Ontogeny and Function of Plasmacytoid Dendritic Cells.

Plasmacytoid dendritic cells (pDCs) represent a unique cell type within the innate immune system. Their defining property is the recognition of pathogen-derived nucleic acids through endosomal Toll-like receptors and the ensuing production of type I interferon and other soluble mediators, which orchestrate innate and adaptive responses. We review several aspects of pDC biology that have recently come to the fore. We discuss emerging questions regarding the lineage affiliation and origin of pDCs and argue that these cells constitute an integral part of the dendritic cell lineage. We emphasize the specific function of pDCs as innate sentinels of virus infection, particularly their recognition of and distinct response to virus-infected cells. This essential evolutionary role of pDCs has been particularly important for the control of coronaviruses, as demonstrated by the recent COVID-19 pandemic. Finally, we highlight the key contribution of pDCs to systemic lupus erythematosus, in which therapeutic targeting of pDCs is currently underway.

Transitional dendritic cells are distinct from conventional DC2 precursors and mediate proinflammatory antiviral responses.

High-dimensional approaches have revealed heterogeneity amongst dendritic cells (DCs), including a population of transitional DCs (tDCs) in mice and humans. However, the origin and relationship of tDCs to other DC subsets has been unclear. Here we show that tDCs are distinct from other well-characterized DCs and conventional DC precursors (pre-cDCs). We demonstrate that tDCs originate from bone marrow progenitors shared with plasmacytoid DCs (pDCs). In the periphery, tDCs contribute to the pool of ESAM+ type 2 DCs (DC2s), and these DC2s have pDC-related developmental features. Different from pre-cDCs, tDCs have less turnover, capture antigen, respond to stimuli and activate antigen-specific naïve T cells, all characteristics of differentiated DCs. Different from pDCs, viral sensing by tDCs results in IL-1ß secretion and fatal immune pathology in a murine coronavirus model. Our findings suggest that tDCs are a distinct pDC-related subset with a DC2 differentiation potential and unique proinflammatory function during viral infections.

Deconvoluting global cytokine signaling networks in natural killer cells.

Cytokine signaling via signal transducer and activator of transcription (STAT) proteins is crucial for optimal antiviral responses of natural killer (NK) cells. However, the pleiotropic effects of both cytokine and STAT signaling preclude the ability to precisely attribute molecular changes to specific cytokine-STAT modules. Here, we employed a multi-omics approach to deconstruct and rebuild the complex interaction of multiple cytokine signaling pathways in NK cells. Proinflammatory cytokines and homeostatic cytokines formed a cooperative axis to commonly regulate global gene expression and to further repress expression induced by type I interferon signaling. These cytokines mediated distinct modes of epigenetic regulation via STAT proteins, and collective signaling best recapitulated global antiviral responses. The most dynamically responsive genes were conserved across humans and mice, which included a cytokine-STAT-induced cross-regulatory program. Thus, an intricate crosstalk exists between cytokine signaling pathways, which governs NK cell responses.

ILC1 Confer Early Host Protection at Initial Sites of Viral Infection.

Infection is restrained by the concerted activation of tissue-resident and circulating immune cells. Whether tissue-resident lymphocytes confer early antiviral immunity at local sites of primary infection prior to the initiation of circulating responses is not well understood. Furthermore, the kinetics of initial antiviral responses at sites of infection remain unclear. Here, we show that tissue-resident type 1 innate lymphoid cells (ILC1) serve an essential early role in host immunity through rapid production of interferon (IFN)-γ following viral infection. Ablation of Zfp683-dependent liver ILC1 lead to increased viral load in the presence of intact adaptive and innate immune cells critical for mouse cytomegalovirus (MCMV) clearance. Swift production of interleukin (IL)-12 by tissue-resident XCR1+ conventional dendritic cells (cDC1) promoted ILC1 production of IFN-γ in a STAT4-dependent manner to limit early viral burden. Thus, ILC1 contribute an essential role in viral immunosurveillance at sites of initial infection in response to local cDC1-derived proinflammatory cytokines.

The IRE1 endoplasmic reticulum stress sensor activates natural killer cell immunity in part by regulating c-Myc.

