Publications by authors named "Nicolás Gonzalo Núñez"

21 Publications

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NASH limits anti-tumour surveillance in immunotherapy-treated HCC.

Authors:
Dominik Pfister Nicolás Gonzalo Núñez Roser Pinyol Olivier Govaere Matthias Pinter Marta Szydlowska Revant Gupta Mengjie Qiu Aleksandra Deczkowska Assaf Weiner Florian Müller Ankit Sinha Ekaterina Friebel Thomas Engleitner Daniela Lenggenhager Anja Moncsek Danijela Heide Kristin Stirm Jan Kosla Eleni Kotsiliti Valentina Leone Michael Dudek Suhail Yousuf Donato Inverso Indrabahadur Singh Ana Teijeiro Florian Castet Carla Montironi Philipp K Haber Dina Tiniakos Pierre Bedossa Simon Cockell Ramy Younes Michele Vacca Fabio Marra Jörn M Schattenberg Michael Allison Elisabetta Bugianesi Vlad Ratziu Tiziana Pressiani Antonio D'Alessio Nicola Personeni Lorenza Rimassa Ann K Daly Bernhard Scheiner Katharina Pomej Martha M Kirstein Arndt Vogel Markus Peck-Radosavljevic Florian Hucke Fabian Finkelmeier Oliver Waidmann Jörg Trojan Kornelius Schulze Henning Wege Sandra Koch Arndt Weinmann Marco Bueter Fabian Rössler Alexander Siebenhüner Sara De Dosso Jan-Philipp Mallm Viktor Umansky Manfred Jugold Tom Luedde Andrea Schietinger Peter Schirmacher Brinda Emu Hellmut G Augustin Adrian Billeter Beat Müller-Stich Hiroto Kikuchi Dan G Duda Fabian Kütting Dirk-Thomas Waldschmidt Matthias Philip Ebert Nuh Rahbari Henrik E Mei Axel Ronald Schulz Marc Ringelhan Nisar Malek Stephan Spahn Michael Bitzer Marina Ruiz de Galarreta Amaia Lujambio Jean-Francois Dufour Thomas U Marron Ahmed Kaseb Masatoshi Kudo Yi-Hsiang Huang Nabil Djouder Katharina Wolter Lars Zender Parice N Marche Thomas Decaens David J Pinato Roland Rad Joachim C Mertens Achim Weber Kristian Unger Felix Meissner Susanne Roth Zuzana Macek Jilkova Manfred Claassen Quentin M Anstee Ido Amit Percy Knolle Burkhard Becher Josep M Llovet Mathias Heikenwalder

Nature 2021 Mar 24. Epub 2021 Mar 24.

Division of Chronic Inflammation and Cancer, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Hepatocellular carcinoma (HCC) can have viral or non-viral causes. Non-alcoholic steatohepatitis (NASH) is an important driver of HCC. Immunotherapy has been approved for treating HCC, but biomarker-based stratification of patients for optimal response to therapy is an unmet need. Here we report the progressive accumulation of exhausted, unconventionally activated CD8PD1 T cells in NASH-affected livers. In preclinical models of NASH-induced HCC, therapeutic immunotherapy targeted at programmed death-1 (PD1) expanded activated CD8PD1 T cells within tumours but did not lead to tumour regression, which indicates that tumour immune surveillance was impaired. When given prophylactically, anti-PD1 treatment led to an increase in the incidence of NASH-HCC and in the number and size of tumour nodules, which correlated with increased hepatic CD8PD1CXCR6, TOX, and TNF T cells. The increase in HCC triggered by anti-PD1 treatment was prevented by depletion of CD8 T cells or TNF neutralization, suggesting that CD8 T cells help to induce NASH-HCC, rather than invigorating or executing immune surveillance. We found similar phenotypic and functional profiles in hepatic CD8PD1 T cells from humans with NAFLD or NASH. A meta-analysis of three randomized phase III clinical trials that tested inhibitors of PDL1 (programmed death-ligand 1) or PD1 in more than 1,600 patients with advanced HCC revealed that immune therapy did not improve survival in patients with non-viral HCC. In two additional cohorts, patients with NASH-driven HCC who received anti-PD1 or anti-PDL1 treatment showed reduced overall survival compared to patients with other aetiologies. Collectively, these data show that non-viral HCC, and particularly NASH-HCC, might be less responsive to immunotherapy, probably owing to NASH-related aberrant T cell activation causing tissue damage that leads to impaired immune surveillance. Our data provide a rationale for stratification of patients with HCC according to underlying aetiology in studies of immunotherapy as a primary or adjuvant treatment.
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http://dx.doi.org/10.1038/s41586-021-03362-0DOI Listing
March 2021

Oxidation inhibits autophagy protein deconjugation from phagosomes to sustain MHC class II restricted antigen presentation.

