Age-related epithelial defects limit thymic function and regeneration.

The thymus is essential for establishing adaptive immunity yet undergoes age-related involution that leads to compromised immune responsiveness. The thymus is also extremely sensitive to acute insult and although capable of regeneration, this capacity declines with age for unknown reasons. We applied single-cell and spatial transcriptomics, lineage-tracing and advanced imaging to define age-related changes in nonhematopoietic stromal cells and discovered the emergence of two atypical thymic epithelial cell (TEC) states. These age-associated TECs (aaTECs) formed high-density peri-medullary epithelial clusters that were devoid of thymocytes; an accretion of nonproductive thymic tissue that worsened with age, exhibited features of epithelial-to-mesenchymal transition and was associated with downregulation of FOXN1. Interaction analysis revealed that the emergence of aaTECs drew tonic signals from other functional TEC populations at baseline acting as a sink for TEC growth factors. Following acute injury, aaTECs expanded substantially, further perturbing trophic regeneration pathways and correlating with defective repair of the involuted thymus. These findings therefore define a unique feature of thymic involution linked to immune aging and could have implications for developing immune-boosting therapies in older individuals.

Transient inhibition of type I interferon enhances CD8 + T cell stemness and vaccine protection.

Developing vaccines that promote CD8 + T cell memory is a challenge for infectious disease and cancer immunotherapy. TCF-1 + stem cell-like memory T (T SCM ) cells are important determinants of long-lived memory. Yet, the developmental requirements for T SCM formation are unclear. Here, we identify the temporal window for type I interferon (IFN-I) receptor (IFNAR) blockade to drive T SCM cell generation. T SCM cells were transcriptionally distinct and emerged from a transitional precursor of exhausted (T PEX ) cellular state concomitant with viral clearance. T SCM differentiation correlated with T cell retention within the lymph node paracortex, due to increased CXCR3 chemokine abundance which disrupted gradient formation. These affects were due a counterintuitive increase in IFNψ, which controlled cell location. Combining IFNAR inhibition with mRNA-LNP vaccination promoted specific T SCM differentiation and enhanced protection against chronic infection. These finding propose a new approach to vaccine design whereby modulation of inflammation promotes memory formation and function. HIGHLIGHTS: Early, transient inhibition of the type I interferon (IFN) receptor (IFNAR) during acute viral infection promotes stem cell-like memory T (T SCM ) cell differentiation without establishing chronic infection. T SCM and precursor of exhausted (T PEX ) cellular states are distinguished transcriptionally and by cell surface markers. Developmentally, T SCM cell differentiation occurs via a transition from a T PEX state coinciding with viral clearance. Transient IFNAR blockade increases IFNψ production to modulate the ligands of CXCR3 and couple T SCM differentiation to cell retention within the T cell paracortex of the lymph node. Specific promotion of T SCM cell differentiation with nucleoside-modified mRNA-LNP vaccination elicits enhanced protection against chronic viral challenge.

GFI1B specifies developmental potential of innate lymphoid cell progenitors in the lungs.

Tissue-resident innate lymphoid cells (ILCs) play a vital role in the frontline defense of various tissues, including the lung. The development of type 2 ILCs (ILC2s) depends on transcription factors such as GATA3, RORα, GFI1, and Bcl11b; however, the factors regulating lung-resident ILC2s remain unclear. Through fate mapping analysis of the paralog transcription factors GFI1 and GFI1B, we show that GFI1 is consistently expressed during the transition from progenitor to mature ILC2s. In contrast, GFI1B expression is limited to specific subsets of bone marrow progenitors and lung-resident ILC progenitors. We found that GFI1B+ lung ILC progenitors represent a multi-lineage subset with tissue-resident characteristics and the potential to form lung-derived ILC subsets and liver-resident ILC1s. Loss of GFI1B in bone marrow progenitors led to the selective loss of lung-resident IL-18R+ ILCs and mature ILC2, subsequently preventing the emergence of effector ILCs that could protect the lung against inflammatory or tumor challenge.

Tumor-derived MMPs regulate cachexia in a Drosophila cancer model.

Cachexia, the wasting syndrome commonly observed in advanced cancer patients, accounts for up to one-third of cancer-related mortalities. We have established a Drosophila larval model of organ wasting whereby epithelial overgrowth in eye-antennal discs leads to wasting of the adipose tissue and muscles. The wasting is associated with fat-body remodeling and muscle detachment and is dependent on tumor-secreted matrix metalloproteinase 1 (Mmp1). Mmp1 can both modulate TGFß signaling in the fat body and disrupt basement membrane (BM)/extracellular matrix (ECM) protein localization in both the fat body and the muscle. Inhibition of TGFß signaling or Mmps in the fat body/muscle using a QF2-QUAS binary expression system rescues muscle wasting in the presence of tumor. Altogether, our study proposes that tumor-derived Mmps are central mediators of organ wasting in cancer cachexia.

