Biological and Molecular Factors Predicting Response to Adoptive Cell Therapies in Cancer

GF is recipient of Marie Skłodowska-Curie Action individual fellowship (H2020-MSCA-IF-2019 - 896403). We thank CERCA Programme/Generalitat de Catalunya for institutional support. Work at ME laboratory is supported by the Health Department PERIS-project no. SLT/002/16/00374 and AGAUR-project no. 2017SGR1080 of the Catalan Government (Generalitat de Catalunya); Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and European Regional Development Fund (ERDF) project no. RTI2018-094049-B-I00; the Cellex Foundation; and "la Caixa" Banking Foundation (LCF/PR/GN18/51140001).Adoptive cell therapy (ACT) constitutes a major breakthrough in cancer management that has expanded in the past years due to impressive results showing durable and even curative responses for some patients with hematological malignancies. ACT leverages antigen specificity and cytotoxic mechanisms of the immune system, particularly relying on the patient's T lymphocytes to target and eliminate malignant cells. This personalized therapeutic approach exemplifies the success of the joint effort of basic, translational, and clinical researchers that has turned the patient's immune system into a great ally in the search for a cancer cure. ACTs are constantly improving to reach a maximum beneficial clinical response. Despite being very promising therapeutic options for certain types of cancers, mainly melanoma and hematological malignancies, these individualized treatments still present several shortcomings, including elevated costs, technical challenges, management of adverse side effects, and a limited population of responder patients. Thus, it is crucial to discover and develop reliable and robust biomarkers to specifically and sensitively pinpoint the patients that will benefit the most from ACT as well as those at higher risk of developing potentially serious toxicities. Although unique readouts of infused cell therapy success have not yet been identified, certain characteristics from the adoptive cells, the tumor, and/or the tumor microenvironment have been recognized to predict patients' outcome on ACT. Here, we comment on the importance of biomarkers to predict ACT chances of success to maximize efficacy of treatments and increase patients' survival

Genetic And Epigenetic Determinants In Autoinflammatory Diseases

The concept of autoinflammation has evolved over the past 20 years, beginning with the discovery that mutations in the Mediterranean Fever (MEFV) gene were causative of Familial Mediterranean Fever. Currently, autoinflammatory diseases comprise a wide range of disorders with the common features of recurrent fever attacks, prevalence of hyperreactive innate immune cells, and signs of inflammation that can be systemic or organ specific in the absence of pathogenic infection of autoimmunity. Innate immune cells from the myeloid compartment are the main effectors of uncontrolled inflammation that is caused in great extent by the overproduction of inflammatory cytokines such as IL-1 beta and IL-18. Defects in several signaling pathways that control innate immune defense, particularly the hyperreactivity of one or more inflammasomes, are at the core of pathologic autoinflammatory phenotypes. Although many of the autoinflammatory syndromes are known to be monogenic, some of them are genetically complex and are impacted by environmental factors. Recently, epigenetic dysregulation has surfaced as an additional contributor to pathogenesis. In the present review, we discuss data that are currently available to describe the contribution of epigenetic mechanisms in autoinflammatory diseases

The Contribution of Epigenetics to Cancer Immunotherapy

Altres ajuts: This work was supported by the the Cellex Foundation; and La Caixa Banking Foundation (LCF/PR/GN18/51140001).Effective anticancer immunotherapy treatments constitute a qualitative leap in cancer management. Nonetheless, not all patients benefit from such therapies because they fail to achieve complete responses, suffer frequent relapses, or develop potentially life-threatening toxicities. Epigenomic signatures in immune and cancer cells appear to be accurate and promising predictors of patient outcomes with immunotherapy. In addition, combined treatments with epigenetic drugs can exploit the dynamic nature of epigenetic changes to potentially modulate responses to immunotherapy. Candidate epigenetic biomarkers may provide a rationale for patient stratification and precision medicine, thus maximizing the chances of treatment success while minimizing unwanted effects. We present a comprehensive up-to-date view of potential epigenetic biomarkers in immunotherapy and discuss their advantages over other indicators

Validation of a DNA methylation microarray for 285,000 CpG sites in the mouse genome