Natural killer (NK) cells are critical mediators of host immunity to pathogens. Here, we demonstrate that the endoplasmic reticulum stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box-binding protein 1 (XBP1) drive NK cell responses against viral infection and tumors in vivo. IRE1α-XBP1 were essential for expansion of activated mouse and human NK cells and are situated downstream of the mammalian target of rapamycin signaling pathway. Transcriptome and chromatin immunoprecipitation analysis revealed c-Myc as a new and direct downstream target of XBP1 for regulation of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, and NK cells with c-Myc haploinsufficency phenocopied IRE1α-XBP1 deficiency. c-Myc overexpression largely rescued the proliferation defect in IRE1α-/- NK cells. Like c-Myc, IRE1α-XBP1 also promotes oxidative phosphorylation in NK cells. Overall, our study identifies a IRE1α-XBP1-cMyc axis in NK cell immunity, providing insight into host protection against infection and cancer.

Mouse cytomegalovirus-experienced ILC1s acquire a memory response dependent on the viral glycoprotein m12.

Innate lymphoid cells (ILCs) are tissue-resident sentinels that are essential for early host protection from pathogens at initial sites of infection. However, whether pathogen-derived antigens directly modulate the responses of tissue-resident ILCs has remained unclear. In the present study, it was found that liver-resident type 1 ILCs (ILC1s) expanded locally and persisted after the resolution of infection with mouse cytomegalovirus (MCMV). ILC1s acquired stable transcriptional, epigenetic and phenotypic changes a month after the resolution of MCMV infection, and showed an enhanced protective effector response to secondary challenge with MCMV consistent with a memory lymphocyte response. Memory ILC1 responses were dependent on the MCMV-encoded glycoprotein m12, and were independent of bystander activation by proinflammatory cytokines after heterologous infection. Thus, liver ILC1s acquire adaptive features in an MCMV-specific manner.

Clonal lineage tracing reveals shared origin of conventional and plasmacytoid dendritic cells.

Developmental origins of dendritic cells (DCs) including conventional DCs (cDCs, comprising cDC1 and cDC2 subsets) and plasmacytoid DCs (pDCs) remain unclear. We studied DC development in unmanipulated adult mice using inducible lineage tracing combined with clonal DNA "barcoding" and single-cell transcriptome and phenotype analysis (CITE-seq). Inducible tracing of Cx3cr1+ hematopoietic progenitors in the bone marrow showed that they simultaneously produce all DC subsets including pDCs, cDC1s, and cDC2s. Clonal tracing of hematopoietic stem cells (HSCs) and of Cx3cr1+ progenitors revealed clone sharing between cDC1s and pDCs, but not between the two cDC subsets or between pDCs and B cells. Accordingly, CITE-seq analyses of differentiating HSCs and Cx3cr1+ progenitors identified progressive stages of pDC development including Cx3cr1+ Ly-6D+ pro-pDCs that were distinct from lymphoid progenitors. These results reveal the shared origin of pDCs and cDCs and suggest a revised scheme of DC development whereby pDCs share clonal relationship with cDC1s.

Epigenetic control of innate and adaptive immune memory.

Clonal expansion and immunological memory are hallmark features of the mammalian adaptive immune response and essential for prolonged host control of pathogens. Recent work demonstrates that natural killer (NK) cells of the innate immune system also exhibit these adaptive traits during infection. Here we demonstrate that differentiating and 'memory' NK cells possess distinct chromatin accessibility states and that their epigenetic profiles reveal a 'poised' regulatory program at the memory stage. Furthermore, we elucidate how individual STAT transcription factors differentially control epigenetic and transcriptional states early during infection. Finally, concurrent chromatin profiling of the canonical CD8+ T cell response against the same infection demonstrated parallel and distinct epigenetic signatures defining NK cells and CD8+ T cells. Overall, our study reveals the dynamic nature of epigenetic modifications during the generation of innate and adaptive lymphocyte memory.

Cytomegalovirus Infection Drives Avidity Selection of Natural Killer Cells.

The process of affinity maturation, whereby T and B cells bearing antigen receptors with optimal affinity to the relevant antigen undergo preferential expansion, is a key feature of adaptive immunity. Natural killer (NK) cells are innate lymphocytes capable of "adaptive" responses after cytomegalovirus (CMV) infection. However, whether NK cells are similarly selected on the basis of their avidity for cognate ligand is unknown. Here, we showed that NK cells with the highest avidity for the mouse CMV glycoprotein m157 were preferentially selected to expand and comprise the memory NK cell pool, whereas low-avidity NK cells possessed greater capacity for interferon-γ (IFN-γ) production. Moreover, we provide evidence for avidity selection occurring in human NK cells during human CMV infection. These results delineate how heterogeneity in NK cell avidity diversifies NK cell effector function during antiviral immunity, and how avidity selection might serve to produce the most potent memory NK cells.

