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Tertiary lymphoid structures (TLS) are ectopic cellular aggregates that resemble secondary lymphoid organs in their composition and structural organization. In contrast to secondary lymphoid organs, TLS are not imprinted during embryogenesis but are formed in non-lymphoid tissues in response to local inflammation. TLS structures exhibiting a variable degree of maturation are found in solid tumors. They are composed of various immune cell types including dendritic cells and antigen-specific B and T lymphocytes, that together, actively drive the immune response against tumor development and progression. This review highlights the successive steps leading to tumor TLS formation and its association with clinical outcomes. We discuss the role played by tumor-infiltrating B lymphocytes and plasma cells, their prognostic value in solid tumors and immunotherapeutic responses and their potential for future targeting.

Peripheral CD8 + T cell tolerance is a checkpoint in both autoimmune disease and anti-cancer immunity. Despite its importance, the relationship between tolerance-induced states and other CD8 + T cell differentiation states remains unclear. Using flow cytometric phenotyping, single-cell RNA sequencing (scRNA-seq), and chromatin accessibility profiling, we demonstrated that in vivo peripheral tolerance to a self-antigen triggered a fundamentally distinct differentiation state separate from exhaustion, memory, and functional effector cells but analogous to cells defectively primed against tumors. Tolerant cells diverged early and progressively from effector cells, adopting a transcriptionally and epigenetically distinct state within 60 h of antigen encounter. Breaching tolerance required the synergistic actions of strong T cell receptor (TCR) signaling and inflammation, which cooperatively induced gene modules that enhanced protein translation. Weak TCR signaling during bystander infection failed to breach tolerance due to the uncoupling of effector gene expression from protein translation. Thus, tolerance engages a distinct differentiation trajectory enforced by protein translation defects., Competing Interests: Declaration of interests I.A.P. receives research funding from AstraZeneca and Bristol-Myers Squibb. H.Y.C. is a co-founder of Accent Therapeutics, Boundless Bio, Cartography Biosciences, and Orbital Therapeutics, and is an advisor for 10× Genomics, Arsenal Biosciences, Chroma Medicine, and Spring Discovery., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Interleukin-(IL) 22 production by intestinal group 3 innate lymphoid cells (ILC3) is critical to maintain gut homeostasis. However, IL-22 needs to be tightly controlled; reduced IL-22 expression is associated with intestinal epithelial barrier defect while its overexpression promotes tumor development. Here, using a single-cell ribonucleic acid sequencing approach, we identified a core set of genes associated with increased IL-22 production by ILC3. Among these genes, programmed cell death 1 (PD-1), extensively studied in the context of cancer and chronic infection, was constitutively expressed on a subset of ILC3. These cells, found in the crypt of the small intestine and colon, displayed superior capacity to produce IL-22. PD-1 expression on ILC3 was dependent on the microbiota and was induced during inflammation in response to IL-23 but, conversely, was reduced in the presence of Notch ligand. PD-1 + ILC3 exhibited distinct metabolic activity with increased glycolytic, lipid, and polyamine synthesis associated with augmented proliferation compared with their PD-1 - counterparts. Further, PD-1 + ILC3 showed increased expression of mitochondrial antioxidant proteins which enable the cells to maintain their levels of reactive oxygen species. Loss of PD-1 signaling in ILC3 led to reduced IL-22 production in a cell-intrinsic manner. During inflammation, PD-1 expression was increased on natural cytotoxicity receptor (NCR) - ILC3 while deficiency in PD-1 expression resulted in increased susceptibility to experimental colitis and failure to maintain gut barrier integrity. Collectively, our findings uncover a new function of the PD-1 and highlight the role of PD-1 signaling in the maintenance of gut homeostasis mediated by ILC3 in mice., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Tissue-resident innate lymphoid cells (ILCs) play a vital role in the frontline defense of various tissues, including the lung. The development of type 2 ILCs (ILC2s) depends on transcription factors such as GATA3, RORα, GFI1, and Bcl11b; however, the factors regulating lung-resident ILC2s remain unclear. Through fate mapping analysis of the paralog transcription factors GFI1 and GFI1B, we show that GFI1 is consistently expressed during the transition from progenitor to mature ILC2s. In contrast, GFI1B expression is limited to specific subsets of bone marrow progenitors and lung-resident ILC progenitors. We found that GFI1B + lung ILC progenitors represent a multi-lineage subset with tissue-resident characteristics and the potential to form lung-derived ILC subsets and liver-resident ILC1s. Loss of GFI1B in bone marrow progenitors led to the selective loss of lung-resident IL-18R + ILCs and mature ILC2, subsequently preventing the emergence of effector ILCs that could protect the lung against inflammatory or tumor challenge.

