Discrete Niches Research Articles

Regulatory T-cells: The Face-off of the Immune Balance.

Regulatory T-cells (Tregs) play a crucial role in maintaining immune homeostasis, ensuring a balanced immune response. Tregs primarily operate in an antigen-specific fashion, facilitated by their distinct distribution within discrete niches. Tregs have been studied extensively, from their point of origin in the thymus origin to their fate in the periphery or organs. Signals received from antigen-presenting cells (APCs) stimulate Tregs to dampen inflammation. Almost all tumors are characterized by a pathological abundance of immune suppression in their microenvironment. Conversely, the lack thereof proves detrimental to immunological disorders. Achieving a balanced expression of Tregs in relation to other immune compartments is important in establishing an effective and adaptable immune tolerance towards cancer cells and autoantigens. In the context of cancer, it is essential to decrease the frequency of Tregs to overcome tumor suppression. A lower survival rate is associated with the presence of excessive exhausted effector immune cells and an increased frequency of regulatory cells. However, when it comes to treating graft rejection and autoimmune diseases, the focus lies on immune tolerance and the transfer of Tregs. Here, we explore the complex mechanisms that Tregs use in human disease to balance effector immune cells.

Safety and reactogenicity of a controlled human infection model of sand fly-transmitted cutaneous leishmaniasis

The leishmaniases are globally important parasitic diseases for which no human vaccines are currently available. To facilitate vaccine development, we conducted an open-label observational study to establish a controlled human infection model (CHIM) of sand fly-transmitted cutaneous leishmaniasis (CL) caused by Leishmania major. Between 24 January and 12 August 2022, we exposed 14 participants to L. major-infected Phlebotomus duboscqi. The primary objective was to demonstrate effectiveness of lesion development (take rate) and safety (absence of CL lesion at 12 months). Secondary and exploratory objectives included rate of lesion development, parasite load and analysis of local immune responses by immunohistology and spatial transcriptomics. Lesion development was terminated by therapeutic biopsy (between days 14 and 42 after bite) in ten participants with clinically compatible lesions, one of which was not confirmed by parasite detection. We estimated an overall take rate for CL development of 64% (9/14). Two of ten participants had one and one of ten participants had two lesion recurrences 4–8 months after biopsy that were treated successfully with cryotherapy. No severe or serious adverse events were recorded, but as expected, scarring due to a combination of CL and the biopsy procedure was evident. All participants were lesion free at >12-month follow-up. We provide the first comprehensive map of immune cell distribution and cytokine/chemokine expression in human CL lesions, revealing discrete immune niches. This CHIM offers opportunities for vaccine candidate selection based on human efficacy data and for a greater understanding of immune-mediated pathology. ClinicalTrials.gov identifier: NCT04512742.

HB-EGF promotes progenitor cell proliferation and sensory neuron regeneration in the zebrafish olfactory epithelium.

Maintenance and regeneration of the zebrafish olfactory epithelium (OE) are supported by two distinct progenitor cell populations that occupy spatially discrete stem cell niches and respond to different tissue conditions. Globose basal cells (GBCs) reside at the inner and peripheral margins of the sensory OE and are constitutively active to replace sporadically dying olfactory sensory neurons (OSNs). In contrast, horizontal basal cells (HBCs) are uniformly distributed across the sensory tissue and are selectively activated by acute injury conditions. Here we show that expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is strongly and transiently upregulated in response to OE injury and signals through the EGF receptor (EGFR), which is expressed by HBCs. Exogenous stimulation of the OE with recombinant HB-EGF promotes HBC expansion and OSN neurogenesis in a pattern that resembles the tissue response to injury. In contrast, pharmacological inhibition of HB-EGF membrane shedding, HB-EGF availability, and EGFR signaling strongly attenuate or delay injury-induced HBC activity and OSN restoration without affecting maintenance neurogenesis by GBCs. Thus, HB-EGF/EGFR signaling appears to be a critical component of the signaling network that controls HBC activity and, consequently, repair neurogenesis in the zebrafish OE.

