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This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs, and various nonlymphoid tissues. Within this article, detailed protocols are presented that allow for the generation of single-cell suspensions from human nonlymphoid tissues including lung, skin, gingiva, intestine as well as from tumors and tumor-draining lymph nodes with a subsequent analysis of dendritic cells by flow cytometry. Further, prepared single-cell suspensions can be subjected to other applications including cellular enrichment procedures, RNA sequencing, functional assays, etc. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

Significance: Novel individual biomarkers are needed to guide therapeutic decisions for patients with head and neck cancer. We report for the first time, granulomas of TREM2-expressing multinucleated giant macrophages in keratin-rich tumor niches, as a biomarker of favorable prognosis and developed a deep-learning model to automate its quantification on routinely stained pathological slides., (©2024 The Authors; Published by the American Association for Cancer Research.)

Dendritic cells (DC) are specialized mononuclear phagocytes that link innate and adaptive immunity. They comprise two principal subsets: plasmacytoid DC (pDC) and conventional DC (cDC). Understanding the generation, differentiation, and migration of cDC is critical for immune homeostasis. Through human in vivo deuterium-glucose labeling, we observed the rapid appearance of AXL+ Siglec6+ DC (ASDC) in the bloodstream. ASDC circulate for ∼2.16 days, while cDC1 and DC2 circulate for ∼1.32 and ∼2.20 days, respectively, upon release from the bone marrow. Interestingly, DC3, a cDC subset that shares several similarities with monocytes, exhibits a labeling profile closely resembling that of DC2. In a human in vivo model of cutaneous inflammation, ASDC were recruited to the inflammatory site, displaying a distinctive effector signature. Taken together, these results quantify the ephemeral circulating lifespan of human cDC and propose functions of cDC and their precursors that are rapidly recruited to sites of inflammation., (© 2024 Lubin et al.)

Macrophages orchestrate tissue homeostasis and immunity. In the tumor microenvironment (TME), macrophage presence is largely associated with poor prognosis because of their reprogramming into immunosuppressive cells. We investigated the effects of hypoxia, a TME-associated feature, on the functional, epigenetic, and transcriptional reprogramming of macrophages and found that hypoxia boosts their immunogenicity. Hypoxic inflammatory macrophages are characterized by a cluster of proinflammatory genes undergoing ten-eleven translocation-mediated DNA demethylation and overexpression. These genes are regulated by NF-κB, while HIF1α dominates the transcriptional reprogramming, demonstrated through ChIP-seq and pharmacological inhibition. In bladder and ovarian carcinomas, hypoxic inflammatory macrophages are enriched in immune-infiltrated tumors, correlating with better patient prognoses. Coculture assays and cell-cell communication analyses support that hypoxic-activated macrophages enhance T cell-mediated responses. The NF-κB-associated hypomethylation signature is displayed by a subset of hypoxic inflammatory macrophages, isolated from ovarian tumors. Our results challenge paradigms regarding the effects of hypoxia on macrophages and highlight actionable target cells to modulate anticancer immune responses.

Tumor-associated macrophages (TAMs) are a heterogeneous population of cells whose phenotypes and functions are shaped by factors that are incompletely understood. Herein, we asked when and where TAMs arise from blood monocytes and how they evolve during tumor development. We initiated pancreatic ductal adenocarcinoma (PDAC) in inducible monocyte fate-mapping mice and combined single-cell transcriptomics and high-dimensional flow cytometry to profile the monocyte-to-TAM transition. We revealed that monocytes differentiate first into a transient intermediate population of TAMs that generates two longer-lived lineages of terminally differentiated TAMs with distinct gene expression profiles, phenotypes, and intratumoral localization. Transcriptome datasets and tumor samples from patients with PDAC evidenced parallel TAM populations in humans and their prognostic associations. These insights will support the design of new therapeutic strategies targeting TAMs in PDAC.

Spatial omics data allow in-depth analysis of tissue architectures, opening new opportunities for biological discovery. In particular, imaging techniques offer single-cell resolutions, providing essential insights into cellular organizations and dynamics. Yet, the complexity of such data presents analytical challenges and demands substantial computing resources. Moreover, the proliferation of diverse spatial omics technologies, such as Xenium, MERSCOPE, CosMX in spatial-transcriptomics, and MACSima and PhenoCycler in multiplex imaging, hinders the generality of existing tools. We introduce Sopa ( https://github.com/gustaveroussy/sopa ), a technology-invariant, memory-efficient pipeline with a unified visualizer for all image-based spatial omics. Built upon the universal SpatialData framework, Sopa optimizes tasks like segmentation, transcript/channel aggregation, annotation, and geometric/spatial analysis. Its output includes user-friendly web reports and visualizer files, as well as comprehensive data files for in-depth analysis. Overall, Sopa represents a significant step toward unifying spatial data analysis, enabling a more comprehensive understanding of cellular interactions and tissue organization in biological systems., (© 2024. The Author(s).)

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. Recent studies have provided evidence for an increasing number of phenotypically distinct conventional DC (cDC) subsets that on one hand exhibit a certain functional plasticity, but on the other hand are characterized by their tissue- and context-dependent functional specialization. Here, we describe a selection of assays for the functional characterization of mouse and human cDC. The first two protocols illustrate analysis of cDC endocytosis and metabolism, followed by guidelines for transcriptomic and proteomic characterization of cDC populations. Then, a larger group of assays describes the characterization of cDC migration in vitro, ex vivo, and in vivo. The final guidelines measure cDC inflammasome and antigen (cross)-presentation activity. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

We developed a phenotypic screening platform for the functional exploration of dendritic cells (DC). Here, we report a genome-wide CRISPR screen that revealed BCL2 as an endogenous inhibitor of DC function. Knockout of BCL2 enhanced DC antigen presentation and activation as well as the capacity of DCs to control tumors and to synergize with PD-1 blockade. The pharmacologic BCL2 inhibitors venetoclax and navitoclax phenocopied these effects and caused a cDC1-dependent regression of orthotopic lung cancers and fibrosarcomas. Thus, solid tumors failed to respond to BCL2 inhibition in mice constitutively devoid of cDC1, and this was reversed by the infusion of DCs. Moreover, cDC1 depletion reduced the therapeutic efficacy of BCL2 inhibitors alone or in combination with PD-1 blockade and treatment with venetoclax caused cDC1 activation, both in mice and in patients. In conclusion, genetic and pharmacologic BCL2 inhibition unveils a DC-specific immune checkpoint that restrains tumor immunosurveillance., Significance: BCL2 inhibition improves the capacity of DCs to stimulate anticancer immunity and restrain cancer growth in an immunocompetent context but not in mice lacking cDC1 or mature T cells. This study indicates that BCL2 blockade can be used to sensitize solid cancers to PD-1/PD-L1-targeting immunotherapy. This article is featured in Selected Articles from This Issue, p. 2293., (©2023 American Association for Cancer Research.)