Mouse has been extensively used as a model organism in many studies to characterize biological pathways and drug effects and to mimic human diseases. Similar DNA sequences between both species facilitate these types of experiments. However, much less is known about the mouse epigenome, particularly for DNA methylation. Progress in delivering mouse DNA methylomes has been slow due to the currently available time-consuming and expensive methodologies. Following the great acceptance of the human DNA methylation microarrays, we have herein validated a newly developed DNA methylation microarray (Infinium Mouse Methylation BeadChip) that interrogates 280,754 unique CpG sites within the mouse genome. The CpGs included in the platform cover CpG Islands, shores, shelves and open sea sequences, and loci surrounding transcription start sites and gene bodies. From a functional standpoint, mouse ENCODE representative DNase hypersensitivity sites (rDHSs) and candidate cis-Regulatory Elements (cCREs) are also included. Herein, we show that the profiled mouse DNA methylation microarray provides reliable values among technical replicates; matched results from fresh frozen versus formalin-fixed samples; detects hemimethylated X-chromosome and imprinted CpG sites; and is able to determine CpG methylation changes in mouse cell lines treated with a DNA demethylating agent or upon genetic disruption of a DNA methyltransferase. Most important, using unsupervised hierarchical clustering and t-SNE approaches, the platform is able to classify all types of normal mouse tissues and organs. These data underscore the great features of the assessed microarray to obtain comprehensive DNA methylation profiles of the mouse genome.We thank the CERCA Programme/Generalitat de Catalunya for institutional support. This work was supported by the Health Department PERIS-project no. SLT/002/16/00374 and AGAUR-project no. 2017SGR1080 of the Catalan Government (Generalitat de Catalunya); Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI), and European Regional Development Fund (ERDF) project no. RTI2018-094049-B-I00 and PID2020-117284RB-I00; the Cellex Foundation; Marie Sklodowska-Curie Fellowship no. 895979 from the European Commission (BNV); and ‘la Caixa’ Banking Foundation (LCF/PR/GN18/51140001).Peer ReviewedPostprint (published version

RXRs control serous macrophage neonatal expansion and identity and contribute to ovarian cancer progression

Tissue-resident macrophages (TRMs) populate all tissues and play key roles in homeostasis, immunity and repair. TRMs express a molecular program that is mostly shaped by tissue cues. However, TRM identity and the mechanisms that maintain TRMs in tissues remain poorly understood. We recently found that serous-cavity TRMs (LPMs) are highly enriched in RXR transcripts and RXR-response elements. Here, we show that RXRs control mouse serous-macrophage identity by regulating chromatin accessibility and the transcriptional regulation of canonical macrophage genes. RXR deficiency impairs neonatal expansion of the LPM pool and reduces the survival of adult LPMs through excess lipid accumulation. We also find that peritoneal LPMs infiltrate early ovarian tumours and that RXR deletion diminishes LPM accumulation in tumours and strongly reduces ovarian tumour progression in mice. Our study reveals that RXR signalling controls the maintenance of the serous macrophage pool and that targeting peritoneal LPMs may improve ovarian cancer outcomes.This work was supported by a HFSP fellowship to M.C-A. (LT000110/2015-L/1), grants from the Spanish Ministerio de Ciencia e Innovación (MCI) (SAF2015-64287R, SAF2017-90604-REDT-NurCaMein, RTI2018-095928-B100), La Marató de TV3 Foundation (201605-32) and Comunidad de Madrid (MOIR-B2017/BMD-3684) to M.R, and the Formación de Profesorado Universitario (FPU17/01731) programme (MCI) to J.P. The CNIC is supported by the MCI and the Pro CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).S

JAK2-STAT Epigenetically Regulates Tolerized Genes in Monocytes in the First Encounter With Gram-Negative Bacterial Endotoxins in Sepsis

