells including dependence on JAK/STAT pathway. We observed that IFNγ signaling increased PD-L1 expression in a JAK2 but not JAK1 dependent fashion. In addition, interferon alpha (IFNα), which signals via JAK1/TYK2 did not upregulate PD-L1 expression while still upregulated HLA class I. Specific JAK2 inhibition downregulated NK cell-derived IFNγ induced PD-L1 expression and enhanced cetuximab mediated ADCC. Our data suggest a crucial role for JAK2/STAT1 in IFNγ mediated PD-L1 upregulation. JAK2 inhibition provides a promising strategy to increase tumor cell lysis through maintaining HLA class I while suppressing tumor cell expressed PD-L1 in combination with anti-EGFR cetuximab therapy

Epidermal growth factor receptor and Janus Kinase 2 regulation of programmed death ligand 1 and immunoescape in head and neck cancer

Co-inhibitory immune checkpoint receptors (ICR) are novel targets for cancer immunotherapy. Programmed death ligand 1 (PD-L1), expressed in many cancers, including head and neck cancers (HNC), interacts with its receptor, programmed death 1 (PD-1), resulting in an exhausted phenotype. As yet, the stimuli and pathways that induce PD-L1 expression in tumor cells are not fully understood. Interferon gamma (IFNγ) and the epidermal growth factor receptor (EGFR) utilize Janus kinase 2 (JAK2) as a common signaling node transmitting tumor cell-mediated extrinsic or intrinsic signals, respectively. We investigated the mechanisms by which these factors upregulate PD-L1 and immunosuppressive cytokine expression in HNC cells in the context of EGFR/JAK/STAT pathway activation. We found that wild type overexpressed EGFR significantly correlated with JAK2 and PD-L1 expression. Furthermore, PD-L1 expression was induced in an EGFR- and JAK2-dependent manner, and specific JAK2 inhibition prevented PD-L1 upregulation in HNC, enhancing their immunogenicity. HNC tumors have higher expression of immunosuppressive cytokines including TGFβ, IL-10, VEGF-A and IDO and lower expression of inflammatory cytokines such as IL-12A and IL-17A than controls. EGFR/JAK2 inhibition downregulated secretion of these STAT3-dependent cytokines in vitro, suggesting that targeting the EGFR/JAK2/STAT3 suppressive pathway may reverse tumor immunoescape. This view is supported by in vivo findings where HNC patients unresponsive to cetuximab therapy had significantly higher concentrations of immunosuppressive cytokines. NK cells are crucial for promoting T cell responses against cancer. However, NK cell PD-1 expression remains largely undefined. Cetuximab-activated NK cells constitute the major effector cell subset that lyses tumor targets via antibody dependent cellular cytotoxicity (ADCC). We demonstrate that expression of PD-1 in HNC tumors correlates with NK cell activation markers. HNC patients exhibit higher levels of circulating PD-1+ NK cells, which are further enriched in the tumor. Interestingly, cetuximab treatment increased this frequency in vitro and in vivo. Inhibition of the PD-L1/PD-1 axis increased cetuximab-mediated NK cell activation and cytotoxicity. Collectively, our findings suggest a novel role for JAK2 in EGFR-mediated PD-L1 upregulation and immunosuppressive cytokine secretion. Importantly, combined inhibition of the EGFR and PD-L1/PD-1 axis presents a potential strategy to reverse cetuximab-resistant immune evasion of HNC by enhancing NK cell cytotoxicity

Reversing EGFR Mediated Immunoescape by Targeted Monoclonal Antibody Therapy

Uncontrolled growth is a signature of carcinogenesis, in part mediated by overexpression or overstimulation of growth factor receptors. The epidermal growth factor receptor (EGFR) mediates activation of multiple oncogenic signaling pathways and escape from recognition by the host immune system. We discuss how EGFR signaling downregulates tumor antigen presentation, upregulates suppressive checkpoint receptor ligand programmed death ligand (PD-L1), induces secretion of inhibitory molecules such as transforming growth factor beta (TGFβ) and reprograms the metabolic pathways in cancer cells to upregulate aerobic glycolysis and lactate secretion that ultimately lead to impaired cellular immunity mediated by natural killer (NK) cell and cytotoxic T lymphocytes (CTL). Ultimately, our understanding of EGFR-mediated escape mechanisms has led us to design EGFR-specific monoclonal antibody therapies that not only inhibit tumor cell metabolic changes and intrinsic oncogenic signaling but also activates immune cells that mediate tumor clearance. Importantly, targeted immunotherapy may also benefit from combination with antibodies that target other immunosuppressive pathways such PD-L1 or TGFβ and ultimately enhance clinical efficacy

