RORα is a critical checkpoint for T cell and ILC2 commitment in the embryonic thymus.

Type 2 innate lymphoid cells (ILC2) contribute to immune homeostasis, protective immunity and tissue repair. Here we demonstrate that functional ILC2 cells can arise in the embryonic thymus from shared T cell precursors, preceding the emergence of CD4+CD8+ (double-positive) T cells. Thymic ILC2 cells migrated to mucosal tissues, with colonization of the intestinal lamina propria. Expression of the transcription factor RORα repressed T cell development while promoting ILC2 development in the thymus. From RNA-seq, assay for transposase-accessible chromatin sequencing (ATAC-seq) and chromatin immunoprecipitation followed by sequencing (ChIP-seq) data, we propose a revised transcriptional circuit to explain the co-development of T cells and ILC2 cells from common progenitors in the thymus. When Notch signaling is present, BCL11B dampens Nfil3 and Id2 expression, permitting E protein-directed T cell commitment. However, concomitant expression of RORα overrides the repression of Nfil3 and Id2 repression, allowing ID2 to repress E proteins and promote ILC2 differentiation. Thus, we demonstrate that RORα expression represents a critical checkpoint at the bifurcation of the T cell and ILC2 lineages in the embryonic thymus.

The Deubiquitinase OTULIN Is an Essential Negative Regulator of Inflammation and Autoimmunity.

Methionine-1 (M1)-linked ubiquitin chains regulate the activity of NF-κB, immune homeostasis, and responses to infection. The importance of negative regulators of M1-linked chains in vivo remains poorly understood. Here, we show that the M1-specific deubiquitinase OTULIN is essential for preventing TNF-associated systemic inflammation in humans and mice. A homozygous hypomorphic mutation in human OTULIN causes a potentially fatal autoinflammatory condition termed OTULIN-related autoinflammatory syndrome (ORAS). Four independent OTULIN mouse models reveal that OTULIN deficiency in immune cells results in cell-type-specific effects, ranging from over-production of inflammatory cytokines and autoimmunity due to accumulation of M1-linked polyubiquitin and spontaneous NF-κB activation in myeloid cells to downregulation of M1-polyubiquitin signaling by degradation of LUBAC in B and T cells. Remarkably, treatment with anti-TNF neutralizing antibodies ameliorates inflammation in ORAS patients and rescues mouse phenotypes. Hence, OTULIN is critical for restraining life-threatening spontaneous inflammation and maintaining immune homeostasis.

Polychromic Reporter Mice Reveal Unappreciated Innate Lymphoid Cell Progenitor Heterogeneity and Elusive ILC3 Progenitors in Bone Marrow.

Innate lymphoid cells (ILCs) play strategic roles in tissue homeostasis and immunity. ILCs arise from lymphoid progenitors undergoing lineage restriction and the development of specialized ILC subsets. We generated "5x polychromILC" transcription factor reporter mice to delineate ILC precursor states by revealing the multifaceted expression of key ILC-associated transcription factors (Id2, Bcl11b, Gata3, RORγt, and RORα) during ILC development in the bone marrow. This approach allowed previously unattained enrichment of rare progenitor subsets and revealed hitherto unappreciated ILC precursor heterogeneity. In vivo and in vitro assays identified precursors with potential to generate all ILC subsets and natural killer (NK) cells, and also permitted discrimination of elusive ILC3 bone marrow antecedents. Single-cell gene expression analysis identified a discrete ILC2-committed population and delineated transition states between early progenitors and a highly heterogeneous ILC1, ILC3, and NK precursor cell cluster. This diversity might facilitate greater lineage potential upon progenitor recruitment to peripheral tissues.

Inflammation-induced formation of fat-associated lymphoid clusters.

Fat-associated lymphoid clusters (FALCs) are a type of lymphoid tissue associated with visceral fat. Here we found that the distribution of FALCs was heterogeneous, with the pericardium containing large numbers of these clusters. FALCs contributed to the retention of B-1 cells in the peritoneal cavity through high expression of the chemokine CXCL13, and they supported B cell proliferation and germinal center differentiation during peritoneal immunological challenges. FALC formation was induced by inflammation, which triggered the recruitment of myeloid cells that expressed tumor-necrosis factor (TNF) necessary for signaling via the TNF receptors in stromal cells. Natural killer T cells (NKT cells) restricted by the antigen-presenting molecule CD1d were likewise required for the inducible formation of FALCs. Thus, FALCs supported and coordinated the activation of innate B cells and T cells during serosal immune responses.

