The Contribution of Epigenetics to Cancer Immunotherapy.

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.

Mast cell interleukin-2 production contributes to suppression of chronic allergic dermatitis.

The incidence of chronic allergic dermatitis is rapidly increasing. Regulatory control of this disease has not been adequately explored. Here we report that mast cell-derived interleukin-2 (IL-2) contributes to the suppression of chronic allergic dermatitis. Mice deficient in IL-2 production, or deficient in mast cells (Kit(W-sh/W-sh)), showed exacerbated dermatitis upon repeated oxazolone challenge when compared to their wild-type counterparts. Adoptive transfer of wild-type, but not Il2(-/-), mast cells into Kit(W-sh/W-sh) mice dampened the inflammatory response. During the course of disease, mast cell expansion occurred at the site of inflammation and also in the spleen, where production of IL-2 by mast cells was markedly enhanced. In the absence of mast cell IL-2 production, the ratio of activated to regulatory T cells at the site of inflammation was increased. Thus, MC-derived IL-2 contributes to the maintenance of suppression in chronic allergic skin inflammation.

DNA demethylation of inflammasome-associated genes is enhanced in patients with cryopyrin-associated periodic syndromes.

BACKGROUND: Inflammasomes are cytosolic multiprotein complexes in macrophages. They assemble after infection- or stress-associated stimuli, activating both caspase-1-mediated inflammatory cytokine secretion and pyroptosis. Increased inflammasome activity resulting from gene mutations is related to monogenic autoinflammatory syndromes. However, variable penetrance among patients with the same gene mutations suggests involvement of additional mechanisms associated with inflammasome gene regulation. OBJECTIVE: We sought to investigate the role of DNA demethylation in activating inflammasome genes during macrophage differentiation and monocyte activation in healthy control subjects and patients with autoinflammatory syndrome. METHODS: Inflammasome-related genes were tested for DNA methylation and mRNA levels by using bisulfite pyrosequencing and quantitative RT-PCR in monocytes in vitro differentiated to macrophages and exposed to inflammatory conditions. The contribution of Tet methylcytosine dioxygenase 2 (TET2) and nuclear factor κB to DNA demethylation was tested by using chromatin immunoprecipitation, small interfering RNA-mediated downregulation, and pharmacologic inhibition. RESULTS: We observed that inflammasome-related genes are rapidly demethylated in both monocyte-to-macrophage differentiation and on monocyte activation. Demethylation associates with increased gene expression, and both mechanisms are impaired when TET2 and nuclear factor κB are downregulated. We analyzed DNA methylation levels of inflammasome-related genes in patients with cryopyrin-associated periodic syndromes (CAPS) and familial Mediterranean fever, 2 archetypical monogenic autoinflammatory syndromes. Under the above conditions, monocytes from untreated patients with CAPS undergo more efficient DNA demethylation than those of healthy subjects. Interestingly, patients with CAPS treated with anti-IL-1 drugs display methylation levels similar to those of healthy control subjects. CONCLUSION: Our study is the first to demonstrate the involvement of DNA methylation-associated alterations in patients with monogenic autoinflammatory disease and opens up possibilities for novel clinical markers.

The adaptor 3BP2 is required for KIT receptor expression and human mast cell survival.

SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein that acts as a positive regulator in mast cell FcεRI-dependent signaling. The KIT receptor whose ligand is the stem cell factor is necessary for mast cell development, proliferation, and survival as well as for optimal IgE-dependent signal. Activating mutations in KIT have been associated with several diseases including mastocytosis. In the present work, we found that 3BP2 silencing impairs KIT signaling pathways, thus affecting phosphoinositide 3-kinase and MAPK pathways in human mast cells (huMCs) from HMC-1, LAD2 (huMC lines), and CD34(+)-derived mast cells. Unexpectedly, silencing of 3BP2 reduces KIT expression in normal huMCs as well as in HMC-1 cells where KIT is mutated, thus increasing cellular apoptosis and caspase-3/7 activity. 3BP2 silencing reduces KIT transcription expression levels. Interestingly, 3BP2 silencing decreased microphthalmia-associated transcription factor (MITF) expression, a transcription factor involved in KIT expression. Reconstitution of 3BP2 in knockdown cells leads to reversal of KIT expression as well as survival phenotype. Accordingly MITF reconstitution enhances KIT expression levels in 3BP2-silenced cells. Moreover, downregulation of KIT expression by miRNA-221 overexpression or the proteasome inhibitor bortezomib also reduced 3BP2 and MITF expression. Furthermore, KIT tyrosine activity inhibition reduced 3BP2 and MITF expression, demonstrating again a tight and reciprocal relationship between these molecules. Taken together, our results show that 3BP2 regulates huMC survival and participates in KIT-mediated signal transduction by directly controlling KIT receptor expression, suggesting its potential as a therapeutic target in mast cell-mediated inflammatory diseases and deregulated KIT disorders.

Cutting edge: persistence of increased mast cell numbers in tissues links dermatitis to enhanced airway disease in a mouse model of atopy.

The development of chronic allergic dermatitis in early life has been associated with increased onset and severity of allergic asthma later in life. However, the mechanisms linking these two diseases are poorly understood. In this study, we report that the development of oxazolone-induced chronic allergic dermatitis, in a mouse model, caused enhanced OVA-induced allergic asthma after the resolution of the former disease. Our findings show that oxazolone-induced dermatitis caused a marked increase in tissue mast cells, which persisted long after the resolution of this disease. Subsequent OVA sensitization and airway challenge of mice that had recovered from dermatitis resulted in increased allergic airway hyperreactivity. The findings demonstrate that the accumulation of mast cells during dermatitis has the detrimental effect of increasing allergic airway hypersensitivity. Importantly, our findings also show that exposure to a given allergen can modify the immune response to an unrelated allergen.

The adaptor 3BP2 is required for early and late events in FcεRI signaling in human mast cells.

Adaptor molecules are essential in organizing signaling molecules and in coordinating and compartmentalizing their activity. SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein mainly expressed by hematopoietic cells that has been shown to act as a positive regulator in T, B, and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells (MCs) similarly degranulate following Ag-dependent aggregation of the FcεRI on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. Thus, MCs participate in both innate and acquired responses. We observed that 3BP2 is expressed in human MCs (huMCs) from diverse origins. Moreover, 3BP2 coimmunoprecipitates with essential MC signaling mediators such as Lyn, Syk, and phospholipase C γ; thus, a role for this adaptor in MC function was postulated. In the present work, we used the short hairpin RNA lentiviral targeting approach to silence 3BP2 expression in huMCs. Our findings point to a requirement for 3BP2 in optimal immediate and late MCs responses such as degranulation and IL-8 or GM-CSF secretion. 3BP2 was determined to be necessary for optimal phosphorylation of Syk, linker for activation of T cells, and phospholipase C γ(1), critical signals for calcium release from intracellular stores. Taken together, our results show that by participating in FcεRI- mediated signal transduction 3BP2 is an important regulator of huMC activation. Thus, 3BP2 could be a potential therapeutic target for IgE-dependent MC-mediated inflammatory disease.

Acute response to pathogens in the early human placenta at single-cell resolution.

The placenta is a selective maternal-fetal barrier that provides nourishment and protection from infections. However, certain pathogens can attach to and even cross the placenta, causing pregnancy complications with potential lifelong impacts on the child's health. Here, we profiled at the single-cell level the placental responses to three pathogens associated with intrauterine complications-Plasmodium falciparum, Listeria monocytogenes, and Toxoplasma gondii. We found that upon exposure to the pathogens, all placental lineages trigger inflammatory responses that may compromise placental function. Additionally, we characterized the responses of fetal macrophages known as Hofbauer cells (HBCs) to each pathogen and propose that they are the probable niche for T. gondii. Finally, we revealed how P. falciparum adapts to the placental microenvironment by modulating protein export into the host erythrocyte and nutrient uptake pathways. Altogether, we have defined the cellular networks and signaling pathways mediating acute placental inflammatory responses that could contribute to pregnancy complications.