Microglia are specialized brain-resident macrophages that arise from primitive macrophages colonizing the embryonic brain 1 . Microglia contribute to multiple aspects of brain development, but their precise roles in the early human brain remain poorly understood owing to limited access to relevant tissues 2-6 . The generation of brain organoids from human induced pluripotent stem cells recapitulates some key features of human embryonic brain development 7-10 . However, current approaches do not incorporate microglia or address their role in organoid maturation 11-21 . Here we generated microglia-sufficient brain organoids by coculturing brain organoids with primitive-like macrophages generated from the same human induced pluripotent stem cells (iMac) 22 . In organoid cocultures, iMac differentiated into cells with microglia-like phenotypes and functions (iMicro) and modulated neuronal progenitor cell (NPC) differentiation, limiting NPC proliferation and promoting axonogenesis. Mechanistically, iMicro contained high levels of PLIN2 + lipid droplets that exported cholesterol and its esters, which were taken up by NPCs in the organoids. We also detected PLIN2 + lipid droplet-loaded microglia in mouse and human embryonic brains. Overall, our approach substantially advances current human brain organoid approaches by incorporating microglial cells, as illustrated by the discovery of a key pathway of lipid-mediated crosstalk between microglia and NPCs that leads to improved neurogenesis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Cytometry enables precise single-cell phenotyping within heterogeneous populations. These cell types are traditionally annotated via manual gating, but this method lacks reproducibility and sensitivity to batch effect. Also, the most recent cytometers-spectral flow or mass cytometers-create rich and high-dimensional data whose analysis via manual gating becomes challenging and time-consuming. To tackle these limitations, we introduce Scyan https://github.com/MICS-Lab/scyan, a Single-cell Cytometry Annotation Network that automatically annotates cell types using only prior expert knowledge about the cytometry panel. For this, it uses a normalizing flow-a type of deep generative model-that maps protein expressions into a biologically relevant latent space. We demonstrate that Scyan significantly outperforms the related state-of-the-art models on multiple public datasets while being faster and interpretable. In addition, Scyan overcomes several complementary tasks, such as batch-effect correction, debarcoding and population discovery. Overall, this model accelerates and eases cell population characterization, quantification and discovery in cytometry., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Conventional dendritic cells (cDCs) are professional antigen-presenting cells that control the adaptive immune response. Their subsets and developmental origins have been intensively investigated but are still not fully understood as their phenotypes, especially in the DC2 lineage and the recently described human DC3s, overlap with monocytes. Here, using LEGENDScreen to profile DC vs. monocyte lineages, we found sustained expression of FLT3 and CD45RB through the whole DC lineage, allowing DCs and their precursors to be distinguished from monocytes. Using fate mapping models, single-cell RNA sequencing and adoptive transfer, we identified a lineage of murine CD16/32 + CD172a + DC3, distinct from DC2, arising from Ly6C + monocyte-DC progenitors (MDPs) through Lyz2 + Ly6C + CD11c - pro-DC3s, whereas DC2s develop from common DC progenitors (CDPs) through CD7 + Ly6C + CD11c + pre-DC2s. Corresponding DC subsets, developmental stages, and lineages exist in humans. These findings reveal DC3 as a DC lineage phenotypically related to but developmentally different from monocytes and DC2s., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

BackgroundSARS-CoV-2 infection in Africa has been characterized by a less severe disease profile than what has been observed elsewhere, but the profile of SARS-CoV-2-specific adaptive immunity in these mainly asymptomatic patients has not, to our knowledge, been analyzed.MethodsWe collected blood samples from residents of rural Kenya (n = 80), who had not experienced any respiratory symptoms or had contact with individuals with COVID-19 and had not received COVID-19 vaccines. We analyzed spike-specific antibodies and T cells specific for SARS-CoV-2 structural (membrane, nucleocapsid, and spike) and accessory (ORF3a, ORF7, ORF8) proteins. Pre-pandemic blood samples collected in Nairobi (n = 13) and blood samples from mild-to-moderately symptomatic COVID-19 convalescent patients (n = 36) living in the urban environment of Singapore were also studied.ResultsAmong asymptomatic Africans, we detected anti-spike antibodies in 41.0% of the samples and T cell responses against 2 or more SARS-CoV-2 proteins in 82.5% of samples examined. Such a pattern was absent in the pre-pandemic samples. Furthermore, distinct from cellular immunity in European and Asian COVID-19 convalescents, we observed strong T cell immunogenicity against viral accessory proteins (ORF3a, ORF8) but not structural proteins, as well as a higher IL-10/IFN-γ cytokine ratio profile.ConclusionsThe high incidence of T cell responses against different SARS-CoV-2 proteins in seronegative participants suggests that serosurveys underestimate SARS-CoV-2 prevalence in settings where asymptomatic infections prevail. The functional and antigen-specific profile of SARS-CoV-2-specific T cells in African individuals suggests that environmental factors can play a role in the development of protective antiviral immunity.FundingUS Centers for Disease Control and Prevention, Division of Global Health Protection; the Singapore Ministry of Health's National Medical Research Council (COVID19RF3-0060, COVID19RF-001, COVID19RF-008, MOH-StaR17Nov-0001).