Metilación del ADN; Tolerancia a la endotoxina; MonocitosDNA methylation; Endotoxin tolerance; MonocytesMetilació de l'ADN; Tolerància a l'endotoxina; MonòcitsMicrobial challenges, such as widespread bacterial infection in sepsis, induce endotoxin tolerance, a state of hyporesponsiveness to subsequent infections. The participation of DNA methylation in this process is poorly known. In this study, we perform integrated analysis of DNA methylation and transcriptional changes following in vitro exposure to gram-negative bacterial lipopolysaccharide, together with analysis of ex vivo monocytes from septic patients. We identify TET2-mediated demethylation and transcriptional activation of inflammation-related genes that is specific to toll-like receptor stimulation. Changes also involve phosphorylation of STAT1, STAT3 and STAT5, elements of the JAK2 pathway. JAK2 pathway inhibition impairs the activation of tolerized genes on the first encounter with lipopolysaccharide. We then confirm the implication of the JAK2-STAT pathway in the aberrant DNA methylome of patients with sepsis caused by gram-negative bacteria. Finally, JAK2 inhibition in monocytes partially recapitulates the expression changes produced in the immunosuppressive cellular state acquired by monocytes from gram-negative sepsis, as described by single cell-RNA-sequencing. Our study evidences both the crucial role the JAK2-STAT pathway in epigenetic regulation and initial response of the tolerized genes to gram-negative bacterial endotoxins and provides a pharmacological target to prevent exacerbated responses.EB was funded by the Spanish Ministry of Science, Innovation and Universities (grant numbers SAF2017-88086-R & PID2020-117212RB-I00), and was cofunded by FEDER funds/European Regional Development Fund (ERDF) - a way to build Europe. OM-P holds an i-PFIS PhD fellowship (grant number IFI17/00034) from Acción Estratégica en Salud 2013-2016 ISCIII, cofinanced by the Fondo Social Europeo

NF-κB-direct activation of microRNAs with repressive effects on monocyte-specific genes is critical for osteoclast differentiation

Monocyte-to-osteoclast conversion is a unique terminal differentiation process that is exacerbated in rheumatoid arthritis and bone metastasis. The mechanisms implicated in upregulating osteoclast-specific genes involve transcription factors, epigenetic regulators and microRNAs (miRNAs). It is less well known how downregulation of osteoclast-inappropriate genes is achieved. RESULTS: In this study, analysis of miRNA expression changes in osteoclast differentiation from human primary monocytes revealed the rapid upregulation of two miRNA clusters, miR-212/132 and miR-99b/let-7e/125a. We demonstrate that they negatively target monocyte-specific and immunomodulatory genes like TNFAIP3, IGF1R and IL15. Depletion of these miRNAs inhibits osteoclast differentiation and upregulates their targets. These miRNAs are also upregulated in other inflammatory monocytic differentiation processes. Most importantly, we demonstrate for the first time the direct involvement of Nuclear Factor kappa B (NF-κB) in the regulation of these miRNAs, as well as with their targets, whereby NF-κB p65 binds the promoters of these two miRNA clusters and NF-κB inhibition or depletion results in impaired upregulation of their expression. CONCLUSIONS:Our results reveal the direct involvement of NF-κB in shutting down certain monocyte-specific genes, including some anti-inflammatory activities, through a miRNA-dependent mechanism for proper osteoclast differentiation

Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsis

Background: Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation. Methods: Bead arrays were used to compare global DNA methylation changes in patients with sepsis, noninfectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming. Results: Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance. Conclusion: We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction

Inflammatory cytokines and organ dysfunction associate with the aberrant DNA methylome of monocytes in sepsis

Sepsis, a life-threatening organ dysfunction caused by a dysregulated systemic immune response to infection, associates with reduced responsiveness to subsequent infections. How such tolerance is acquired is not well understood but is known to involve epigenetic and transcriptional dysregulation. Bead arrays were used to compare global DNA methylation changes in patients with sepsis, non-infectious systemic inflammatory response syndrome, and healthy controls. Bioinformatic analyses were performed to dissect functional reprogramming and signaling pathways related to the acquisition of these specific DNA methylation alterations. Finally, in vitro experiments using human monocytes were performed to test the induction of similar DNA methylation reprogramming. Here, we focused on DNA methylation changes associated with sepsis, given their potential role in stabilizing altered phenotypes. Tolerized monocytes from patients with sepsis display changes in their DNA methylomes with respect to those from healthy controls, affecting critical monocyte-related genes. DNA methylation profiles correlate with IL-10 and IL-6 levels, significantly increased in monocytes in sepsis, as well as with the Sequential Organ Failure Assessment score; the observed changes associate with TFs and pathways downstream to toll-like receptors and inflammatory cytokines. In fact, in vitro stimulation of toll-like receptors in monocytes results in similar gains and losses of methylation together with the acquisition of tolerance. We have identified a DNA methylation signature associated with sepsis that is downstream to the response of monocytes to inflammatory signals associated with the acquisition of a tolerized phenotype and organic dysfunction