Nat Commun 2021 03 8;12(1):1508. Epub 2021 Mar 8.

Viral Immunobiology, Institute of Experimental Immunology, University of Zürich, Zürich, Switzerland.

LC3-associated phagocytosis (LAP) contributes to a wide range of cellular processes and notably to immunity. The stabilization of phagosomes by the macroautophagy machinery in human macrophages can maintain antigen presentation on MHC class II molecules. However, the molecular mechanisms involved in the formation and maturation of the resulting LAPosomes are not completely understood. Here, we show that reactive oxygen species (ROS) produced by NADPH oxidase 2 (NOX2) stabilize LAPosomes by inhibiting LC3 deconjugation from the LAPosome cytosolic surface. NOX2 residing in the LAPosome membrane generates ROS to cause oxidative inactivation of the protease ATG4B, which otherwise releases LC3B from LAPosomes. An oxidation-insensitive ATG4B mutant compromises LAP and thereby impedes sustained MHC class II presentation of exogenous Candida albicans antigens. Redox regulation of ATG4B is thereby an important mechanism for maintaining LC3 decoration of LAPosomes to support antigen processing for MHC class II presentation.
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http://dx.doi.org/10.1038/s41467-021-21829-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940406PMC
March 2021

IL-17 controls central nervous system autoimmunity through the intestinal microbiome.

Sci Immunol 2021 Feb;6(56)

Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany.

Interleukin-17A- (IL-17A) and IL-17F-producing CD4 T helper cells (T17 cells) are implicated in the development of chronic inflammatory diseases, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). T17 cells also orchestrate leukocyte invasion of the central nervous system (CNS) and subsequent tissue damage. However, the role of IL-17A and IL-17F as effector cytokines is still confused with the encephalitogenic function of the cells that produce these cytokines, namely, T17 cells, fueling a long-standing debate in the neuroimmunology field. Here, we demonstrated that mice deficient for IL-17A/F lose their susceptibility to EAE, which correlated with an altered composition of their gut microbiota. However, loss of IL-17A/F in T cells did not diminish their encephalitogenic capacity. Reconstitution of a wild-type-like intestinal microbiota or reintroduction of IL-17A specifically into the gut epithelium of IL-17A/F-deficient mice reestablished their susceptibility to EAE. Thus, our data demonstrated that IL-17A and IL-17F are not encephalitogenic mediators but rather modulators of intestinal homeostasis that indirectly alter CNS-directed autoimmunity.
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http://dx.doi.org/10.1126/sciimmunol.aaz6563DOI Listing
February 2021

Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients.

Nat Commun 2020 06 29;11(1):3272. Epub 2020 Jun 29.

Institut Curie, PSL Research University, INSERM U932, F-75005, Paris, France.

Tumor-draining lymph node (TDLN) invasion by metastatic cells in breast cancer correlates with poor prognosis and is associated with local immunosuppression, which can be partly mediated by regulatory T cells (Tregs). Here, we study Tregs from matched tumor-invaded and non-invaded TDLNs, and breast tumors. We observe that Treg frequencies increase with nodal invasion, and that Tregs express higher levels of co-inhibitory/stimulatory receptors than effector cells. Also, while Tregs show conserved suppressive function in TDLN and tumor, conventional T cells (Tconvs) in TDLNs proliferate and produce Th1-inflammatory cytokines, but are dysfunctional in the tumor. We describe a common transcriptomic signature shared by Tregs from tumors and nodes, including CD80, which is significantly associated with poor patient survival. TCR RNA-sequencing analysis indicates trafficking between TDLNs and tumors and ongoing Tconv/Treg conversion. Overall, TDLN Tregs are functional and express a distinct pattern of druggable co-receptors, highlighting their potential as targets for cancer immunotherapy.
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http://dx.doi.org/10.1038/s41467-020-17046-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324591PMC
June 2020

Single-Cell Mapping of Human Brain Cancer Reveals Tumor-Specific Instruction of Tissue-Invading Leukocytes.