Tissue-Restricted Adaptive Type 2 Immunity Is Orchestrated by Expression of the Costimulatory Molecule OX40L on Group 2 Innate Lymphoid Cells.

The local regulation of type 2 immunity relies on dialog between the epithelium and the innate and adaptive immune cells. Here we found that alarmin-induced expression of the co-stimulatory molecule OX40L on group 2 innate lymphoid cells (ILC2s) provided tissue-restricted T cell co-stimulation that was indispensable for Th2 and regulatory T (Treg) cell responses in the lung and adipose tissue. Interleukin (IL)-33 administration resulted in organ-specific surface expression of OX40L on ILC2s and the concomitant expansion of Th2 and Treg cells, which was abolished upon deletion of OX40L on ILC2s (Il7raCre/+Tnfsf4fl/fl mice). Moreover, Il7raCre/+Tnfsf4fl/fl mice failed to mount effective Th2 and Treg cell responses and corresponding adaptive type 2 pulmonary inflammation arising from Nippostrongylus brasiliensis infection or allergen exposure. Thus, the increased expression of OX40L in response to IL-33 acts as a licensing signal in the orchestration of tissue-specific adaptive type 2 immunity, without which this response fails to establish.

The transcription factor T-bet is essential for the development of NKp46+ innate lymphocytes via the Notch pathway.

NKp46+ innate lymphoid cells (ILCs) serve important roles in regulating the intestinal microbiota and defense against pathogens. Whether NKp46+ ILCs arise directly from lymphoid tissue-inducer (LTi) cells or represent a separate lineage remains controversial. We report here that the transcription factor T-bet (encoded by Tbx21) was essential for the development of NKp46+ ILCs but not of LTi cells or nuocytes. Deficiency in interleukin 22 (IL-22)-producing NKp46+ ILCs resulted in greater susceptibility of Tbx21-/- mice to intestinal infection. Haploinsufficient T-bet expression resulted in lower expression of the signaling molecule Notch, and Notch signaling was necessary for the transition of LTi cells into NKp46+ ILCs. Furthermore, NKp46+ ILCs differentiated solely from the CD4- LTi population, not the CD4+ LTi population. Our results pinpoint the regulation of Notch signaling by T-bet as a distinct molecular pathway that guides the development of NKp46+ ILCs.

Identification of aceNKPs, a committed common progenitor population of the ILC1 and NK cell continuum.

The development of innate lymphoid cell (ILC) transcription factor reporter mice has shown a previously unexpected complexity in ILC hematopoiesis. Using novel polychromic mice to achieve higher phenotypic resolution, we have characterized bone marrow progenitors that are committed to the group 1 ILC lineage. These common ILC1/NK cell progenitors (ILC1/NKP), which we call "aceNKPs", are defined as lineage-Id2+IL-7Rα+CD25-α4ß7-NKG2A/C/E+Bcl11b-. In vitro, aceNKPs differentiate into group 1 ILCs, including NK-like cells that express Eomes without the requirement for IL-15, and produce IFN-γ and perforin upon IL-15 stimulation. Following reconstitution of Rag2-/-Il2rg-/- hosts, aceNKPs give rise to a spectrum of mature ILC1/NK cells (regardless of their tissue location) that cannot be clearly segregated into the traditional ILC1 and NK subsets, suggesting that group 1 ILCs constitute a dynamic continuum of ILCs that can develop from a common progenitor. In addition, aceNKP-derived ILC1/NK cells effectively ameliorate tumor burden in a model of lung metastasis, where they acquired a cytotoxic NK cell phenotype. Our results identify the primary ILC1/NK progenitor that lacks ILC2 or ILC3 potential and is strictly committed to ILC1/NK cell production irrespective of tissue homing.

Transcription factor RORα is critical for nuocyte development.

Nuocytes are essential in innate type 2 immunity and contribute to the exacerbation of asthma responses. Here we found that nuocytes arose in the bone marrow and differentiated from common lymphoid progenitors, which indicates they are distinct, previously unknown members of the lymphoid lineage. Nuocytes required interleukin 7 (IL-7), IL-33 and Notch signaling for development in vitro. Pro-T cell progenitors at double-negative stage 1 (DN1) and DN2 maintained nuocyte potential in vitro, although the thymus was not essential for nuocyte development. Notably, the transcription factor RORα was critical for the development of nuocytes and their role in the expulsion of parasitic worms.

