Genome-wide screening identifies Trim33 as an essential regulator of dendritic cell differentiation.

The development of dendritic cells (DCs), including antigen-presenting conventional DCs (cDCs) and cytokine-producing plasmacytoid DCs (pDCs), is controlled by the growth factor Flt3 ligand (Flt3L) and its receptor Flt3. We genetically dissected Flt3L-driven DC differentiation using CRISPR-Cas9-based screening. Genome-wide screening identified multiple regulators of DC differentiation including subunits of TSC and GATOR1 complexes, which restricted progenitor growth but enabled DC differentiation by inhibiting mTOR signaling. An orthogonal screen identified the transcriptional repressor Trim33 (TIF-1γ) as a regulator of DC differentiation. Conditional targeting in vivo revealed an essential role of Trim33 in the development of all DCs, but not of monocytes or granulocytes. In particular, deletion of Trim33 caused rapid loss of DC progenitors, pDCs, and the cross-presenting cDC1 subset. Trim33-deficient Flt3+ progenitors up-regulated pro-inflammatory and macrophage-specific genes but failed to induce the DC differentiation program. Collectively, these data elucidate mechanisms that control Flt3L-driven differentiation of the entire DC lineage and identify Trim33 as its essential regulator.

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.

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.

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.

Direct PCR with the CDC 2019 SARS-CoV-2 assay: optimization for limited-resource settings.

PCR-based diagnostics generally require nucleic acid extraction from patient specimens prior to amplification. As highlighted early in the COVID-19 pandemic, extraction steps may be difficult to scale during times of massive demand and limited reagent supply. Forgoing an extraction step, we previously reported that the N1 primer/probe-set of the widespread CDC COVID-19 assay maintains high categorical sensitivity (95%) and specificity (100%) with direct inoculation of viral transport media (VTM) into qRT-PCR reactions. In contrast, the N2 set demonstrated a prominent Ct delay and low sensitivity (33%) without extraction. In the current study, we have improved the performance of this modified CDC assay (in particular the N2 set) by incorporating N1/N2/RNase P multiplexing and dissecting the effects of annealing temperature, VTM interference, and inoculum volume. The latter two factors exerted a more prominent effect on the performance of N2 than N1, although these effects were largely overcome through elevated annealing temperature. This unextracted/multiplex protocol was evaluated with 41 SARS-CoV-2 positive and 43 negative clinical samples, demonstrating a categorical sensitivity of 92.7% and specificity of 100% versus the unmodified CDC methodology. Overall, this work offers a generalizable strategy to maximize testing capabilities for COVID-19 or other emerging pathogens when resources are constrained.

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.

Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test.

Among nucleic acid diagnostic strategies, non-enzymatic tests are the most promising for application at the point of care in low-resource settings. They remain relatively under-utilized, however, due to inadequate sensitivity. Inspired by a recent demonstration of a highly-sensitive dumbbell DNA amplification strategy, we developed an automated, self-contained assay for detection of target DNA. In this new diagnostic platform, called the automated Pi-powered looping oligonucleotide transporter, magnetic beads capture the target DNA and are then loaded into a microfluidic reaction cassette along with the other reaction solutions. A stepper motor controls the motion of the cassette relative to an external magnetic field, which moves the magnetic beads through the reaction solutions automatically. Real-time fluorescence is used to measure the accumulation of dumbbells on the magnetic bead surface. Left-handed DNA dumbbells produce a distinct signal which reflects the level of non-specific amplification, acting as an internal control. The autoPiLOT assay detected as little as 5 fM target DNA, and was also successfully applied to the detection of S. mansoni DNA. The autoPiLOT design is a novel step forward in the development of a sensitive, user-friendly, low-resource, non-enzymatic diagnostic test.

Diurnal Variation in SARS-CoV-2 PCR Test Results: Test Accuracy May Vary by Time of Day.

False negative tests for SARS-CoV-2 are common and have important public health and medical implications. We tested the hypothesis of diurnal variation in viral shedding by assessing the proportion of positive versus negative SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) tests and cycle time (Ct) values among positive samples by the time of day. Among 86,342 clinical tests performed among symptomatic and asymptomatic patients in a regional health care network in the southeastern United States from March to August 2020, we found evidence for diurnal variation in the proportion of positive SARS-CoV-2 tests, with a peak around 1400 h and 1.7-fold variation over the day after adjustment for age, sex, race, testing location, month, and day of week and lower Ct values during the day for positive samples. These findings have important implications for public health testing and vaccination strategies.

Addition of mirror-image L-DNA elements to DNA amplification circuits to distinguish leakage from target signal.

DNA amplification circuits that rely on thermodynamically-driven hybridization events triggered by a target nucleic acid are becoming increasingly utilized due to their relative simplicity. A drawback of these circuits is that non-specific amplification, or circuit leakage, must be estimated using a separate "no-target" control reaction to eliminate false positives. Aside from requiring an additional reaction, the problem with this approach is the difficulty of creating a no-target control for biological specimens. To overcome this limitation, we propose a strategy that combines both reactions into the same tube using naturally-occurring right-handed D-DNA circuit elements for the target detection reaction and identical synthetic mirror-image left-handed L-DNA circuit elements for the no-target control reaction. We illustrate this approach using catalyzed hairpin assembly (CHA), one of the most studied DNA amplification circuits. In a dual-chirality CHA design, the right-handed circuit signal is produced by target-specific amplification and circuit leakage, whereas the left-handed circuit signal is produced only by circuit leakage. The target-specific amplification is calculated as the difference between the two signals. The limit of detection of this dual-chirality CHA reaction was found to be similar to that of traditional CHA (81 vs 92 pM, respectively). Furthermore, the left-handed no-target signal matched the right-handed leakage across a wide range of sample conditions including background DNA, increased salt concentration, increased temperature, and urine. These results demonstrate the robustness of a dual-chirality design and the potential utility of left-handed DNA in the development of new DNA amplification circuits better-suited for target detection applications in biological samples.