Cell 2020 06 28;181(7):1626-1642.e20. Epub 2020 May 28.

Institute of Experimental Immunology, University of Zurich, Zurich 8057, Switzerland. Electronic address:

Brain malignancies can either originate from within the CNS (gliomas) or invade from other locations in the body (metastases). A highly immunosuppressive tumor microenvironment (TME) influences brain tumor outgrowth. Whether the TME is predominantly shaped by the CNS micromilieu or by the malignancy itself is unknown, as is the diversity, origin, and function of CNS tumor-associated macrophages (TAMs). Here, we have mapped the leukocyte landscape of brain tumors using high-dimensional single-cell profiling (CyTOF). The heterogeneous composition of tissue-resident and invading immune cells within the TME alone permitted a clear distinction between gliomas and brain metastases (BrM). The glioma TME presented predominantly with tissue-resident, reactive microglia, whereas tissue-invading leukocytes accumulated in BrM. Tissue-invading TAMs showed a distinctive signature trajectory, revealing tumor-driven instruction along with contrasting lymphocyte activation and exhaustion. Defining the specific immunological signature of brain tumors can facilitate the rational design of targeted immunotherapy strategies.
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http://dx.doi.org/10.1016/j.cell.2020.04.055DOI Listing
June 2020

Tumor endothelial cell up-regulation of IDO1 is an immunosuppressive feed-back mechanism that reduces the response to CD40-stimulating immunotherapy.

Oncoimmunology 2020 9;9(1):1730538. Epub 2020 Mar 9.

Department of Immunology, Genetics and Pathology, Science for Life Laboratory, The Rudbeck Laboratory, Uppsala University, Uppsala, Sweden.

CD40-stimulating immunotherapy can elicit potent anti-tumor responses by activating dendritic cells and enhancing T-cell priming. Tumor vessels orchestrate T-cell recruitment during immune response, but the effect of CD40-stimulating immunotherapy on tumor endothelial cells has not been evaluated. Here, we have investigated how tumor endothelial cells transcriptionally respond to CD40-stimulating immunotherapy by isolating tumor endothelial cells from agonistic CD40 mAb- or isotype-treated mice bearing B16-F10 melanoma, and performing RNA-sequencing. Gene set enrichment analysis revealed that agonistic CD40 mAb therapy increased interferon (IFN)-related responses in tumor endothelial cells, including up-regulation of the immunosuppressive enzyme Indoleamine 2, 3-Dioxygenase 1 (IDO1). IDO1 was predominantly expressed in endothelial cells within the tumor microenvironment, and its expression in tumor endothelium was positively correlated to T-cell infiltration and to increased intratumoral expression of IFNγ. , endothelial cells up-regulated IDO1 in response to T-cell-derived IFNγ, but not in response to CD40-stimulation. Combining agonistic CD40 mAb therapy with the IDO1 inhibitor epacadostat delayed tumor growth in B16-F10 melanoma, associated with increased activation of tumor-infiltrating T-cells. Hereby, we show that the tumor endothelial cells up-regulate IDO1 upon CD40-stimulating immunotherapy in response to increased IFNγ-secretion by T-cells, revealing a novel immunosuppressive feedback mechanism whereby tumor vessels limit T-cell activation.
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http://dx.doi.org/10.1080/2162402X.2020.1730538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094447PMC
March 2020

Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas.

Nat Commun 2020 02 18;11(1):931. Epub 2020 Feb 18.

DKTK Clinical Cooperation Unit Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany.

Intrinsic malignant brain tumors, such as glioblastomas are frequently resistant to immune checkpoint blockade (ICB) with few hypermutated glioblastomas showing response. Modeling patient-individual resistance is challenging due to the lack of predictive biomarkers and limited accessibility of tissue for serial biopsies. Here, we investigate resistance mechanisms to anti-PD-1 and anti-CTLA-4 therapy in syngeneic hypermutated experimental gliomas and show a clear dichotomy and acquired immune heterogeneity in ICB-responder and non-responder tumors. We made use of this dichotomy to establish a radiomic signature predicting tumor regression after pseudoprogression induced by ICB therapy based on serial magnetic resonance imaging. We provide evidence that macrophage-driven ICB resistance is established by CD4 T cell suppression and T expansion in the tumor microenvironment via the PD-L1/PD-1/CD80 axis. These findings uncover an unexpected heterogeneity of response to ICB in strictly syngeneic tumors and provide a rationale for targeting PD-L1-expressing tumor-associated macrophages to overcome resistance to ICB.
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http://dx.doi.org/10.1038/s41467-020-14642-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028933PMC
February 2020

Immunization against poly--acetylglucosamine reduces neutrophil activation and GVHD while sparing microbial diversity.