A rapid and simple method for the determination of psychoactive alkaloids by CE-UV: application to Peganum Harmala seed infusions

The β-carboline alkaloids of the harmala (HAlks) group are compounds widely spread in many natural sources, but found at relatively high levels in some specific plants like Peganum harmala (Syrian rue) or Banisteriopsis caapi. HAlks are a reversible Mono Amino Oxidase type A Inhibitor (MAOI) and, as a consequence, these plants or their extracts can be used to produce psychotropic effects when are combined with psychotropic drugs based on amino groups. Since the occurrence and the levels of the HAlks in natural sources are subject to significant variability, more widespread use is not clinical but recreational or ritual, for example B. caapi is a known part of the Ayahuasca ritual mixture. The lack of simple methods to control the variable levels of these compounds in natural sources restricts the possibilities to dose in strict quantities and, as a consequence, limits its use with pharmacological or clinical purposes. In this work, we present a fast, simple, and robust method of quantifying simultaneously the six HAlksmore frequently found in plants, i.e., harmine, harmaline, harmol, harmalol, harmane, and norharmane, by capillary electrophoresis instruments equipped with the more common detector UV. The method is applied to analyze these HAlks in P. Harmala seeds infusion which is a frequent intake form for these HAlks. The method is validated in three different instruments in order to evaluate the transferability and to compare the performances between them. In this case, harmaline, harmine, and harmol were found in the infusion samples.Laboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA

A high performance system to study the influence of temperature in on-line solid-phase extraction capillary electrophoresis

A novel high performance system to control the temperature of the microcartridge in on-line solid phase extraction capillary electrophoresis (SPE–CE) is introduced. The mini-device consists in a thermostatic bath that fits inside of the cassette of any commercial CE instrument, while its temperature is controlled from an external circuit of liquid connecting three differentwater baths. The circuits are controlled from a switchboard connected to an array of electrovalves that allow to rapidly alternate the water circulation through the mini-thermostatic-bath between temperatures from 5 to 90 ºC. The combination of the mini-device and the forced-air thermostatization system of the commercial CE instrument allows to optimize independently the temperature of the sample loading, the clean-up, the analyte elution and the electrophoretic separation steps. The system is used to study the effect of temperature on the C18-SPE–CE analysis of the opioid peptides, Dynorphin A (Dyn A), Endomorphin1 (END) and Met-enkephalin (MET), in both standard solutions and in spiked plasma samples. Extraction recoveries demonstrated to depend, with a non-monotonous trend, on the microcartridge temperature during the sample loading and became maximum at 60 ºC. Results prove the potential of temperature control to further enhance sensitivity in SPE–CE when analytes are thermally stable.Facultad de Ciencias ExactasLaboratorio de Investigación y Desarrollo de Métodos Analíticos (LIDMA)Centro de Investigación y Desarrollo en Tecnología de Pintura

EGFR-mediated tumor immunoescape: The imbalance between phosphorylated STAT1 and phosphorylated STAT3

The epidermal growth factor receptor (EGFR) supports the escape of malignant cells from immunosurveillance by inhibiting the activation of signal transducer and activator of transcription 1 (STAT1) while promoting that of STAT3. We have recently demonstrated that protein tyrosine phosphatase, non-receptor type 11 (PTNP11, best known as SHP2), a phosphatase that operates downstream of EGFR, is responsible for the dephosphorylation of active STAT1 and for the inhibition of the antigen-processing machinery (APM), hence favoring tumor immunoescape. Thus, EGFR signaling may skew the tumor microenvironment to suppress cellular immune responses