CD4 + T cells play a key role in γ-herpesvirus infection control. However, the mechanisms involved are unclear. Murine herpesvirus type 4 (MuHV-4) allows relevant immune pathways to be dissected experimentally in mice. In the lungs, it colonizes myeloid cells, which can express MHC class II (MHCII), and type 1 alveolar epithelial cells (AEC1), which lack it. Nevertheless, CD4 + T cells can control AEC1 infection, and this control depends on MHCII expression in myeloid cells. Interferon-gamma (IFNγ) is a major component of CD4 + T cell-dependent MuHV-4 control. Here, we show that the action of IFNγ is also indirect, as CD4 + T cell-mediated control of AEC1 infection depended on IFNγ receptor (IFNγR1) expression in CD11c + cells. Indirect control also depended on natural killer (NK) cells. Together, the data suggest that the activation of MHCII + CD11c + antigen-presenting cells is key to the CD4 + T cell/NK cell protection axis. By contrast, CD8 + T cell control of AEC1 infection appeared to operate independently., Importance: CD4 + T cells are critical for the control of gamma-herpesvirus infection; they act indirectly, by recruiting natural killer (NK) cells to attack infected target cells. Here, we report that the CD4 + T cell/NK cell axis of gamma-herpesvirus control requires interferon-γ engagement of CD11c + dendritic cells. This mechanism of CD4 + T cell control releases the need for the direct engagement of CD4 + T cells with virus-infected cells and may be a common strategy for host control of immune-evasive pathogens., Competing Interests: The authors declare no conflict of interest.

Objective: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear., Design: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation., Results: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes., Conclusion: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Fibroblastic reticular cells (FRCs) construct microanatomical niches that support lymph node (LN) homeostasis and coordination of immune responses. Transcription factors regulating the functionality of FRCs remain poorly understood. Here, we investigated the role of the transcription factor SpiB that is expressed in LN FRCs. Conditional ablation of SpiB in FRCs impaired the FRC network in the T-cell zone of LNs, leading to reduced numbers of FRCs and altered homeostatic functions including reduced CCL21 and interleukin-7 expression. The size and cellularity of LNs remained intact in the absence of SpiB but the space between the reticular network increased, indicating that although FRCs were reduced in number they stretched to maintain network integrity. Following virus infection, antiviral CD8 + T-cell responses were impaired, suggesting a role for SpiB expression in FRCs in orchestrating immune responses. Together, our findings reveal a new role for SpiB as an important regulator of FRC functions and immunity in LNs., (© 2024 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

Objectives: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus infection in pregnancy is associated with higher incidence of placental dysfunction, referred to by a few studies as a 'preeclampsia-like syndrome'. However, the mechanisms underpinning SARS-CoV-2-induced placental malfunction are still unclear. Here, we investigated whether the transcriptional architecture of the placenta is altered in response to SARS-CoV-2 infection., Methods: We utilised whole-transcriptome, digital spatial profiling, to examine gene expression patterns in placental tissues from participants who contracted SARS-CoV-2 in the third trimester of their pregnancy ( n  = 7) and those collected prior to the start of the coronavirus disease 2019 (COVID-19) pandemic ( n  = 9)., Results: Through comprehensive spatial transcriptomic analyses of the trophoblast and villous core stromal cell subpopulations in the placenta, we identified SARS-CoV-2 to promote signatures associated with hypoxia and placental dysfunction. Notably, genes associated with vasodilation ( NOS3 ), oxidative stress ( GDF15 , CRH ) and preeclampsia ( FLT1 , EGFR , KISS1 , PAPPA2 ) were enriched with SARS-CoV-2. Pathways related to increased nutrient uptake, vascular tension, hypertension and inflammation were also enriched in SARS-CoV-2 samples compared to uninfected controls., Conclusions: Our findings demonstrate the utility of spatially resolved transcriptomic analysis in defining the underlying pathogenic mechanisms of SARS-CoV-2 in pregnancy, particularly its role in placental dysfunction. Furthermore, this study highlights the significance of digital spatial profiling in mapping the intricate crosstalk between trophoblasts and villous core stromal cells, thus shedding light on pathways associated with placental dysfunction in pregnancies with SARS-CoV-2 infection., Competing Interests: Andy Nam and Liuliu Pan are employed by Nanostring Technologies. Nicolas Matigian is employed by QCIF Bioinformatics., (© 2024 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Antibody-secreting plasma cells (PCs) are generated in secondary lymphoid organs but are reported to reside in an emerging range of anatomical sites. Analysis of the transcriptome of different tissue-resident (Tr)PC populations revealed that they each have their own transcriptional signature indicative of functional adaptation to the host tissue environment. In contrast to expectation, all TrPCs were extremely long-lived, regardless of their organ of residence, with longevity influenced by intrinsic factors like the immunoglobulin isotype. Analysis at single-cell resolution revealed that the bone marrow is unique in housing a compendium of PCs generated all over the body that retain aspects of the transcriptional program indicative of their tissue of origin. This study reveals that extreme longevity is an intrinsic property of TrPCs whose transcriptome is imprinted by signals received both at the site of induction and within the tissue of residence., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Individuals with autism often experience gastrointestinal issues but the cause is unknown. Many gene mutations that modify neuronal synapse function are associated with autism and therefore may impact the enteric nervous system that regulates gastrointestinal function. A missense mutation in the Nlgn3 gene encoding the cell adhesion protein Neuroligin-3 was identified in two brothers with autism who both experienced severe gastrointestinal dysfunction. Mice expressing this mutation ( Nlgn3 R451C mice) are a well-studied preclinical model of autism and show autism-relevant characteristics, including impaired social interaction and communication, as well as repetitive behaviour. We previously showed colonic dysmotility in response to GABAergic inhibition and increased myenteric neuronal numbers in the small intestine in Nlgn3 R451C mice bred on a mixed genetic background. Here, we show that gut dysfunction is a persistent phenotype of the Nlgn3 R451C mutation in mice backcrossed onto a C57BL/6 background. We report that Nlgn3 R451C mice show a 30.9% faster gastrointestinal transit ( p = 0.0004) in vivo and have 6% longer small intestines ( p = 0.04) compared to wild-types due to a reduction in smooth muscle tone. In Nlgn3 R451C mice, we observed a decrease in resting jejunal diameter (proximal jejunum: 10.6% decrease, p = 0.02; mid: 9.8%, p = 0.04; distal: 11.5%, p = 0.009) and neurally regulated dysmotility as well as shorter durations of contractile complexes (mid: 25.6% reduction in duration, p = 0.009; distal: 30.5%, p = 0.004) in the ileum. In Nlgn3 R451C mouse colons, short contractions were inhibited to a greater extent (57.2% by the GABA A antagonist, gabazine, compared to 40.6% in wild-type mice ( p = 0.007). The inhibition of nitric oxide synthesis decreased the frequency of contractile complexes in the jejunum (WT p = 0.0006, Nlgn3 R451C p = 0.002), but not the ileum, in both wild-type and Nlgn3 R451C mice. These findings demonstrate that changes in enteric nervous system function contribute to gastrointestinal dysmotility in mice expressing the autism-associated R451C missense mutation in the Neuroligin-3 protein.

