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The liver macrophage population comprises resident Kupffer cells (KCs) and monocyte-derived macrophages with distinct pro- or anti-inflammatory properties that affect the severity and course of liver diseases. The mechanisms underlying macrophage differentiation and functions in metabolic dysfunction-associated steatotic liver disease and/or steatohepatitis (MASLD/MASH) remain mostly unknown. Using single-cell RNA sequencing (scRNA-seq) and fate mapping of hepatic macrophage subpopulations, we unraveled the temporal and spatial dynamics of distinct monocyte and monocyte-derived macrophage subsets in MASH. We revealed a crucial role for the Notch-Recombination signal binding protein for immunoglobulin kappa J region (RBPJ) signaling pathway in controlling the monocyte-to-macrophage transition, with Rbpj deficiency blunting inflammatory macrophages and monocyte-derived KC differentiation and conversely promoting the emergence of protective Ly6C lo monocytes. Mechanistically, Rbpj deficiency promoted lipid uptake driven by elevated CD36 expression in Ly6C lo monocytes, enhancing their protective interactions with endothelial cells. Our findings uncover the crucial role of Notch-RBPJ signaling in monocyte-to-macrophage transition and will aid in the design of therapeutic strategies for MASH treatment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Conventional dendritic cells (cDC) are antigen-presenting cells comprising cDC1 and cDC2, responsible for priming naive CD8 + and CD4 + T cells, respectively. Recent studies have unveiled cDC2 heterogeneity and identified various cDC2 progenitors beyond the common DC progenitor (CDP), hinting at distinct cDC2 lineages. By generating Cd300c iCre-hCD2 R26 tdTomato reporter mice, we identified a bone marrow pro-cDC2 progenitor exclusively generating cDC2 in vitro and in vivo. Single-cell analyses and multiparametric flow cytometry demonstrated that pro-cDC2 encompasses myeloid-derived pre-cDC2 and lymphoid-derived plasmacytoid DC (pDC)-like precursors differentiating into a transcriptionally convergent cDC2 phenotype. Cd300c-traced cDC2 had distinct transcriptomic profiles, phenotypes, and tissue distributions compared with Ms4a3 Cre R26 tdTomato lineage-traced DC3, a monocyte-DC progenitor (MDP)-derived subset that bypasses CDP. Mice with reduced Cd300c-traced cDC2 showed impaired humoral responses to T cell-dependent antigens. We conclude that progenitors of distinct lineages shape the diversity of mature cDC2 across tissues. Thus, ontogenesis may impact tissue immune responses., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Acute stroke triggers extensive changes to myeloid immune cell populations in the brain that may be targets for limiting brain damage and enhancing repair. Immunomodulatory approaches will be most effective with precise manipulation of discrete myeloid cell phenotypes in time and space. Here, we investigate how stroke alters mononuclear myeloid cell composition and phenotypes at single-cell resolution and key spatial patterns. Our results show that multiple reactive microglial states and monocyte-derived populations contribute to an extensive myeloid cell repertoire in post-stroke brains. We identify important overlaps and distinctions among different cell types/states that involve ontogeny- and spatial-related properties. Notably, brain connectivity with infarcted tissue underpins the pattern of local and remote altered cell accumulation and reactivity. Our discoveries suggest a global but anatomically governed brain myeloid cell response to stroke that comprises diverse phenotypes arising through intrinsic cell ontogeny factors interacting with exposure to spatially organized brain damage and neuro-axonal cues., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells., Competing Interests: Declaration of interests J.-L.C. serves on the scientific advisory boards of ADMA Biologics Inc., Kymera Therapeutics, and Elixiron Immunotherapeutics., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer is a progressive disease that can develop and evolve over decades, with inflammation playing a central role at each of its stages, from tumor initiation to metastasis. In this context, macrophages represent well-established bridges reciprocally linking inflammation and cancer via an array of diverse functions that have spurred efforts to classify them into subtypes. Here, we discuss the intertwines between macrophages, inflammation, and cancer with an emphasis on temporal dynamics of macrophage diversity and functions in pre-malignancy and cancer. By instilling temporal dynamism into the more static classic view of tumor-associated macrophage biology, we propose a new framework to better contextualize their significance in the inflammatory processes that precede and result from the onset of cancer and shape its evolution., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Microglia (MG), the brain-resident macrophages, play major roles in health and disease via a diversity of cellular states. While embryonic MG display a large heterogeneity of cellular distribution and transcriptomic states, their functions remain poorly characterized. Here, we uncovered a role for MG in the maintenance of structural integrity at two fetal cortical boundaries. At these boundaries between structures that grow in distinct directions, embryonic MG accumulate, display a state resembling post-natal axon-tract-associated microglia (ATM) and prevent the progression of microcavities into large cavitary lesions, in part via a mechanism