Proc Natl Acad Sci U S A 2019 10 16;116(41):20700-20706. Epub 2019 Sep 16.

Department of Hematology, Oncology and Stem Cell Transplantation, Freiburg University Medical Center, Faculty of Medicine, 79106 Freiburg, Germany;

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly--acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.
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http://dx.doi.org/10.1073/pnas.1908549116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789638PMC
October 2019

Development, application and computational analysis of high-dimensional fluorescent antibody panels for single-cell flow cytometry.

Nat Protoc 2019 07 3;14(7):1946-1969. Epub 2019 Jun 3.

Laboratory of Translational Immunology, Humanitas Clinical and Research Center, Rozzano, Milan, Italy.

The interrogation of single cells is revolutionizing biology, especially our understanding of the immune system. Flow cytometry is still one of the most versatile and high-throughput approaches for single-cell analysis, and its capability has been recently extended to detect up to 28 colors, thus approaching the utility of cytometry by time of flight (CyTOF). However, flow cytometry suffers from autofluorescence and spreading error (SE) generated by errors in the measurement of photons mainly at red and far-red wavelengths, which limit barcoding and the detection of dim markers. Consequently, development of 28-color fluorescent antibody panels for flow cytometry is laborious and time consuming. Here, we describe the steps that are required to successfully achieve 28-color measurement capability. To do this, we provide a reference map of the fluorescence spreading errors in the 28-color space to simplify panel design and predict the success of fluorescent antibody combinations. Finally, we provide detailed instructions for the computational analysis of such complex data by existing, popular algorithms (PhenoGraph and FlowSOM). We exemplify our approach by designing a high-dimensional panel to characterize the immune system, but we anticipate that our approach can be used to design any high-dimensional flow cytometry panel of choice. The full protocol takes a few days to complete, depending on the time spent on panel design and data analysis.
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http://dx.doi.org/10.1038/s41596-019-0166-2DOI Listing
July 2019

TLR3 Activation of Intratumoral CD103 Dendritic Cells Modifies the Tumor Infiltrate Conferring Anti-tumor Immunity.

Front Immunol 2019 20;10:503. Epub 2019 Mar 20.

Department of Clinical Biochemistry, Faculty of Chemical Sciences, Center for Research in Clinical Biochemistry and Immunology, National University of Cordoba, Cordoba, Argentina.

An important challenge in cancer immunotherapy is to expand the number of patients that benefit from immune checkpoint inhibitors (CI), a fact that has been related to the pre-existence of an efficient anti-tumor immune response. Different strategies are being proposed to promote tumor immunity and to be used in combined therapies with CI. Recently, we reported that intratumoral administration of naked poly A:U, a dsRNA mimetic empirically used in early clinical trials with some success, delays tumor growth and prolongs mice survival in several murine cancer models. Here, we show that CD103 cDC1 and, to a much lesser extent CD11b cDC2, are the only populations expressing TLR3 at the tumor site, and consequently could be potential targets of poly A:U. Upon poly A:U administration these cells become activated and elicit profound changes in the composition of the tumor immune infiltrate, switching the immune suppressive tumor environment to anti-tumor immunity. The sole administration of naked poly A:U promotes striking changes within the lymphoid compartment, with all the anti-tumoral parameters being enhanced: a higher frequency of CD8 Granzyme B T cells, (lower Treg/CD8 ratio) and an important expansion of tumor-antigen specific CD8 T cells. Also, PD1/PDL1 showed an increased expression indicating that neutralization of this axis could be exploited in combination with poly A:U. Our results shed new light to promote further assays in this dsRNA mimetic to the clinical field.
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http://dx.doi.org/10.3389/fimmu.2019.00503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6435583PMC
September 2020

Targeted Delivery of IL2 to the Tumor Stroma Potentiates the Action of Immune Checkpoint Inhibitors by Preferential Activation of NK and CD8 T Cells.