Memory T cells are generated from naïve precursors undergoing proliferation during the initial immune response. Both naïve and memory T cells are maintained in a resting, quiescent state and respond to activation with a controlled proliferative burst and differentiation into effector cells. This similarity in the maintenance and response dynamics points to the preservation of key cellular fate programs; however, whether memory T cells have acquired intrinsic changes in these programs that may contribute to the enhanced immune protection in a recall response is not fully understood. Here we used a quantitative model-based analysis of proliferation and survival kinetics of in vitro-stimulated murine naïve and memory CD8 + T cells in response to homeostatic and activating signals to establish intrinsic similarities or differences within these cell types. We show that resting memory T cells display heightened sensitivity to homeostatic cytokines, responding to interleukin (IL)-2 in addition to IL-7 and IL-15. The proliferative response to αCD3 was equal in size and kinetics, demonstrating that memory T cells undergo the same controlled division burst and automated return to quiescence as naïve T cells. However, perhaps surprisingly, we observed reduced expansion of αCD3-stimulated memory T cells in response to activating signals αCD28 and IL-2 compared with naïve T cells. Overall, we demonstrate that although sensitivities to cytokine and costimulatory signals have shifted, fate programs regulating the scale of the division burst are conserved in memory T cells., (© 2023 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of the Australian and New Zealand Society for Immunology, Inc.)

The SARS-CoV-2 (COVID-19) virus has caused a devastating global pandemic of respiratory illness. To understand viral pathogenesis, methods are available for studying dissociated cells in blood, nasal samples, bronchoalveolar lavage fluid and similar, but a robust platform for deep tissue characterization of molecular and cellular responses to virus infection in the lungs is still lacking. We developed an innovative spatial multi-omics platform to investigate COVID-19-infected lung tissues. Five tissue-profiling technologies were combined by a novel computational mapping methodology to comprehensively characterize and compare the transcriptome and targeted proteome of virus infected and uninfected tissues. By integrating spatial transcriptomics data (Visium, GeoMx and RNAScope) and proteomics data (CODEX and PhenoImager HT) at different cellular resolutions across lung tissues, we found strong evidence for macrophage infiltration and defined the broader microenvironment surrounding these cells. By comparing infected and uninfected samples, we found an increase in cytokine signalling and interferon responses at different sites in the lung and showed spatial heterogeneity in the expression level of these pathways. These data demonstrate that integrative spatial multi-omics platforms can be broadly applied to gain a deeper understanding of viral effects on cellular environments at the site of infection and to increase our understanding of the impact of SARS-CoV-2 on the lungs., (© 2023 The Authors. Immunology published by John Wiley & Sons Ltd.)

Intraepithelial lymphocytes (IELs), including αβ and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/ TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is known to present with pulmonary and extra-pulmonary organ complications. In comparison with the 2009 pandemic (pH1N1), SARS-CoV-2 infection is likely to lead to more severe disease, with multi-organ effects, including cardiovascular disease. SARS-CoV-2 has been associated with acute and long-term cardiovascular disease, but the molecular changes that govern this remain unknown. In this study, we investigated the host transcriptome landscape of cardiac tissues collected at rapid autopsy from seven SARS-CoV-2, two pH1N1, and six control patients using targeted spatial transcriptomics approaches. Although SARS-CoV-2 was not detected in cardiac tissue, host transcriptomics showed upregulation of genes associated with DNA damage and repair, heat shock, and M1-like macrophage infiltration in the cardiac tissues of COVID-19 patients. The DNA damage present in the SARS-CoV-2 patient samples, were further confirmed by γ-H2Ax immunohistochemistry. In comparison, pH1N1 showed upregulation of interferon-stimulated genes, in particular interferon and complement pathways, when compared with COVID-19 patients. These data demonstrate the emergence of distinct transcriptomic profiles in cardiac tissues of SARS-CoV-2 and pH1N1 influenza infection supporting the need for a greater understanding of the effects on extra-pulmonary organs, including the cardiovascular system of COVID-19 patients, to delineate the immunopathobiology of SARS-CoV-2 infection, and long term impact on health., (© 2022 The Authors. Immunology published by John Wiley & Sons Ltd.)