Spatial omics and multiplexed imaging to explore cancer biology.

Understanding intratumoral heterogeneity-the molecular variation among cells within a tumor-promises to address outstanding questions in cancer biology and improve the diagnosis and treatment of specific cancer subtypes. Single-cell analyses, especially RNA sequencing and other genomics modalities, have been transformative in revealing novel biomarkers and molecular regulators associated with tumor growth, metastasis and drug resistance. However, these approaches fail to provide a complete picture of tumor biology, as information on cellular location within the tumor microenvironment is lost. New technologies leveraging multiplexed fluorescence, DNA, RNA and isotope labeling enable the detection of tens to thousands of cancer subclones or molecular biomarkers within their native spatial context. The expeditious growth in these techniques, along with methods for multiomics data integration, promises to yield a more comprehensive understanding of cell-to-cell variation within and between individual tumors. Here we provide the current state and future perspectives on the spatial technologies expected to drive the next generation of research and diagnostic and therapeutic strategies for cancer.

The site of breast cancer metastases dictates their clonal composition and reversible transcriptomic profile.

Intratumoral heterogeneity is a driver of breast cancer progression, but the nature of the clonal interactive network involved in this process remains unclear. Here, we optimized the use of optical barcoding to visualize and characterize 31 cancer subclones in vivo. By mapping the clonal composition of thousands of metastases in two clinically relevant sites, the lungs and liver, we found that metastases were highly polyclonal in lungs but not in the liver. Furthermore, the transcriptome of the subclones varied according to their metastatic niche. We also identified a reversible niche-driven signature that was conserved in lung and liver metastases collected during patient autopsies. Among this signature, we found that the tumor necrosis factor-α pathway was up-regulated in lung compared to liver metastases, and inhibition of this pathway affected metastasis diversity. These results highlight that the cellular and molecular heterogeneity observed in metastases is largely dictated by the tumor microenvironment.

A functional genetic screen identifies aurora kinase b as an essential regulator of Sox9-positive mouse embryonic lung progenitor cells.

Development of a branching tree in the embryonic lung is crucial for the formation of a fully mature functional lung at birth. Sox9+ cells present at the tip of the primary embryonic lung endoderm are multipotent cells responsible for branch formation and elongation. We performed a genetic screen in murine primary cells and identified aurora kinase b (Aurkb) as an essential regulator of Sox9+ cells ex vivo. In vivo conditional knockout studies confirmed that Aurkb was required for lung development but was not necessary for postnatal growth and the repair of the adult lung after injury. Deletion of Aurkb in embryonic Sox9+ cells led to the formation of a stunted lung that retained the expression of Sox2 in the proximal airways, as well as Sox9 in the distal tips. Although we found no change in cell polarity, we showed that loss of Aurkb or chemical inhibition of Aurkb caused Sox9+ cells to arrest at G2/M, likely responsible for the lack of branch bifurcation. This work demonstrates the power of genetic screens in identifying novel regulators of Sox9+ progenitor cells and lung branching morphogenesis.

Effector and stem-like memory cell fates are imprinted in distinct lymph node niches directed by CXCR3 ligands.

T cells dynamically interact with multiple, distinct cellular subsets to determine effector and memory differentiation. Here, we developed a platform to quantify cell location in three dimensions to determine the spatial requirements that direct T cell fate. After viral infection, we demonstrated that CD8+ effector T cell differentiation is associated with positioning at the lymph node periphery. This was instructed by CXCR3 signaling since, in its absence, T cells are confined to the lymph node center and alternatively differentiate into stem-like memory cell precursors. By mapping the cellular sources of CXCR3 ligands, we demonstrated that CXCL9 and CXCL10 are expressed by spatially distinct dendritic and stromal cell subsets. Unlike effector cells, retention of stem-like memory precursors in the paracortex is associated with CCR7 expression. Finally, we demonstrated that T cell location can be tuned, through deficiency in CXCL10 or type I interferon signaling, to promote effector or stem-like memory fates.

Macrophages provide a transient muscle stem cell niche via NAMPT secretion.

