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Supplemental figures S1-S11

Supplementary patient data, supplementary materials and methods

Supplemental patient tables (1-3) and mass cytometry reagent table (4)

The composition of the gut microbiota in early childhood is linked to asthma risk but the role of the gut microbiota in older patients with established asthma is less clear. Here, we used a cohort of 38 school-aged children (19 with asthma) and 57 adults (17 with asthma) to develop a model that aids in the design of mechanistic experiments in gnotobiotic mice. These experiments show that enterotoxigenic Bacteroides fragilis (ETBF) is associated with increased gut permeability, oxidative stress, and markers of Th17-mediated inflammation in the lungs of mice following ovalbumin sensitization and challenge (OSC). Further, ETBF is enriched in a human population with asthma compared to healthy controls. Our results provide evidence that ETBF has the potential to alter the phenotype of airway inflammation in a subset of patients with asthma outside of early childhood which suggests that therapies targeting the gut microbiota may be helpful tools for asthma control.

The signature hallmark of adaptive immunity is the evolution of somatically rearranged antigen receptors, which confer both diversity and specificity to T and B lymphocytes. For decades, immunologists have observed cells which possess lymphoid characteristics yet lack such antigen-specific receptors. Collectively, these populations are referred to as innate lymphoid cells (ILCs) (Vivier et al. in Cell 174(5):1054-1066, 2018). Cytotoxic natural killer (NK) cells and lymphoid tissue-inducing cells (LTi), which contribute to the formation of lymphoid organs during embryogenesis, are the earliest described ILCs. Subsequently, diverse populations of ILCs have been described based on the signature cytokines they produce. Group 1 ILCs (ILC1) produce IFNγ, group 2 ILCs (ILC2) produce IL-5 and IL-13, and group 3 ILCs (ILC3) produce IL-22 and IL-17. In contrast to adaptive lymphocytes which take several days to undergo clonal expansion and acquire effector functions, ILCs secrete cytokines rapidly in response to activating signals in their tissue of residence. ILCs may also directly regulate adaptive lymphocytes and myeloid cells through co-stimulatory molecules and soluble factors. Thus, ILCs play important roles in both the initiation and amplification of the immune response. When properly regulated, ILCs maintain intestinal homeostasis and protect the host from infection by various pathogens. However, dysregulation of mucosal immunity drives intestinal inflammation and contributes to pathology, such as inflammatory bowel disease (IBD). In this review, we outline the roles that ILCs play in amplifying or regulating intestinal inflammation as well as ongoing efforts to target these disease mechanisms for IBD therapy.

Natural killer (NK) cells are an emerging cancer cellular therapy and potent mediators of antitumor immunity. Cytokine-induced memory-like (ML) NK cellular therapy is safe and induces remissions in patients with acute myeloid leukemia (AML). However, the dynamic changes in phenotype that occur after NK-cell transfer that affect patient outcomes remain unclear. Here, we report comprehensive multidimensional correlates from ML NK cell–treated patients with AML using mass cytometry. These data identify a unique in vivo differentiated ML NK–cell phenotype distinct from conventional NK cells. Moreover, the inhibitory receptor NKG2A is a dominant, transcriptionally induced checkpoint important for ML, but not conventional NK-cell responses to cancer. The frequency of CD8α+ donor NK cells is negatively associated with AML patient outcomes after ML NK therapy. Thus, elucidating the multidimensional dynamics of donor ML NK cells in vivo revealed critical factors important for clinical response, and new avenues to enhance NK-cell therapeutics. Significance: Mass cytometry reveals an in vivo memory-like NK-cell phenotype, where NKG2A is a dominant checkpoint, and CD8α is associated with treatment failure after ML NK–cell therapy. These findings identify multiple avenues for optimizing ML NK–cell immunotherapy for cancer and define mechanisms important for ML NK–cell function. This article is highlighted in the In This Issue feature, p. 1775