Innate lymphoid cells (ILCs) comprise a number of different subsets, including natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and lymphoid tissue-inducer (LTi) cells that express receptors and signaling pathways that are highly responsive to continuously changing microenvironmental cues. In this Review, we highlight the key features of innate cells that define their capacity to respond rapidly to different environments, how this ability can drive both tumor protection (limiting tumor development) or, alternatively, tumor progression, promoting tumor dissemination and resistance to immunotherapy. We discuss how understanding the regulation of ILCs that can detect tumor cells early in a response opens the possibility of exploiting this functional plasticity to develop rational therapeutic strategies to bolster adaptive immune responses and improve patient outcomes., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Crown Copyright © 2022. Published by Elsevier Inc. All rights reserved.)

Purpose: Robust biomarkers that predict disease outcomes amongst COVID-19 patients are necessary for both patient triage and resource prioritisation. Numerous candidate biomarkers have been proposed for COVID-19. However, at present, there is no consensus on the best diagnostic approach to predict outcomes in infected patients. Moreover, it is not clear whether such tools would apply to other potentially pandemic pathogens and therefore of use as stockpile for future pandemic preparedness., Methods: We conducted a multi-cohort observational study to investigate the biology and the prognostic role of interferon alpha-inducible protein 27 ( IFI27 ) in COVID-19 patients., Results: We show that IFI27 is expressed in the respiratory tract of COVID-19 patients and elevated IFI27 expression in the lower respiratory tract is associated with the presence of a high viral load. We further demonstrate that the systemic host response, as measured by blood IFI27 expression, is associated with COVID-19 infection. For clinical outcome prediction (e.g., respiratory failure), IFI27 expression displays a high sensitivity (0.95) and specificity (0.83), outperforming other known predictors of COVID-19 outcomes. Furthermore, IFI27 is upregulated in the blood of infected patients in response to other respiratory viruses. For example, in the pandemic H1N1/09 influenza virus infection, IFI27- like genes were highly upregulated in the blood samples of severely infected patients., Conclusion: These data suggest that prognostic biomarkers targeting the family of IFI27 genes could potentially supplement conventional diagnostic tools in future virus pandemics, independent of whether such pandemics are caused by a coronavirus, an influenza virus or another as yet-to-be discovered respiratory virus., Competing Interests: FS-F-G is a consultant for Biotheus Inc. KS is a consultant for Sanofi, Roche and NovoNordisk. The opinions and data presented in this manuscript are of the authors and are independent of these relationships. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Shojaei, Shamshirian, Monkman, Grice, Tran, Tan, Teo, Rodrigues Rossi, McCulloch, Nalos, Raei, Razavi, Ghasemian, Gheibi, Roozbeh, Sly, Spann, Chew, Zhu, Xia, Wells, Senegaglia, Kuniyoshi, Franck, dos Santos, Noronha, Motamen, Valadan, Amjadi, Gogna, Madan, Alizadeh-Navaei, Lamperti, Zuñiga, Nova-Lamperti, Labarca, Knippenberg, Herwanto, Wang, Phu, Chew, Kwan, Kim, Teoh, Pelaia, Kuan, Jee, Iredell, O’Byrne, Fraser, Davis, Belz, Warkiani, Gallo, Souza-Fonseca-Guimaraes, Nguyen, Mclean, Kulasinghe, Short and Tang.)

Innate and adaptive immune cells are found in distinct tissue niches where they orchestrate immune responses. This requires intrinsic and temporal metabolic adaptability to coordinately activate the immune response cascade. Dysregulation of this program is a key feature of immunosuppression. Direct or indirect metabolic immune cell reprogramming may offer new approaches to modulate immune cells behavior for therapy to overcome dysregulation. In this review, we explored how metabolism regulates lymphocytes beyond the classical T cell subsets. We focus on the innate lymphoid cell (ILC) family, highlighting the distinct metabolic characteristics of these cells, the impact of environmental factors, and the receptors that could alter immune cell functions through manipulation of metabolic pathways to potentially prevent or treat various diseases., (© 2022 Crown copyright. The government of Australia, Canada, or the UK ("the Crown") owns the copyright interests of authors who are government employees. The Crown Copyright is not transferable.)

Innate lymphoid cells (ILCs) and adaptive T cells remain a challenge to study because of a significant overlap in their transcriptomic profiles. Here, we describe the adoptive transfer of ILC progenitors into mice genetically deficient in innate and adaptive immune cells to allow detailed study of the development and function of ILCs and gene regulation in an in vivo setting. For complete details on the use and execution of this protocol, please refer to Jacquelot et al. (2021) and Seillet et al. (2016)., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cancer is a complex disease, and despite incredible progress over the last decade, it remains the leading cause of death worldwide. Liver cancers, including hepatocellular carcinoma (HCC), and liver metastases are distinct from other cancers in that they typically emerge as a consequence of long-term low-grade inflammation. Understanding the mechanisms that underpin inflammation-driven tissue remodeling of the hepatic immune environment is likely to provide new insights into much needed treatments for this devastating disease. Group 1 innate lymphoid cells (ILCs), which include natural killer (NK) cells and ILC1s, are particularly enriched in the liver and thought to contribute to the pathogenesis of a number of liver diseases, including cancer. NK cells are an attractive, but underexplored, therapeutic target in hepatic disease due to their role in immunosurveillance and their ability to recognize and eliminate malignant cells. ILC1s are closely related to and share many phenotypic features with NK cells but are less well studied. Thus, their utility in immunotherapeutic approaches is not yet well understood. Here, we review our current understanding of ILCs in cancer with a particular focus on liver and liver-related diseases., (© 2022. Crown.)