CD84 negatively regulates IgE high-affinity receptor signaling in human mast cells.

CD84 is a self-binding receptor from the CD150 (or signaling lymphocyte activation molecule [SLAM]) family that is broadly expressed in hematopoietic cells. It has been described that the adaptors SLAM-associated protein (SAP) and EWS-FLI1-activated transcript 2 (EAT-2) are critical for CD150 family members' signaling and function. We observed that human mast cells express CD84 but lack SAP or EAT-2, that CD84 is tyrosine phosphorylated upon FcεRI engagement, and that the release of granule contents is reduced when FcεRI is coengaged with CD84 in LAD2 and human CD34(+)-derived mast cells. In addition, we observed that the release of IL-8 and GM-CSF was also reduced in FcεRI/CD84-costimulated cells as compared with FcεRI/Ig control. To understand how CD84 downregulates FcεRI-mediated function, we analyzed signaling pathways affected by CD84 in human mast cells. Our results showed that CD84 dampens FcεRI-mediated calcium mobilization after its co-cross-linking with the receptor. Furthermore, FcεRI-mediated Syk-linker for activation of T cells-phospholipase C-γ1 axis activity is downregulated after CD84 stimulation, compared with FcεRI/Ig control. The inhibitory kinase Fes phosphorylates mainly the inhibitory motif for CD84. Moreover, Fes, which has been described to become phosphorylated after substrate binding, also gets phosphorylated when coexpressed with CD84. Consistently, Fes was observed to be more phosphorylated after CD84 and FcεRI co-cross-linking. The phosphorylation of the protein phosphatase Src homology region 2 domain-containing phosphatase-1 also increases after CD84 and FcεRI coengagement. Taken together, our results show that CD84 is highly expressed in mast cells and that it contributes to the regulation of FcεRI signaling in SAP- and EAT-2-independent and Fes- and Src homology region 2 domain-containing phosphatase-1-dependent mechanisms.

Adapters in the organization of mast cell signaling.

Mast cells are pivotal in innate immunity and play an important role in amplifying adaptive immunity. Nonetheless, they have long been known to be central to the initiation of allergic disorders. This results from the dysregulation of the immune response whereby normally innocuous substances are recognized as non-self, resulting in the production of IgE antibodies to these 'allergens'. Preformed and newly synthesized inflammatory (allergic) mediators are released from the mast cell following allergen-mediated aggregation of allergen-specific IgE bound to the high-affinity receptors for IgE (FcepsilonRI). Thus, the process by which the mast cell is able to interpret the engagement of FcepsilonRI into the molecular events necessary for release of their allergic mediators is of considerable therapeutic interest. Unraveling these molecular events has led to the discovery of a functional class of proteins that are essential in organizing activated signaling molecules and in coordinating and compartmentalizing their activity. These so-called 'adapters' bind multiple signaling proteins and localize them to specific cellular compartments, such as the plasma membrane. This organization is essential for normal mast cell responses. Here, we summarize the role of adapter proteins in mast cells focusing on the most recent advances toward understanding how these molecules work upon FcepsilonRI engagement.

Validation of the new EPIC DNA methylation microarray (900K EPIC v2) for high-throughput profiling of the human DNA methylome.

DNA methylation, one of the best characterized epigenetic marks in the human genome, plays a pivotal role in gene transcription regulation and other biological processes in humans. On top of that, the DNA methylome undergoes profound changes in cancer and other disorders. However, large-scale and population-based studies are limited by high costs and the need for considerable expertise in data analysis for whole-genome bisulphite-sequencing methodologies. Following the success of the EPIC DNA methylation microarray, the newly developed Infinium HumanMethylationEPIC version 2.0 (900K EPIC v2) is now available. This new array contains more than 900,000 CpG probes covering the human genome and excluding masked probes from the previous version. The 900K EPIC v2 microarray adds more than 200,000 probes covering extra DNA cis-regulatory regions such as enhancers, super-enhancers and CTCF binding regions. Herein, we have technically and biologically validated the new methylation array to show its high reproducibility and consistency among technical replicates and with DNA extracted from FFPE tissue. In addition, we have hybridized primary normal and tumoural tissues and cancer cell lines from different sources and tested the robustness of the 900K EPIC v2 microarray when analysing the different DNA methylation profiles. The validation highlights the improvements offered by the new array and demonstrates the versatility of this updated tool for characterizing the DNA methylome in human health and disease.