Transcription Factor IRF8 Orchestrates the Adaptive Natural Killer Cell Response.

Natural killer (NK) cells are innate lymphocytes that display features of adaptive immunity during viral infection. Biallelic mutations in IRF8 have been reported to cause familial NK cell deficiency and susceptibility to severe viral infection in humans; however, the precise role of this transcription factor in regulating NK cell function remains unknown. Here, we show that cell-intrinsic IRF8 was required for NK-cell-mediated protection against mouse cytomegalovirus infection. During viral exposure, NK cells upregulated IRF8 through interleukin-12 (IL-12) signaling and the transcription factor STAT4, which promoted epigenetic remodeling of the Irf8 locus. Moreover, IRF8 facilitated the proliferative burst of virus-specific NK cells by promoting expression of cell-cycle genes and directly controlling Zbtb32, a master regulator of virus-driven NK cell proliferation. These findings identify the function and cell-type-specific regulation of IRF8 in NK-cell-mediated antiviral immunity and provide a mechanistic understanding of viral susceptibility in patients with IRF8 mutations.

Clonal barcoding of endogenous adult hematopoietic stem cells reveals a spectrum of lineage contributions.

The hierarchical model of hematopoiesis posits that self-renewing, multipotent hematopoietic stem cells (HSCs) give rise to all blood cell lineages. While this model accounts for hematopoiesis in transplant settings, its applicability to steady-state hematopoiesis remains to be clarified. Here, we used inducible clonal DNA barcoding of endogenous adult HSCs to trace their contribution to major hematopoietic cell lineages in unmanipulated animals. While the majority of barcodes were unique to a single lineage, we also observed frequent barcode sharing between multiple lineages, specifically between lymphocytes and myeloid cells. These results suggest that both single-lineage and multilineage contributions by HSCs collectively drive continuous hematopoiesis, and highlight a close relationship of myeloid and lymphoid development.

Stem cell decoupling underlies impaired lymphoid development during aging.

Mammalian aging is associated with multiple defects of hematopoiesis, most prominently with the impaired development of T and B lymphocytes. This defect is thought to originate in hematopoietic stem cells (HSCs) of the bone marrow, specifically due to the age-dependent accumulation of HSCs with preferential megakaryocytic and/or myeloid potential ("myeloid bias"). Here, we tested this notion using inducible genetic labeling and tracing of HSCs in unmanipulated animals. We found that the endogenous HSC population in old mice shows reduced differentiation into all lineages including lymphoid, myeloid, and megakaryocytic. Single-cell RNA sequencing and immunophenotyping (CITE-Seq) showed that HSC progeny in old animals comprised balanced lineage spectrum including lymphoid progenitors. Lineage tracing using the aging-induced HSC marker Aldh1a1 confirmed the low contribution of old HSCs across all lineages. Competitive transplantations of total bone marrow cells with genetically marked HSCs revealed that the contribution of old HSCs was reduced, but compensated by other donor cells in myeloid cells but not in lymphocytes. Thus, the HSC population in old animals becomes globally decoupled from hematopoiesis, which cannot be compensated in lymphoid lineages. We propose that this partially compensated decoupling, rather than myeloid bias, is the primary cause of the selective impairment of lymphopoiesis in older mice.

Adipose-Resident Group 1 Innate Lymphoid Cells Promote Obesity-Associated Insulin Resistance.

Innate lymphoid cells (ILCs) function to protect epithelial barriers against pathogens and maintain tissue homeostasis in both barrier and non-barrier tissues. Here, utilizing Eomes reporter mice, we identify a subset of adipose group 1 ILC (ILC1) and demonstrate a role for these cells in metabolic disease. Adipose ILC1s were dependent on the transcription factors Nfil3 and T-bet but phenotypically and functionally distinct from adipose mature natural killer (NK) and immature NK cells. Analysis of parabiotic mice revealed that adipose ILC1s maintained long-term tissue residency. Diet-induced obesity drove early production of interleukin (IL)-12 in adipose tissue depots and led to the selective proliferation and accumulation of adipose-resident ILC1s in a manner dependent on the IL-12 receptor and STAT4. ILC1-derived interferon-γ was necessary and sufficient to drive proinflammatory macrophage polarization to promote obesity-associated insulin resistance. Thus, adipose-resident ILC1s contribute to obesity-related pathology in response to dysregulated local proinflammatory cytokine production.