High-sensitive spatially resolved T cell receptor sequencing with SPTCR-seq

Spatial resolution of the T cell repertoire is essential for deciphering cancer-associated immune dysfunction. Current spatially resolved transcriptomic technologies are unable to directly annotate T cell receptors (TCR). We present spatially resolved T cell receptor sequencing (SPTCR-seq), which integrates optimized target enrichment and long-read sequencing for highly sensitive TCR sequencing. The SPTCR computational pipeline achieves yield and coverage per TCR comparable to alternative single-cell TCR technologies. Our comparison of PCR-based and SPTCR-seq methods underscores SPTCR-seq’s superior ability to reconstruct the entire TCR architecture, including V, D, J regions and the complementarity-determining region 3 (CDR3). Employing SPTCR-seq, we assess local T cell diversity and clonal expansion across spatially discrete niches. Exploration of the reciprocal interaction of the tumor microenvironmental and T cells discloses the critical involvement of NK and B cells in T cell exhaustion. Integrating spatially resolved omics and TCR sequencing provides as a robust tool for exploring T cell dysfunction in cancers and beyond.

Laminin-coated electronic scaffolds with vascular topography for tracking and promoting the migration of brain cells after injury.

In the adult brain, neural stem cells are largely restricted into spatially discrete neurogenic niches, and hence areas of neuron loss during neurodegenerative disease or following a stroke or traumatic brain injury do not typically repopulate spontaneously. Moreover, understanding neural activity accompanying the neural repair process is hindered by a lack of minimally invasive devices for the chronic measurement of the electrophysiological dynamics in damaged brain tissue. Here we show that 32 individually addressable platinum microelectrodes integrated into laminin-coated branched polymer scaffolds stereotaxically injected to span a hydrogel-filled cortical lesion and deeper regions in the brains of mice promote neural regeneration while allowing for the tracking of migrating host brain cells into the lesion. Chronic measurements of single-unit activity and neural-circuit analyses revealed the establishment of spiking activity in new neurons in the lesion and their functional connections with neurons deeper in the brain. Electronic implants mimicking the topographical and surface properties of brain vasculature may aid the stimulation and tracking of neural-circuit restoration following injury.

Novel pelagiphage isolate Polarivirus skadi is a polar specialist that dominates SAR11-associated bacteriophage communities at high latitudes

The SAR11 clade are the most abundant members of surface marine bacterioplankton and a critical component of global biogeochemical cycles. Similarly, pelagiphages that infect SAR11 are ubiquitous and highly abundant in the oceans. Pelagiphages are predicted to shape SAR11 community structures and increase carbon turnover throughout the oceans. Yet, ecological drivers of host and niche specificity of pelagiphage populations are poorly understood. Here we report the global distribution of a novel pelagiphage called “Polarivirus skadi”, which is the sole representative of a novel genus. P. skadi was isolated from the Western English Channel using a cold-water ecotype of SAR11 as bait. P. skadi is closely related to the globally dominant pelagiphage HTVC010P. Along with other HTVC010P-type viruses, P. skadi belongs to a distinct viral family within the order Caudovirales, for which we propose the name Ubiqueviridae. Metagenomic read recruitment identified P. skadi as one of the most abundant pelagiphages on Earth. P. skadi is a polar specialist, replacing HTVC010P at high latitudes. Experimental evaluation of P. skadi host range against cold- and warm-water SAR11 ecotypes supported cold-water specialism. Relative abundance of P. skadi in marine metagenomes correlated negatively with temperature, and positively with nutrients, available oxygen, and chlorophyll concentrations. In contrast, relative abundance of HTVC010P correlated negatively with oxygen and positively with salinity, with no significant correlation to temperature. The majority of other pelagiphages were scarce in most marine provinces, with a few representatives constrained to discrete ecological niches. Our results suggest that pelagiphage populations persist within a global viral seed bank, with environmental parameters and host availability selecting for a few ecotypes that dominate ocean viromes.

The Hematopoietic Microenvironment: A Network of Niches for the Development of All Blood Cell Lineages.

