Systematic identification of gene combinations to target in innate immune cells to enhance T cell activation.

Genetic engineering of immune cells has opened new avenues for improving their functionality but it remains a challenge to pinpoint which genes or combination of genes are the most beneficial to target. Here, we conduct High Multiplicity of Perturbations and Cellular Indexing of Transcriptomes and Epitopes (HMPCITE-seq) to find combinations of genes whose joint targeting improves antigen-presenting cell activity and enhances their ability to activate T cells. Specifically, we perform two genome-wide CRISPR screens in bone marrow dendritic cells and identify negative regulators of CD86, that participate in the co-stimulation programs, including Chd4, Stat5b, Egr2, Med12, and positive regulators of PD-L1, that participate in the co-inhibitory programs, including Sptlc2, Nckap1l, and Pi4kb. To identify the genetic interactions between top-ranked genes and find superior combinations to target, we perform high-order Perturb-Seq experiments and we show that targeting both Cebpb and Med12 results in a better phenotype compared to the single perturbations or other combinations of perturbations.

Loss of function of ENT3 drives histiocytosis and inflammation through TLR-MAPK signaling.

Histiocytoses are inflammatory myeloid neoplasms often driven by somatic activating mutations in mitogen-activated protein kinase (MAPK) cascade genes. H syndrome is an inflammatory genetic disorder caused by germ line loss-of-function mutations in SLC29A3, encoding the lysosomal equilibrative nucleoside transporter 3 (ENT3). Patients with H syndrome are predisposed to develop histiocytosis, yet the mechanism is unclear. Here, through phenotypic, molecular, and functional analysis of primary cells from a cohort of patients with H syndrome, we reveal the molecular pathway leading to histiocytosis and inflammation in this genetic disorder. We show that loss of function of ENT3 activates nucleoside-sensing toll-like receptors (TLR) and downstream MAPK signaling, inducing cytokine secretion and inflammation. Importantly, MEK inhibitor therapy led to resolution of histiocytosis and inflammation in a patient with H syndrome. These results demonstrate a yet-unrecognized link between a defect in a lysosomal transporter and pathological activation of MAPK signaling, establishing a novel pathway leading to histiocytosis and inflammation.

Human Dendritic Cell Enrichment and Their Activation of T Cells.

Dendritic cells (DCs) enable the immune system to mount and modulate precisely targeted responses to various threats across the organism by bridging innate and adaptive immunity. Historically, DCs have been classified as conventional (cDC) and plasmacytoid (pDC). More recently, cDCs were acknowledged as a heterogenous population composed of several subsets. Examining the functional diversity of cDCs in healthy homeostasis and pathology requires a robust experimental pipeline, beginning with an efficient enrichment protocol in preparation for cell sorting. Unfortunately, several commercial DC enrichment kits were developed before the discovery of the more recently described DC populations. Here, we detail two approaches to enrich human blood DCs or certain DC subsets and an in vitro protocol to examine DC stimulation of naïve T cells. The methods employed here overcome many hurdles encountered while enriching human DC subsets. Basic Protocol 1 describes a method that will enrich pDC, Axl Siglec6-DC (AS-DC), cDC1, DC2, DC3, monocytes, and human HLA+ cells by crosslinking unwanted cells to erythrocytes. Basic Protocol 2 describes the enrichment of pDC, AS-DC, cDC1, and DC2 but not DC3 via a highly efficient negative magnetic selection that is valuable in circumstances where DC3 is not required. Finally, Basic Protocol 3 describes a conventional protocol to perform a Mixed leucocyte Reaction (MLR) following the isolation of these DC subsets. These methods detail the advantages and pitfalls when isolating a heterogeneous population of cells. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Human peripheral mononuclear phagocyte enrichment Basic Protocol 2: DC enrichment of pDC, cDC1, AS-DC, and DC2 but not DC3 Basic Protocol 3: Basic mixed lymphocyte reaction protocol with sorted human DC subsets.

Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia.

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

Increased monocyte distribution width in COVID-19 and sepsis arises from a complex interplay of altered monocyte cellular size and subset frequency.