Cutting Edge: Heterogeneity in Cell Age Contributes to Functional Diversity of NK Cells.

Heterogeneity among naive adaptive lymphocytes determines their individual functions and fate decisions during an immune response. NK cells are innate lymphocytes capable of generating "adaptive" responses during infectious challenges. However, the factors that govern various NK cell functions are not fully understood. In this study, we use a reporter mouse model to permanently "time stamp" NK cells and type 1 innate lymphoid cells (ILC1s) to characterize the dynamics of their homeostatic turnover. We found that the homeostatic turnover of tissue-resident ILC1s is much slower than that of circulating NK cells. NK cell homeostatic turnover is further accelerated without the transcription factor Eomes. Finally, heterogeneity in NK cell age diversifies NK cell function, with "older" NK cells exhibiting more potent IFN-γ production to activating stimuli and more robust adaptive responses during CMV infection. These results provide insight into how the functional response of an NK cell varies over its lifespan.

Spatial and temporal coordination of antiviral responses by group 1 ILCs.

Group 1 innate lymphocytes consist of a phenotypically, spatially, and functionally heterogeneous population of NK cells and ILC1s that are engaged during pathogen invasion. We are only beginning to understand the context-dependent roles that different subsets of group 1 innate lymphocytes play during homeostatic perturbations. With a focus on viral infection, this review highlights the organization and regulation of spatially and temporally distinct waves of NK cell and ILC1 responses that collectively serve to achieve optimal viral control.

COVID-19 diagnostics for resource-limited settings: Evaluation of "unextracted" qRT-PCR.

The coronavirus disease 2019 (COVID-19) pandemic has created a precipitous increase in the need for molecular diagnostics. Unfortunately, access to RNA extraction reagents can represent a bottleneck for quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR)-based methodologies, stemming from both extraordinary supply-chain stresses and the global reach of the virus into resource-limited settings. To provide flexible diagnostic options for such environments, we report here an "unextracted modification" for qRT-PCR using the Centers for Disease Control's (CDC's) widely utilized primers/probe sets for severe acute respiratory syndrome coronavirus 2 (N1/N2/N3 targeting viral nucleocapsid and RP-control targeting human RNase P). This approach replaces RNA extraction/purification with a heat-inactivation step of viral transport media (VTM), followed by direct inoculation-with or without VTM spin concentration-into PCR master mixes. Using derivatives of care from our clinical workflow, we compared traditional and unextracted CDC methodologies. Although some decrease in analytic sensitivity was evident (by higher Ct values) without extraction, in particular for the N2 primer/probe-set, we observed high categorical positive agreement between extracted and unextracted results for N1 (unconcentrated VTM-38/40; concentrated VTM-39/41), N3 (unconcentrated VTM-38/40; concentrated VTM-41/41), and RP (unconcentrated and concentrated VTM-81/81). The negative categorical agreement for N1/N2/N3 was likewise high. Overall, these results suggest that laboratories could adapt and validate unextracted qRT-PCR protocols as a contingency to overcome supply limitations, with minimal impact on categorical results.

NK Cell Responses Redefine Immunological Memory.

Immunological memory has traditionally been regarded as a unique trait of the adaptive immune system. Nevertheless, there is evidence of immunological memory in lower organisms and invertebrates, which lack an adaptive immune system. Despite their innate ability to rapidly produce effector cytokines and kill virally infected or transformed cells, NK cells also exhibit adaptive characteristics such as clonal expansion, longevity, self-renewal, and robust recall responses to antigenic or nonantigenic stimuli. In this review, we highlight the intracellular and extracellular requirements for memory NK cell generation and describe the emerging evidence for memory precursor NK cells and their derivation.

Adaptive PCR Based on Hybridization Sensing of Mirror-Image l-DNA.