Cancer Immunol Res 2019 04 19;7(4):572-583. Epub 2019 Feb 19.

Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology (ETH Zurich), Zurich, Switzerland.

Recombinant human IL2 is being considered as a combination partner for immune checkpoint inhibitors in cancer therapy, but the product only has a narrow therapeutic window. Therefore, we used F8-IL2, an antibody-IL2 fusion protein capable of selective localization to the tumor site, in combination with antibodies against murine CTLA-4, PD-1, and PD-L1. In immunocompetent mice bearing CT26 tumors, the combination of F8-IL2 with CTLA-4 blockade was efficacious, leading to increased progression-free survival and protective immunity against subsequent tumor rechallenges. The combination with anti-PD-1 induced substantial tumor growth retardation, but tumor clearance was rare, whereas the combination with anti-PD-L1 exhibited the lowest activity. A detailed high-parametric single-cell analysis of the tumor leukocyte composition revealed that F8-IL2 had a strong impact on NK-cell activity without collateral immune activation in the systemic immune compartment, whereas CTLA-4 blockade led to significant changes in the T-cell compartment. Leukocyte depletion studies revealed that CD8 T and NK cells were the main drivers of the therapeutic activity. We extended the experimental observations to a second model, treating MC38 tumor-bearing mice with F8-IL2 and/or CTLA-4 blockade. Only the combination treatment displayed potent anticancer activity, characterized by an increase in cytolytic CD8 T and NK cells in tumors and draining lymph nodes. A decrease in the regulatory T cell frequency, within the tumors, was also observed. The results provide a rationale for the combined use of engineered IL2 therapeutics with immune checkpoint inhibitors for cancer therapy.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0566DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978143PMC
April 2019

The end of omics? High dimensional single cell analysis in precision medicine.

Eur J Immunol 2019 02 25;49(2):212-220. Epub 2019 Jan 25.

Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland.

High-dimensional single-cell (HDcyto) technologies, such as mass cytometry (CyTOF) and flow cytometry, are the key techniques that hold a great promise for deciphering complex biological processes. During the last decade, we witnessed an exponential increase of novel HDcyto technologies that are able to deliver an in-depth profiling in different settings, such as various autoimmune diseases and cancer. The concurrent advance of custom data-mining algorithms has provided a rich substrate for the development of novel tools in translational medicine research. HDcyto technologies have been successfully used to investigate cellular cues driving pathophysiological conditions, and to identify disease-specific signatures that may serve as diagnostic biomarkers or therapeutic targets. These technologies now also offer the possibility to describe a complete cellular environment, providing unanticipated insights into human biology. In this review, we present an update on the current cutting-edge HDcyto technologies and their applications, which are going to be fundamental in providing further insights into human immunology and pathophysiology of various diseases. Importantly, we further provide an overview of the main algorithms currently available for data mining, together with the conceptual workflow for high-dimensional cytometric data handling and analysis. Overall, this review aims to be a handy overview for immunologists on how to design, develop and read HDcyto data.
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http://dx.doi.org/10.1002/eji.201847758DOI Listing
February 2019

IL2/Anti-IL2 Complex Combined with CTLA-4, But Not PD-1, Blockade Rescues Antitumor NK Cell Function by Regulatory T-cell Modulation.

Cancer Immunol Res 2019 03 16;7(3):443-457. Epub 2019 Jan 16.

Institut Curie, PSL Research University, INSERM U932, TransImm Team, Paris, France.

High-dose IL2 immunotherapy can induce long-lasting cancer regression but is toxic and insufficiently efficacious. Improvements are obtained with IL2/anti-IL2 complexes (IL2Cx), which redirect IL2 action to CD8 T and natural killer (NK) cells. Here, we evaluated the efficacy of combining IL2Cx with blockade of inhibitory immune pathways. In an autochthonous lung adenocarcinoma model, we show that the IL2Cx/anti-PD-1 combination increases CD8 T-cell infiltration of the lung and controls tumor growth. In the B16-OVA model, which is resistant to checkpoint inhibition, combination of IL2Cx with PD-1 or CTLA-4 pathway blockade reverses that resistance. Both combinations work by reinvigorating exhausted intratumoral CD8 T cells and by increasing the breadth of tumor-specific T-cell responses. However, only the IL2Cx/anti-CTLA-4 combination is able to rescue NK cell antitumor function by modulating intratumoral regulatory T cells. Overall, association of IL2Cx with PD-1 or CTLA-4 pathway blockade acts by different cellular mechanisms, paving the way for the rational design of combinatorial antitumor therapies.
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http://dx.doi.org/10.1158/2326-6066.CIR-18-0697DOI Listing
March 2019