Group 3 innate lymphoid cells (ILC3s) are distributed along the gastrointestinal tract at the interface between the immune system and the gut lumen, which carries a significant microbial burden. In a new study, Zhou et al. investigated the expression of transcription factor ZBTB46, normally thought to be restricted to classical dendritic cells (cDCs), and discovered that ZBTB46 expression by ILC3s in the mouse colon forms an essential part of the gastrointestinal armory to calibrate inflammatory responses., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Crown Copyright © 2022. Published by Elsevier Ltd. All rights reserved.)

Immunotherapy has revolutionized cancer therapy by reactivating tumour-resident cytotoxic lymphocytes. More recently, immunotherapy has emerged to restore immunity against infectious agents, including bacterial infections. Immunotherapy primarily targets inhibitory pathways in T cells, however interest in other effector populations, such as natural killer (NK) cells, is growing. We have previously discovered that NK cell metabolism, proliferation and activation can be neutralized through the immunosuppressive transforming growth factor (TGF)-β pathway by inducing plasticity of NK cells and differentiation into innate lymphoid cell (ILC)1-like subsets. NK cells are also regulated through cytokine-inducible SH2-containing protein (CIS), which is induced by interleukin (IL)-15 and is a potent intracellular checkpoint suppressing NK cell survival and function. Targeting these two distinct pathways to restore NK cell function has shown promise in cancer models, but their application in bacterial infection remains unknown. Here, we investigate whether enhancement of NK cell function can improve anti-bacterial immunity, using Salmonella Typhimurium as a model. We identified conversion of NK cells to ILC1-like for the first time in the context of bacterial infection, where TGF-β signalling contributed to this plasticity. Future study should focus on identifying further drivers of ILC1 plasticity and its functional implication in bacterial infection model. We further describe that CIS-deficient mice displayed enhanced pro-inflammatory function and dramatically enhanced anti-bacterial immunity. Inhibition of CIS may present as a viable therapeutic option to enhance immunity towards bacterial infection., (© 2022 The Authors. Immunology published by John Wiley & Sons Ltd.)

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which emerged in late 2019 has spread globally, causing a pandemic of respiratory illness designated coronavirus disease 2019 (COVID-19). A better definition of the pulmonary host response to SARS-CoV-2 infection is required to understand viral pathogenesis and to validate putative COVID-19 biomarkers that have been proposed in clinical studies., Methods: Here, we use targeted transcriptomics of formalin-fixed paraffin-embedded tissue using the NanoString GeoMX platform to generate an in-depth picture of the pulmonary transcriptional landscape of COVID-19, pandemic H1N1 influenza and uninfected control patients., Results: Host transcriptomics showed a significant upregulation of genes associated with inflammation, type I interferon production, coagulation and angiogenesis in the lungs of COVID-19 patients compared to non-infected controls. SARS-CoV-2 was non-uniformly distributed in lungs (emphasising the advantages of spatial transcriptomics) with the areas of high viral load associated with an increased type I interferon response. Once the dominant cell type present in the sample, within patient correlations and patient-patient variation, had been controlled for, only a very limited number of genes were differentially expressed between the lungs of fatal influenza and COVID-19 patients. Strikingly, the interferon-associated gene IFI27 , previously identified as a useful blood biomarker to differentiate bacterial and viral lung infections, was significantly upregulated in the lungs of COVID-19 patients compared to patients with influenza., Conclusion: Collectively, these data demonstrate that spatial transcriptomics is a powerful tool to identify novel gene signatures within tissues, offering new insights into the pathogenesis of SARS-COV-2 to aid in patient triage and treatment., Competing Interests: Conflict of interest: Fernando Souza-Fonseca-Guimaraes is a consultant for Biotheus Inc. However, all opinions and reviews presented in this manuscript belong to the authors alone and are independent of the relationships with Biotheus. All other authors declare no competing interests., (Copyright ©The authors 2022.)

Targeting the potent immunosuppressive properties of FOXP3 + regulatory T cells (T regs ) has substantial therapeutic potential for treating autoimmune and inflammatory diseases. Yet, the molecular mechanisms controlling T reg homeostasis, particularly during inflammation, remain unclear. We report that caspase-8 is a central regulator of T reg homeostasis in a context-specific manner that is decisive during immune responses. In mouse genetic models, targeting caspase-8 in T regs led to accumulation of effector T regs resistant to apoptotic cell death. Conversely, inflammation induced the MLKL-dependent necroptosis of caspase-8-deficient lymphoid and tissue T regs , which enhanced immunity to a variety of chronic infections to promote clearance of viral or parasitic pathogens. However, improved immunity came at the risk of lethal inflammation in overwhelming infections. Caspase-8 inhibition using a clinical-stage compound revealed that human T regs have heightened sensitivity to necroptosis compared with conventional T cells. These findings reveal a fundamental mechanism in T regs that could be targeted to manipulate the balance between immune tolerance versus response for therapeutic benefit.