Polymerase chain reaction (PCR) is dependent on two key hybridization events during each cycle of amplification, primer annealing and product melting. To ensure that these hybridization events occur, current PCR approaches rely on temperature set points and reaction contents that are optimized and maintained using rigid thermal cycling programs and stringent sample preparation procedures. This report describes a fundamentally simpler and more robust PCR design that dynamically controls thermal cycling by more directly monitoring the two key hybridization events during the reaction. This is achieved by optically sensing the annealing and melting of mirror-image l-DNA analogs of the reaction's primers and targets. Because the properties of l-DNA enantiomers parallel those of natural d-DNAs, the l-DNA reagents indicate the cycling conditions required for effective primer annealing and product melting during each cycle without interfering with the reaction. This hybridization-sensing approach adapts in real time to variations in reaction contents and conditions that impact primer annealing and product melting and eliminates the requirement for thermal calibrations and cycling programs. Adaptive PCR is demonstrated to amplify DNA targets with high efficiency and specificity under both controlled conditions and conditions that are known to cause traditional PCR to fail. The advantages of this approach promise to make PCR-based nucleic acid analysis simpler, more robust, and more accessible outside of well-controlled laboratory settings.

Metal Affinity-Enabled Capture and Release Antibody Reagents Generate a Multiplex Biomarker Enrichment System that Improves Detection Limits of Rapid Diagnostic Tests.

Multi-antigen rapid diagnostic tests (RDTs) are highly informative, simple, mobile, and inexpensive, making them valuable point-of-care (POC) diagnostic tools. However, these RDTs suffer from several technical limitations-the most significant being the failure to detect low levels of infection. To overcome this, we have developed a magnetic bead-based multiplex biomarker enrichment strategy that combines metal affinity and immunospecific capture to purify and enrich multiple target biomarkers. Modifying antibodies to contain histidine-rich peptides enables reversible loading onto immobilized metal affinity magnetic beads, generating a novel class of antibodies coined "Capture and Release" (CaR) antibody reagents. This approach extends the specificity of immunocapture to metal affinity magnetic beads while also maintaining a common trigger for releasing multiple biomarkers. Multiplex biomarker enrichment is accomplished by adding magnetic beads equipped with CaR antibody reagents to a large sample volume to capture biomarkers of interest. Once captured, these biomarkers are magnetically purified, concentrated, and released into a RDT-compatible volume. This system was tailored to enhance a popular dual-antigen lateral flow malaria RDT that targets Plasmodium falciparum histidine-rich protein-II (HRPII) and Plasmodium lactate dehydrogenase (pLDH). A suite of pLDH CaR antibody reagents were synthesized, characterized, and the optimal CaR antibody reagent was loaded onto magnetic beads to make a multiplex magnetic capture bead that simultaneously enriches pLDH and HRPII from Plasmodium falciparum parasitized blood samples. This system achieves a 17.5-fold improvement in the dual positive HRPII/pan-pLDH detection limits enabling visual detection of both antigens at levels correlating to 5 p/µL. This front-end sample processing system serves as an efficient strategy to improve the sensitivity of RDTs without the need for modifications or remanufacturing.

Magnetically-enabled biomarker extraction and delivery system: towards integrated ASSURED diagnostic tools.

Diagnosis of asymptomatic malaria poses a great challenge to global disease elimination efforts. Healthcare infrastructure in rural settings cannot support existing state-of-the-art tools necessary to diagnose asymptomatic malaria infections. Instead, lateral flow immunoassays (LFAs) are widely used as a diagnostic tool in malaria endemic areas. While LFAs are simple and easy to use, they are unable to detect low levels of parasite infection. We have developed a field deployable Magnetically-enabled Biomarker Extraction And Delivery System (mBEADS) that significantly improves limits of detection for several commercially available LFAs. Integration of mBEADS with leading commercial Plasmodium falciparum malaria LFAs improves detection limits to encompass an estimated 95% of the disease reservoir. This user-centered mBEADS platform makes significant improvements to a previously cumbersome malaria biomarker enrichment strategy by improving reagent stability, decreasing the processing time 10-fold, and reducing the assay cost 10-fold. The resulting mBEADS process adds just three minutes and less than $0.25 to the total cost of a single LFA, thus balancing sensitivity and practicality to align with the World Health Organization's ASSURED criteria for point-of-care (POC) testing.