involving the ATM-factor Spp1. MG and Spp1 furthermore contribute to the rapid repair of lesions, collectively highlighting protective functions that preserve the fetal brain from physiological morphogenetic stress and injury. Our study thus highlights key major roles for embryonic MG and Spp1 in maintaining structural integrity during morphogenesis, with major implications for our understanding of MG functions and brain development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Microglial reactivity to injury and disease is emerging as a heterogeneous, dynamic, and crucial determinant in neurological disorders. However, the plasticity and fate of disease-associated microglia (DAM) remain largely unknown. We established a lineage tracing system, leveraging the expression dynamics of secreted phosphoprotein 1（Spp1） to label and track DAM-like microglia during brain injury and recovery. Fate mapping of Spp1 + microglia during stroke in juvenile mice revealed an irreversible state of DAM-like microglia that were ultimately eliminated from the injured brain. By contrast, DAM-like microglia in the neonatal stroke models exhibited high plasticity, regaining a homeostatic signature and integrating into the microglial network after recovery. Furthermore, neonatal injury had a lasting impact on microglia, rendering them intrinsically sensitized to subsequent immune challenges. Therefore, our findings highlight the plasticity and innate immune memory of neonatal microglia, shedding light on the fate of DAM-like microglia in various neuropathological conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Adipose tissues (ATs) are innervated by sympathetic nerves, which drive reduction of fat mass via lipolysis and thermogenesis. Here, we report a population of immunomodulatory leptin receptor-positive (LepR + ) sympathetic perineurial barrier cells (SPCs) present in mice and humans, which uniquely co-express Lepr and interleukin-33 (Il33) and ensheath AT sympathetic axon bundles. Brown ATs (BATs) of mice lacking IL-33 in SPCs (SPC ΔIl33 ) had fewer regulatory T (Treg) cells and eosinophils, resulting in increased BAT inflammation. SPC ΔIl33 mice were more susceptible to diet-induced obesity, independently of food intake. Furthermore, SPC ΔIl33 mice had impaired adaptive thermogenesis and were unresponsive to leptin-induced rescue of metabolic adaptation. We therefore identify LepR + SPCs as a source of IL-33, which orchestrate an anti-inflammatory BAT environment, preserving sympathetic-mediated thermogenesis and body weight homeostasis. LepR + IL-33 + SPCs provide a cellular link between leptin and immune regulation of body weight, unifying neuroendocrinology and immunometabolism as previously disconnected fields of obesity research., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Deciphering the cell-state transitions underlying immune adaptation across time is fundamental for advancing biology. Empirical in vivo genomic technologies that capture cellular dynamics are currently lacking. We present Zman-seq, a single-cell technology recording transcriptomic dynamics across time by introducing time stamps into circulating immune cells, tracking them in tissues for days. Applying Zman-seq resolved cell-state and molecular trajectories of the dysfunctional immune microenvironment in glioblastoma. Within 24 hours of tumor infiltration, cytotoxic natural killer cells transitioned to a dysfunctional program regulated by TGFB1 signaling. Infiltrating monocytes differentiated into immunosuppressive macrophages, characterized by the upregulation of suppressive myeloid checkpoints Trem2, Il18bp, and Arg1, over 36 to 48 hours. Treatment with an antagonistic anti-TREM2 antibody reshaped the tumor microenvironment by redirecting the monocyte trajectory toward pro-inflammatory macrophages. Zman-seq is a broadly applicable technology, enabling empirical measurements of differentiation trajectories, which can enhance the development of more efficacious immunotherapies., Competing Interests: Declaration of interests A.W. and I.A. are inventors of a patent related to TREM2 antibodies. I.A. is a member of the Cell Advisory Board. A patent application has been filed related to this work., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Neurodegenerative diseases (ND) are characterized by progressive loss of neuronal function. Mechanisms of ND pathogenesis are incompletely understood, hampering the development of effective therapies. Langerhans cell histiocytosis (LCH) is an inflammatory neoplastic disorder caused by hematopoietic progenitors expressing mitogen-activated protein kinase (MAPK)-activating mutations that differentiate into senescent myeloid cells that drive lesion formation. Some individuals with LCH subsequently develop progressive and incurable neurodegeneration (LCH-ND). Here, we showed that LCH-ND was caused by myeloid cells that were clonal with peripheral LCH cells. Circulating BRAFV600E + myeloid cells caused the breakdown of the blood-brain barrier (BBB), enhancing migration into the brain parenchyma where they differentiated into senescent, inflammatory CD11a + macrophages that accumulated in the brainstem and cerebellum. Blocking MAPK activity and senescence programs reduced peripheral inflammation, brain parenchymal infiltration, neuroinflammation, neuronal damage and improved neurological outcome in preclinical LCH-ND. MAPK activation and senescence programs in circulating myeloid cells represent targetable mechanisms of LCH-ND., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023. Published by Elsevier Inc.)

Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.)

Memory CD8 + T cells can be broadly divided into circulating (T CIRCM ) and tissue-resident memory T (T RM ) populations. Despite well-defined migratory and transcriptional differences, the phenotypic and functional delineation of T CIRCM and T RM cells, particularly across tissues, remains elusive. Here, we utilized an antibody screening platform and machine learning prediction pipeline (InfinityFlow) to profile >200 proteins in T CIRCM and T RM cells in solid organs and barrier locations. High-dimensional analyses revealed unappreciated heterogeneity within T CIRCM and T RM cell lineages across nine different organs after either local or systemic murine infection models. Additionally, we demonstrated the relative effectiveness of strategies allowing for the selective ablation of T CIRCM or T RM populations across organs and identified CD55, KLRG1, CXCR6, and CD38 as stable markers for characterizing memory T cell function during inflammation. Together, these data and analytical framework provide an in-depth resource for memory T cell classification in both steady-state and inflammatory conditions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Therapeutic neoantigen cancer vaccines have limited clinical efficacy to date. Here, we identify a heterologous prime-boost vaccination strategy using a self-assembling peptide nanoparticle TLR-7/8 agonist (SNP) vaccine prime and a chimp adenovirus (ChAdOx1) vaccine boost that elicits potent CD8 T cells and tumor regression. ChAdOx1 administered intravenously (i.v.) had 4-fold higher antigen-specific CD8 T cell responses than mice boosted by the intramuscular (i.m.) route. In the therapeutic MC38 tumor model, i.v. heterologous prime-boost vaccination enhances regression compared with ChAdOx1 alone. Remarkably, i.v. boosting with a ChAdOx1 vector encoding an irrelevant antigen also mediates tumor regression, which is dependent on type I IFN signaling. Single-cell RNA sequencing of the tumor myeloid compartment shows that i.v. ChAdOx1 reduces the frequency of immunosuppressive Chil3 monocytes and activates cross-presenting type 1 conventional dendritic cells (cDC1s). The dual effect of i.v. ChAdOx1 vaccination enhancing CD8 T cells and modulating the TME represents a translatable paradigm for enhancing anti-tumor immunity in humans., 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. C.S.K.L. and B.J.V.d.E. are inventors on a patent that covers viral vectors and methods for the prevention and treatment of cancer. F.B. is an employee of Genentech, a member of the Roche group, which develops and markets drugs for profit., (Published by Elsevier Inc.)