INTRODUCTION: Monocyte distribution width (MDW), a parameter generated alongside full blood counts (FBC) in new-generation haematology analysers, has been proposed as a diagnostic test for severe infection/sepsis. It represents the standard deviation (SD) of the monocyte mean volume (MMV). METHODS: This study aimed to compare monocyte volumetric parameters retrieved by the UniCel DxH 900 haematology analyser (MMV and MDW) against corresponding parameters from the same sample measured using flow cytometry (forward scatter [FSC] mean and SD) in combination with phenotypic characterization of monocyte subtypes. We analysed blood samples from healthy individuals (n = 11) and patients with conditions associated with elevated MDW: sepsis (n = 26) and COVID-19 (n = 15). RESULTS: Between-instrument comparisons of monocyte volume parameters (MMV vs. FSC-mean) showed relatively good levels of correlation, but comparisons across volume variability parameters (MDW vs. FSC-SD) were poor. Stratification on sample type revealed this lack of correlation only within the sepsis group. Flow cytometry analysis revealed that in healthy controls intermediate monocytes are the largest and non-classical the smallest cells. In each disease state, however, each monocyte subset undergoes different changes in volume and frequency that together determine the overall configuration of the monocyte population. Increased MDW was associated with reduced classical monocyte frequency and increased intermediate monocyte size. In COVID-19, the range of monocyte sizes (smallest to largest) reduced, whereas in sepsis it increased. CONCLUSION: Increased MDW in COVID-19 and sepsis has no single flow cytometric phenotypic correlate. It represents-within a single value-the delicate equipoise between monocyte subset frequency and size.

Tongue immune compartment analysis reveals spatial macrophage heterogeneity.

The tongue is a unique muscular organ situated in the oral cavity where it is involved in taste sensation, mastication, and articulation. As a barrier organ, which is constantly exposed to environmental pathogens, the tongue is expected to host an immune cell network ensuring local immune defence. However, the composition and the transcriptional landscape of the tongue immune system are currently not completely defined. Here, we characterised the tissue-resident immune compartment of the murine tongue during development, health and disease, combining single-cell RNA-sequencing with in situ immunophenotyping. We identified distinct local immune cell populations and described two specific subsets of tongue-resident macrophages occupying discrete anatomical niches. Cx3cr1+ macrophages were located specifically in the highly innervated lamina propria beneath the tongue epidermis and at times in close proximity to fungiform papillae. Folr2+ macrophages were detected in deeper muscular tissue. In silico analysis indicated that the two macrophage subsets originate from a common proliferative precursor during early postnatal development and responded differently to systemic LPS in vivo. Our description of the under-investigated tongue immune system sets a starting point to facilitate research on tongue immune-physiology and pathology including cancer and taste disorders.

Dntt expression reveals developmental hierarchy and lineage specification of hematopoietic progenitors.

Intrinsic and extrinsic cues determine developmental trajectories of hematopoietic stem cells (HSCs) towards erythroid, myeloid and lymphoid lineages. Using two newly generated transgenic mice that report and trace the expression of terminal deoxynucleotidyl transferase (TdT), transient induction of TdT was detected on a newly identified multipotent progenitor (MPP) subset that lacked self-renewal capacity but maintained multilineage differentiation potential. TdT induction on MPPs reflected a transcriptionally dynamic but uncommitted stage, characterized by low expression of lineage-associated genes. Single-cell CITE-seq indicated that multipotency in the TdT+ MPPs is associated with expression of the endothelial cell adhesion molecule ESAM. Stable and progressive upregulation of TdT defined the lymphoid developmental trajectory. Collectively, we here identify a new multipotent progenitor within the MPP4 compartment. Specification and commitment are defined by downregulation of ESAM which marks the progressive loss of alternative fates along all lineages.

Early antitumor activity of oral Langerhans cells is compromised by a carcinogen.

Early diagnosis of oral squamous cell carcinoma (OSCC) remains an unmet clinical need. Therefore, elucidating the initial events of OSCC preceding tumor development could benefit OSCC prognosis. Here, we define the Langerhans cells (LCs) of the tongue and demonstrate that LCs protect the epithelium from carcinogen-induced OSCC by rapidly priming αßT cells capable of eliminating γH2AX+ epithelial cells, whereas γδT and natural killer cells are dispensable. The carcinogen, however, dysregulates the epithelial resident mononuclear phagocytes, reducing LC frequencies, while dendritic cells (DCs), macrophages, and plasmacytoid DCs (pDCs) populate the epithelium. Single-cell RNA-sequencing analysis indicates that these newly differentiated cells display an immunosuppressive phenotype accompanied by an expansion of T regulatory (Treg) cells. Accumulation of the Treg cells was regulated, in part, by pDCs and precedes the formation of visible tumors. This suggests LCs play an early protective role during OSCC, yet the capacity of the carcinogen to dysregulate the differentiation of mononuclear phagocytes facilitates oral carcinogenesis.