Blood cell formation (hematopoiesis) takes place mainly in the bone marrow, within the hematopoietic microenvironment, composed of a number of different cell types and their molecular products that together shape spatially organized and highly specialized microstructures called hematopoietic niches. From the earliest developmental stages and throughout the myeloid and lymphoid lineage differentiation pathways, hematopoietic niches play a crucial role in the preservation of cellular integrity and the regulation of proliferation and differentiation rates. Current evidence suggests that each blood cell lineage develops under specific, discrete niches that support committed progenitor and precursor cells, and potentially cooperate with transcriptional programs determining the gradual lineage commitment and specification. This review aims to discuss recent advances on the cellular identity and structural organization of lymphoid, granulocytic, monocytic, megakaryocytic and erythroid niches throughout the hematopoietic microenvironment, and the mechanisms by which they interconnect and regulate viability, maintenance, maturation, and function of the developing blood cells.

Dietary Niche of Three Omnivorous Turtle Species in a Northern Florida River: Insights from Stable Isotope Analysis

Macrochelys suwanniensis (Suwannee Alligator Snapping Turtle) coexists with 2 other native large omnivorous turtle species (Chelydra serpentina [Snapping Turtle] and Trachemys scripta scripta [Yellow-bellied Slider]) in a 9-km section of the Santa Fe River in northern Florida. A major shift in dominant submersed aquatic vegetation prompted us to quantify trophic position and niche overlap among these 3 species. Here, we examine carbon and nitrogen isotopic values of these turtles and their potential food resources within the changing riverine system. We provide evidence of low isotopic niche overlap between M. suwanniensis and C. serpentina, whereas T. s. scripta occupied a discrete niche having lower carbon and nitrogen values.

Intratumoral dendritic cell-CD4+ T helper cell niches enable CD8+ T cell differentiation following PD-1 blockade in hepatocellular carcinoma.

Despite no apparent defects in T cell priming and recruitment to tumors, a large subset of T cell rich tumors fail to respond to immune checkpoint blockade (ICB). We leveraged a neoadjuvant anti-PD-1 trial in patients with hepatocellular carcinoma (HCC), as well as additional samples collected from patients treated off-label, to explore correlates of response to ICB within T cell-rich tumors. We show that ICB response correlated with the clonal expansion of intratumoral CXCL13+CH25H+IL-21+PD-1+CD4+ T helper cells ("CXCL13+ TH") and Granzyme K+ PD-1+ effector-like CD8+ T cells, whereas terminally exhausted CD39hiTOXhiPD-1hiCD8+ T cells dominated in nonresponders. CD4+ and CD8+ T cell clones that expanded post-treatment were found in pretreatment biopsies. Notably, PD-1+TCF-1+ (Progenitor-exhausted) CD8+ T cells shared clones mainly with effector-like cells in responders or terminally exhausted cells in nonresponders, suggesting that local CD8+ T cell differentiation occurs upon ICB. We found that these Progenitor CD8+ T cells interact with CXCL13+ TH within cellular triads around dendritic cells enriched in maturation and regulatory molecules, or "mregDC". These results suggest that discrete intratumoral niches that include mregDC and CXCL13+ TH control the differentiation of tumor-specific Progenitor exhasuted CD8+ T cells following ICB.

Restoring carboxypeptidase E rescues BDNF maturation and neurogenesis in aged brains

Abstract Adult neurogenesis declines with age due to the less functional neural stem cells (NSCs) and niches, but the underlying molecular bases for this impaired condition remain unclear. Here we analyzed >55,000 single-cell transcriptomes from two discrete neurogenic niches across the mouse lifespan, and identified new features and populations in NSCs, new markers, and neurogenic regional-specific alternations during aging. Intercellular communication analysis revealed defects in brain-derived neurotrophic factor (BDNF)-TrkB signaling cascade in old NSCs. Carboxypeptidase E (CPE) was found to be highly enriched in NSCs, and played a crucial role in mature/proBDNF balance and adult neurogenesis. Diminishment of CPE with aging resulted in impaired generation of BDNF, thus limiting the neurogenesis in old neurogenic niches. Restoring CPE expression markedly rescued the adult neurogenesis by increasing the production of mature BDNF, offering an attractive therapeutic strategy for the treatment of certain disorders in regions associated with constitutive neurogenesis.