Antibiotics (ABX) compromise the efficacy of programmed cell death protein 1 (PD-1) blockade in cancer patients, but the mechanisms underlying their immunosuppressive effects remain unknown. By inducing the down-regulation of mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the ileum, post-ABX gut recolonization by Enterocloster species drove the emigration of enterotropic α4β7 + CD4 + regulatory T 17 cells into the tumor. These deleterious ABX effects were mimicked by oral gavage of Enterocloster species, by genetic deficiency, or by antibody-mediated neutralization of MAdCAM-1 and its receptor, α4β7 integrin. By contrast, fecal microbiota transplantation or interleukin-17A neutralization prevented ABX-induced immunosuppression. In independent lung, kidney, and bladder cancer patient cohorts, low serum levels of soluble MAdCAM-1 had a negative prognostic impact. Thus, the MAdCAM-1-α4β7 axis constitutes an actionable gut immune checkpoint in cancer immunosurveillance.

Langerhans cell histiocytosis (LCH) is a potentially fatal neoplasm characterized by the aberrant differentiation of mononuclear phagocytes, driven by mitogen-activated protein kinase (MAPK) pathway activation. LCH cells may trigger destructive pathology yet remain in a precarious state finely balanced between apoptosis and survival, supported by a unique inflammatory milieu. The interactions that maintain this state are not well known and may offer targets for intervention. Here, we used single-cell RNA-seq and protein analysis to dissect LCH lesions, assessing LCH cell heterogeneity and comparing LCH cells with normal mononuclear phagocytes within lesions. We found LCH discriminatory signatures pointing to senescence and escape from tumor immune surveillance. We also uncovered two major lineages of LCH with DC2- and DC3/monocyte-like phenotypes and validated them in multiple pathological tissue sites by high-content imaging. Receptor-ligand analyses and lineage tracing in vitro revealed Notch-dependent cooperativity between DC2 and DC3/monocyte lineages during expression of the pathognomonic LCH program. Our results present a convergent dual origin model of LCH with MAPK pathway activation occurring before fate commitment to DC2 and DC3/monocyte lineages and Notch-dependent cooperativity between lineages driving the development of LCH cells.

Therapeutic cancer vaccines are designed to increase tumor-specific T cell immunity. However, suppressive mechanisms within the tumor microenvironment (TME) may limit T cell function. Here, we assessed how the route of vaccination alters intratumoral myeloid cells. Using a self-assembling nanoparticle vaccine that links tumor antigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we treated tumor-bearing mice subcutaneously (SNP-SC) or intravenously (SNP-IV). Both routes generated antigen-specific CD8 + T cells that infiltrated tumors. However, only SNP-IV mediated tumor regression, dependent on systemic type I interferon at the time of boost. Single-cell RNA-sequencing revealed that intratumoral monocytes expressing an immunoregulatory gene signature (Chil3, Anxa2, Wfdc17) were reduced after SNP-IV boost. In humans, the Chil3 + monocyte gene signature is enriched in CD16 - monocytes and associated with worse outcomes. Our results show that the generation of tumor-specific CD8 + T cells combined with remodeling of the TME is a promising approach for tumor immunotherapy., Competing Interests: Declaration of interests A.S.I., G.M.L., and R.A.S. are listed as inventors on patents describing polymer-based vaccines. A.S.I. and G.M.L. are employees of Vaccitech North America, which is commercializing polymer-based drug delivery technologies for immunotherapeutic applications. F.B. and S.M. are employees of Genentech, a member of the Roche group, which develops and markets drugs for profit., (Published by Elsevier Inc.)