Induction of anergic or regulatory tumor-specific CD4 T cells in the tumor-draining lymph node.

Nat Commun 2018 05 29;9(1):2113. Epub 2018 May 29.

Inserm U932, PSL University, Institut Curie, Paris, 75005, France.

CD4 T cell antitumor responses have mostly been studied in transplanted tumors expressing secreted model antigens (Ags), while most mutated proteins in human cancers are not secreted. The fate of Ag-specific CD4 T cells recognizing a cytoplasmic Ag in mice bearing autochthonous tumors is still unclear. Here we show, using a genetically engineered lung adenocarcinoma mouse model, that naive tumor-specific CD4 T cells are activated and proliferate in the tumor-draining lymph node (TdLN) but do not differentiate into effectors or accumulate in tumors. Instead, these CD4 T cells are driven toward anergy or peripherally-induced Treg (pTreg) differentiation, from the early stage of tumor development. This bias toward immune suppression is restricted to the TdLN, and is maintained by Tregs enriched in the tumor Ag-specific cell population. Thus, tumors may enforce a dominant inhibition of the anti-tumor CD4 response in the TdLN by recapitulating peripheral self-tolerance mechanisms.
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http://dx.doi.org/10.1038/s41467-018-04524-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5974295PMC
May 2018

Downregulation of adaptor protein MyD88 compromises the angiogenic potential of B16 murine melanoma.

PLoS One 2017 29;12(6):e0179897. Epub 2017 Jun 29.

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

The mechanisms that link inflammatory responses to cancer development remain a subject of intense investigation, emphasizing the need to better understand the cellular and molecular pathways that create a tumor promoting microenvironment. The myeloid differentiation primary response protein MyD88 acts as a main adaptor molecule for the signaling cascades initiated from Toll-like receptors (TLRs) and the interleukin 1 receptor (IL-1R). MyD88 has been shown to contribute to tumorigenesis in many inflammation-associated cancer models. In this study, we sought to better define the role of MyD88 in neoplastic cells using a murine melanoma model. Herein, we have demonstrated that MyD88 expression is required to maintain the angiogenic switch that supports B16 melanoma growth. By knocking down MyD88 we reduced TLR-mediated NF-κB activation with no evident effects over cell proliferation and survival. In addition, MyD88 downregulation was associated with a decrease of HIF1α levels and its target gene VEGF, in correlation with an impaired capability to induce capillary sprouting and tube formation of endothelial cells. Melanomas developed from cells lacking MyD88 showed an enhanced secretion of chemoattractant ligands such as CCL2, CXCL10 and CXCL1 and have an improved infiltration of macrophages to the tumor site. Our results imply that cell-autonomous signaling through MyD88 is required to sustain tumor growth and underscore its function as an important positive modulator of tumor angiogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179897PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491060PMC
September 2017

Phenotype of NK-Like CD8(+) T Cells with Innate Features in Humans and Their Relevance in Cancer Diseases.

Front Immunol 2017 27;8:316. Epub 2017 Mar 27.

INSERM 1082, Poitiers, France; CHU de Poitiers, Poitiers, France; Université de Poitiers, Poitiers, France; Service d'Immunologie et Inflammation, CHU de Poitiers, Poitiers, France.