Stem cell-independent reprogramming of differentiated cells has recently been identified as an important paradigm for repairing injured tissues. Following periportal injury, mature hepatocytes re-activate reprogramming/progenitor-related genes (RRGs) and dedifferentiate into liver progenitor-like cells (LPLCs) in both mice and humans, which contribute remarkably to regeneration. However, it remains unknown which and how external factors trigger hepatocyte reprogramming. Here, by employing single-cell transcriptional profiling and lineage-specific deletion tools, we uncovered that periportal-specific LPLC formation was initiated by regionally activated Kupffer cells but not peripheral monocyte-derived macrophages. Unexpectedly, using in vivo screening, the proinflammatory factor IL-6 was identified as the niche signal repurposed for RRG induction via STAT3 activation, which drove RRG expression through binding to their pre-accessible enhancers. Notably, RRGs were activated through injury-specific rather than liver embryogenesis-related enhancers. Collectively, these findings depict an injury-specific niche signal and the inflammation-mediated transcription in driving the conversion of hepatocytes into a progenitor phenotype., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sensing of incoming viruses is a pivotal task of dendritic cells (DCs). Human primary blood DCs encompass various subsets that are diverse in their susceptibility and response to HIV-1. The recent identification of the blood Axl + DC subset, endowed with unique capacities to bind, replicate, and transmit HIV-1 prompted us to evaluate its anti-viral response. We demonstrate that HIV-1 induced two main broad and intense transcriptional programs in different Axl + DCs potentially induced by different sensors; an NF-κB-mediated program that led to DC maturation and efficient CD4 + T cell activation, and a program mediated by STAT1/2 that activated type I IFN and ISG responses. These responses were absent from cDC2 exposed to HIV-1 except when viral replication was allowed. Finally, Axl + DCs actively replicating HIV-1 identified by quantification of viral transcripts exhibited a mixed NF-κB/ISG innate response. Our results suggest that the route of HIV-1 entry may dictate different innate sensing pathways by DCs., Competing Interests: All authors declare no conflict of interests., (© 2023 The Authors.)

The NLRP3 inflammasome plays a central role in antimicrobial defense as well as in the context of sterile inflammatory conditions. NLRP3 activity is governed by two independent signals: the first signal primes NLRP3, rendering it responsive to the second signal, which then triggers inflammasome formation. Our understanding of how NLRP3 priming contributes to inflammasome activation remains limited. Here, we show that IKKβ, a kinase activated during priming, induces recruitment of NLRP3 to phosphatidylinositol-4-phosphate (PI4P), a phospholipid enriched on the trans-Golgi network. NEK7, a mitotic spindle kinase that had previously been thought to be indispensable for NLRP3 activation, was redundant for inflammasome formation when IKKβ recruited NLRP3 to PI4P. Studying iPSC-derived human macrophages revealed that the IKKβ-mediated NEK7-independent pathway constitutes the predominant NLRP3 priming mechanism in human myeloid cells. Our results suggest that PI4P binding represents a primed state into which NLRP3 is brought by IKKβ activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.)

The heterogeneity of tissue macrophages, in health and in disease, has become increasingly transparent over the last decade. But with the plethora of data comes a natural need for organization and the design of a conceptual framework for how we can better understand the origins and functions of different macrophages. We propose that the ontogeny of a macrophage-beyond its fundamental derivation as either embryonically or bone marrow-derived, but rather inclusive of the course of its differentiation, amidst steady-state cues, disease-associated signals, and time-constitutes a critical piece of information about its contribution to homeostasis or the progression of disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