The innate lymphoid cell (ILC) family is composed of natural killer (NK) cells, ILC1, ILC2 and ILC3, which participate in immune responses to virus, bacteria, parasites and transformed cells. ILC1, ILC2 and ILC3 subsets are mostly tissue-resident, and are profoundly imprinted by their organ of residence. They exhibit pleiotropic effects, driving seemingly paradoxical responses such as tissue repair and, alternatively, immunopathology toward allergens and promotion of tumorigenesis. Despite this, a trickle of studies now suggests that non-NK ILCs may not be overwhelmingly tumorigenic and could potentially be harnessed to drive anti-tumor responses. Here, we examine the pleiotropic behavior of ILCs in cancer and begin to unravel the gap in our knowledge that exposes a new horizon for thinking about modifying ILCs and targeting them for immunotherapy., (© 2022. Crown.)

The spleen is a compartmentalized organ that serves as a blood filter and safeguard of systemic immune surveillance. Labyrinthine networks of fibroblastic stromal cells construct complex niches within the white pulp and red pulp that are important for tissue homeostasis and immune activation. However, the identity and roles of the global splenic fibroblastic stromal cells in homeostasis and immune responses are poorly defined. Here, we performed a cellular and molecular dissection of the splenic reticular stromal cell landscape. We found that white pulp fibroblastic reticular cells (FRCs) responded robustly during acute viral infection, but this program of gene regulation was suppressed during persistent viral infection. Single-cell transcriptomic analyses in mice revealed diverse fibroblast cell niches and unexpected heterogeneity among podoplanin-expressing cells that include glial, mesothelial, and adventitial cells in addition to FRCs. We found analogous fibroblastic stromal cell diversity in the human spleen. In addition, we identify the transcription factor SpiB as a critical regulator required to support white pulp FRC differentiation, homeostatic chemokine expression, and antiviral T cell responses. Together, our study provides a comprehensive map of fibroblastic stromal cell types in the spleen and defines roles for red and white pulp fibroblasts for splenic function and orchestration of immune responses.

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein-coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ-activated eosinophils facilitated CD4 + and CD8 + T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics. SIGNIFICANCE: These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer., (©2021 American Association for Cancer Research.)

CD4 + T-helper 22 (Th22) cells are a phenotypically distinct lymphocyte subset that produces high levels of interleukin (IL)-22 without co-production of IL-17A. However, the developmental origin and lineage classification of Th22 cells, their interrelationship to Th17 cells, and potential for plasticity at sites of infection and inflammation remain largely undefined. An improved understanding of the mechanisms underpinning the outgrowth of Th22 cells will provide insights into their regulation during homeostasis, infection, and disease. To address this knowledge gap we generated 'IL-17A-fate-mapping IL-17A/IL-22 reporter transgenic mice' and show that Th22 cells develop in the gastrointestinal tract and lung during bacterial infection without transitioning via an Il17a-expressing intermediate, although in some compartments alternative transition pathways exist. Th22-cell development was not dependent on T-bet; however, this transcription factor functioned as a promiscuous T-cell-intrinsic regulator of IL-17A and IL-22 production, in addition to regulating the outgrowth, phenotypic stability, and plasticity of Th22 cells. Thus, we demonstrate that at sites of mucosal bacterial infection Th22 cells develop as a distinct lineage independently of Th17 cells; though both lineages exhibit bidirectional phenotypic flexibility within infected tissues and their draining lymph nodes, and that T-bet plays a critical regulatory role in Th22-cell function and identity., (© 2021. The Author(s), under exclusive licence to Society for Mucosal Immunology.)

Tissue-resident memory T (T RM ) cells are non-recirculating cells that exist throughout the body. Although T RM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze T RM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate T RM cell function, durability and malleability. We find that unequal responsiveness to TGFβ is a major driver of this diversity. Notably, dampened TGFβ signaling results in CD103 - T RM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFβ-responsive CD103 + T RM counterparts. Furthermore, whereas CD103 - T RM cells readily modified their phenotype upon relocation, CD103 + T RM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for T RM cell development, tissue adaptation of these cells confers discrete functional properties such that T RM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1) are specific innate lymphoid cell subsets that are key for the detection and elimination of pathogens and cancer cells. In liver, while they share a number of characteristics, they differ in many features. These include their developmental pathways, tissue distribution, phenotype and functions. NK cells and ILC1 contribute to organ homeostasis through the production of key cytokines and chemokines and the elimination of potential harmful bacteria and viruses. In addition, they are equipped with a wide range of receptors, allowing them to detect "stressed cells' such as cancer cells. Our understanding of the role of innate lymphoid cells in hepatocellular carcinoma (HCC) is growing owing to the development of mouse models, the progress in immunotherapeutic treatment and the recent use of scRNA sequencing analyses. In this review, we summarize the current understanding of NK cells and ILC1 in hepatocellular carcinoma and discuss future strategies to take advantage of these innate immune cells in anti-tumor immunity. Immunotherapies hold great promise in HCC, and a better understanding of the role and function of NK cells and ILC1 in liver cancer could pave the way for new NK cell and/or ILC1-targeted treatment.