Intradermal lipopolysaccharide challenge as an acute in vivo inflammatory model in healthy volunteers.

AIMS: Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies, systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers, and as such qualify the method as local inflammation model for clinical pharmacology studies. METHODS: Eighteen healthy male volunteers received 2 or 4 intradermal 5 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion, skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate. RESULTS: LPS elicited a visible response and returned to baseline at 48 hours. Erythema, perfusion and temperature were statistically significant (P < .0001) over a 24-hour time course compared to saline. The protein response was dominated by an acute interleukin (IL)-6, IL-8 and tumour necrosis factor response followed by IL-1ß, IL-10 and interferon-γ. The cellular response consisted of an acute neutrophil influx followed by different monocyte subsets and dendritic cells. DISCUSSION: Intradermal LPS administration in humans causes an acute, localized and transient inflammatory reaction that is well-tolerated by healthy volunteers. This may be a valuable inflammation model for evaluating the pharmacological activity of anti-inflammatory investigational compounds in proof of pharmacology studies.

Monocytes, macrophages, dendritic cells and neutrophils: an update on lifespan kinetics in health and disease.

Phagocytes form a family of immune cells that play a crucial role in tissue maintenance and help orchestrate the immune response. This family of cells can be separated by their nuclear morphology into mononuclear and polymorphonuclear phagocytes. The generation of these cells in the bone marrow, to the blood and finally into tissues is a tightly regulated process. Ensuring the adequate production of these cells and their timely removal is key for both the initiation and resolution of inflammation. Insight into the kinetic profiles of innate myeloid cells during steady state and pathology will permit the rational development of therapies to boost the production of these cells in times of need or reduce them when detrimental.

Longevity and replenishment of human liver-resident memory T cells and mononuclear phagocytes.

The human liver contains specialized subsets of mononuclear phagocytes (MNPs) and T cells, but whether these have definitive features of tissue residence (long-term retention, lack of egress) and/or can be replenished from the circulation remains unclear. Here we addressed these questions using HLA-mismatched liver allografts to discriminate the liver-resident (donor) from the infiltrating (recipient) immune composition. Allografts were rapidly infiltrated by recipient leukocytes, which recapitulated the liver myeloid and lymphoid composition, and underwent partial reprogramming with acquisition of CD68/CD206 on MNPs and CD69/CD103 on T cells. The small residual pool of donor cells persisting in allografts for over a decade contained CX3CR1hi/CD163hi/CD206hi Kupffer cells (KCs) and CXCR3hi tissue-resident memory T cells (TRM). CD8+ TRM were found in the local lymph nodes but were not detected egressing into the hepatic vein. Our findings inform organ transplantation and hepatic immunotherapy, revealing remarkably long-lived populations of KCs and TRM in human liver, which can be additionally supplemented by their circulating counterparts.

Author Correction: Cxcl10+ monocytes define a pathogenic subset in the central nervous system during autoimmune neuroinflammation.

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

Publisher Correction: Cxcl10+ monocytes define a pathogenic subset in the central nervous system during autoimmune neuroinflammation.

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

Cxcl10+ monocytes define a pathogenic subset in the central nervous system during autoimmune neuroinflammation.

Multiple sclerosis (MS) is characterized by pathological inflammation that results from the recruitment of lymphoid and myeloid immune cells from the blood into the brain. Due to subset heterogeneity, defining the functional roles of the various cell subsets in acute and chronic stages of MS has been challenging. Here, we used index and transcriptional single-cell sorting to characterize the mononuclear phagocytes that infiltrate the central nervous system from the periphery in mice with experimentally induced autoimmune encephalomyelitis, a model of MS. We identified eight monocyte and three dendritic cell subsets at acute and chronic disease stages in which the defined transcriptional programs pointed toward distinct functions. Monocyte-specific cell ablation identified Cxcl10+ and Saa3+ monocytic subsets with a pathogenic potential. Transfer experiments with different monocyte and precursor subsets indicated that these Cxcl10+ and Saa3+ pathogenic cells were not derived from Ly6C+ monocytes but from early myeloid cell progenitors. These results suggest that blocking specific pathogenic monocytic subsets, including Cxcl10+ and Saa3+ monocytes, could be used for targeted therapeutic interventions.