The surface of the central nervous system (CNS) is protected by the meninges, which contain a dense network of meningeal macrophages (MMs). Here, we examined the role of tissue-resident MM in viral infection. MHC-II - MM were abundant neonatally, whereas MHC-II + MM appeared over time. These barrier macrophages differentially responded to in vivo peripheral challenges such as LPS, SARS-CoV-2, and lymphocytic choriomeningitis virus (LCMV). Peripheral LCMV infection, which was asymptomatic, led to a transient infection and activation of the meninges. Mice lacking macrophages but conserving brain microglia, or mice bearing macrophage-specific deletion of Stat1 or Ifnar, exhibited extensive viral spread into the CNS. Transcranial pharmacological depletion strategies targeting MM locally resulted in several areas of the meninges becoming infected and fatal meningitis. Low numbers of MHC-II + MM, which is seen upon LPS challenge or in neonates, corelated with higher viral load upon infection. Thus, MMs protect against viral infection and may present targets for therapeutic manipulation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Microglial research has advanced considerably in recent decades yet has been constrained by a rolling series of dichotomies such as "resting versus activated" and "M1 versus M2." This dualistic classification of good or bad microglia is inconsistent with the wide repertoire of microglial states and functions in development, plasticity, aging, and diseases that were elucidated in recent years. New designations continuously arising in an attempt to describe the different microglial states, notably defined using transcriptomics and proteomics, may easily lead to a misleading, although unintentional, coupling of categories and functions. To address these issues, we assembled a group of multidisciplinary experts to discuss our current understanding of microglial states as a dynamic concept and the importance of addressing microglial function. Here, we provide a conceptual framework and recommendations on the use of microglial nomenclature for researchers, reviewers, and editors, which will serve as the foundations for a future white paper., Competing Interests: Declaration of interests B.A. is the shareholder and member of scientific advisory board of Tranquis Therapeutics. K.B. is an employee and shareholder of AbbVie. M.C. receives research support from Vigil, is a member of the scientific advisory board of Vigil, and has a patent on TREM2. S.C. is a recipient of research funding from Eli Lilly and Company. C.C. is a member of the advisory board of Exalys Therapeutics and is the recipient of a research grant from IONIS therapeutics. B.D.S. is occasionally consulting for different companies. He is founding scientist of Augustin TX and of Muna TX. He is also shareholder of Muna TX. C.H. collaborates with Denali Therapeutics. C.H. is chief advisor of ISAR Bioscience and a member of the advisory board of AviadoBio. J.K. is a scientific advisor and collaborator with PureTech. T.M. is a cofounder of REGAIN Therapeutics, owner of a provisional patent on compositions and methods for treatment and/or prophylaxis of proteinopathies, and owner of a provisional patent on preventing or reverting abnormal amyloid deposition. R.M. has scientific collaborations with Alector, Nodthera, and Alchemab and is a consultant for Sanofi. B.M. has received consultancy fees from AstraZeneca. A. Sierra is a recipient of a research grant from Hoffmann La Roche., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.)

Despite the ubiquitous function of macrophages across the body, the diversity, origin, and function of adrenal gland macrophages remain largely unknown. We define the heterogeneity of adrenal gland immune cells using single-cell RNA sequencing and use genetic models to explore the developmental mechanisms yielding macrophage diversity. We define populations of monocyte-derived and embryonically seeded adrenal gland macrophages and identify a female-specific subset with low major histocompatibility complex (MHC) class II expression. In adulthood, monocyte recruitment dominates adrenal gland macrophage maintenance in female mice. Adrenal gland macrophage sub-tissular distribution follows a sex-dimorphic pattern, with MHC class II low macrophages located at the cortico-medullary junction. Macrophage sex dimorphism depends on the presence of the cortical X-zone. Adrenal gland macrophage depletion results in altered tissue homeostasis, modulated lipid metabolism, and decreased local aldosterone production during stress exposure. Overall, these data reveal the heterogeneity of adrenal gland macrophages and point toward sex-restricted distribution and functions of these cells., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by 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.)

Here, we provide detailed protocols for the isolation of mouse Kupffer cells - the liver-resident macrophages - for phenotypic (e.g., via flow cytometry, mass cytometry, or RNA-sequencing) analyses or for functional experiments involving cell culture. The procedures presented can be adapted for the isolation of other hepatic cell populations. For complete details on the use and execution of this protocol, please refer to De Simone et al. (2021)., Competing Interests: M.I. participates in advisory boards/consultancies for Gilead Sciences, Roche, Third Rock Ventures, Amgen, Allovir. M.I. is an inventor on patents filed, owned, and managed by San Raffaele Scientific Institute, Vita-Salute San Raffaele University and Telethon Foundation on technology related to work discussed in this manuscript (WO2020/016434, WO2020/016427, WO2020/030781, WO2020/234483, EU patent applications n. 19211249.8 and n 20156716.1, and UK patent application n. 1907493.9). F.G. is a member of the Immunity advisory board., (© 2021 The Author(s).)