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.

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.

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.

Clonal expansion of innate and adaptive lymphocytes.

One of the hallmarks of the vertebrate adaptive immune system is the prolific expansion of individual cell clones that encounter their cognate antigen. More recently, however, there is growing evidence for the clonal expansion of innate lymphocytes, particularly in the context of pathogen challenge. Clonal expansion not only serves to amplify the number of specific lymphocytes to mount a robust protective response to the pathogen at hand but also results in selection and differentiation of the responding lymphocytes to generate a multitude of cell fates. Here, we summarize the evidence for clonal expansion in innate lymphocytes, which has primarily been observed in natural killer (NK) cells responding to cytomegalovirus infection, and consider the requirements for such a response in NK cells in light of those for T cells. Furthermore, we discuss multiple aspects of heterogeneity that both contribute to and result from the fundamental immunological process of clonal expansion, highlighting the parallels between innate and adaptive lymphocytes, with a particular focus on NK cells and CD8+ T cells.

Low-Resource Nucleic Acid Extraction Method Enabled by High-Gradient Magnetic Separation.

Nucleic acid-based diagnostic tests often require isolation and concentration of nucleic acids from biological samples. Commercial purification kits are difficult to use in low-resource settings because of their cost and insufficient laboratory infrastructure. Several recent approaches based on the use of magnetic beads offer a potential solution but remain limited to small volume samples. We have developed a simple and low-cost nucleic acid extraction method suitable for isolation and concentration of nucleic acids from small or large sample volumes. The method uses magnetic beads, a transfer pipette, steel wool, and an external magnet to implement high-gradient magnetic separation (HGMS) to retain nucleic acid-magnetic bead complexes within the device's steel wool matrix for subsequent processing steps. We demonstrate the method's utility by extracting tuberculosis DNA from both sputum and urine, two typical large volume sample matrices (5-200 mL), using guanidine-based extraction chemistry. Our HGMS-enabled extraction method is statistically indistinguishable from commercial extraction kits when detecting a spiked 123-base DNA sequence. For our HGMS-enabled extraction method, we obtained extraction efficiencies for sputum and urine of approximately 10 and 90%, whereas commercial kits obtained 10-17 and 70-96%, respectively. We also used this method previously in a blinded sample preparation comparison study published by Beall et al., 2019. Our manual extraction method is insensitive to high flow rates and sample viscosity, with capture of ∼100% for flow rates up to 45 mL/min and viscosities up to 55 cP, possibly making it suitable for a wide variety of sample volumes and types and point-of-care users. This HGMS-enabled extraction method provides a robust instrument-free method for magnetic bead-based nucleic acid extraction, potentially suitable for field implementation of nucleic acid testing.

Multiplexed Adaptive RT-PCR Based on L-DNA Hybridization Monitoring for the Detection of Zika, Dengue, and Chikungunya RNA.

Reverse transcription polymerase chain reaction (RT-PCR) is the gold standard for the molecular diagnosis of many infectious diseases, including RNA viruses, but is generally limited to settings with access to trained personnel and laboratory resources. We have previously reported a fundamentally simpler thermal cycling platform called Adaptive PCR, which dynamically controls thermal cycling conditions during each cycle by optically monitoring the annealing and melting of mirror-image L-DNA surrogates of the PCR primers and targets. In this report, we integrate optically-controlled reverse transcription and single-channel monitoring of L-DNAs to develop a multiplexed Adaptive RT-PCR instrument and assay for the detection of Zika, dengue, and chikungunya virus RNA with high target specific and low limits of detection. The assay is demonstrated to detect as low as 5 copies/reaction of Zika or chikungunya RNA and 50 copies/reaction of dengue RNA. The multiplexed Adaptive RT-PCR instrument is robust and has many of the features required to implement diagnostic assays for RNA viruses in settings that lack traditional laboratory resources.

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.

Detection of Single-Nucleotide Polymorphism Markers of Antimalarial Drug Resistance Directly from Whole Blood.

Monitoring of antimalarial resistance is important to prevent its further spread, but the available options for assessing resistance are less than ideal for field settings. Although molecular detection is perhaps the most efficient method, it is also the most complex because it requires DNA extraction and PCR instrumentation. To develop a more deployable approach, we designed new probes, which, when used in combination with an inhibitor-tolerant Taq polymerase, enable single-nucleotide polymorphism genotyping directly from whole blood. The probes feature two strategic design elements: locked nucleic acids to enhance specificity and the reporter dyes Cy5 and TEX615, which have less optical overlap with the blood absorbance spectra than other commonly used dyes. Probe performance was validated on a traditional laboratory-based instrument and then further tested on a field-deployable Adaptive PCR instrument to develop a point-of-care platform appropriate for use in malaria settings. The probes discriminated between wild-type Plasmodium falciparum and the chloroquine-resistant CRT PF3D7_0709000:c.227A>C (p.Lys76Thr) mutant in the presence of 2% blood. Additionally, in allelic discrimination plots with the new probes, samples clustered more closely to their respective axes compared with samples using minor groove binder probes with 6-FAM and VIC reporter dyes. Our strategy greatly simplifies single-nucleotide polymorphism detection and provides a more accessible alternative for antimalarial resistance surveillance in the field.

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.

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.