Skeletal muscle regenerates through the activation of resident stem cells. Termed satellite cells, these normally quiescent cells are induced to proliferate by wound-derived signals1. Identifying the source and nature of these cues has been hampered by an inability to visualize the complex cell interactions that occur within the wound. Here we use muscle injury models in zebrafish to systematically capture the interactions between satellite cells and the innate immune system after injury, in real time, throughout the repair process. This analysis revealed that a specific subset of macrophages 'dwell' within the injury, establishing a transient but obligate niche for stem cell proliferation. Single-cell profiling identified proliferative signals that are secreted by dwelling macrophages, which include the cytokine nicotinamide phosphoribosyltransferase (Nampt, which is also known as visfatin or PBEF in humans). Nampt secretion from the macrophage niche is required for muscle regeneration, acting through the C-C motif chemokine receptor type 5 (Ccr5), which is expressed on muscle stem cells. This analysis shows that in addition to their ability to modulate the immune response, specific macrophage populations also provide a transient stem-cell-activating niche, directly supplying proliferation-inducing cues that govern the repair process that is mediated by muscle stem cells. This study demonstrates that macrophage-derived niche signals for muscle stem cells, such as NAMPT, can be applied as new therapeutic modalities for skeletal muscle injury and disease.

Severe Impairment of TNF Post-transcriptional Regulation Leads to Embryonic Death.

Post-transcriptional regulation mechanisms control mRNA stability or translational efficiency via ribosomes, and recent evidence indicates that it is a major determinant of the accurate levels of cytokine mRNAs. Transcriptional regulation of Tnf has been well studied and found to be important for the rapid induction of Tnf mRNA and regulation of the acute phase of inflammation, whereas study of its post-transcriptional regulation has been largely limited to the role of the AU-rich element (ARE), and to a lesser extent, to that of the constitutive decay element (CDE). We have identified another regulatory element (NRE) in the 3' UTR of Tnf and demonstrate that ARE, CDE, and NRE cooperate in vivo to efficiently downregulate Tnf expression and prevent autoimmune inflammatory diseases. We also show that excessive TNF may lead to embryonic death.

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Self-assembling influenza nanoparticle vaccines drive extended germinal center activity and memory B cell maturation.

Protein-based, self-assembling nanoparticles elicit superior immunity compared with soluble protein vaccines, but the immune mechanisms underpinning this effect remain poorly defined. Here, we investigated the immunogenicity of a prototypic ferritin-based nanoparticle displaying influenza hemagglutinin (HA) in mice and macaques. Vaccination of mice with HA-ferritin nanoparticles elicited higher serum antibody titers and greater protection against experimental influenza challenge compared with soluble HA protein. Germinal centers in the draining lymph nodes were expanded and persistent following HA-ferritin vaccination, with greater deposition of antigen that colocalized with follicular dendritic cells. Our findings suggest that a highly ordered and repetitive antigen array may directly drive germinal centers through a B cell-intrinsic mechanism that does not rely on ferritin-specific T follicular helper cells. In contrast to mice, enhanced immunogenicity of HA-ferritin was not observed in pigtail macaques, where antibody titers and lymph node immunity were comparable to soluble vaccination. An improved understanding of factors that drive nanoparticle vaccine immunogenicity in small and large animal models will facilitate the clinical development of nanoparticle vaccines for broad and durable protection against diverse pathogens.

Author Correction: The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

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

The neuropeptide VIP confers anticipatory mucosal immunity by regulating ILC3 activity.

Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.

A Novel Ultra-Stable, Monomeric Green Fluorescent Protein For Direct Volumetric Imaging of Whole Organs Using CLARITY.

Recent advances in thick tissue clearing are enabling high resolution, volumetric fluorescence imaging of complex cellular networks. Fluorescent proteins (FPs) such as GFP, however, can be inactivated by the denaturing chemicals used to remove lipids in some tissue clearing methods. Here, we solved the crystal structure of a recently engineered ultra-stable GFP (usGFP) and propose that the two stabilising mutations, Q69L and N164Y, act to improve hydrophobic packing in the core of the protein and facilitate hydrogen bonding networks at the surface, respectively. usGFP was found to dimerise strongly, which is not desirable for some applications. A point mutation at the dimer interface, F223D, generated monomeric usGFP (muGFP). Neurons in whole mouse brains were virally transduced with either EGFP or muGFP and subjected to Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel (CLARITY) clearing. muGFP fluorescence was retained after CLARITY whereas EGFP fluorescence was highly attenuated, thus demonstrating muGFP is a novel FP suitable for applications where high fluorescence stability and minimal self-association are required.

Promises and Pitfalls of Metal Imaging in Biology.

