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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

TH2 cell development and function.

T helper 2 (TH2) cells orchestrate protective type 2 immune responses, such as those that target helminths and facilitate tissue repair, but also contribute to chronic inflammatory diseases, such as asthma and allergy. Here, we review recent insights into how diverse molecular signals from cellular sources, including dendritic cells, innate lymphoid cells and the epithelium, are integrated by T cells to guide the transcriptional and epigenetic changes necessary for TH2 cell differentiation. Our improved understanding of these pathways has opened new avenues for therapeutically targeting TH2 cells in asthma and allergy. The advent of comprehensive single-cell transcriptomics along with improvements in single-cell proteomics and the generation of novel in vivo cell fate mapping techniques promise to expand our understanding of T cell diversity and offer new insight into disease-related heterogeneity and plasticity of TH cell responses.

Type-2 innate lymphoid cells control the development of atherosclerosis in mice.

Type-2 innate lymphoid cells (ILC2) are a prominent source of type II cytokines and are found constitutively at mucosal surfaces and in visceral adipose tissue. Despite their role in limiting obesity, how ILC2s respond to high fat feeding is poorly understood, and their direct influence on the development of atherosclerosis has not been explored. Here, we show that ILC2 are present in para-aortic adipose tissue and lymph nodes and display an inflammatory-like phenotype atypical of adipose resident ILC2. High fat feeding alters both the number of ILC2 and their type II cytokine production. Selective genetic ablation of ILC2 in Ldlr-/- mice accelerates the development of atherosclerosis, which is prevented by reconstitution with wild type but not Il5-/- or Il13-/- ILC2. We conclude that ILC2 represent a major innate cell source of IL-5 and IL-13 required for mounting atheroprotective immunity, which can be altered by high fat diet.

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.

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.

Bcl11b is essential for group 2 innate lymphoid cell development.

Group 2 innate lymphoid cells (ILC2s) are often found associated with mucosal surfaces where they contribute to protective immunity, inappropriate allergic responses, and tissue repair. Although we know they develop from a common lymphoid progenitor in the bone marrow (BM), the specific lineage path and transcriptional regulators that are involved are only starting to emerge. After ILC2 gene expression analysis we investigated the role of Bcl11b, a factor previously linked to T cell commitment, in ILC2 development. Using combined Bcl11b-tom and Id2-gfp reporter mice, we show that Bcl11b is expressed in ILC2 precursors in the BM and maintained in mature ILC2s. In vivo deletion of Bcl11b, by conditional tamoxifen-induced depletion or by Bcl11b(-/-) fetal liver chimera reconstitution, demonstrates that ILC2s are wholly dependent on Bcl11b for their development. Notably, in the absence of Bcl11b there is a concomitant expansion of the RORγt(+) ILC3 population, suggesting that Bcl11b may negatively regulate this lineage. Using Nippostrongylus brasiliensis infection, we reveal that the absence of Bcl11b leads to impaired worm expulsion, caused by a deficit in ILC2s, whereas Citrobacter rodentium infection is cleared efficiently. These data clearly establish Bcl11b as a new factor in the differentiation of ILC2s.

The tyrosine kinase Lyn limits the cytokine responsiveness of plasma cells to restrict their accumulation in mice.

Maintenance of an appropriate number of plasma cells, long-lived antibody-producing cells that are derived from B cells, is essential for maintaining immunological memory while limiting disease. Plasma cell survival relies on extrinsic factors, the limited availability of which determines the size of the plasma cell population. Mice deficient in the nonreceptor tyrosine kinase Lyn are prone to an autoimmune disease that is characterized by inflammation and an excess of plasma cells (plasmacytosis). We demonstrated that the plasmacytosis was intrinsic to B cells and independent of inflammation. We also showed that Lyn attenuated signaling by signal transducer and activator of transcription 3 (STAT3) and STAT5 in response to the cytokines interleukin-6 (IL-6) and IL-3, respectively, in two previously uncharacterized plasma cell signaling pathways. Thus, in the absence of Lyn, the survival of plasma cells was improved, which enabled the plasma cells to become established in excess numbers in niches in vivo. These data identify Lyn as a key regulator of survival signaling in plasma cells, limiting plasma cell accumulation and autoimmune disease susceptibility.

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.

Development and function of group 2 innate lymphoid cells.

The innate lymphoid cell (ILC) family has recently expanded with the discovery of type-2 innate lymphoid cells (ILC2). These cells arise from lymphoid progenitors in the bone marrow and, under the control of the transcriptional regulators RORα and Gata3, they mature to give rise to IL-5, IL-9 and IL-13 producing ILC2. These cells are critical components of the innate immune response to parasitic worm infections and have also been implicated in the pathogenesis of asthma and allergy. Recent advances in our understanding of the molecular regulation of ILC2 development and function now present the opportunity to develop new genetic models to assess ILC2 immune function and to investigate possible therapeutic interventions.

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.