Gene Regulatory Circuits in Innate and Adaptive Immune Cells.

Cell identity and function largely rely on the programming of transcriptomes during development and differentiation. Signature gene expression programs are orchestrated by regulatory circuits consisting of cis-acting promoters and enhancers, which respond to a plethora of cues via the action of transcription factors. In turn, transcription factors direct epigenetic modifications to revise chromatin landscapes, and drive contacts between distal promoter-enhancer combinations. In immune cells, regulatory circuits for effector genes are especially complex and flexible, utilizing distinct sets of transcription factors and enhancers, depending on the cues each cell type receives during an infection, after sensing cellular damage, or upon encountering a tumor. Here, we review major players in the coordination of gene regulatory programs within innate and adaptive immune cells, as well as integrative omics approaches that can be leveraged to decipher their underlying circuitry.

The transcription factor Aiolos restrains the activation of intestinal intraepithelial lymphocytes.

Intestinal intraepithelial lymphocytes (IELs) exhibit prompt innate-like responses to microenvironmental cues and require strict control of effector functions. Here we showed that Aiolos, an Ikaros zinc-finger family member encoded by Ikzf3, acted as a regulator of IEL activation. Ikzf3-/- CD8αα+ IELs had elevated expression of NK receptors, cytotoxic enzymes, cytokines and chemokines. Single-cell RNA sequencing of Ikzf3-/- and Ikzf3+/+ IELs showed an amplified effector machinery in Ikzf3-/- CD8αα+ IELs compared to Ikzf3+/+ counterparts. Ikzf3-/- CD8αα+ IELs had increased responsiveness to interleukin-15, which explained a substantial part, but not all, of the observed phenotypes. Aiolos binding sites were close to those for the transcription factors STAT5 and RUNX, which promote interleukin-15 signaling and cytolytic programs, and Ikzf3 deficiency partially increased chromatin accessibility and histone acetylation in these regions. Ikzf3 deficiency in mice enhanced susceptibility to colitis, underscoring the relevance of Aiolos in regulating the effector function in IELs.

Whole-genome profiling of DNA methylation and hydroxymethylation identifies distinct regulatory programs among innate lymphocytes.

Innate lymphocytes encompass a diverse array of phenotypic identities with specialized functions. DNA methylation and hydroxymethylation are essential for epigenetic fidelity and fate commitment. The landscapes of these modifications are unknown in innate lymphocytes. Here, we characterized the whole-genome distribution of methyl-CpG and 5-hydroxymethylcytosine (5hmC) in mouse innate lymphoid cell 3 (ILC3), ILC2 and natural killer (NK) cells. We identified differentially methylated regions (DMRs) and differentially hydroxymethylated regions (DHMRs) between ILC and NK cell subsets and correlated them with transcriptional signatures. We associated lineage-determining transcription factors (LDTFs) with demethylation and demonstrated unique patterns of DNA methylation/hydroxymethylation in relationship to open chromatin regions (OCRs), histone modifications and TF-binding sites. We further identified an association between hydroxymethylation and NK cell superenhancers (SEs). Using mice lacking the DNA hydroxymethylase TET2, we showed the requirement for TET2 in optimal production of hallmark cytokines by ILC3s and interleukin-17A (IL-17A) by inflammatory ILC2s. These findings provide a powerful resource for studying innate lymphocyte epigenetic regulation and decode the regulatory logic governing their identity.

Gene Regulatory Programs Conferring Phenotypic Identities to Human NK Cells.

Natural killer (NK) cells develop from common progenitors but diverge into distinct subsets, which differ in cytokine production, cytotoxicity, homing, and memory traits. Given their promise in adoptive cell therapies for cancer, a deeper understanding of regulatory modules controlling clinically beneficial NK phenotypes is of high priority. We report integrated "-omics" analysis of human NK subsets, which revealed super-enhancers associated with gene cohorts that may coordinate NK functions and localization. A transcription factor-based regulatory scheme also emerged, which is evolutionarily conserved and shared by innate and adaptive lymphocytes. For both NK and T lineages, a TCF1-LEF1-MYC axis dominated the regulatory landscape of long-lived, proliferative subsets that traffic to lymph nodes. In contrast, effector populations circulating between blood and peripheral tissues shared a PRDM1-dominant landscape. This resource defines transcriptional modules, regulated by feedback loops, which may be leveraged to enhance phenotypes for NK cell-based therapies.

Publisher Correction: Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Subsets of ILC3-ILC1-like cells generate a diversity spectrum of innate lymphoid cells in human mucosal tissues.