Functional analysis of Lyn kinase A and B isoforms reveals redundant and distinct roles in Fc epsilon RI-dependent mast cell activation.

Engagement of FcepsilonRI causes its phosphorylation by Lyn kinase. Two alternatively spliced variants, Lyn A and B, are expressed in mast cells, and both isoforms interact with FcepsilonRI. Unlike Lyn A, Lyn B lacks a 21-aa region in the N-terminal unique domain. In this study, we investigated the role of Lyn A and B isoforms in mast cell signaling and responses. Lyn B was found to be a poor inducer of mast cell degranulation and was less potent in both inositol 1,4,5-triphosphate production and calcium responses. Expression of Lyn B alone showed reduced phosphorylation of both phospholipase Cgamma-1 and -2 and decreased interaction of phospholipase Cgamma-1 with the phosphorylated linker for activation of T cells. Lyn B also showed increased binding of tyrosine-phosphorylated proteins, which included the negative regulatory lipid phosphatase SHIP-1. In contrast, both Lyn A and B caused similar total cellular tyrosine phosphorylation and FcepsilonRI phosphorylation and neither Lyn A nor Lyn B alone could completely restore mast cell degranulation or dampen the excessive cytokine production seen in the absence of Lyn. However, expression of both isoforms showed complementation and normalized responses. These findings demonstrate that Lyn B differs from Lyn A in its association with SHIP-1 and in the regulation of calcium responses. However, complementation of both isoforms is required in mast cell activation.

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

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.

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.

Robust temporal map of human in vitro myelopoiesis using single-cell genomics.

Myeloid cells are central to homeostasis and immunity. Characterising in vitro myelopoiesis protocols is imperative for their use in research, immunotherapies, and understanding human myelopoiesis. Here, we generate a >470K cells molecular map of human induced pluripotent stem cells (iPSC) differentiation into macrophages. Integration with in vivo single-cell atlases shows in vitro differentiation recapitulates features of yolk sac hematopoiesis, before definitive hematopoietic stem cells (HSC) emerge. The diversity of myeloid cells generated, including mast cells and monocytes, suggests that HSC-independent hematopoiesis can produce multiple myeloid lineages. We uncover poorly described myeloid progenitors and conservation between in vivo and in vitro regulatory programs. Additionally, we develop a protocol to produce iPSC-derived dendritic cells (DC) resembling cDC2. Using CRISPR/Cas9 knock-outs, we validate the effects of key transcription factors in macrophage and DC ontogeny. This roadmap of myeloid differentiation is an important resource for investigating human fetal hematopoiesis and new therapeutic opportunities.

Perspectives on Epigenetics and Cancer Immunotherapy: A Preface to Special Issue.

The interaction of tumor cells with immune cells within the tumor microenvironment (TME) is the basis for several strategies of tumor evasion from immune surveillance, which nurture cancer development [...].

Single-cell technologies and analyses in hematopoiesis and hematological malignancies.