La perspectiva post-cualitativa en la investigación educativa: genealogía, movimientos, posibilidades y tensiones

This article aims to establish the genealogy of the strands that are linked to the so-called post-qualitative approach in research. These strands have emerged as a response to the attempts, since the 1990s, to positivize and objectify qualitative research by applying to it standards and rubrics which seek to define researcher training and the conceptualization and practice of research. This tendency, in the case of the United States where it is acquiring great relevance, is reaffirmed in 2002 when the National Research Council publishes a document that establishes how scientific research in education should be: replicable, generalizable, empirical and preferably experimental. This decision is part of a neoliberal form of governance that seeks to establish a unified vision of what reality should be and the role that research should have in this reality. The post-qualitative approach, which feeds on differentreferents and projects itself in various directions, questions the onto-espistemological-methodological and ethical foundations that drive this normative attempt, at the same time as it avoids ways of doing research that predetermine the open and unpredictable meanings to which processes of inquiry can lead. In doing so, this study proposes other ways of conceptualizing what a research process may become.En este artículo se trata de situar una cierta genealogía de algunos de los movimientos que se vinculan en torno a la denominada perspectiva post-cualitativa en investigación. Estos movimientos han ido surgiendo como respuesta a los intentos, desde la década de los años 90 del pasado siglo, de positivizar, disciplinar y objetivar la investigación cualitativa bajo estándares y rúbricas que fijan los caminos a seguir tanto en la formación de investigadores como en la conceptualización y la práctica de la investigación. Esta tendencia, en el caso de Estados Unidos, que es donde está adquiriendo una mayor relevancia, queda reafirmada cuando en 2002 el NationalResearch Council publica un documento en el que se establece cómo ha de ser la investigación científica en educación: replicable, generalizable, empírica y preferentemente experimental. Decisión que forma parte de una gobernabilidad neoli beral que pretende establecer una visión unificada respecto a lo que ha de ser la realidad y el papel que la investigación ha de jugar en este propósito. Este movimiento, que se nutre de diferentes referentesy que se proyecta en varias direcciones cuestiona con argumentos la fundamentación onto-espistemológica-metodológica y ética que rige este intento normativizador, al tiempo que rehúye la explicitación de modos de hacer investigación que predetermine los sentidos abiertos e imprevisibles de todo proceso de indagación y plantea otras maneras de conceptualizar lo que un proceso de investigación puede llegar a ser

On-line coupling of aptamer affinity solid-phase extraction and immobilized enzyme microreactor capillary electrophoresis-mass spectrometry for the sensitive targeted bottom-up analysis of protein biomarkers

In this paper, we present a fully integrated valve-free method for the sensitive targeted bottom-up analysis of proteins through on-line aptamer affinity solid-phase extraction and immobilized enzyme microreactor capillary electrophoresis-mass spectrometry (AA-SPE-IMER-CE-MS). The method was developed analyzing α-synuclein (α-syn), which is a protein biomarker related to different neurodegenerative disorders, including Parkinson's disease. Under optimized conditions, on-line purification and preconcentration of α-syn, enzymatic digestion, electrophoretic separation, and identification of the tryptic peptides by mass spectrometry was achieved in less than 35 min. The limit of detection was 0.02 μg mL-1 of digested protein (66.7% of coverage, i.e., 8 out of 12 expected tryptic peptides were detected). This value was 125 and 10 times lower than for independent on-line digestion by IMER-CE-MS (2.5 μg mL-1) and on-line preconcentration by AA-SPE-CE-MS (0.2 μg mL-1). The repeatability of AA-SPE-IMER-CE-MS was adequate (at 0.5 μg mL-1,% RSD ranged from 3.7 to 16.9% for peak areas and 3.5 to 7.7% for migration times of the tryptic peptides), and the modified capillary could be reused up to 10 analyses with optimum performance, similarly to IMER-CE-MS. The method was subsequently applied to the analysis of endogenous α-syn from red blood cell lysates. Ten α-syn tryptic peptides were detected (83.3% of coverage), enabling the characterization and localization of post-translational modifications of blood α-syn (i.e., N-terminal acetylation)