Este estudo apresenta uma perspectiva sobre investigações de acidentes na indústria de óleo e gás por meio da análise de dezesseis relatórios de investigação de acidentes. Os acidentes foram representados utilizando método AcciMap, onde cada um dos fatorescontribuintes identificados foi traçado em seu nível sociotécnico relacionado ao sistema.Após, os fatores contribuintes foram classificados em categorias genéricas permitindo a abstração dos dados e a análise da distribuição de frequência entre os diferentes níveis. Dos589 fatores contribuintes identificados, 55% aparecem nos níveis operacionais, 37% nos níveis de companhia e 8% nos níveis de regulação e governo. Os resultados indicam que aindependência dos comitês de investigação e os métodos adotados são determinantes para o nível de abordagem sistêmica e de aprendizagem em qualquer investigação.

BACKGROUNDS For patients with medically refractory ulcerative colitis (UC), total proctocolectomy and ileal pouch-anal anastomosis (IPAA) has been the definitive surgery of choice. Approximately 10% of patients who were initially diagnosed with UC and underwent an IPAA, subsequently develop a condition referred to as Crohn’s disease-like phenotype of ileoanal pouch (CDP). CDP is typified by ulcers in the pre-pouch ileum, stricture involving the pouch or pre-pouch ileum, and/or fistulas. The etiology of CDP remains unknown and remains difficult to diagnose and treat. To define the mucosal immunology of CDP, we analyzed ileal samples from patients with CDP and patients with a normal pouch and pre-pouch ileum using mass cytometry (CyTOF). METHODS Ileal mucosal biopsies from patients with a history of CDP with long-segmental pre-pouch ileitis (n=10) and those with a normal pouch and pre-pouch ileum after IPAA for UC (n=9) were collected during routine pouchoscopy, frozen in medium, and processed, barcoded and analyzed in one CyTOF experiment. Data were analyzed by using viSNE for dimensionality reduction and CITRUS for clustering in Cytobank. RESULTS Compared with patients with a normal pouch/ileum, we observed the following in ileal samples from CDP based on viSNE analysis (Figure 1): 1) A decreased abundance of CD8+CD103+ tissue-resident T cells (TRM). This result is similar to data from colonic mucosal samples in patients with UC (our internal data not shown here and others), but unlike data from prior studies derived from inflamed ileal mucosal specimens in patients with CD (native bowel anatomy without IPAA) which had increased CD8+ TRM. 2) An increase in CD4+CD103-CD161+ Th17 cells previously linked to an activated Th17 phenotype in Crohn’s disease specimens. 3) Increased CD4+CD103-CD45RA+ T cells associated with reduced IL-10/IL-4 and increased TNF-a in IBD. 4) A higher abundance of CD4+CD103-TIGIT+ T cells that were previously shown to predict severity of pediatric IBD. 5) A decrease in CD8+CD103+CD127+ TRM in CDP, although its role in IBD is unclear. The findings from viSNE were confirmed by CITRUS (Figure 2). Further analyses of pouch samples as well as ileal samples from UC and CD with native GI anatomy (for comparison) using CyTOF and scRNAseq are currently underway. CONCLUSIONS We used mass cytometry to identify immune cells specific to ileal mucosa from patients with CDP. Interestingly, immune cells from the inflamed pre-pouch ileum share some features of those from either UC or CD with native bowel anatomy. We also identified an altered immune cell population which appears to be specific to CDP. These data reveal a unique immune landscape of CDP which may serve as a foundation for identifying therapeutic approaches that target specific cell populations for medical management of CDP.