In recent years, single-cell technologies have emerged as breakthrough techniques that enable the characterization of hematopoietic cell populations of normal and malignant tissue samples and will be combined in the near future with bulk technologies, currently used in clinical practice, to improve diagnosis, prognosis, and the search for novel molecular targets. These single-cell methods have the advantage of not masking cell-to-cell variation features and involve the study of genetic, epigenetic, transcriptional, and proteomic landscapes from a single-cell perspective. Latest advances in this field have enabled the development of novel strategies that significantly increase both sensitivity and high throughput. In this review, we emphasize emerging techniques aimed at assessing individual or multiomic parameters at single-cell resolution and analyze how these technologies have helped us understand hematopoietic variability and identify unknown and/or rare subpopulations. We also summarize the impact of these single-cell profiling strategies on the characterization of cell diversity within the tumor and the clonal evolution of multiple hematological malignancies in samples from untreated and treated patients, which provide valuable information for diagnosis, prognosis, and future treatments and explain why current therapies may fail. However, despite these improvements, new challenges lie ahead.

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

Microbial 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.

Epigenome-wide association study of COVID-19 severity with respiratory failure.

BACKGROUND: Patients infected with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the coronavirus disease 2019 (COVID-19), exhibit a wide spectrum of disease behaviour. Since DNA methylation has been implicated in the regulation of viral infections and the immune system, we performed an epigenome-wide association study (EWAS) to identify candidate loci regulated by this epigenetic mark that could be involved in the onset of COVID-19 in patients without comorbidities. METHODS: Peripheral blood samples were obtained from 407 confirmed COVID-19 patients ≤ 61 years of age and without comorbidities, 194 (47.7%) of whom had mild symptomatology that did not involve hospitalization and 213 (52.3%) had a severe clinical course that required respiratory support. The set of cases was divided into discovery (n = 207) and validation (n = 200) cohorts, balanced for age and sex of individuals. We analysed the DNA methylation status of 850,000 CpG sites in these patients. FINDINGS: The DNA methylation status of 44 CpG sites was associated with the clinical severity of COVID-19. Of these loci, 23 (52.3%) were located in 20 annotated coding genes. These genes, such as the inflammasome component Absent in Melanoma 2 (AIM2) and the Major Histocompatibility Complex, class I C (HLA-C) candidates, were mainly involved in the response of interferon to viral infection. We used the EWAS-identified sites to establish a DNA methylation signature (EPICOVID) that is associated with the severity of the disease. INTERPRETATION: We identified DNA methylation sites as epigenetic susceptibility loci for respiratory failure in COVID-19 patients. These candidate biomarkers, combined with other clinical, cellular and genetic factors, could be useful in the clinical stratification and management of patients infected with the SARS-CoV-2. FUNDING: The Unstoppable campaign of the Josep Carreras Leukaemia Foundation, the Cellex Foundation and the CERCA Programme/Generalitat de Catalunya.

Integrative Analysis Reveals a Molecular Stratification of Systemic Autoimmune Diseases.

OBJECTIVE: Clinical heterogeneity, a hallmark of systemic autoimmune diseases, impedes early diagnosis and effective treatment, issues that may be addressed if patients could be classified into groups defined by molecular pattern. This study was undertaken to identify molecular clusters for reclassifying systemic autoimmune diseases independently of clinical diagnosis. METHODS: Unsupervised clustering of integrated whole blood transcriptome and methylome cross-sectional data on 955 patients with 7 systemic autoimmune diseases and 267 healthy controls was undertaken. In addition, an inception cohort was prospectively followed up for 6 or 14 months to validate the results and analyze whether or not cluster assignment changed over time. RESULTS: Four clusters were identified and validated. Three were pathologic, representing "inflammatory," "lymphoid," and "interferon" patterns. Each included all diagnoses and was defined by genetic, clinical, serologic, and cellular features. A fourth cluster with no specific molecular pattern was associated with low disease activity and included healthy controls. A longitudinal and independent inception cohort showed a relapse-remission pattern, where patients remained in their pathologic cluster, moving only to the healthy one, thus showing that the molecular clusters remained stable over time and that single pathogenic molecular signatures characterized each individual patient. CONCLUSION: Patients with systemic autoimmune diseases can be jointly stratified into 3 stable disease clusters with specific molecular patterns differentiating different molecular disease mechanisms. These results have important implications for future clinical trials and the study of nonresponse to therapy, marking a paradigm shift in our view of systemic autoimmune diseases.