Bats are reservoir hosts of many zoonotic viruses with pandemic potential. We utilized single-cell transcriptome sequencing (scRNA-seq) to analyze the immune response in bat lungs upon in vivo infection with a double-stranded RNA virus, Pteropine orthoreovirus PRV3M. Bat neutrophils were distinguished by high basal IDO1 expression. NK cells and T cells were the most abundant immune cells in lung tissue. Three distinct CD8 + effector T cell populations could be delineated by differential expression of KLRB1, GFRA2, and DPP4. Select NK and T clusters increased expression of genes involved in T cell activation and effector function early after viral infection. Alveolar macrophages and classical monocytes drove antiviral interferon signaling. Infection expanded a CSF1R + population expressing collagen-like genes, which became the predominant myeloid cell type post-infection. This work uncovers features relevant to viral disease tolerance in bats, lays a foundation for future experimental work, and serves as a resource for comparative immunology studies., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background and Aims: Hypoxia is one of the central players in shaping the immune context of the tumor microenvironment (TME). However, the complex interplay between immune cell infiltrates within the hypoxic TME of HCC remains to be elucidated., Approach and Results: We analyzed the immune landscapes of hypoxia-low and hypoxia-high tumor regions using cytometry by time of light, immunohistochemistry, and transcriptomic analyses. The mechanisms of immunosuppression in immune subsets of interest were further explored using in vitro hypoxia assays. Regulatory T cells (Tregs) and a number of immunosuppressive myeloid subsets, including M2 macrophages and human leukocyte antigen-DR isotype (HLA-DR lo ) type 2 conventional dendritic cell (cDC2), were found to be significantly enriched in hypoxia-high tumor regions. On the other hand, the abundance of active granzyme B hi PD-1 lo CD8 + T cells in hypoxia-low tumor regions implied a relatively active immune landscape compared with hypoxia-high regions. The up-regulation of cancer-associated genes in the tumor tissues and immunosuppressive genes in the tumor-infiltrating leukocytes supported a highly pro-tumorigenic network in hypoxic HCC. Chemokine genes such as CCL20 (C-C motif chemokine ligand 20) and CXCL5 (C-X-C motif chemokine ligand 5) were associated with recruitment of both Tregs and HLA-DR lo cDC2 to hypoxia-high microenvironments. The interaction between Tregs and cDC2 under a hypoxic TME resulted in a loss of antigen-presenting HLA-DR on cDC2., Conclusions: We uncovered the unique immunosuppressive landscapes and identified key immune subsets enriched in hypoxic HCC. In particular, we identified a potential Treg-mediated immunosuppression through interaction with a cDC2 subset in HCC that could be exploited for immunotherapies., (© 2022 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Brain macrophage populations include parenchymal microglia, border-associated macrophages, and recruited monocyte-derived cells; together, they control brain development and homeostasis but are also implicated in aging pathogenesis and neurodegeneration. The phenotypes, localization, and functions of each population in different contexts have yet to be resolved. We generated a murine brain myeloid scRNA-seq integration to systematically delineate brain macrophage populations. We show that the previously identified disease-associated microglia (DAM) population detected in murine Alzheimer's disease models actually comprises two ontogenetically and functionally distinct cell lineages: embryonically derived triggering receptor expressed on myeloid cells 2 (TREM2)-dependent DAM expressing a neuroprotective signature and monocyte-derived TREM2-expressing disease inflammatory macrophages (DIMs) accumulating in the brain during aging. These two distinct populations appear to also be conserved in the human brain. Herein, we generate an ontogeny-resolved model of brain myeloid cell heterogeneity in development, homeostasis, and disease and identify cellular targets for the treatment of neurodegeneration., Competing Interests: Declaration of interests A.S. and F.G. are inventors on a patent filed, owned, and managed by A∗ccelerate technologies Pte Ltd, A(∗)STAR, Singapore, on technology related to the work presented in this manuscript., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Macrophage infiltration is a hallmark of solid cancers, and overall macrophage infiltration correlates with lower patient survival and resistance to therapy. Tumor-associated macrophages, however, are phenotypically and functionally heterogeneous. Specific subsets of tumor-associated macrophage might be endowed with distinct roles on cancer progression and antitumor immunity. Here, we identify a discrete population of FOLR2 + tissue-resident macrophages in healthy mammary gland and breast cancer primary tumors. FOLR2 + macrophages localize in perivascular areas in the tumor stroma, where they interact with CD8 + T cells. FOLR2 + macrophages efficiently prime effector CD8 + T cells ex vivo. The density of FOLR2 + macrophages in tumors positively correlates with better patient survival. This study highlights specific roles for tumor-associated macrophage subsets and paves the way for subset-targeted therapeutic interventions in macrophages-based cancer therapies., Competing Interests: Declaration of interests The authors J.H., R.N.R., E.P., and P.G. and their institutions have filed a patent related to this work., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The immune system is a complex, dynamic, and plastic ecosystem composed of multiple cell types that constantly sense and interact with their local microenvironment to protect from infection and maintain homeostasis. For over a century, great efforts and ingenuity have been applied to the characterization of immune cells and their microenvironments, but traditional marker-based and bulk technologies left key questions unanswered. In the past decade, the advent of single-cell genomic approaches has revolutionized our knowledge of the cellular and molecular makeup of the immune system. In this perspective, we outline the past, present, and future applications of single-cell genomics in immunology and discuss how the integration of multiomics at the single-cell level will pave the way for future advances in immunology research and clinical translation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system underpinned by partially understood genetic risk factors and environmental triggers and their undefined interactions 1,2 . Here we investigated the peripheral immune signatures of 61 monozygotic twin pairs discordant for MS to dissect the influence of genetic predisposition and environmental factors. Using complementary multimodal high-throughput and high-dimensional single-cell technologies in conjunction with data-driven computational tools, we identified an inflammatory shift in a monocyte cluster of twins with MS, coupled with the emergence of a population of IL-2 hyper-responsive transitional naive helper T cells as MS-related immune alterations. By integrating data on the immune profiles of healthy monozygotic and dizygotic twin pairs, we estimated the variance in CD25 expression by helper T cells displaying a naive phenotype to be largely driven by genetic and shared early environmental influences. Nonetheless, the expanding helper T cells of twins with MS, which were also elevated in non-twin patients with MS, emerged independent of the individual genetic makeup. These cells expressed central nervous system-homing receptors, exhibited a dysregulated CD25-IL-2 axis, and their proliferative capacity positively correlated with MS severity. Together, our matched-pair analysis of the extended twin approach allowed us to discern genetically and environmentally determined features of an MS-associated immune signature., (© 2022. The Author(s).)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., (© 2021 Wiley-VCH GmbH.)

Modern immunologic research increasingly requires high-dimensional analyses to understand the complex milieu of cell types that comprise the tissue microenvironments of disease. To achieve this, we developed Infinity Flow combining hundreds of overlapping flow cytometry panels using machine learning to enable the simultaneous analysis of the coexpression patterns of hundreds of surface-expressed proteins across millions of individual cells. In this study, we demonstrate that this approach allows the comprehensive analysis of the cellular constituency of the steady-state murine lung and the identification of previously unknown cellular heterogeneity in the lungs of melanoma metastasis–bearing mice. We show that by using supervised machine learning, Infinity Flow enhances the accuracy and depth of clustering or dimensionality reduction algorithms. Infinity Flow is a highly scalable, low-cost, and accessible solution to single-cell proteomics in complex tissues.

Inflammatory skin diseases including atopic dermatitis (AD) and psoriasis (PSO) are underpinned by dendritic cell (DC)-mediated T cell responses. Currently, the heterogeneous human cutaneous DC population is incompletely characterized, and its contribution to these diseases remains unclear. Here, we performed index-sorted single-cell flow cytometry and RNA sequencing of lesional and nonlesional AD and PSO skin to identify macrophages and all DC subsets, including the newly described mature LAMP3+BIRC3+ DCs enriched in immunoregulatory molecules (mregDC) and CD14+ DC3. By integrating our indexed data with published skin datasets, we generated a myeloid cell universe of DC and macrophage subsets in healthy and diseased skin. Importantly, we found that CD14+ DC3s increased in PSO lesional skin and co-produced IL1B and IL23A, which are pathological in PSO. Our study comprehensively describes the molecular characteristics of macrophages and DC subsets in AD and PSO at single-cell resolution, and identifies CD14+ DC3s as potential promoters of inflammation in PSO., Competing Interests: Disclosures:   E. Guttman-Yassky is an employee of Mount Sinai and has received research funds (grants paid to the institution) from Abbvie, Almirall, Amgen, AnaptysBio, Asana Biosciences, AstraZeneca, Boerhinger-Ingelhiem, Celgene, Dermavant, DS Biopharma, Eli Lilly, Galderma,Glenmark/Ichnos Sciences, Innovaderm, Janssen, Kiniksa, Kyowa Kirin, Leo Pharma, Novan, Novartis, Pfizer, Ralexar, Regeneron Pharmaceuticals, Inc., Sienna Biopharma, UCB, and Union Therapeutics/Antibiotx; and is a consultant for Abbvie, Aditum Bio, Almirall, Alpine, Amgen, Arena, Asana Biosciences, AstraZeneca, Bluefin Biomedicine, Boerhinger-Ingelhiem, Boston Pharmaceuticals, Botanix, Bristol-Meyers Squibb, Cara Therapeutics, Celgene, Clinical Outcome Solutions, Concert, DBV, Dermavant, Dermira, Douglas Pharmaceutical, DS Biopharma, Eli Lilly, EMD Serono, Evelo Bioscience, Evidera, FIDE, Galderma, GSK, Haus Bioceuticals, Ichnos Sciences, Incyte, Kyowa Kirin, Larrk Bio, Leo Pharma, Medicxi, Medscape, Neuralstem, Noble Insights, Novan, Novartis, Okava Pharmaceuticals, Pandion Therapeutics, Pfizer, Principia Biopharma, RAPT Therapeutics, Realm, Regeneron Pharmaceuticals, Inc., Sanofi, SATO Pharmaceutical, Sienna Biopharma, Seanegy Dermatology, Seelos Therapeutics, Serpin Pharma, Siolta Therapeutics, Sonoma Biotherapeutics, Sun Pharma, Target PharmaSolutions and Union Therapeutics, Vanda Pharmaceuticals, Ventyx Biosciences, and Vimalan. No other disclosures were reported., (© 2021 Nakamizo et al.)

