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.

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.

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.

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.

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.

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.

'Neonatal' Nav1.2 reduces neuronal excitability and affects seizure susceptibility and behaviour.

Developmentally regulated alternative splicing produces 'neonatal' and 'adult' isoforms of four Na(+) channels in human brain, NaV1.1, NaV1.2, NaV1.3 and NaV1.6. Heterologously expressed 'neonatal' NaV1.2 channels are less excitable than 'adult' channels; however, functional importance of this difference is unknown. We hypothesized that the 'neonatal' NaV1.2 may reduce neuronal excitability and have a seizure-protective role during early brain development. To test this hypothesis, we generated NaV1.2(adult) mice expressing only the 'adult' NaV1.2, and compared the firing properties of pyramidal cortical neurons, as well as seizure susceptibility, between the NaV1.2(adult) and wild-type (WT) mice at postnatal day 3 (P3), when the 'neonatal' isoform represents 65% of the WT NaV1.2. We show significant increases in action potential firing in NaV1.2(adult) neurons and in seizure susceptibility of NaV1.2(adult) mice, supporting our hypothesis. At postnatal day 15 (P15), when 17% of the WT NaV1.2 is 'neonatal', the firing properties of NaV1.2(adult) and WT neurons converged. However, inhibitory postsynaptic currents in NaV1.2(adult) neurons were larger and the expression level of Scn2a mRNA was 24% lower compared with the WT. The enhanced seizure susceptibility of the NaV1.2(adult) mice persisted into adult age. The adult NaV1.2(adult) mice also exhibited greater risk-taking behaviour. Overall, our data reveal a significant impact of 'neonatal' NaV1.2 on neuronal excitability, seizure susceptibility and behaviour and may contribute to our understanding of NaV1.2 roles in health and diseases such as epilepsy and autism.

Comparative strength and dendritic organization of thalamocortical and corticocortical synapses onto excitatory layer 4 neurons.

Thalamus is a potent driver of cortical activity even though cortical synapses onto excitatory layer 4 neurons outnumber thalamic synapses 10 to 1. Previous in vitro studies have proposed that thalamocortical (TC) synapses are stronger than corticocortical (CC) synapses. Here, we investigated possible anatomical and physiological differences between these inputs in the rat in vivo. We developed a high-throughput light microscopy method, validated by electron microscopy, to completely map the locations of synapses across an entire dendritic tree. This demonstrated that TC synapses are slightly more proximal to the soma than CC synapses, but detailed compartmental modeling predicted that dendritic filtering does not appreciably favor one synaptic class over another. Measurements of synaptic strength in intact animals confirmed that both TC and CC synapses are weak and approximately equivalent. We conclude that thalamic effectiveness does not rely on enhanced TC strength, but rather on coincident activation of converging inputs.

Reduced dendritic arborization and hyperexcitability of pyramidal neurons in a Scn1b-based model of Dravet syndrome.

Epileptic encephalopathies, including Dravet syndrome, are severe treatment-resistant epilepsies with developmental regression. We examined a mouse model based on a human ß1 sodium channel subunit (Scn1b) mutation. Homozygous mutant mice shared phenotypic features and pharmaco-sensitivity with Dravet syndrome. Patch-clamp analysis showed that mutant subicular and layer 2/3 pyramidal neurons had increased action potential firing rates, presumably as a consequence of their increased input resistance. These changes were not seen in L5 or CA1 pyramidal neurons. This raised the concept of a regional seizure mechanism that was supported by data showing increased spontaneous synaptic activity in the subiculum but not CA1. Importantly, no changes in firing or synaptic properties of gamma-aminobutyric acidergic interneurons from mutant mice were observed, which is in contrast with Scn1a-based models of Dravet syndrome. Morphological analysis of subicular pyramidal neurons revealed reduced dendritic arborization. The antiepileptic drug retigabine, a K+ channel opener that reduces input resistance, dampened action potential firing and protected mutant mice from thermal seizures. These results suggest a novel mechanism of disease genesis in genetic epilepsy and demonstrate an effective mechanism-based treatment of the disease.

Visualization of mouse barrel cortex using ex-vivo track density imaging.

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo T1- and T2-weighted imaging and gradient echo T1/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3mm)(3) block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system.

Inhibitory interneurons in a cortical column form hot zones of inhibition in layers 2 and 5A.