MHCII-mediated dialog between group 2 innate lymphoid cells and CD4(+) T cells potentiates type 2 immunity and promotes parasitic helminth expulsion.

Group 2 innate lymphoid cells (ILC2s) release interleukin-13 (IL-13) during protective immunity to helminth infection and detrimentally during allergy and asthma. Using two mouse models to deplete ILC2s in vivo, we demonstrate that T helper 2 (Th2) cell responses are impaired in the absence of ILC2s. We show that MHCII-expressing ILC2s interact with antigen-specific T cells to instigate a dialog in which IL-2 production from T cells promotes ILC2 proliferation and IL-13 production. Deletion of MHCII renders IL-13-expressing ILC2s incapable of efficiently inducing Nippostrongylus brasiliensis expulsion. Thus, during transition to adaptive T cell-mediated immunity, the ILC2 and T cell crosstalk contributes to their mutual maintenance, expansion and cytokine production. This interaction appears to augment dendritic-cell-induced T cell activation and identifies a previously unappreciated pathway in the regulation of type-2 immunity.

Plasma cell development and survival.

Plasma cells have long been recognized as the basis of humoral immunity, yet we are only now beginning to appreciate the complexities of plasma cell development and the fact that not all plasma cells are created equal. In vivo, plasma cells can arise from two developmental routes: one occurring outside the follicle and another within the germinal center. A B cell's decision to follow one of these pathways is in part determined by the phenotypic subset to which it belongs and is also influenced by the nature of the antigen eliciting the response and the affinity of the B-cell receptor for that antigen. Once a plasma cell has chosen one of these pathways, the outcome of differentiation is relatively hard-wired. However, the phenotype of the plasma cells arising from these two pathways is distinct in terms of survival, location, and the quantity and quality of antibody they secrete. The extra-follicular pathway represents a relatively unchecked route to differentiation resulting in the generation of short-lived plasma cells that secrete low-affinity antibody. The germinal center response, however, allows the integration of external signals to delay plasma cell differentiation, eventually generating a plasma cell that secretes high-affinity antibody of an appropriate class, and that persists for a lifetime. The means by which these varying properties are conferred to a developing plasma cell are the subject of intense investigation.

Innate lymphoid cells in the airways.

The airways, similar to other mucosal surfaces, are continuously exposed to the outside environment and a barrage of antigens, allergens, and microorganisms. Of critical importance therefore is the ability to mount rapid and effective immune responses to control commensal and pathogenic microbes, while simultaneously limiting the extent of these responses to prevent immune pathology and chronic inflammation. The function of the adaptive immune response in controlling these processes at mucosal surfaces has been well documented but the important role of the innate immune system, particularly the recently identified family of innate lymphoid cells, has only lately become apparent. In this review, we give an overview of the innate lymphoid cells that exist in the airways and examine the evidence pertaining to their emerging roles in airways immunity, inflammation, and homeostasis.

Increased red cell turnover in a line of CD22-deficient mice is caused by Gpi1c: a model for hereditary haemolytic anaemia.

CD22, an inhibitory co-receptor of the BCR, has been identified as a potential candidate gene for the development of autoimmune haemolytic anaemia in mice. In this study, we have examined Cd22(tm1Msn) CD22-deficient mice and identified an increase in RBC turnover and stress erythropoiesis, which might be consistent with haemolysis. We then, however, eliminated CD22 deficiency as the cause of accelerated RBC turnover and established that enhanced RBC turnover occurs independently of B cells and anti-RBC autoanti-bodies. Accelerated RBC turnover in this particular strain of CD22-deficient mice is red cell intrinsic and appears to be the consequence of a defective allele of glucose phosphate isomerase, Gpi1(c). This form of Gpi1 was originally derived from wild mice and results in a substantial reduction in enzyme activity. We have identified the polymorphism that causes impaired catalytic activity in the Gpi1(c) allele, and biochemically confirmed an approximate 75% reduction of GPI1 activity in Cd22(-/-) RBCs. The Cd22(-/-).Gpi1(c) congenic mouse provides a novel animal model of GPI1-deficiency, which is one of the most common causes of chronic non-spherocytic haemolytic anaemia in humans.