Innate lymphoid cells (ILCs) are tissue-resident lymphocytes categorized on the basis of their core regulatory programs and the expression of signature cytokines. Human ILC3s that produce the cytokine interleukin-22 convert into ILC1-like cells that produce interferon-γ in vitro, but whether this conversion occurs in vivo remains unclear. In the present study we found that ILC3s and ILC1s in human tonsils represented the ends of a spectrum that included additional discrete subsets. RNA velocity analysis identified an intermediate ILC3-ILC1 cluster, which had strong directionality toward ILC1s. In humanized mice, the acquisition of ILC1 features by ILC3s showed tissue dependency. Chromatin studies indicated that the transcription factors Aiolos and T-bet cooperated to repress regulatory elements active in ILC3s. A transitional ILC3-ILC1 population was also detected in the human intestine. We conclude that ILC3s undergo conversion into ILC1-like cells in human tissues in vivo, and that tissue factors and Aiolos were required for this process.

Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells.

Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts. Super-enhancers demarcate cohorts of cell-identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC-Th subsets.

The Colonic Crypt Protects Stem Cells from Microbiota-Derived Metabolites.

In the mammalian intestine, crypts of Leiberkühn house intestinal epithelial stem/progenitor cells at their base. The mammalian intestine also harbors a diverse array of microbial metabolite compounds that potentially modulate stem/progenitor cell activity. Unbiased screening identified butyrate, a prominent bacterial metabolite, as a potent inhibitor of intestinal stem/progenitor proliferation at physiologic concentrations. During homeostasis, differentiated colonocytes metabolized butyrate likely preventing it from reaching proliferating epithelial stem/progenitor cells within the crypt. Exposure of stem/progenitor cells in vivo to butyrate through either mucosal injury or application to a naturally crypt-less host organism led to inhibition of proliferation and delayed wound repair. The mechanism of butyrate action depended on the transcription factor Foxo3. Our findings indicate that mammalian crypt architecture protects stem/progenitor cell proliferation in part through a metabolic barrier formed by differentiated colonocytes that consume butyrate and stimulate future studies on the interplay of host anatomy and microbiome metabolism.

Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes.

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

Weakened APC/C activity at mitotic exit drives cancer vulnerability to KIF18A inhibition.

The efficacy of current antimitotic cancer drugs is limited by toxicity in highly proliferative healthy tissues. A cancer-specific dependency on the microtubule motor protein KIF18A therefore makes it an attractive therapeutic target. Not all cancers require KIF18A, however, and the determinants underlying this distinction remain unclear. Here, we show that KIF18A inhibition drives a modest and widespread increase in spindle assembly checkpoint (SAC) signaling from kinetochores which can result in lethal mitotic delays. Whether cells arrest in mitosis depends on the robustness of the metaphase-to-anaphase transition, and cells predisposed with weak basal anaphase-promoting complex/cyclosome (APC/C) activity and/or persistent SAC signaling through metaphase are uniquely sensitive to KIF18A inhibition. KIF18A-dependent cancer cells exhibit hallmarks of this SAC:APC/C imbalance, including a long metaphase-to-anaphase transition, and slow mitosis overall. Together, our data reveal vulnerabilities in the cell division apparatus of cancer cells that can be exploited for therapeutic benefit.

Cytotoxic Programming of CD4+ T Cells Is Regulated by Opposing Actions of the Related Transcription Factors Eos and Aiolos.

In contrast to the "helper" activities of most CD4+ T effector subsets, CD4+ cytotoxic T lymphocytes (CD4-CTLs) perform functions normally associated with CD8+ T and NK cells. Specifically, CD4-CTLs secrete cytotoxic molecules and directly target and kill compromised cells in an MHC class II-restricted fashion. The functions of these cells have been described in diverse immunological contexts, including their ability to provide protection during antiviral and antitumor responses, as well as being implicated in autoimmunity. Despite their significance to human health, the complete mechanisms that govern their programming remain unclear. In this article, we identify the Ikaros zinc finger transcription factor Eos (Ikzf4) as a positive regulator of CD4-CTL differentiation during murine immune responses against influenza virus infection. We find that the frequency of Eos+ cells is elevated in lung CD4-CTL populations and that the cytotoxic gene program is compromised in Eos-deficient CD4+ T cells. Consequently, we observe a reduced frequency and number of lung-residing, influenza virus-responsive CD4-CTLs in the absence of Eos. Mechanistically, we determine that this is due, at least in part, to reduced expression of IL-2 and IL-15 cytokine receptor subunits on the surface of Eos-deficient CD4+ T cells, both of which support the CD4-CTL program. Finally, we find that Aiolos, a related Ikaros family member and known CD4-CTL antagonist, represses Eos expression by antagonizing STAT5-dependent activation of the Ikzf4 promoter. Collectively, our findings reveal a mechanism wherein Eos and Aiolos act in opposition to regulate cytotoxic programming of CD4+ T cells.

Provider Perceptions Regarding Cardiopulmonary Resuscitation in Surgical Patients with Frailty.