A picture may speak a thousand words, but if those words fail to form a coherent sentence there is little to be learned. As cutting-edge imaging technology now provides us the tools to decipher the multitude of roles played by metals and metalloids in molecular, cellular, and developmental biology, as well as health and disease, it is time to reflect on the advances made in imaging, the limitations discovered, and the future of a burgeoning field. In this Perspective, the current state of the art is discussed from a self-imposed contrarian position, as we not only highlight the major advances made over the years but use them as teachable moments to zoom in on challenges that remain to be overcome. We also describe the steps being taken toward being able to paint a completely undisturbed picture of cellular metal metabolism, which is, metaphorically speaking, the Holy Grail of the discipline.

High-resolution complementary chemical imaging of bio-elements in Caenorhabditis elegans.

Here, we present a sub-µm multimodal approach to image essential elements in Caenorhabditis elegans. A combination of chemical imaging technologies reveals total metal concentration, chemical state and the protein to which an element is associated. This application of distinct yet complementary chemical imaging techniques provided unique insight into essential and trace elements at the subcellular level.

Loss of synaptic Zn2+ transporter function increases risk of febrile seizures.

Febrile seizures (FS) are the most common seizure syndrome and are potentially a prelude to more severe epilepsy. Although zinc (Zn(2+)) metabolism has previously been implicated in FS, whether or not variation in proteins essential for Zn(2+) homeostasis contributes to susceptibility is unknown. Synaptic Zn(2+) is co-released with glutamate and modulates neuronal excitability. SLC30A3 encodes the zinc transporter 3 (ZNT3), which is primarily responsible for moving Zn(2+) into synaptic vesicles. Here we sequenced SLC30A3 and discovered a rare variant (c.892C > T; p.R298C) enriched in FS populations but absent in population-matched controls. Functional analysis revealed a significant loss-of-function of the mutated protein resulting from a trafficking deficit. Furthermore, mice null for ZnT3 were more sensitive than wild-type to hyperthermia-induced seizures that model FS. Together our data suggest that reduced synaptic Zn(2+) increases the risk of FS and more broadly support the idea that impaired synaptic Zn(2+) homeostasis can contribute to neuronal hyperexcitability.

Cortical microarchitecture changes in genetic epilepsy.

OBJECTIVE: The human GABAAγ2(R43Q) mutation is associated with genetic epilepsy. Because of the role of γ-aminobutyric acid (GABA) in brain development, we asked whether this epilepsy mutation might affect excitability by changing cortical cytoarchitecture. METHODS: We used a mouse model harboring a heterozygous R43Q missense mutation in the GABAA receptor subunit γ2, as identified in a family with absence epilepsy and febrile seizures. Three-dimensional quantification of immunostained neurons (NeuN), inhibitory neurons (GABA), and inhibitory neuron subpopulations (calretinin, parvalbumin, and calbindin) was performed in fiducial somatosensory cortical columns of seizure-naive GABAAγ2(R43Q) and control mice. RESULTS: Of note, the densities of GABA-, calretinin-, parvalbumin-, and calbindin-containing neurons were increased, and somewhat perplexing, the ratio between putative excitatory and inhibitory neurons was decreased in GABAAγ2(R43Q) mice. Differences were detected in a layer-specific manner with greater overall effects in layers 2/3, 5, and 6, as compared with layers 1 and 4. CONCLUSIONS: Our results suggest that the γ2(R43Q) mutation significantly affects cortical microcircuitry in the cortex of this model of human genetic epilepsy.

Sodium channel ß1 subunit localizes to axon initial segments of excitatory and inhibitory neurons and shows regional heterogeneity in mouse brain.

The ß1 subunit of voltage-gated sodium channels, Nav ß1, plays multiple roles in neurons spanning electrophysiological modulation of sodium channel α subunits to cell adhesion and neurite outgrowth. This study used immunohistochemistry to investigate Nav ß1 subneuronal and regional expression. Nav ß1 was enriched at axon initial segments (AIS) and nodes of Ranvier. Nav ß1 expression at the AIS was detected throughout the brain, predominantly in the hippocampus, cortex, and cerebellum. Despite expression of Nav ß1 in both excitatory and inhibitory AIS, it displayed a marked and fine-grained heterogeneity of expression. Such heterogeneity could have important implications for the tuning of single neuronal and regional excitability, especially in view of the fact that Nav ß1 coexpressed with Nav 1.1, Nav 1.2, and Nav 1.6 subunits. The disruption of Nav ß1 AIS expression by a human epilepsy-causing C121W genetic mutation in Nav ß1 was also investigated using a mouse model. AIS expression of Nav ß1 was reduced by approximately 50% in mice heterozygous for the C121W mutation and was abolished in homozygotes, suggesting that loss of Nav α subunit modulation by Nav ß1 contributes to the mechanism of epileptogenesis in these animals as well as in patients.