Group 2 innate lymphoid cells (ILC2s) are essential to maintain tissue homeostasis. In cancer, ILC2s can harbor both pro-tumorigenic and anti-tumorigenic functions, but we know little about their underlying mechanisms or whether they could be clinically relevant or targeted to improve patient outcomes. Here, we found that high ILC2 infiltration in human melanoma was associated with a good clinical prognosis. ILC2s are critical producers of the cytokine granulocyte-macrophage colony-stimulating factor, which coordinates the recruitment and activation of eosinophils to enhance antitumor responses. Tumor-infiltrating ILC2s expressed programmed cell death protein-1, which limited their intratumoral accumulation, proliferation and antitumor effector functions. This inhibition could be overcome in vivo by combining interleukin-33-driven ILC2 activation with programmed cell death protein-1 blockade to significantly increase antitumor responses. Together, our results identified ILC2s as a critical immune cell type involved in melanoma immunity and revealed a potential synergistic approach to harness ILC2 function for antitumor immunotherapies.

Immunity to melanoma is thought to be mainly mediated by adaptive immune cells. To what extent innate immunity, particularly innate lymphoid cells, drive the immune response and impact melanoma prognosis and therapeutic responsiveness is not well understood. In a recent article published in Nature Immunology , we uncovered a critical role that ILC2 play in the control of melanoma. Using both complementary mouse models and human samples, we showed that ILC2-derived granulocyte macrophage-colony stimulating factor (GM-CSF) drives eosinophil tumor recruitment and activation. We found that ILC2 express PD-1 which inhibits ILC2 effector function and impairs anti-tumor responses. We further demonstrated that the combination of IL-33 and anti-PD-1 blocking antibodies improved anti-tumor responses through the expansion of splenic and tumor-infiltrating ILC2 and eosinophils. These findings have revealed an essential mechanism involving ILC2 and eosinophils necessary for anti-melanoma immunity and immunotherapy responses., (© 2021 The Author(s). Published with license by Taylor & Francis Group, LLC.)

Innate lymphoid cells (ILCs) are resident in the lung and are involved in both the maintenance of homeostasis and the pathogenesis of respiratory diseases. In this study, murine lung ILCs were characterized using flow cytometry and the impact of mouse age, sex and strain were assessed. Lung ILCs were found as early as postnatal day 4 and numbers peaked at 2 weeks, and then decreased as the lung matured. During postnatal lung development, ILC expressed differential amounts of group 2 ILC (ILC2)-associated cell surface antigens including ST2, CD90.2 and ICOS. Using Il5 venus Il13 td-tomato dual reporter mice, neonates were found to have increased constitutive interleukin (IL)-13 expression compared with adult mice. Neonates and adults had similar ratios of IL-5 + CD45 + leukocytes; however, these cells were mostly composed of ILCs in neonates and T cells in adults. Sex-specific differences in ILC numbers were also observed, with females having greater numbers of lung ILCs than males in both neonatal and adult mice. Female lung ILCs also expressed higher levels of ICOS and decreased KLRG1. Mouse strain also impacted on lung ILCs with BALB/c mice having more ILCs in the lung and increased expression of ST2 and ICOS compared with C57BL/6J mice. Collectively, these data show that lung ILC numbers, cell surface antigen expression, IL-5 and IL-13 levels differed between neonatal and adult lung ILCs. In addition, cell surface antigens commonly used for ILC2 quantification, such as ST2, CD90.2 and ICOS, differ depending on age, sex and strain and these are important considerations for consistent universal identification of lung ILC2s., (© 2021 Australian and New Zealand Society for Immunology, Inc.)

The Earth's rotation around its axis, is one of the parameters that never changed since life emerged. Therefore, most of the organisms from the cyanobacteria to humans have conserved natural oscillations to regulate their physiology. These daily oscillations define the circadian rhythms that set the biological clock for almost all physiological processes of an organism. They allow the organisms to anticipate and respond behaviorally and physiologically to changes imposed by the day/night cycle. As other physiological systems, the immune system is also regulated by circadian rhythms and while diurnal variation in host immune responses to lethal infection have been observed for many decades, the underlying mechanisms that affect immune function and health have only just started to emerge. These oscillations are generated by the central clock in our brain, but neuroendocrine signals allow the synchronization of the clocks in peripheral tissues. In this review, we discuss how the neuroimmune interactions create a rhythmic activity of the innate lymphoid cells. We highlight how the disruption of these rhythmic regulations of immune cells can disturb homeostasis and lead to the development of chronic inflammation in murine models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jacquelot, Belz and Seillet.)

Irritable bowel syndrome often appears following gastrointestinal infection and is marked by diarrhea, dysbiosis, fever, and intestinal pain following eating. A recent study by Aguilera-Lizarraga et al. now demonstrates that a breakdown in intestinal immunotolerance sparks an inflammatory response to typically tolerated food antigens and causes visceral pain., Competing Interests: Declaration of Interests The authors declare no conflicts of interest., (Crown Copyright © 2021. Published by Elsevier Ltd. All rights reserved.)