OBJECTIVE: To characterize the perceptions of surgeons, anesthesiologists, and geriatricians regarding perioperative CPR in surgical patients with frailty. SUMMARY BACKGROUND DATA: The population of patients undergoing surgery is growing older and more frail. Despite a growing focus on goal-concordant care, frailty assessment, and debate regarding the appropriateness of cardiopulmonary resuscitation (CPR) in patients with frailty, providers' views regarding frailty and perioperative CPR are unknown. METHODS: We performed qualitative thematic analysis of transcripts from semi-structured interviews of anesthesiologists (8), surgeons (10), and geriatricians (9) who care for high-risk surgical patients at two academic medical centers in Boston, MA. The interview guide elicited clinicians' understanding of frailty, approach to decision-making regarding perioperative CPR, and perceptions of perioperative CPR in frail surgical patients. RESULTS: We identified 5 themes: perceptions of perioperative CPR in patients with frailty vary by provider specialty; judgments regarding appropriateness of CPR in surgical patients with frailty are typically multifactorial and include patient goals, age, comorbidities, and arrest etiology; resuscitation in patients with frailty is sometimes associated with moral distress; biases such as ableism and ageism may skew clinicians' perceptions of appropriateness of perioperative CPR in patients with frailty; and evidence to guide risk stratification for patients with frailty undergoing perioperative CPR is inadequate. CONCLUSIONS: Anesthesiologists, surgeons, and geriatricians offer different accounts of frailty's relevance to judgments regarding CPR in surgical patients. Divergent views regarding frailty and perioperative CPR may impede efforts to deliver goal-concordant care and suggest a need for research to inform risk stratification, predict patient-centered outcomes, and understand the role of potential biases such as ageism and ableism.

State of the art: Monitoring of the respiratory system during veno-venous extracorporeal membrane oxygenation.

Monitoring the patient receiving veno-venous extracorporeal membrane oxygenation (VV ECMO) is challenging due to the complex physiological interplay between native and membrane lung. Understanding these interactions is essential to understand the utility and limitations of different approaches to respiratory monitoring during ECMO. We present a summary of the underlying physiology of native and membrane lung gas exchange and describe different tools for titrating and monitoring gas exchange during ECMO. However, the most important role of VV ECMO in severe respiratory failure is as a means of avoiding further ergotrauma. Although optimal respiratory management during ECMO has not been defined, over the last decade there have been advances in multimodal respiratory assessment which have the potential to guide care. We describe a combination of imaging, ventilator-derived or invasive lung mechanic assessments as a means to individualise management during ECMO.

The intricate physiology of veno-venous extracorporeal membrane oxygenation: an overview for clinicians.

During veno-venous extracorporeal membrane oxygenation (V-V ECMO), blood is drained from the central venous circulation to be oxygenated and decarbonated by an artificial lung. It is then reinfused into the right heart and pulmonary circulation where further gas-exchange occurs. Each of these steps is characterized by a peculiar physiology that this manuscript analyses, with the aim of providing bedside tools for clinical care: we begin by describing the factors that affect the efficiency of blood drainage, such as patient and cannulae position, fluid status, cardiac output and ventilatory strategies. We then dig into the complexity of extracorporeal gas-exchange, with particular reference to the effects of extracorporeal blood-flow (ECBF), fraction of delivered oxygen (FdO2) and sweep gas-flow (SGF) on oxygenation and decarbonation. Subsequently, we focus on the reinfusion of arterialized blood into the right heart, highlighting the effects on recirculation and, more importantly, on right ventricular function. The importance and challenges of haemodynamic monitoring during V-V ECMO are also analysed. Finally, we detail the interdependence between extracorporeal circulation, native lung function and mechanical ventilation in providing adequate arterial blood gases while allowing lung rest. In the absence of evidence-based strategies to care for this particular group of patients, clinical practice is underpinned by a sound knowledge of the intricate physiology of V-V ECMO.

[Unusual gluteal localization of unicentric Castleman's disease: A case report and review of the literature]. / Localisation intramusculaire d'une maladie de Castleman unicentrique : cas clinique et revue de la littérature.

BACKGROUND: Castleman's disease is a rare and benign lymphoproliferative disorder which can be unicentric (UCD) or multicentric (MCD). UCD usually involves a single lymph node or less frequently a group of lymph nodes. The most common sites of nodal UCD presentation are the mediastinum, neck, abdomen and retroperitoneum. Rarely extranodal involvement has been reported. The intramuscular location is very unusual with only about 10 cases described in medical literature so far. CASE REPORT: We present a case of atypical localization of Castleman's disease occurring in the right gluteal area in a 40-years-old female patient. The patient was asymptomatic and clinical examination was unremarkable except for a right gluteal palpable mass. The CT scanner-guided needle core biopsy was inconclusive. A surgical excision was then performed that revealed a hyaline-vascular type of Castleman's disease. The patient has an uneventful post-operative course. CONCLUSION: The present case is instructive in the work-up of primary soft tissue tumors, for which Castleman's disease is extremely rare and not considered in the differential diagnosis of clinicians. Pathologists must be aware of its existence so that it can be evoked in the presence of a lymphoid population on histological examination.