Unconventional T cells are defined by their capacity to respond to signals other than the well-known complex of peptides and major histocompatibility complex proteins. Among the burgeoning family of unconventional T cells, innate-like CD8(+) T cells in the mouse were discovered in the early 2000s. This subset of CD8(+) T cells bears a memory phenotype without having encountered a foreign antigen and can respond to innate-like IL-12 + IL-18 stimulation. Although the concept of innate memory CD8(+) T cells is now well established in mice, whether an equivalent memory NK-like T-cell population exists in humans remains under debate. We recently reported that CD8(+) T cells responding to innate-like IL-12 + IL-18 stimulation and co-expressing the transcription factor Eomesodermin (Eomes) and KIR/NKG2A membrane receptors with a memory/EMRA phenotype may represent a new, functionally distinct innate T cell subset in humans. In this review, after a summary on the known innate CD8(+) T-cell features in the mouse, we propose Eomes together with KIR/NKG2A and CD49d as a signature to standardize the identification of this innate CD8(+) T-cell subset in humans. Next, we discuss IL-4 and IL-15 involvement in the generation of innate CD8(+) T cells and particularly its possible dependency on the promyelocytic leukemia zinc-finger factor expressing iNKT cells, an innate T cell subset well documented for its susceptibility to tumor immune subversion. After that, focusing on cancer diseases, we provide new insights into the potential role of these innate CD8(+) T cells in a physiopathological context in humans. Based on empirical data obtained in cases of chronic myeloid leukemia, a myeloproliferative syndrome controlled by the immune system, and in solid tumors, we observe both the possible contribution of innate CD8(+) T cells to cancer disease control and their susceptibility to tumor immune subversion. Finally, we note that during tumor progression, innate CD8(+) T lymphocytes could be controlled by immune checkpoints. This study significantly contributes to understanding of the role of NK-like CD8(+) T cells and raises the question of the possible involvement of an iNKT/innate CD8(+) T cell axis in cancer.
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http://dx.doi.org/10.3389/fimmu.2017.00316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5366313PMC
March 2017

Loss of immune tolerance to IL-2 in type 1 diabetes.

Nat Commun 2016 10 6;7:13027. Epub 2016 Oct 6.

Sorbonne Universités, Pierre and Marie Curie University Paris 06, Paris 75005, France.

Type 1 diabetes (T1D) is characterized by a chronic, progressive autoimmune attack against pancreas-specific antigens, effecting the destruction of insulin-producing β-cells. Here we show interleukin-2 (IL-2) is a non-pancreatic autoimmune target in T1D. Anti-IL-2 autoantibodies, as well as T cells specific for a single orthologous epitope of IL-2, are present in the peripheral blood of non-obese diabetic (NOD) mice and patients with T1D. In NOD mice, the generation of anti-IL-2 autoantibodies is genetically determined and their titre increases with age and disease onset. In T1D patients, circulating IgG memory B cells specific for IL-2 or insulin are present at similar frequencies. Anti-IL-2 autoantibodies cloned from T1D patients demonstrate clonality, a high degree of somatic hypermutation and nanomolar affinities, indicating a germinal centre origin and underscoring the synergy between cognate autoreactive T and B cells leading to defective immune tolerance.
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http://dx.doi.org/10.1038/ncomms13027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059699PMC
October 2016

In Vivo Visualizing the IFN-β Response Required for Tumor Growth Control in a Therapeutic Model of Polyadenylic-Polyuridylic Acid Administration.

J Immunol 2016 Mar 15;196(6):2860-9. Epub 2016 Feb 15.

Department of Clinical Biochemistry, Faculty of Chemical Sciences, Center for Research in Clinical Biochemistry and Immunology, National University of Cordoba, X5000HUA Córdoba, Argentina;

The crucial role that endogenously produced IFN-β plays in eliciting an immune response against cancer has recently started to be elucidated. Endogenous IFN-β has an important role in immune surveillance and control of tumor development. Accordingly, the role of TLR agonists as cancer therapeutic agents is being revisited via the strategy of intra/peritumoral injection with the idea of stimulating the production of endogenous type I IFN inside the tumor. Polyadenylic-polyuridylic acid (poly A:U) is a dsRNA mimetic explored empirically in cancer immunotherapy a long time ago with little knowledge regarding its mechanisms of action. In this work, we have in vivo visualized the IFN-β required for the antitumor immune response elicited in a therapeutic model of poly A:U administration. In this study, we have identified the role of host type I IFNs, cell populations that are sources of IFN-β in the tumor microenvironment, and other host requirements for tumor control in this model. One single peritumoral dose of poly A:U was sufficient to induce IFN-β, readily visualized in vivo. IFN-β production relied mainly on the activation of the transcription factor IFN regulatory factor 3 and the molecule UNC93B1, indicating that TLR3 is required for recognizing poly A:U. CD11c(+) cells were an important, but not the only source of IFN-β. Host type I IFN signaling was absolutely required for the reduced tumor growth, prolonged mice survival, and the strong antitumor-specific immune response elicited upon poly A:U administration. These findings add new perspectives to the use of IFN-β-inducing compounds in tumor therapy.
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http://dx.doi.org/10.4049/jimmunol.1501044DOI Listing
March 2016

Krüppel-like factor 6 interferes with cellular transformation induced by the H-ras oncogene.