Inherited and Environmental Factors Influence Human Monocyte Heterogeneity.

Blood monocytes develop in the bone marrow before being released into the peripheral circulation. The circulating monocyte pool is composed of multiple subsets, each with specialized functions. These cells are recruited to repopulate resident monocyte-derived cells in the periphery and also to sites of injury. Several extrinsic factors influence the function and quantity of monocytes in the blood. Here, we outline the impact of sex, ethnicity, age, sleep, diet, and exercise on monocyte subsets and their function, highlighting that steady state is not a single physiological condition. A clearer understanding of the relationship between these factors and the immune system may allow for improved therapeutic strategies.

Variations in the Phagosomal Environment of Human Neutrophils and Mononuclear Phagocyte Subsets.

The phagosome microenvironment maintains enzyme activity and function. Here we compared the phagosomal pH of human neutrophils, monocytes, dendritic cells (DC), and monocyte-derived cells. An unexpected observation was the striking difference in phagosomal environment between the three monocytes subsets. Classical monocytes and neutrophils exhibited alkaline phagosomes, yet non-classical monocytes had more acidic phagosomes, while intermediate monocytes had a phenotype in-between. We next investigated the differences between primary naïve DC vs. in vitro monocyte-derived DC (MoDC) and established that both these cells had acidic phagosomal environments. Across all phagocytes, alkalinization was dependent upon the activity of the NADPH oxidase activity, demonstrated by the absence of NADPH oxidase from a patient with chronic granulomatous disease (CGD) or the use of a pharmacological inhibitor, diphenylene iodonium (DPI). Interestingly, MoDC stimulated with bacterial lipopolysaccharide had increased phagosomal pH. Overall, the increase in alkalinity within the phagosome was associated with increased oxidase activity. These data highlight the heterogeneous nature and potential function of phagocytic vacuoles within the family of mononuclear phagocytes.

A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFα-Mediated Recruitment of Neutrophils.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor α (VEGF-α) by a minor subset of activated EpCAM+CD59+Ly-6D+ cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guérin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Fine needle aspirates comprehensively sample intrahepatic immunity.

OBJECTIVE: In order to refine new therapeutic strategies in the pipeline for HBV cure, evaluation of virological and immunological changes compartmentalised at the site of infection will be required. We therefore investigated if liver fine needle aspirates (FNAs) could comprehensively sample the local immune landscape in parallel with viable hepatocytes. DESIGN: Matched blood, liver biopsy and FNAs from 28 patients with HBV and 15 without viral infection were analysed using 16-colour multiparameter flow cytometry. RESULTS: The proportion of CD4 T, CD8 T, Mucosal Associated Invariant T cell (MAIT), Natural Killer (NK) and B cells identified by FNA correlated with that in liver biopsies from the same donors. Populations of Programmed Death-1 (PD-1)hiCD39hi tissue-resident memory CD8 T cells (CD69+CD103+) and liver-resident NK cells (CXCR6+T-betloEomeshi), were identified by both FNA and liver biopsy, and not seen in the blood. Crucially, HBV-specific T cells could be identified by FNAs at similar frequencies to biopsies and enriched compared with blood. FNAs could simultaneously identify populations of myeloid cells and live hepatocytes expressing albumin, Scavenger Receptor class B type 1 (SR-B1), Programmed Death-Ligand 1 (PD-L1), whereas hepatocytes were poorly viable after the processing required for liver biopsies. CONCLUSION: We demonstrate for the first time that FNAs identify a range of intrahepatic immune cells including locally resident sentinel HBV-specific T cells and NK cells, together with PD-L1-expressing hepatocytes. In addition, we provide a scoring tool to estimate the extent to which an individual FNA has reliably sampled intrahepatic populations rather than contaminating blood. The broad profiling achieved by this less invasive, rapid technique makes it suitable for longitudinal monitoring of the liver to optimise new therapies for HBV.

Good things come in threes.

Ectopic expression of PU.1, IRF8, and BATF3 reprograms mouse and human fibroblasts into dendritic cells. See related Research Article by Rosa et al.