An Unusual Case of Acute Scrotal Pain.

Patients frequently present to the emergency department with complaints of scrotal or testicular pain. Generally, there is an algorithmic approach to workup, which includes assessment for torsion, infection, or vascular causes, and musculoskeletal causes of pain are also sometimes considered. Spinal cord pathology, however, is less often explored as a cause of testicular pain. Here, we present a case of a 45-year-old man with end-stage renal disease and hypertension who presented with acute testicular pain. After a comprehensive workup, however, the source of pain was not initially found. Progression of the patient's symptoms led to the diagnosis of spontaneous spinal subdural hematoma. This atypical presentation of a rare diagnosis is also interesting due to the patient's concomitant diagnosis of an otherwise asymptomatic COVID-19 infection. While our case represents an atypical combination of clinical features, it also illustrates the importance of continued vigilance and ongoing workup when patients present with severe pain and unclear causes of their symptoms.

Age-induced changes in anti-tumor immunity alter the tumor immune infiltrate and impact response to immuno-oncology treatments.

Introduction: Immuno-oncology (IO) research relies heavily on murine syngeneic tumor models. However, whilst the average age for a cancer diagnosis is 60 years or older, for practical purposes the majority of preclinical studies are conducted in young mice, despite the fact that ageing has been shown to have a significant impact on the immune response. Methods: Using aged (60-72 weeks old) mice bearing CT26 tumors, we investigated the impact of ageing on tumor growth as well as the immune composition of the tumor and peripheral lymphoid organs. Results: We found many differences in the immune cell composition of both the tumor and tumor-draining lymph node between aged and young mice, such as a reduction in the naïve T cell population and a decreased intratumoral CD8/Treg ratio in aged animals. We hypothesized that these differences may contribute to impaired anti-cancer immune responses in aged mice and therefore assessed the anti-tumor efficacy of different IO therapies in aged mice, including both co-stimulation (using an anti-OX40 antibody) and immune checkpoint blockade (using anti-PD-L1 and anti-CTLA-4 antibodies). Whilst aged mice retained the capacity to generate anti-tumor immune responses, these were significantly attenuated when compared to the responses observed in young mice. Discussion: These differences highlight the importance of age-related immunological changes in assessing and refining the translational insights gained from preclinical mouse models.

Trend of Outcome Metrics in Recent Out-of-Hospital-Cardiac-Arrest Research: A Narrative Review of Clinical Trials.

Cardiopulmonary resuscitation (CPR) research traditionally focuses on survival. In 2018, the International Liaison Committee on Resuscitation (ILCOR) proposed more patient-centered outcomes. Our narrative review assessed clinical trials after 2018 to identify the trends of outcome metrics in the field OHCA research. We performed a search of the PubMed database from 1 January 2019 to 22 September 2023. Prospective clinical trials involving adult humans were eligible. Studies that did not report any patient-related outcomes or were not available in full-text or English language were excluded. The articles were assessed for demographic information and primary and secondary outcomes. We included 89 studies for analysis. For the primary outcome, 31 (35%) studies assessed neurocognitive functions, and 27 (30%) used survival. For secondary outcomes, neurocognitive function was present in 20 (22%) studies, and survival was present in 10 (11%) studies. Twenty-six (29%) studies used both survival and neurocognitive function. Since the publication of the COSCA guidelines in 2018, there has been an increased focus on neurologic outcomes. Although survival outcomes are used frequently, we observed a trend toward fewer studies with ROSC as a primary outcome. There were no quality-of-life assessments, suggesting a need for more studies with patient-centered outcomes that can inform the guidelines for cardiac-arrest management.

Group 2 Innate Lymphoid Cells Exhibit Tissue-Specific Dynamic Behaviour During Type 2 Immune Responses.