Getting around the blood–brain barrier The meninges comprise three membranes that surround and protect the central nervous system (CNS). Recent studies have noted the existence of myeloid cells resident there, but little is known about their ontogeny and function, and whether other meningeal immune cell populations have important roles remains unclear (see the Perspective by Nguyen and Kubes). Cugurra et al. found in mice that a large proportion of continuously replenished myeloid cells in the dura mater are not blood derived, but rather transit from cranial bone marrow through specialized channels. In models of CNS injury and neuroinflammation, the authors demonstrated that these myeloid cells have an immunoregulatory phenotype compared with their more inflammatory blood-derived counterparts. Similarly, Brioschi et al. show that the meninges host B cells that are also derived from skull bone marrow, mature locally, and likely acquire a tolerogenic phenotype. They further found that the brains of aging mice are infiltrated by a second population of age-associated B cells, which come from the periphery and may differentiate into autoantibody-secreting plasma cells after encountering CNS antigens. Together, these two studies may inform future treatment of neurological diseases. Science , abf7844, abf9277, this issue p. eabf7844 , p. eabf9277 ; see also abj8183, p. 396

Checkpoint immunotherapy unleashes T cell control of tumors, but is undermined by immunosuppressive myeloid cells. TREM2 is a myeloid receptor that transmits intracellular signals that sustain microglial responses during Alzheimer’s disease. TREM2 is also expressed by tumor-infiltrating macrophages. Here, we found that Trem2-/- mice are more resistant to growth of various cancers than wild-type mice and are more responsive to anti-PD-1 immunotherapy. Furthermore, treatment with anti-TREM2 mAb curbed tumor growth and fostered regression when combined with anti-PD-1. scRNA-seq revealed that both TREM2 deletion and anti-TREM2 are associated with scant MRC1+ and CX3CR1+ macrophages in the tumor infiltrate, paralleled by expansion of myeloid subsets expressing immunostimulatory molecules that promote improved T cell responses. TREM2 was expressed in tumor macrophages in over 200 human cancer cases and inversely correlated with prolonged survival for two types of cancer. Thus, TREM2 might be targeted to modify tumor myeloid infiltrates and augment checkpoint immunotherapy. Citation Format: Martina Molgora, Blanda Di Luccia, Darya Khantakova, Natalia Jaeger, Rafael Sanguinetti Czepielewski, Cristiane Secca da Silva, Jennifer Bando, Susan Gilfillan, Marina Cella, Robert Schreiber, Marco Colonna. TREM2+ macrophages mediate immunosuppression in tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3615.

SUMMARY Homeostatic mucosal immune responses are fine-tuned by naturally evolved interactions with native microbes, and integrating these relationships into experimental models can provide new insights into human diseases. Here, we leverage a murine-adapted airway microbe, Bordetella pseudohinzii (Bph), to investigate how chronic colonization impacts mucosal immunity and the development of allergic airway inflammation (AAI). Colonization with Bph induces the differentiation of interleukin-17A (IL-17A)-secreting T-helper cells that aid in controlling bacterial abundance. Bph colonization protects from AAI and is associated with increased production of secretory leukocyte protease inhibitor (SLPI), an antimicrobial peptide with anti-inflammatory properties. These findings are additionally supported by clinical data showing that higher levels of upper respiratory SLPI correlate both with greater asthma control and the presence of Haemophilus, a bacterial genus associated with AAI. We propose that SLPI could be used as a biomarker of beneficial host-commensal relationships in the airway., In Brief Asthma is known to be modified by airway microbes. Jaeger et al. use a murine-adapted bacterium to show that airway colonization evokes a Th17 response associated with increased SLPI, an antimicrobial peptide, and protection from lung inflammation. In people, SLPI was correlated with airway microbiota composition., Graphical Abstract

Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Here, we profile, via multiple single-cell technologies, T cells purified from the intestinal epithelium and lamina propria (LP) from terminal ileum resections of adult severe CD cases. We find that intraepithelial lymphocytes (IEL) contain several unique T cell subsets, including NKp30+γδT cells expressing RORγt and producing IL-26 upon NKp30 engagement. Further analyses comparing tissues from non-inflamed and inflamed regions of patients with CD versus healthy controls show increased activated TH17 but decreased CD8+T, γδT, TFH and Treg cells in inflamed tissues. Similar analyses of LP find increased CD8+, as well as reduced CD4+T cells with an elevated TH17 over Treg/TFH ratio. Our analyses of CD tissues thus suggest a potential link, pending additional validations, between transmural inflammation, reduced IEL γδT cells and altered spatial distribution of IEL and LP T cell subsets., Crohn’s disease results from transmural inflammation in the gut, but analyses of local immune populations are still lacking. Here, the authors show, by combining multiple single-cell approaches, that intraepithelial and lamina propria T cells are heterogenous, show unique phenotypes, and exhibit altered subsets upon inflammation.