Tissue macrophages are immune cells whose phenotypes and functions are dictated by origin and niches. However, tissues are complex environments, and macrophage heterogeneity within the same organ has been overlooked so far. Here, we used high-dimensional approaches to characterize macrophage populations in the murine liver. We identified two distinct populations among embryonically derived Kupffer cells (KCs) sharing a core signature while differentially expressing numerous genes and proteins: a major CD206 lo ESAM - population (KC1) and a minor CD206 hi ESAM + population (KC2). KC2 expressed genes involved in metabolic processes, including fatty acid metabolism both in steady-state and in diet-induced obesity and hepatic steatosis. Functional characterization by depletion of KC2 or targeted silencing of the fatty acid transporter Cd36 highlighted a crucial contribution of KC2 in the liver oxidative stress associated with obesity. In summary, our study reveals that KCs are more heterogeneous than anticipated, notably describing a subpopulation wired with metabolic functions., Competing Interests: Declaration of interests C.B., M.A., 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 © 2021 Elsevier Inc. All rights reserved.)

Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8 + T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8 + T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity., Competing Interests: Declaration of interests M.I. participates in advisory boards and consultancies for Gilead Sciences, Roche, Third Rock Ventures, Amgen, and Allovir. L.G.G is a member of the board of directors at Genenta Science and Epsilon Bio and participates in advisory boards and consultancies for Gilead Sciences, Roche, and Arbutus Biopharma. M.I. and L.G.G. are inventors on patents filed, owned, and managed by San Raffaele Scientific Institute, Vita-Salute San Raffaele University, and the Telethon Foundation on technology related to work discussed in this manuscript (WO2020/016434, WO2020/016427, WO2020/030781, WO2020/234483, European Union [EU] patent applications 19211249.8 and 20156716.1, and U.K. patent application 1907493.9). F.G. is a member of the Immunity advisory board., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.)

Despite considerable recent advances in understanding and treating many other cancers, malignant brain tumors remain associated with low survival or severe long-term sequelae. Limited progress, including development of immunotherapies, relates in part to difficulties in accurately reproducing brain microenvironment with current preclinical models. The cellular interactions among resident microglia, recruited tumor-associated macrophages, stromal cells, glial cells, neurons, and cancer cells and how they affect tumor growth or behavior are emerging, yet many questions remain. The role of the blood-brain barrier, extracellular matrix components, and heterogeneity among tumor types and within different regions of a single tumor further complicate the matter. Here, we focus on brain microenvironment features impacted by tumor biology. We also discuss limits of current preclinical models and how complementary models, such as humanized animals and organoids, will allow deeper mechanistic insights on cancer biology, allowing for more efficient testing of therapeutic strategies, including immunotherapy, for brain cancers., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Classically considered short-lived and purely defensive leukocytes, neutrophils are unique in their fast and moldable response to stimulation. This plastic behavior may underlie variable and even antagonistic functions during inflammation or cancer, yet the full spectrum of neutrophil properties as they enter healthy tissues remains unexplored. Using a new model to track neutrophil fates, we found short but variable lifetimes across multiple tissues. Through analysis of the receptor, transcriptional, and chromatin accessibility landscapes, we identify varying neutrophil states and assign non-canonical functions, including vascular repair and hematopoietic homeostasis. Accordingly, depletion of neutrophils compromised angiogenesis during early age, genotoxic injury, and viral infection, and impaired hematopoietic recovery after irradiation. Neutrophils acquired these properties in target tissues, a process that, in the lungs, occurred in CXCL12-rich areas and relied on CXCR4. Our results reveal that tissues co-opt neutrophils en route for elimination to induce programs that support their physiological demands., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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.)