Although physiological data on microcircuits involving a few inhibitory neurons in the mammalian cerebral cortex are available, data on the quantitative relation between inhibition and excitation in cortical circuits involving thousands of neurons are largely missing. Because the distribution of neurons is very inhomogeneous in the cerebral cortex, it is critical to map all neurons in a given volume rather than to rely on sparse sampling methods. Here, we report the comprehensive mapping of interneurons (INs) in cortical columns of rat somatosensory cortex, immunolabeled for neuron-specific nuclear protein and glutamate decarboxylase. We found that a column contains ~2,200 INs (11.5% of ~19,000 neurons), almost a factor of 2 less than previously estimated. The density of GABAergic neurons was inhomogeneous between layers, with peaks in the upper third of L2/3 and in L5A. IN density therefore defines a distinct layer 2 in the sensory neocortex. In addition, immunohistochemical markers of IN subtypes were layer-specific. The "hot zones" of inhibition in L2 and L5A match the reported low stimulus-evoked spiking rates of excitatory neurons in these layers, suggesting that these inhibitory hot zones substantially suppress activity in the neocortex.

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.

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.

Short- and long-term plasticity at the axon initial segment.

The axon initial segment (AIS) is a highly specialized neuronal subregion that is the site of action potential initiation and the boundary between axonal and somatodendritic compartments. In recent years, our understanding of the molecular structure of the AIS, its maturation, and its multiple fundamental roles in neuronal function has seen major advances. We are beginning to appreciate that the AIS is dynamically regulated, both over short timescales via adaptations in ion channel function, and long timescales via activity-dependent structural reorganization. Here, we review results from this emerging field highlighting how structural and functional plasticity relate to the development of the initial segment, and to neuronal disorders linked to AIS dysfunction.

Dimensions of a projection column and architecture of VPM and POm axons in rat vibrissal cortex.

This is the first article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). S1 receives 2 major types of TC inputs, lemiscal and paralemniscal. Lemiscal axons arise from the ventral posteromedial nucleus (VPM) of the thalamus, whereas paralemniscal fibers originate in the posteromedial nucleus (POm). While these 2 TC projections are largely complementary in L4, overlap in other cortical layers is still a matter of debate. VPM and POm axons were specifically labeled in the same rat by virus-mediated expression of different fluorescent proteins. We show that columnar and septal projection patterns are maintained throughout most of the cortical depth with a lower degree of separation in infragranular layers, where TC axons form bands along rows. Finally, we present anatomical dimensions of "TC projection domains" for a standard column in S1.

Cell type-specific thalamic innervation in a column of rat vibrissal cortex.

This is the concluding article in a series of 3 studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). We used viral synaptophysin-enhanced green fluorescent protein expression in thalamic neurons and reconstructions of biocytin-labeled cortical neurons in TC slices to quantify the number and distribution of boutons from the ventral posterior medial (VPM) and posteromedial (POm) nuclei potentially innervating dendritic arbors of excitatory neurons located in layers (L)2-6 of a cortical column in rat somatosensory cortex. We found that 1) all types of excitatory neurons potentially receive substantial TC input (90-580 boutons per neuron); 2) pyramidal neurons in L3-L6 receive dual TC input from both VPM and POm that is potentially of equal magnitude for thick-tufted L5 pyramidal neurons (ca. 300 boutons each from VPM and POm); 3) L3, L4, and L5 pyramidal neurons have multiple (2-4) subcellular TC innervation domains that match the dendritic compartments of pyramidal cells; and 4) a subtype of thick-tufted L5 pyramidal neurons has an additional VPM innervation domain in L4. The multiple subcellular TC innervation domains of L5 pyramidal neurons may partly explain their specific action potential patterns observed in vivo. We conclude that the substantial potential TC innervation of all excitatory neuron types in a cortical column constitutes an anatomical basis for the initial near-simultaneous representation of a sensory stimulus in different neuron types.

Number and laminar distribution of neurons in a thalamocortical projection column of rat vibrissal cortex.

This is the second article in a series of three studies that investigate the anatomical determinants of thalamocortical (TC) input to excitatory neurons in a cortical column of rat primary somatosensory cortex (S1). Here, we report the number and distribution of NeuN-positive neurons within the C2, D2, and D3 TC projection columns in P27 rat somatosensory barrel cortex based on an exhaustive identification of 89,834 somata in a 1.15 mm(3) volume of cortex. A single column contained 19,109 ± 444 neurons (17,560 ± 399 when normalized to a standard-size projection column). Neuron density differences along the vertical column axis delineated "cytoarchitectonic" layers. The resulting neuron numbers per layer in the average column were 63 ± 10 (L1), 2039 ± 524 (L2), 3735 ± 905 (L3), 4447 ± 439 (L4), 1737 ± 251 (L5A), 2235 ± 99 (L5B), 3786 ± 168 (L6A), and 1066 ± 170 (L6B). These data were then used to derive the layer-specific action potential (AP) output of a projection column. The estimates confirmed previous reports suggesting that the ensembles of spiny L4 and thick-tufted pyramidal neurons emit the major fraction of APs of a column. The number of APs evoked in a column by a sensory stimulus (principal whisker deflection) was estimated as 4441 within 100 ms post-stimulus.

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.

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.