FASEB J 2014 Dec 11;28(12):5262-76. Epub 2014 Sep 11.

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI), Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina

KLF6 is a member of the Krüppel-like factor family of transcription factors, with diverse roles in the regulation of cell physiology, including proliferation, signal transduction, and apoptosis. Mutations or down-regulation of KLF6 have been described in several human cancers. In this work, we found that KLF6-knockdown resulted in the formation of transformed foci and allowed the spontaneous conversion of NIH3T3 cells to a tumorigenic state. We further assessed the role of KLF6 in the context of oncogenic Ras. We showed that KLF6 was up-regulated by H-Ras(G12V) expression in a Jun N-terminal kinase (JNK)-dependent manner, correlated with enhanced klf6 promoter activity. We found that ectopic KLF6 expression induced a G1-phase cell cycle arrest, thereby decreasing the cell proliferation rate. In addition, constitutive KLF6 expression impaired H-Ras(G12V)-mediated loss of density-dependent growth inhibition and anchorage-independent growth. Moreover, growth of H-Ras(G12V)-driven tumors was reduced in mice challenged with cells stably expressing KLF6. KLF6 expression correlated with the up-regulation of p21, whereas neither p53 induction nor apoptotic cell death was detected. Further, p21 knockdown impaired KLF6-induced cell cycle arrest. These findings provide novel evidence highlighting KLF6 function in response to malignant transformation, suggesting the relevance of KLF6 in controlling cell proliferation and hindering tumorigenesis.
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http://dx.doi.org/10.1096/fj.14-251884DOI Listing
December 2014

Direct effect of dsRNA mimetics on cancer cells induces endogenous IFN-β production capable of improving dendritic cell function.

Eur J Immunol 2013 Jul 28;43(7):1849-61. Epub 2013 May 28.

Centro de Investigaciones en Bioquímica Clínica e Inmunología (CIBICI-CONICET), Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Viral double-stranded RNA (dsRNA) mimetics have been explored in cancer immunotherapy to promote antitumoral immune response. Polyinosine-polycytidylic acid (poly I:C) and polyadenylic-polyuridylic acid (poly A:U) are synthetic analogs of viral dsRNA and strong inducers of type I interferon (IFN). We describe here a novel effect of dsRNA analogs on cancer cells: besides their potential to induce cancer cell apoptosis through an IFN-β autocrine loop, dsRNA-elicited IFN-β production improves dendritic cell (DC) functionality. Human A549 lung and DU145 prostate carcinoma cells significantly responded to poly I:C stimulation, producing IFN-β at levels that were capable of activating STAT1 and enhancing CXCL10, CD40, and CD86 expression on human monocyte-derived DCs. IFN-β produced by poly I:C-activated human cancer cells increased the capacity of monocyte-derived DCs to stimulate IFN-γ production in an allogeneic stimulatory culture in vitro. When melanoma murine B16 cells were stimulated in vitro with poly A:U and then inoculated into TLR3(-/-) mice, smaller tumors were elicited. This tumor growth inhibition was abrogated in IFNAR1(-/-) mice. Thus, dsRNA compounds are effective adjuvants not only because they activate DCs and promote strong adaptive immunity, but also because they can directly act on cancer cells to induce endogenous IFN-β production and contribute to the antitumoral response.
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http://dx.doi.org/10.1002/eji.201242902DOI Listing
July 2013

IFNβ produced by TLR4-activated tumor cells is involved in improving the antitumoral immune response.

Cancer Res 2012 Feb 2;72(3):592-603. Epub 2011 Dec 2.

Centro de Investigaciones en Bioquímica Clínica e Inmunología, CIBICI-CONICET, Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Córdoba, Argentina.

Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 (lps-del)). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNβ was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow-derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNβ-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-0534DOI Listing
February 2012