Group 2 innate lymphoid cells (ILC2s) are early effectors of mucosal type 2 immunity, producing cytokines such as interleukin (IL)-13 to mediate responses to helminth infection and allergen-induced inflammation. ILC2s are also present in lymph nodes (LNs) and can express molecules required for antigen presentation, but to date there are limited data on their dynamic behaviour. We used a CD2/IL-13 dual fluorescent reporter mouse for in vivo imaging of ILC2s and Th2 T cells in real time following a type 2 priming helminth infection or egg injection. After helminth challenge, we found that ILC2s were the main source of IL-13 in lymphoid organs (Peyer's patches and peripheral LNs), and were located in T cell areas. Intravital imaging demonstrated an increase in IL-13+ ILC2 size and movement following helminth infection, but reduced duration of interactions with T cells compared with those in homeostasis. In contrast, in the intestinal mucosa, we observed an increase in ILC2-T cell interactions post-infection, including some of prolonged duration, as well as increased IL-13+ ILC2 movement. These data suggest that ILC2 activation enhances cell motility, with the potential to increase the area of distribution of cytokines to optimise the early generation of type 2 responses. The prolonged ILC2 interactions with T cells within the intestinal mucosa are consistent with the conclusion that contact-based T cell activation may occur within inflamed tissues rather than lymphoid organs. Our findings have important implications for our understanding of the in vivo biology of ILC2s and the way in which these cells facilitate adaptive immune responses.

Group-2 innate lymphoid cell-dependent regulation of tissue neutrophil migration by alternatively activated macrophage-secreted Ear11.

Type-2 immunity is characterised by interleukin (IL)-4, IL-5 and IL-13, eosinophilia, mucus production, IgE, and alternatively activated macrophages (AAM). However, despite the lack of neutrophil chemoattractants such as CXCL1, neutrophils, a feature of type-1 immunity, are observed in type-2 responses. Consequently, alternative mechanisms must exist to ensure that neutrophils can contribute to type-2 immune reactions without escalation of deleterious inflammation. We now demonstrate that type-2 immune-associated neutrophil infiltration is regulated by the mouse RNase A homologue, eosinophil-associated ribonuclease 11 (Ear11), which is secreted by AAM downstream of IL-25-stimulated ILC2. Transgenic overexpression of Ear11 resulted in tissue neutrophilia, whereas Ear11-deficient mice have fewer resting tissue neutrophils, whilst other type-2 immune responses are not impaired. Notably, administration of recombinant mouse Ear11 increases neutrophil motility and recruitment. Thus, Ear11 helps maintain tissue neutrophils at homoeostasis and during type-2 reactions when chemokine-producing classically activated macrophages are infrequently elicited.

Mapping Rora expression in resting and activated CD4+ T cells.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

Reproductive parameters in female yellow-blotched map turtles (Graptemys flavimaculata) from a historically contaminated site vs. a reference site.

Graptemys flavimaculata, the yellow-blotched map turtle, is a long-lived, threatened, species, endemic to the Pascagoula River drainage in Mississippi. During the 1980s, one branch of the drainage (i.e. the Leaf River) was impacted by effluent from a wood pulp processing plant known to contain endocrine disrupters. A decade later, we examined seasonal reproductive parameters (i.e. monthly plasma estradiol-17beta (E(2)), testosterone (T), vitellogenin (VTG) and follicular development) in adult female turtles from historically polluted and reference sites in the drainage to determine if legacy exposure to pollution impacts reproduction . We found no seasonal patterns in E(2) or T and these patterns did not differ between sites. However, E(2) differed significantly among ovarian stages for the reference, but not pollutant exposed females. A significantly greater percentage of reference site females were able to produce a second clutch than females from the historically polluted site (50% and 17%). Additionally, there was a significant positive correlation between E(2) with VTG levels for reference, but not pollutant exposed females. Body and yolk tissue contaminant analysis indicated that exposure to pollutants is presently minimal and unlikely the cause of the reproductive differences observed between sites; instead, differences are potentially due to exposure history.

A stromal cell niche sustains ILC2-mediated type-2 conditioning in adipose tissue.

Group-2 innate lymphoid cells (ILC2), type-2 cytokines, and eosinophils have all been implicated in sustaining adipose tissue homeostasis. However, the interplay between the stroma and adipose-resident immune cells is less well understood. We identify that white adipose tissue-resident multipotent stromal cells (WAT-MSCs) can act as a reservoir for IL-33, especially after cell stress, but also provide additional signals for sustaining ILC2. Indeed, we demonstrate that WAT-MSCs also support ICAM-1-mediated proliferation and activation of LFA-1-expressing ILC2s. Consequently, ILC2-derived IL-4 and IL-13 feed back to induce eotaxin secretion from WAT-MSCs, supporting eosinophil recruitment. Thus, MSCs provide a niche for multifaceted dialogue with ILC2 to sustain a type-2 immune environment in WAT.