Cloning and characterization of a novel inhibitory receptor expressed by myeloid cells

El presente trabajo describe el clonaje y caracterización de un nuevo receptor inhibidor de la superfamilia de las Ig, que hemos denominado IREM-1 (Immune Receptor Expressed by Myeloid Cells). El analisis de su secuencia demuestra que esta molécula forma parte de la familia multigénica conocida como CMRF35 o CD300. IREM-1 fue clonado a partir de cDNA proveniente de células mononucleares de sangre periférica (PBMCs), y es una glucoproteina tipo I de membrana, con un único dominio Ig extracelular, un fragmento transmembrana y una cola citoplasmática con cinco residuos tirosina que cosntituyen diversos motivos de señalización intracelular. Dos de estos residuos, las tirosinas 205 y 249, corresponden a ITIMs, mientras que las tirosinas 236 y 263 son parte de motivos YxxM asociados al reclutamiento de PI3K. La tirosina más distal (Y284) podría ser considerada como parte de un motivo ITIM-like. IREM-1 es capaz de asociarse a la fosfatasa SHP-1, y hemos identificado al residuo Y205 como principal responsable de dicha interacción. IREM-1 es capaz de producir señales inhibitorias sobre receptores activadores como el FcRI, en un modelo heterólogo de rata con la línea basofílica RBL. Esta inhibición está mediada por ambos ITIMs. IREM-1 también es capaz de unir la subunidad p85 de la PI3K a través de sus dos motivos YxxM. Ambos motivo poseen la capacidad de unir esta molécula. El triple mutante de IREM-1 de los 2 sitios ITIMs y el ITIM-like, que no une SHP-1, es capaz de producir señales activadoras como la inducción de la degranulación en RBLs. Esta respuesta es dependiente de PI3K y es anulada con inhibidores de esta enzima. La expresión de IREM-1 se restringe a células hematopoiéticas de linaje mieloide incluyendo precursores de médula ósea CD34+. En conclusión, presentamos la identificación y caracterización molecular y funcional del receptor IREM-1.The present work describes the cloning and characterization of a novel inhibitory receptor belonging to the Ig superfamily, that we termed IREM-1 (Immune Receptor Expressed by Myeloid Cells). The analysy of IREM-1 sequence demonstrated that this molecule belongs to the multigenic family known as CMRF35 or CD300.. IREM-1 was cloned from peripheral blood mononuclear cells (PBMCs) cDNA, and it is a type I glycoprotein, with a single extracellular Ig domain, a transmembrane region and a cytoplasmic tail with five tyrosine residues in the context of several intracellular signaling motifs. Two of these residues, including tyrosines 205 and 249, correspond to ITIMs, whereas tyrosines 236 and 263 are part of YxxM motifs associated to the recruitment of PI3K. The most distal tyrosine (Y284) could be considered as part of an ITIM-like motif. IREM-1 is able to associate to SHP-1 phosphatase, and we identified the residue Y205, as the main responsible for the interaction. IREM-1 delivers inhibitory signal over activating receptors as FcRI, in a heterologous model as the rat basophilic cell line, RBL. This inhibition is mediated by both ITIMs. IREM-1 also binds the p85 subunit of PI3K through its YxxM motifs. Both motifs have the capacity of binding this molecule.A mutated form of IREM-1 mutant lacking the two ITIMs and the ITIM-like, is able to produce activating signals like the induction of of RBL degranulation. This responses are produced in the absence of SHP-1 recruitment and are dependent on PI3K, because are abrogated with inhibitors of this enzyme. IREM-1 expression is restricted to hematopoietic cells of myeloid lineage, including CD34+ precursors from bone marrow. In conclusion, we herein present the identification and molecular and functional characterization of the receptor IREM-1