The prevalence of chronic immune and metabolic disorders is increasing rapidly. In particular, inflammatory bowel diseases, obesity, diabetes, asthma and chronic obstructive pulmonary disease have become major healthcare and economic burdens worldwide. Recent advances in microbiome research have led to significant discoveries of associative links between alterations in the microbiome and health, as well as these chronic supposedly noncommunicable, immune/metabolic disorders. Importantly, the interplay between diet, microbiome and the mucous barrier in these diseases has gained significant attention. Diet modulates the mucous barrier via alterations in gut microbiota, resulting in either disease onset/exacerbation due to a "poor" diet or protection against disease with a "healthy" diet. In addition, many mucosa-associated disorders possess a specific gut microbiome fingerprint associated with the composition of the mucous barrier, which is further influenced by host-microbiome and inter-microbial interactions, dietary choices, microbe immigration and antimicrobials. Our review focuses on the interactions of diet (macronutrients and micronutrients), gut microbiota and mucous barriers (gastrointestinal and respiratory tract) and their importance in the onset and/or progression of major immune/metabolic disorders. We also highlight the key mechanisms that could be targeted therapeutically to prevent and/or treat these disorders., (© 2020 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Chronic inflammation of the gastrointestinal (GI) tract contributes to colorectal cancer (CRC) progression. While the role of adaptive T cells in CRC is now well established, the role of innate immune cells, specifically innate lymphoid cells (ILCs), is not well understood. To define the role of ILCs in CRC we employed complementary heterotopic and chemically-induced CRC mouse models. We discovered that ILCs were abundant in CRC tumours and contributed to anti-tumour immunity. We focused on ILC2 and showed that ILC2-deficient mice developed a higher tumour burden compared with littermate wild-type controls. We generated an ILC2 gene signature and using machine learning models revealed that CRC patients with a high intratumor ILC2 gene signature had a favourable clinical prognosis. Collectively, our results highlight a critical role for ILC2 in CRC, suggesting a potential new avenue to improve clinical outcomes through ILC2-agonist based therapeutic approaches.

Type 2 innate lymphoid cells (ILC2s) are important producers of type 2 cytokines whose role in hematological cancers remains unclear. ILC2s are a heterogeneous population encompassing distinct subsets with different tissue localization and cytokine responsiveness. In this study, we investigated the role of bone marrow (BM) ILC2s and interleukin (IL)-33-stimulated ILC2s in multiple myeloma, a plasma cell malignancy that develops in the BM. We found that myeloma growth was associated with phenotypic and functional alterations of BM ILC2s, characterized by an increased expression of maturation markers and reduced cytokine response to IL-2/IL-33. We identified a population of KLRG1 hi ILC2s that preferentially accumulated in the liver and spleen of Il2rg -/- Rag2 -/- mice reconstituted with BM ILC2s. A similar population of KLRG1 hi ILC2s was observed in the blood, liver and spleen of IL-33-treated wild-type mice. The presence of KLRG1 hi ILC2s in ILC2-reconstituted Il2rg -/- Rag2 -/- mice or in IL-33-treated wild-type mice was associated with increased eosinophil numbers but had no effect on myeloma progression. Interestingly, while decreased myeloma growth was observed following treatment of Rag-deficient mice with the type 1 cytokines IL-12 and IL-18, this protection was reversed when mice received a combined treatment of IL-33 together with IL-12 and IL-18. In summary, our data indicate that IL-33 treatment induces a population of circulating inflammatory KLRG1 hi ILC2s and inhibits type 1 immunity against multiple myeloma. These results argue against therapeutic administration of IL-33 to myeloma patients., (© 2020 Australian and New Zealand Society for Immunology Inc.)

Natural killer (NK) cells play critical roles in protection against hematological malignancies but can acquire a dysfunctional state, which limits antitumor immunity. However, the underlying reasons for this impaired NK cell function remain to be uncovered. We found that NK cells in aggressive B-cell lymphoma underwent substantial transcriptional reprogramming associated with increased lipid metabolism, including elevated expression of the transcriptional regulator peroxisome activator receptor-γ (PPAR-γ). Exposure to fatty acids in the lymphoma environment potently suppressed NK cell effector response and cellular metabolism. NK cells from both diffuse large B-cell lymphoma patients and Eµ-myc B-cell lymphoma-bearing mice displayed reduced interferon-γ (IFN-γ) production. Activation of PPAR-γ partially restored mitochondrial membrane potential and IFN-γ production. Overall, our data indicate that increased lipid metabolism, while impairing their function, is a functional adaptation of NK cells to the fatty-acid rich lymphoma environment., (© 2020 by The American Society of Hematology.)

The dynamics of CD4 + T cell memory development remain to be examined at genome scale. In malaria-endemic regions, antimalarial chemoprevention protects long after its cessation and associates with effects on CD4 + T cells. We applied single-cell RNA sequencing and computational modelling to track memory development during Plasmodium infection and treatment. In the absence of central memory precursors, two trajectories developed as T helper 1 (T H 1) and follicular helper T (T FH ) transcriptomes contracted and partially coalesced over three weeks. Progeny of single clones populated T H 1 and T FH trajectories, and fate-mapping suggested that there was minimal lineage plasticity. Relationships between T FH and central memory were revealed, with antimalarials modulating these responses and boosting T H 1 recall. Finally, single-cell epigenomics confirmed that heterogeneity among effectors was partially reset in memory. Thus, the effector-to-memory transition in CD4 + T cells is gradual during malaria and is modulated by antiparasitic drugs. Graphical user interfaces are presented for examining gene-expression dynamics and gene-gene correlations ( http://haquelab.mdhs.unimelb.edu.au/cd4_memory/ ).