Breast cancers are complex cellular ecosystems where heterotypic interactions play central roles in disease progression and response to therapy. However, our knowledge of their cellular composition and organization is limited. Here we present a single-cell and spatially resolved transcriptomics analysis of human breast cancers. We developed a single-cell method of intrinsic subtype classification (SCSubtype) to reveal recurrent neoplastic cell heterogeneity. Immunophenotyping using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) provides high-resolution immune profiles, including new PD-L1/PD-L2 + macrophage populations associated with clinical outcome. Mesenchymal cells displayed diverse functions and cell-surface protein expression through differentiation within three major lineages. Stromal-immune niches were spatially organized in tumors, offering insights into antitumor immune regulation. Using single-cell signatures, we deconvoluted large breast cancer cohorts to stratify them into nine clusters, termed 'ecotypes', with unique cellular compositions and clinical outcomes. This study provides a comprehensive transcriptional atlas of the cellular architecture of breast cancer., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Hepatocellular carcinoma (HCC) often develops following chronic hepatitis B virus (HBV) infection and responds poorly to immune checkpoint blockade. Here, we examined the antigen specificities of HCC-infiltrating T cells and their relevance to tumor control. Using highly multiplexed peptide-MHC tetramer staining of unexpanded cells from blood, liver, and tumor tissues from 46 HCC patients, we detected 91 different antigen-specific CD8 + T cell populations targeting HBV, neoantigen, tumor-associated, and disease-unrelated antigens. Parallel high-dimensional analysis delineated five distinct antigen-specific tissue-resident memory T (Trm) cell populations. Intratumoral and intrahepatic HBV-specific T cells were enriched for two Trm cell subsets that were PD-1 lo TOX lo , despite being clonally expanded. High frequencies of intratumoral terminally exhausted T cells were uncommon. Patients with tumor-infiltrating HBV-specific CD8 + Trm cells exhibited longer-term relapse-free survival. Thus, non-terminally exhausted HBV-specific CD8 + Trm cells show hallmarks of active involvement and effective antitumor response, implying that these cells could be harnessed for therapeutic purposes., Competing Interests: Declaration of interests Y.C. and E.W.N. are inventors on US patent application (pending) held by A(∗)STAR, which includes the uses of tumor-reactive epitope sequences and the epitope-reactive TCRs for treatment against HBV-associated HCC. S.G.L. received research grants from Gilead Science, Merck, and Abbott Diagnostics. S.G.L. served as the advisory board member of Gilead Science, Abbvie, Abbott Diagnostics, Merck, Springbank, and Roche. E.W.N. is the co-founder, shareholder, and an advisor and is on the board of directors of ImmunoScape Pte. Ltd. and is an advisor for Neogene Therapeutics and Nanostring Technologies. P.K.H.C. has received honoraria from Sirtex Medical, Ipsen, Bristol Myers Squibb, Oncosoil, Bayer, Roche, New B Innovation, MSD, EISA, Abbott, IQVIA, Genetech, L.E.K. Consulting, and AstraZeneca in an advisory role., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mononuclear phagocytes (MNPs) encompass dendritic cells, monocytes, and macrophages (MoMac), which exhibit antimicrobial, homeostatic, and immunoregulatory functions. We integrated 178,651 MNPs from 13 tissues across 41 datasets to generate a MNP single-cell RNA compendium (MNP-VERSE), a publicly available tool to map MNPs and define conserved gene signatures of MNP populations. Next, we generated a MoMac-focused compendium that revealed an array of specialized cell subsets widely distributed across multiple tissues. Specific pathological forms were expanded in cancer and inflammation. All neoplastic tissues contained conserved tumor-associated macrophage populations. In particular, we focused on IL4I1 + CD274(PD-L1) + IDO1 + macrophages, which accumulated in the tumor periphery in a T cell-dependent manner via interferon-γ (IFN-γ) and CD40/CD40L-induced maturation from IFN-primed monocytes. IL4I1_Macs exhibited immunosuppressive characteristics through tryptophan degradation and promoted the entry of regulatory T cell into tumors. This integrated analysis provides a robust online-available platform for uniform annotation and dissection of specific macrophage functions in healthy and pathological states., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2 nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14 th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2 nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