Updated phylogeny of Vestimentifera (Siboglinidae, Polychaeta, Annelida) based on mitochondrial genomes, with a new species.

Siboglinid tubeworms are found at chemosynthetic environments worldwide and the Vestimentifera clade is particularly well known for their reliance on chemoautotrophic bacterial symbionts for nutrition. The mitochondrial genomes have been published for nine vestimentiferan species to date. This study provides new complete mitochondrial genomes for ten further Vestimentifera, including the first mitochondrial genomes sequenced for Alaysia spiralis, Arcovestia ivanovi, Lamellibrachia barhami, Lamellibrachia columna, Lamellibrachia donwalshi, and unnamed species of Alaysia and Oasisia. Phylogenetic analyses combining fifteen mitochondrial genes and the nuclear 18S rRNA gene recovered Lamellibrachia as sister to the remaining Vestimentifera and Riftia pachyptila as separate from the other vent-endemic taxa. Implications and auxiliary analyses regarding differing phylogenetic tree topologies, substitution saturation, ancestral state reconstruction, and divergence estimates are also discussed. Additionally, a new species of Alaysia is described from the Manus Basin.

Causes of Death in Cutaneous T-Cell Lymphoma Patients.

BACKGROUND: The advancing evolution toward a Th2 immune environment confers a progressive immunosuppression in patients with longstanding cutaneous T-cell lymphoma (CTCL). The conjunction of the disease-related immunosuppression as well as the immunosuppressive character of some CTCL treatments increase the risk of infectious and neoplastic diseases, sometimes with fatal outcomes. OBJECTIVES: The aim of the study was to prospectively study the causes of death in a cohort of CTCL patients, in a tertiary university skin cancer center. METHODS: All CTCL patients who died between 2008 and 2020 were included. The cause of the death was classified as directly or indirectly related or unrelated to CTCL. RESULTS: Over the study period, 31 (13F/18m) patients with CTCL died (mean age: 75.2 years), mean delay between diagnosis and death: 3.2 years (min: 1, max: 12 years), 58.1% of death causes were classified as indirect (infection), 12.9% directly related (blastic transformation), 22.5% unrelated, and 6.5% of unknown cause. 51.6% of mycosis fungoides (MF) patients who died had early-stage disease (1A-2A) or were on remission. 45.2% of dead patients had advanced-stage MF (2B-4B). Mean CRP level is increased in patients who died from infection whereas LDH level increased in patients with blastosis. A tertiary center is expected to manage of a higher proportion of CTCL patients with advanced-stage disease. CONCLUSIONS: As infection represented more than 50% of the causes of death in CTCL patients, particular attention should be given to preventive measures such as anti-infective vaccination. Regular surveillance of CRP and LDH levels could be helpful for follow-up of MF patients, respectively, with regards to infection and blastosis.

A holistic and comprehensive data approach validates the distribution of the critically endangered flapper skate (Dipturus intermedius).

Morphological similarities between skates of the genus Dipturus in the north-eastern Atlantic and Mediterranean have resulted in longstanding confusion, misidentification and misreporting. Current evidence indicates that the common skate is best explained as two species, the flapper skate (Dipturus intermedius) and the common blue skate (D. batis). However, some management and conservation initiatives developed prior to the separation continue to refer to common skate (as 'D. batis'). This taxonomic uncertainty can lead to errors in estimating population viability, distribution range, and impact on fisheries management and conservation status. Here, we demonstrate how a concerted taxonomic approach, using molecular data and a combination of survey, angler and fisheries data, in addition to expert witness statements, can be used to build a higher resolution picture of the current distribution of D. intermedius. Collated data indicate that flapper skate has a more constrained distribution compared to the perceived distribution of the 'common skate', with most observations recorded from Norway and the western and northern seaboards of Ireland and Scotland, with occasional specimens from Portugal and the Azores. Overall, the revised spatial distribution of D. intermedius has significantly reduced the extant range of the species, indicating a possibly fragmented distribution range.

The first records of sea star wasting disease in Crossaster papposus in Europe.

Sea star wasting disease (SSWD) refers to a suite of gross pathological signs observed in Asteroidea species. It presents to varying degrees as abnormal posture, epidermal ulceration, arm autotomy and eversion of viscera. We report observations of SSWD in the sunstar Crossaster papposus, the first observations of its kind in Europe. While the exact cause of SSWD remains unknown, studies have proposed pathogenic and environmental-stress pathways for disease outbreaks. Although the present observations do not support a precise aetiology, the presence of SSWD in a keystone predator may have wide reaching ecological and management implications.