Single-cell analysis reveals prognostic fibroblast subpopulations linked to molecular and immunological subtypes of lung cancer

Fibroblasts are poorly characterised cells that variably impact tumour progression. Here, we use single cell RNA-sequencing, multiplexed immunohistochemistry and digital cytometry (CIBERSORTx) to identify and characterise three major fibroblast subpopulations in human non-small cell lung cancer: adventitial, alveolar and myofibroblasts. Alveolar and adventitial fibroblasts (enriched in control tissue samples) localise to discrete spatial niches in histologically normal lung tissue and indicate improved overall survival rates when present in lung adenocarcinomas (LUAD). Trajectory inference identifies three phases of control tissue fibroblast activation, leading to myofibroblast enrichment in tumour samples: initial upregulation of inflammatory cytokines, followed by stress-response signalling and ultimately increased expression of fibrillar collagens. Myofibroblasts correlate with poor overall survival rates in LUAD, associated with loss of epithelial differentiation, TP53 mutations, proximal molecular subtypes and myeloid cell recruitment. In squamous carcinomas myofibroblasts were not prognostic despite being transcriptomically equivalent. These findings have important implications for developing fibroblast-targeting strategies for cancer therapy.

Congeneric predators fill discrete niches created by the relative abundances of their prey species

To what degree is niche partitioning driven by underlying patterns in resources such as food, rather than by competition itself? Do discrete niches exist? We address these questions in the context of Cooper's and sharp‐shinned hawks, two broadly sympatric, North American, bird‐eating raptors in the genus Accipiter. We find that the resource base, as quantified by body masses of birds at bird feeders, is approximately lognormal (smallest birds are most abundant), with lesser modes (peaks) in abundance at larger body mass. The predators appear to exploit peaks in the resource base, with sharp‐shinned hawks focusing on small prey items (median of 26.5 g), and Cooper's hawks taking prey from the two most abundant peaks (both the small body mass peak and a lesser peak at medium body mass ~90 g). We tested the ability of citizen scientists to distinguish these notoriously similar species, and we determined the influence of potential false positive detections on our conclusions. We find that citizen scientists struggle to distinguish these predators from one another, and 18% of Cooper's hawks were identified as sharp‐shinned hawks, while 27% of sharp‐shinned hawks were identified as Cooper's hawks. Yet, simulations show that this uncertainty did not jeopardize our qualitative conclusions.

Tongue immune compartment analysis reveals spatial macrophage heterogeneity.

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

Distinct phenotypic states and spatial distribution of CD8+ T cell clonotypes in human brain metastases

SummaryMetastatic disease in the brain is difficult to control and predicts poor prognosis. Here, we analyze human brain metastases and demonstrate their robust infiltration by CD8+ T cell subsets with distinct antigen specificities, phenotypic states, and spatial localization within the tumor microenvironment. Brain metastases are densely infiltrated by T cells; the majority of infiltrating CD8+ T cells express PD-1. Single-cell RNA sequencing shows significant clonal overlap between proliferating and exhausted CD8+ T cells, but these subsets have minimal clonal overlap with circulating and other tumor-infiltrating CD8+ T cells, including bystander CD8+ T cells specific for microbial antigens. Using spatial transcriptomics and spatial T cell receptor (TCR) sequencing, we show these clonally unrelated, phenotypically distinct CD8+ T cell populations occupy discrete niches within the brain metastasis tumor microenvironment. Together, our work identifies signaling pathways within CD8+ T cells and in their surrounding environment that may be targeted for immunotherapy of brain metastases.

NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation.

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here, we used rational engineering and cross-species screening to develop a NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Amino Acid Sensing and Assimilation by the Fungal Pathogen Candida albicans in the Human Host.

Nutrient uptake is essential for cellular life and the capacity to perceive extracellular nutrients is critical for coordinating their uptake and metabolism. Commensal fungal pathogens, e.g., Candida albicans, have evolved in close association with human hosts and are well-adapted to using diverse nutrients found in discrete host niches. Human cells that cannot synthesize all amino acids require the uptake of the “essential amino acids” to remain viable. Consistently, high levels of amino acids circulate in the blood. Host proteins are rich sources of amino acids but their use depends on proteases to cleave them into smaller peptides and free amino acids. C. albicans responds to extracellular amino acids by pleiotropically enhancing their uptake and derive energy from their catabolism to power opportunistic virulent growth. Studies using Saccharomyces cerevisiae have established paradigms to understand metabolic processes in C. albicans; however, fundamental differences exist. The advent of CRISPR/Cas9-based methods facilitate genetic analysis in C. albicans, and state-of-the-art molecular biological techniques are being applied to directly examine growth requirements in vivo and in situ in infected hosts. The combination of divergent approaches can illuminate the biological roles of individual cellular components. Here we discuss recent findings regarding nutrient sensing with a focus on amino acid uptake and metabolism, processes that underlie the virulence of C. albicans.