The efficacy of virus-specific T cells in clearing pathogens involves a fine balance between antiviral and inflammatory features. SARS-CoV-2-specific T cells in individuals who clear SARS-CoV-2 without symptoms could reveal nonpathological yet protective characteristics. We longitudinally studied SARS-CoV-2-specific T cells in a cohort of asymptomatic (n = 85) and symptomatic (n = 75) COVID-19 patients after seroconversion. We quantified T cells reactive to structural proteins (M, NP, and Spike) using ELISpot and cytokine secretion in whole blood. Frequencies of SARS-CoV-2-specific T cells were similar between asymptomatic and symptomatic individuals, but the former showed an increased IFN-γ and IL-2 production. This was associated with a proportional secretion of IL-10 and proinflammatory cytokines (IL-6, TNF-α, and IL-1β) only in asymptomatic infection, while a disproportionate secretion of inflammatory cytokines was triggered by SARS-CoV-2-specific T cell activation in symptomatic individuals. Thus, asymptomatic SARS-CoV-2-infected individuals are not characterized by weak antiviral immunity; on the contrary, they mount a highly functional virus-specific cellular immune response., Competing Interests: Disclosures: N. Le Bert and A.T. Tan reported a patent for a method to monitor SARS-CoV-2-specific T cells in biological samples pending. W.N. Chia reported a patent for a sublicense agreement with GenScript for the surrogate virus neutralization test pending (Duke-NUS). P. Tambyah reported grants from Arcturus, Roche, Shionogi, Sanofi-Pasteur, and Aj Biologics outside the submitted work. L. Wang reported a patent application on sVNT pending. A. Bertoletti reported personal fees from Oxford Immunotech and Qiagen outside the submitted work; in addition, A. Bertoletti had a patent for the use of peptide pools in whole blood for detection of SARS-CoV-2 T cells pending. C.C. Tam reported grants from Roche and personal fees from Verivax outside the submitted work. No other disclosures were reported., (© 2021 Le Bert et al.)

Acquisition of cell-associated tumor antigens by type 1 dendritic cells (cDC1) is essential to induce and sustain tumor specific CD8 + T cells via cross-presentation. Here we show that capture and engulfment of cell associated antigens by tissue resident lung cDC1 is inhibited during progression of mouse lung tumors. Mechanistically, loss of phagocytosis is linked to tumor-mediated downregulation of the phosphatidylserine receptor TIM4, that is highly expressed in normal lung resident cDC1. TIM4 receptor blockade and conditional cDC1 deletion impair activation of tumor specific CD8 + T cells and promote tumor progression. In human lung adenocarcinomas, TIM4 transcripts increase the prognostic value of a cDC1 signature and predict responses to PD-1 treatment. Thus, TIM4 on lung resident cDC1 contributes to immune surveillance and its expression is suppressed in advanced tumors.

Background: Atopic dermatitis (AD) is a common skin disease affecting up to 20% of the global population, with significant clinical heterogeneity and limited information about molecular subtypes and actionable biomarkers. Although alterations in the skin microbiome have been described in subjects with AD during progression to flare state, the prognostic value of baseline microbiome configurations has not been explored., Objective: Our aim was to identify microbial signatures on AD skin that are predictive of disease fate., Methods: Nonlesional skin of patients with AD and healthy control subjects were sampled at 2 time points separated by at least 4 weeks. Using whole metagenome analysis of skin microbiomes of patients with AD and control subjects (n = 49 and 189 samples), we identified distinct microbiome configurations (dermotypes A and B). Blood was collected for immunophenotyping, and skin surface samples were analyzed for correlations with natural moisturizing factors and antimicrobial peptides., Results: Dermotypes were robust and validated across 2 additional cohorts (63 individuals), with strong enrichment of subjects with AD in dermotype B. Dermotype B was characterized by reduced microbial richness, depletion of Cutibacterium acnes, Dermacoccus and Methylobacterium species, individual-specific outlier abundance of Staphylococcus species (eg, S epidermidis, S capitis, S aureus), and enrichment in metabolic pathways (eg, branched chain amino acids and arginine biosynthesis) and virulence genes (eg, β-toxin, δ-toxin) that defined a pathogenic ecology. Skin surface and circulating host biomarkers exhibited a distinct microbial-associated signature that was further reflected in more severe itching, frequent flares, and increased disease severity in patients harboring the dermotype B microbiome., Conclusion: We report distinct clusters of microbial profiles that delineate the role of microbiome configurations in AD heterogeneity, highlight a mechanism for ongoing inflammation, and provide prognostic utility toward microbiome-based disease stratification., (Copyright © 2020 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

For anthracycline-based chemotherapy to be immunogenic, dying cancer cells must release annexin A1 (ANXA1) that subsequently interacts with the pattern recognition receptor, formyl peptide receptor 1 (FPR1), on the surface of dendritic cells (DC). Approximately 30% of individuals bear loss-of-function alleles of FPR1 , calling for strategies to ameliorate their anticancer immune response. Here, we show that immunotherapy with a ligand of Toll-like receptor-3, polyinosinic:polycytidylic acid (pIC), restores the deficient response to chemotherapy of tumors lacking ANXA1 developing in immunocompetent mice or those of normal cancers growing in FPR1-deficient mice. This effect was accompanied by improved DC- and T-lymphocyte-mediated anticancer immunity. Of note, carcinogen-induced breast cancers precociously developed in FPR1-deficient mice as compared with wild-type controls. A similar tendency for earlier cancer development was found in patients carrying the loss-of-function allele of FPR1 . These findings have potential implications for the clinical management of FPR1-deficient patients. SIGNIFICANCE: The loss-of-function variant rs867228 in FPR1 , harbored by approximately 30% of the world population, is associated with the precocious manifestation of breast, colorectal, esophageal, and head and neck carcinomas. pIC restores deficient chemotherapeutic responses in mice lacking Fpr1 , suggesting a personalized strategy for compensating for the FPR1 defect. This article is highlighted in the In This Issue feature, p. 211 ., (©2020 American Association for Cancer Research.)

Dendritic cells (DCs) develop in the bone marrow from haematopoietic progenitors that have numerous shared characteristics between mice and humans. Human counterparts of mouse DC progenitors have been identified by their shared transcriptional signatures and developmental potential. New findings continue to revise models of DC ontogeny but it is well accepted that DCs can be divided into two main functional groups. Classical DCs include type 1 and type 2 subsets, which can detect different pathogens, produce specific cytokines and present antigens to polarize mainly naive CD8 + or CD4 + T cells, respectively. By contrast, the function of plasmacytoid DCs is largely innate and restricted to the detection of viral infections and the production of type I interferon. Here, we discuss genetic models of mouse DC development and function that have aided in correlating ontogeny with function, as well as how these findings can be translated to human DCs and their progenitors.