Drug-induced liver injury (DILI) is a major cause of acute liver failure (ALF), where hepatocyte necrotic products trigger liver inflammation, release of CXC chemokine receptor 2 (CXCR2) ligands (IL-8) and other neutrophil chemotactic molecules. Liver infiltration by neutrophils is a major cause of the life-threatening tissue damage that ensues. A GRPR (gastrin-releasing peptide receptor) antagonist impairs IL-8-induced neutrophil chemotaxis in vitro. We investigated its potential to reduce acetaminophen-induced ALF, neutrophil migration, and mechanisms underlying this phenomenon. We found that acetaminophen-overdosed mice treated with GRPR antagonist had reduced DILI and neutrophil infiltration in the liver. Intravital imaging and cell tracking analysis revealed reduced neutrophil mobility within the liver. Surprisingly, GRPR antagonist inhibited CXCL2-induced migration in vivo, decreasing neutrophil activation through CD11b and CD62L modulation. Additionally, this compound decreased CXCL8-driven neutrophil chemotaxis in vitro independently of CXCR2 internalization, induced activation of MAPKs (p38 and ERK1/2) and downregulation of neutrophil adhesion molecules CD11b and CD66b. In silico analysis revealed direct binding of GRPR antagonist and CXCL8 to the same binding spot in CXCR2. These findings indicate a new potential use for GRPR antagonist for treatment of DILI through a mechanism involving adhesion molecule modulation and possible direct binding to CXCR2.

In-depth immune profiling can guide the development and targeting of cancer immunotherapies. In this issue, Gherardin and colleagues find a strong signature of γδ T cells in the immune infiltrate of Merkel cell carcinoma that correlates with a more favorable prognosis and heightened responsiveness to checkpoint inhibitors. This may lead to future γδ T cell–targeted immunotherapies. See article by Gherardin et al., p. 612

Crohn’s disease (CD) is a chronic transmural inflammation of intestinal segments caused by dysregulated interaction between microbiome and gut immune system. Recurrent/relapsing CD and resistance to medical treatments result in complications requiring surgery. High-dimensional single-cell profiling approaches, such as scRNA-seq and mass cytometry, have been recently performed on intestinal specimens from patients with IBD and controls. However, most of these studies have analyzed whole mucosal biopsies or the lamina propria (LP) compartment, while few have addressed the intraepithelial lymphocytes (IEL) compartment. Here, we profiled T cells purified from the IEL and LP from terminal ileum resections of adult severe CD cases by single cell technologies. Our study defined a vast heterogeneity of T cell lineages in the IEL compartment. IEL included, among others, unique γδT cell subsets: NKp30+γδ T cells expressing RORγt, which produced IL-26 upon NKp30 engagement and a subset expressing PDGFD and CSF1, which may act on epithelial cells, IEL ILC1s, and macrophages, respectively. We have also observed long-lived memory TCF7+CD8+ T cells expressing DC chemoattractants and TFH subsets that may respond to distinct glutathione-conjugated lipids. CD IEL showed a significant increase of activated TH17, coupled with decreased CD8+ T cells, γδT cells, TFH, and Treg. Conversely, the LP showed increased CD8+ T cells and reduced CD4+ T cells with a relative increase of TH17 over Treg/TFH. Results provide an unbiased view of diversity of cell lineages and their functional states in the intestinal mucosa of controls and CD and identify an altered spatial distribution of T cell subsets between the IEL and the LP compartments.