Blood myeloid cells are known to be dysregulated in coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2. It is unknown whether the innate myeloid response differs with disease severity and whether markers of innate immunity discriminate high-risk patients. Thus, we performed high-dimensional flow cytometry and single-cell RNA sequencing of COVID-19 patient peripheral blood cells and detected disappearance of non-classical CD14 Low CD16 High monocytes, accumulation of HLA-DR Low classical monocytes (Human Leukocyte Antigen - DR isotype), and release of massive amounts of calprotectin (S100A8/S100A9) in severe cases. Immature CD10 Low CD101 - CXCR4 +/- neutrophils with an immunosuppressive profile accumulated in the blood and lungs, suggesting emergency myelopoiesis. Finally, we show that calprotectin plasma level and a routine flow cytometry assay detecting decreased frequencies of non-classical monocytes could discriminate patients who develop a severe form of COVID-19, suggesting a predictive value that deserves prospective evaluation., Competing Interests: Declaration of Interests A. Silvin, N.C., M.F., E. Solary, and F. Ginhoux are inventors of patent EP 20305624.7, “Methods for detecting and treating COVID patients requiring intensive care,” submitted on June 9, 2020 under Gustave Roussy., (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.)

Psoriasis is a chronic inflammatory disease whose etiology is multifactorial. The contributions of cellular metabolism to psoriasis are unclear. Here, we report that interleukin-17 (IL-17) downregulated Protein Phosphatase 6 (PP6) in psoriatic keratinocytes, causing phosphorylation and activation of the transcription factor C/EBP-β and subsequent generation of arginase-1. Mice lacking Pp6 in keratinocytes were predisposed to psoriasis-like skin inflammation. Accumulation of arginase-1 in Pp6-deficient keratinocytes drove polyamine production from the urea cycle. Polyamines protected self-RNA released by psoriatic keratinocytes from degradation and facilitated the endocytosis of self-RNA by myeloid dendritic cells to promote toll-like receptor-7 (TLR7)-dependent RNA sensing and IL-6 production. An arginase inhibitor improved skin inflammation in murine and non-human primate models of psoriasis. Our findings suggest that urea cycle hyperreactivity and excessive polyamine generation in psoriatic keratinocytes promote self-RNA sensation and PP6 deregulation in keratinocytes is a pivotal event that amplifies the inflammatory circuits in psoriasis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Resident tissue macrophages (RTMs) have a broad spectrum of immune- and non-immune-related tissue-supporting activities. The roots of this heterogeneity and versatility are only beginning to be understood. Here, we propose a conceptual framework for considering the RTM heterogeneity that organizes the factors shaping RTM identity within four cardinal points: (1) ontogeny and the view that adult RTM populations comprise a defined mixture of cells that arise from either embryonic precursors or adult monocytes; (2) local factors unique to the niche of residence, evolving during development and aging; (3) inflammation status; and (4) the cumulative effect of time spent in a specific tissue that contributes to the resilient adaptation of macrophages to their dynamic environment. We review recent findings within this context and discuss the technological advances that are revolutionizing the study of macrophage biology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Myeloid cells, including dendritic cells (DCs), granulocytes, monocytes, monocyte-derived cells and macrophages, are important players in the immune response, but their identification is not as clear as lymphocytes, especially in tissues. This protocol details the step-by-step procedure for the analysis of myeloid populations in various mouse tissues by flow cytometry. For complete details on the use and execution of this protocol, please refer to Liu et al. (2019)., Competing Interests: The authors declare no competing interests., (© 2020 The Author(s).)

Central nervous system (CNS) macrophages comprise microglia and border-associated macrophages (BAMs) residing in the meninges, the choroid plexus, and the perivascular spaces. Most CNS macrophages emerge during development, with the exception of choroid plexus and dural macrophages, which are replaced by monocytes in adulthood. Whether microglia and BAMs share a developmental program or arise from separate lineages remains unknown. Here, we identified two phenotypically, transcriptionally, and locally distinct brain macrophages throughout development, giving rise to either microglia or BAMs. Two macrophage populations were already present in the yolk sac suggesting an early segregation. Fate-mapping models revealed that BAMs mostly derived from early erythro-myeloid progenitors in the yolk sac. The development of microglia was dependent on TGF-β, whereas the genesis of BAMs occurred independently of this cytokine. Collectively, our data show that developing parenchymal and non-parenchymal brain macrophages are separate entities in terms of ontogeny, gene signature, and requirement for TGF-β., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)