Personalized cancer vaccines are a promising approach for inducing T cell immunity to tumor neoantigens. Using a self-assembling nanoparticle vaccine that links neoantigen peptides to a Toll-like receptor 7/8 agonist (SNP-7/8a), we show how the route and dose alter the magnitude and quality of neoantigen-specific CD8 + T cells. Intravenous vaccination (SNP-IV) induced a higher proportion of TCF1 + PD-1 + CD8 + T cells as compared to subcutaneous immunization (SNP-SC). Single-cell RNA sequencing showed that SNP-IV induced stem-like genes (Tcf7, Slamf6, Xcl1) whereas SNP-SC enriched for effector genes (Gzmb, Klrg1, Cx3cr1). Stem-like cells generated by SNP-IV proliferated and differentiated into effector cells upon checkpoint blockade, leading to superior antitumor response as compared to SNP-SC in a therapeutic model. The duration of antigen presentation by dendritic cells controlled the magnitude and quality of CD8 + T cells. These data demonstrate how to optimize antitumor immunity by modulating vaccine parameters for specific generation of effector or stem-like CD8 + T cells.

Mast cells (MCs) are central effector cells in allergic reactions that are often mediated by immunoglobulin E (IgE). Allergies commonly start at an early age, and both MCs and IgE are detectable in fetuses. However, the origin of fetal IgE and whether fetal MCs can degranulate in response to IgE-dependent activation are presently unknown. Here, we show that human and mouse fetal MCs phenotypically mature through pregnancy and can be sensitized by maternal IgE. IgE crossed the placenta, dependent on the fetal neonatal Fc receptor (FcRN), and sensitized fetal MCs for allergen-specific degranulation. Both passive and active prenatal sensitization conferred allergen sensitivity, resulting in postnatal skin and airway inflammation after the first allergen encounter. We report a role for MCs within the developing fetus and demonstrate that fetal MCs may contribute to antigen-specific vertical transmission of allergic disease., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

We employed scRNA sequencing to extensively characterize the cellular landscape of human liver from development to disease. Analysis of ∼212,000 cells representing human fetal, hepatocellular carcinoma (HCC), and mouse liver revealed remarkable fetal-like reprogramming of the tumor microenvironment. Specifically, the HCC ecosystem displayed features reminiscent of fetal development, including re-emergence of fetal-associated endothelial cells (PLVAP/VEGFR2) and fetal-like (FOLR2) tumor-associated macrophages. In a cross-species comparative analysis, we discovered remarkable similarity between mouse embryonic, fetal-liver, and tumor macrophages. Spatial transcriptomics further revealed a shared onco-fetal ecosystem between fetal liver and HCC. Furthermore, gene regulatory analysis, spatial transcriptomics, and in vitro functional assays implicated VEGF and NOTCH signaling in maintaining onco-fetal ecosystem. Taken together, we report a shared immunosuppressive onco-fetal ecosystem in fetal liver and HCC. Our results unravel a previously unexplored onco-fetal reprogramming of the tumor ecosystem, provide novel targets for therapeutic interventions in HCC, and open avenues for identifying similar paradigms in other cancers and disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Granulocyte-monocyte progenitors (GMPs) have been previously defined for their potential to generate various myeloid progenies such as neutrophils and monocytes. Although studies have proposed lineage heterogeneity within GMPs, it is unclear if committed progenitors already exist among these progenitors and how they may behave differently during inflammation. By combining single-cell transcriptomic and proteomic analyses, we identified the early committed progenitor within the GMPs responsible for the strict production of neutrophils, which we designate as proNeu1. Our dissection of the GMP hierarchy led us to further identify a previously unknown intermediate proNeu2 population. Similar populations could be detected in human samples. proNeu1s, but not proNeu2s, selectively expanded during the early phase of sepsis at the expense of monocytes. Collectively, our findings help shape the neutrophil maturation trajectory roadmap and challenge the current definition of GMPs., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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

Classical dendritic cells (cDCs) are rare sentinel cells specialized in the regulation of adaptive immunity. Modeling cDC development is crucial to study cDCs and harness their therapeutic potential. Here we address whether cDCs could differentiate in response to trophic cues delivered by mesenchymal components of the hematopoietic niche. We find that mesenchymal stromal cells engineered to express membrane-bound FLT3L and stem cell factor (SCF) together with CXCL12 induce the specification of human cDCs from CD34 + hematopoietic stem and progenitor cells (HSPCs). Engraftment of engineered mesenchymal stromal cells (eMSCs) together with CD34 + HSPCs creates an in vivo synthetic niche in the dermis of immunodeficient mice driving the differentiation of cDCs and CD123 + AXL + CD327 + pre/AS-DCs. cDC2s generated in vivo display higher levels of resemblance with human blood cDCs unattained by in vitro-generated subsets. Altogether, eMSCs provide a unique platform recapitulating the full spectrum of cDC subsets enabling their functional characterization in vivo.

Bats are asymptomatic reservoir hosts for several highly pathogenic viruses. Understanding this enigmatic relationship between bats and emerging zoonotic viruses requires tools and approaches which enable the comparative study of bat immune cell populations and their functions. We show that bat genomes have a conservation of immune marker genes which delineate phagocyte populations in humans, while lacking key mouse surface markers such as Ly6C and Ly6G. Cross-reactive antibodies against CD44, CD11b, CD14, MHC II, and CD206 were multiplexed to characterize circulating monocytes, granulocytes, bone-marrow derived macrophages (BMDMs) and lung alveolar macrophages (AMs) in the cave nectar bat Eonycteris spelaea. Transcriptional profiling of bat monocytes and BMDMs identified additional markers - including MARCO, CD68, CD163, CD172α, and CD88 - which can be used to further characterize bat myeloid populations. Bat cells often resembled their human counterparts when comparing immune parameters that are divergent between humans and mice, such as the expression patterns of certain immune cell markers. A genome-wide comparison of immune-related genes also revealed a much closer phylogenetic relationship between bats and humans compared to rodents. Taken together, this study provides a set of tools and a comparative framework which will be important for unravelling viral disease tolerance mechanisms in bats.