A pan-metazoan concept for adult stem cells: the wobbling Penrose landscape.

ABSTRACTAdult stem cells (ASCs) in vertebrates and model invertebrates (e.g. Drosophila melanogaster) are typically long‐lived, lineage‐restricted, clonogenic and quiescent cells with somatic descendants and tissue/organ‐restricted activities. Such ASCs are mostly rare, morphologically undifferentiated, and undergo asymmetric cell division. Characterized by ‘stemness’ gene expression, they can regulate tissue/organ homeostasis, repair and regeneration. By contrast, analysis of other animal phyla shows that ASCs emerge at different life stages, present both differentiated and undifferentiated phenotypes, and may possess amoeboid movement. Usually pluri/totipotent, they may express germ‐cell markers, but often lack germ‐line sequestering, and typically do not reside in discrete niches. ASCs may constitute up to 40% of animal cells, and participate in a range of biological phenomena, from whole‐body regeneration, dormancy, and agametic asexual reproduction, to indeterminate growth. They are considered legitimate units of selection. Conceptualizing this divergence, we present an alternative stemness metaphor to the Waddington landscape: the ‘wobbling Penrose’ landscape. Here, totipotent ASCs adopt ascending/descending courses of an ‘Escherian stairwell’, in a lifelong totipotency pathway. ASCs may also travel along lower stemness echelons to reach fully differentiated states. However, from any starting state, cells can change their stemness status, underscoring their dynamic cellular potencies. Thus, vertebrate ASCs may reflect just one metazoan ASC archetype.

Distribution of Aldh1L1-CreERT2 Recombination in Astrocytes Versus Neural Stem Cells in the Neurogenic Niches of the Adult Mouse Brain.

In the adult central nervous system, neural stem cells (NSCs) reside in two discrete niches: the subependymal zone (SEZ) of the lateral ventricle and the subgranular zone (SGZ) of the dentate gyrus (DG). Here, NSCs represent a population of highly specialized astrocytes that are able to proliferate and give rise to neuronal and glial progeny. This process, termed adult neurogenesis, is extrinsically regulated by other niche cells such as non-stem cell astrocytes. Studying these non-stem cell niche astrocytes and their role during adult neuro- and gliogenesis has been hampered by the lack of genetic tools to discriminate between transcriptionally similar NSCs and niche astrocytes. Recently, Aldh1L1 has been shown to be a pan-astrocyte marker and that its promoter can be used to specifically target astrocytes using the Cre-loxP system. In this study we explored whether the recently described Aldh1L1-CreERT2 mouse line (Winchenbach et al., 2016) can serve to specifically target niche astrocytes without inducing recombination in NSCs in adult neurogenic niches. Using short- and long-term tamoxifen protocols we revealed high recombination efficiency and specificity in non-stem cell astrocytes and little to no recombination in NSCs of the adult DG. However, in the SEZ we observed recombination in ependymal cells, astrocytes, and NSCs, the latter giving rise to neuronal progeny of the rostral migratory stream and olfactory bulb. Thus, we recommend the here described Aldh1L1-CreERT2 mouse line for predominantly studying the functions of non-stem cell astrocytes in the DG under physiological and pathological conditions.

An intimate encounter: DC3s empower anti-tumor CTLs.

Discrete tissue niches are emerging as essential prerequisites enabling cell communication and function in both homeostasis and disease. In a recent Cell paper, Di Pilato etal. identify a unique dendritic cell-cytotoxic Tcell crosstalk within the perivascular space that facilitates Tcell survival and proliferation and drives anti-tumor activity.

CXCR6 positions cytotoxic T cells to receive critical survival signals in the tumor microenvironment

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.