Summary Natural killer (NK) cells are cytotoxic innate lymphoid cells (ILCs) that mediate antiviral and antitumor responses and require the transcriptional regulator Eomesodermin (Eomes) for early development. However, the role of Eomes and its molecular program in mature NK cell biology is unclear. To address this, we develop a tamoxifen-inducible, type-1-ILC-specific (Ncr1-targeted) cre mouse and combine this with Eomes-floxed mice. Eomes deletion after normal NK cell ontogeny results in a rapid loss of NK cells (but not ILC1s), with a particularly profound effect on penultimately mature stage III NK cells. Mechanisms responsible for stage III reduction include increased apoptosis and impaired maturation from stage II precursors. Induced Eomes deletion also decreases NK cell cytotoxicity and abrogates in vivo rejection of major histocompatibility complex (MHC)-class-I-deficient cells. However, other NK cell functional responses, and stage IV NK cells, are largely preserved. These data indicate that mature NK cells have distinct Eomes-dependent and -independent stages., Graphical Abstract, Highlights • Induced Eomes deletion results in a rapid decrease in NK cell numbers • Eomes-deleted stage III NK cells exhibit increased apoptosis • Eomes-deleted stage II and III NK cells exhibit differentiation defects • Induced Eomes deletion compromises NK cytotoxicity and MHCI−/− rejection in vivo, The transcription factor Eomes is important for early natural killer (NK) cell development. Wagner et al. utilize an inducible, type 1 ILC-specific cre model to demonstrate a stage-specific role for Eomes in NK cell survival and homeostasis as well as a persistent requirement for Eomes in promoting NK cell cytotoxicity.

O setor de assistência à saúde atualmente, carece de técnicas de gerenciamento que visem a melhoria dos processos, permitindo a redução de custos ao otimizar os recursos existentes. O sistema de saúde, classificado como um sistema complexo devido a diversidade de interações não-lineares que apresenta, vem sendo estudado sob a ótica da engenharia de resiliência que enfatiza a necessidade de regular o seu funcionamento diante de uma situação adversa. Por sua vez, a resiliência é facilitada por meio do uso de folgas, que fornecem recursos reservas para lidar com as variabilidades. Ao passo que as folgas são importantes para manter a resiliência, também podem ser recursos ociosos que tendem a mascarar as variabilidades, quando em excesso. Portanto, um equilíbrio deve ser considerado entre o mínimo necessário para manter a resiliência do sistema e o máximo que não venha a se enquadrar como desperdício de recursos. Para isso, esse trabalho desenvolve e aplica uma framework que visa analisar qualitativa e quantitativamente as folgas a fim de reprojetar o sistema sócio-técnico complexo. O estudo foi realizado numa maternidade, referência no atendimento privado. Os processos de atravessamento do fluxo de valor da paciente foram mapeados, permitindo a identificação de 17 fontes de variabilidade e 20 recursos de folga. O reprojeto do sistema de trabalho envolveu a classificação das folgas entre os requisitos levantados, de maneira a priorizar as variabilidades menos cobertas pelas folgas. A framework se mostrou eficaz ao elencar as prioridades no redesenho das folgas e variabilidades, a fim de tornar o sistema estudado mais resiliente. The healthcare sector currently lacks management techniques that aim to improve processes, allowing cost reduction by optimizing existing resources. The health system, classified as a complex system due to the diversity of nonlinear interactions that it presents, has been studied from the point of view of resilience engineering that emphasizes the need to regulate its functioning in the face of an adverse situation. In turn, resilience is facilitated through the use of slack, which provide reserve resources to deal with variability. While slack are important to maintaining resilience, it can also be idle resources that tend to mask variability when in excess. Therefore, a balance must be considered between the minimum necessary to maintain the resilience of the system and the maximum that doesn’t fit as a waste of resources. For this, this work develops and applies a framework that aims to analyze qualitatively and quantitatively the slack in order to redesign the complex socio-technical system. The study was performed in a maternity hospital, that is a reference in private care. The processes of flow of the patient's value were mapped, allowing the identification of 17 sources of variability and 20 resources of slack. The redesign of the work system involved the classification of slack between the requisites raised, in order to prioritize the variabilities less covered by the slack. The framework proved to be effective in highlighting the priorities in the redesign of the slack and variabilities, in order to make the studied system more resilient.