Background & Aims: Knowledge about the regulation of anti-HBV humoral immunity during natural HBV infection is limited. We recently utilized dual fluorochrome-conjugated HBsAg to demonstrate, in patients with chronic HBV (CHB) infection, the functional impairment of their HBsAg-specific B cells. However, the features of their HBcAg-specific B cells are unknown. Here we developed a method to directly visualize, select and characterize HBcAg-specific B cells in parallel with HBsAg-specific B cells., Methods: Fluorochrome-conjugated HBcAg reagents were synthesized and utilized to directly detect ex vivo HBcAg-specific B cells in 36 patients with CHB. The frequency, phenotype, functional maturation and transcriptomic profile of HBcAg-specific B cells was studied by flow cytometry, in vitro maturation assays and NanoString-based detection of expression of immune genes, which we compared with HBsAg-specific B cells and total B cells., Results: HBcAg-specific B cells are present at a higher frequency than HBsAg-specific B cells in patients with CHB and, unlike HBsAg-specific B cells, they mature efficiently into antibody-secreting cells in vitro. Their phenotypic and transcriptomic profiles show that HBcAg-specific B cells are preferentially IgG+ memory B cells. However, despite their phenotypic and functional differences, HBcAg- and HBsAg-specific B cells from patients with CHB share an mRNA expression pattern that differs from global memory B cells and is characterized by high expression of genes indicative of cross-presentation and innate immune activity., Conclusions: During chronic HBV infection, a direct relation exists between serological detection of anti-HBs and anti-HBc antibodies, and the quantity and function of their respective specific B cells. However, the transcriptomic analysis performed in HBsAg- and HBcAg-specific B cells suggests additional roles of HBV-specific B cells beyond the production of antibodies., Lay Summary: Protection of viral infection necessitates the production of antibodies that are generated by specialized cells of the immune system called B cells. During chronic HBV infection, antibodies against the internal part of the virus (core or HBcAg) are detectable while the antibodies directed against the virus envelope (surface or HBsAg) are not present. Here we developed a method that allows us to directly visualize ex vivo the B cells specific for these 2 viral components, highlighting their differences and similarities, and showing how 2 components of the same virus can have different impacts on the function of antiviral B cells., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer's and Parkinson's disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background & Aims: Chronic liver inflammation leads to fibrosis and cirrhosis and is associated with an accumulation of intrahepatic TNFα-secreting CD206 + macrophages, which may participate in maintaining chronic liver disease in a GM-CSF-dependent manner. We aimed to elucidate the exact role of GM-CSF in the development and progression of chronic liver disease., Methods: Liver immunohistochemistry and serum quantification were performed in patients with viral and non-viral-related liver disease to compare CD206 + monocyte/macrophages, fibrosis and GM-CSF. This was followed by functional validations in vitro and in vivo in humanised mice., Results: Using multiplex immunofluorescence and histo-cytometry, we show that highly fibrotic livers had a greater density of CD206 + macrophages that produced more TNFα and GM-CSF in the non-tumour liver regions of patients with hepatocellular carcinoma (n = 47), independent of aetiology. In addition, the absolute number of CD206 + macrophages strongly correlated with the absolute number of GM-CSF-producing macrophages. In non-HCC chronic HCV + patients (n = 40), circulating GM-CSF levels were also increased in proportion to the degree of liver fibrosis and serum viral titres. We then demonstrated in vitro that monocytes converted to TNFα-producing CD206 + macrophage-like cells in response to bacterial products (lipopolysaccharide) in a GM-CSF-dependent manner, confirming the in vivo normalisation of serum GM-CSF concentration following oral antibiotic treatment observed in HBV-infected humanised mice. Finally, anti-GM-CSF neutralising antibody treatment reduced intrahepatic CD206 + macrophage accumulation and abolished liver fibrosis in HBV-infected humanised mice., Conclusions: While the direct involvement of CD206 + macrophages in liver fibrosis remains to be demonstrated, these findings show that GM-CSF may play a central role in liver fibrosis and could guide the development of anti-GM-CSF antibody-based therapy for the management of patients with chronic liver disease., Lay Summary: Liver fibrosis is a major driver of liver disease progression. Herein, we have shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the development of liver fibrosis. Our findings support the use of anti-GM-CSF neutralising antibodies for the management of patients with chronic liver disease resulting from both viral and non-viral causes., (© 2019 The Author(s).)

The human dendritic cell (DC) lineage has recently been unraveled by high-dimensional mapping, revealing the existence of a discrete new population of blood circulating DC precursors (pre-DCs). Whether this new DC population possesses specific functional features as compared to the other blood DC subset upon pathogen encounter remained to be evaluated. A unique feature of pre-DCs among blood DCs is their constitutive expression of the viral adhesion receptor Siglec-1. Here, we show that pre-DCs, but not other blood DC subsets, are susceptible to infection by HIV-1 in a Siglec-1-dependent manner. Siglec-1 mediates pre-DC infection of CCR5- and CXCR4-tropic strains. Infection of pre-DCs is further enhanced in the presence of HIV-2/SIVmac Vpx, indicating that Siglec-1 does not counteract restriction factors such as SAMHD1. Instead, Siglec-1 promotes attachment and fusion of viral particles. HIV-1-infected pre-DCs produce new infectious viral particles that accumulate in intracellular compartments reminiscent of the virus-containing compartment of macrophages. Pre-DC activation by toll-like receptor (TLR) ligands induces an antiviral state that inhibits HIV-1 fusion and infection, but Siglec-1 remains functional and mediates replication-independent transfer of HIV-1 to activated primary T lymphocytes. Altogether, Siglec-1-mediated susceptibility to HIV-1 infection of pre-DCs constitutes a unique functional feature that might represent a preferential relationship of this emerging cell type with viruses., Competing Interests: The authors declare no competing interest.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Human mononuclear phagocytes comprise phenotypically and functionally overlapping subsets of dendritic cells (DCs) and monocytes, but the extent of their heterogeneity and distinct markers for subset identification remains elusive. By integrating high-dimensional single-cell protein and RNA expression data, we identified distinct markers to delineate monocytes from conventional DC2 (cDC2s). Using CD88 and CD89 for monocytes and HLA-DQ and FcεRIα for cDC2s allowed for their specific identification in blood and tissues. We also showed that cDC2s could be subdivided into phenotypically and functionally distinct subsets based on CD5, CD163, and CD14 expression, including a distinct subset of circulating inflammatory CD5 - CD163 + CD14 + cells related to previously defined DC3s. These inflammatory DC3s were expanded in systemic lupus erythematosus patients and correlated with disease activity. These findings further unravel the heterogeneity of DC subpopulations in health and disease and may pave the way for the identification of specific DC subset-targeting therapies., (Copyright © 2019 Elsevier Inc. All rights reserved.)