Kidney collecting duct cell type composition is regulated by Notch signaling via modulation of mTORC1.

The plasticity and diversity of cell types with specialized functions likely defines the capacity of multicellular organisms to adapt to physiologic stressors. The kidney collecting ducts contribute to water, electrolyte, and pH homeostasis and are composed of mature intermingled epithelial cell types that are susceptible to transdifferentiate. The conversion of kidney collecting duct principal cells to intercalated cells is actively inhibited by Notch signaling to ensure urine concentrating capability. Here we identify Hes1, a target of Notch signaling, allows for maintenance of functionally distinct epithelial cell types within the same microenvironment by regulating mechanistic target of rapamycin complex 1 (mTORC1) activity. Hes1 directly represses the expression of insulin receptor substrate 1 ( Irs1 ), an upstream component of mTOR pathway and suppresses mTORC1 activity in principal cells. Genetic inactivation of tuberous sclerosis complex 2 ( Tsc2 ) to increase mTORC1 activity in mature principal cells is sufficient to promote acquisition of intercalated cell properties, while inhibition of mTORC1 in adult kidney epithelia suppresses intercalated cell properties. Considering that mTORC1 integrates environmental cues, the linkage of functionally distinct epithelial cell types to mTORC1 activity levels likely allows for cell plasticity to be regulated by physiologic and metabolic signals and the ability to sense/transduce these signals.

Enabling methanol fixation of pediatric nasal wash during respiratory illness for single cell sequencing in comparison with fresh samples.

BACKGROUND: Lower respiratory tract infection (LRTI) including pneumonia, bronchitis, and bronchiolitis is the sixth leading cause of mortality around the world and leading cause of death in children under 5 years. Systemic immune response to viral infection is well characterized. However, there is little data regarding the immune response at the upper respiratory tract mucosa. The upper respiratory mucosa is the site of viral entry, initial replication and the first barrier against respiratory infections. Lower respiratory tract samples can be challenging to obtain and require more invasive procedures. However, nasal wash (NW) samples from the upper respiratory tract can be obtained with minimal discomfort to the patient. METHOD: In a pilot study, we developed a protocol using NW samples obtained from hospitalized children with LRTI that enables single cell RNA sequencing (scRNA-seq) after the NW sample is methanol-fixed. RESULTS: We found no significant changes in scRNA-seq qualitative and quantitative parameters between methanol-fixed and fresh NW samples. CONCLUSIONS: We present a novel protocol to enable scRNA-seq in NW samples from children admitted with LRTI. With the inherent challenges associated with clinical samples, the protocol described allows for processing flexibility as well as multicenter collaboration. IMPACT: There are no significant differences in scRNA-seq qualitative and quantitative parameters between methanol fixed and fresh Pediatric Nasal wash samples. The study demonstrates the effectiveness of methanol fixation process on preserving respiratory samples for single cell sequencing. This enables Pediatric Nasal wash specimen for single cell RNA sequencing in pediatric patients with respiratory tract infection and allows processing flexibility and multicenter collaboration.

Development and Characterization of an In Vitro Cell-Based Assay to Predict Potency of mRNA-LNP-Based Vaccines.

Messenger RNA (mRNA) vaccines have emerged as a flexible platform for vaccine development. The evolution of lipid nanoparticles as effective delivery vehicles for modified mRNA encoding vaccine antigens was demonstrated by the response to the COVID-19 pandemic. The ability to rapidly develop effective SARS-CoV-2 vaccines from the spike protein genome, and to then manufacture multibillions of doses per year was an extraordinary achievement and a vaccine milestone. Further development and application of this platform for additional pathogens is clearly of interest. This comes with the associated need for new analytical tools that can accurately predict the performance of these mRNA vaccine candidates and tie them to an immune response expected in humans. Described here is the development and characterization of an imaging based in vitro assay able to quantitate transgene protein expression efficiency, with utility to measure lipid nanoparticles (LNP)-encapsulated mRNA vaccine potency, efficacy, and stability. Multiple biologically relevant adherent cell lines were screened to identify a suitable cell substrate capable of providing a wide dose-response curve and dynamic range. Biologically relevant assay attributes were examined and optimized, including cell monolayer morphology, antigen expression kinetics, and assay sensitivity to LNP properties, such as polyethylene glycol-lipid (or PEG-lipid) composition, mRNA mass, and LNP size. Collectively, this study presents a strategy to quickly optimize and develop a robust cell-based potency assay for the development of future mRNA-based vaccines.

Sterol dysregulation in Smith-Lemli-Opitz syndrome causes astrocyte immune reactivity through microglia crosstalk.

Owing to the need for de novo cholesterol synthesis and cholesterol-enriched structures within the nervous system, cholesterol homeostasis is critical to neurodevelopment. Diseases caused by genetic disruption of cholesterol biosynthesis, such as Smith-Lemli-Opitz syndrome, which is caused by mutations in 7-dehydrocholesterol reductase (DHCR7), frequently result in broad neurological deficits. Although astrocytes regulate multiple neural processes ranging from cell migration to network-level communication, immunological activation of astrocytes is a hallmark pathology in many diseases. However, the impact of DHCR7 on astrocyte function and immune activation remains unknown. We demonstrate that astrocytes from Dhcr7 mutant mice display hallmark signs of reactivity, including increased expression of glial fibrillary acidic protein (GFAP) and cellular hypertrophy. Transcript analyses demonstrate extensive Dhcr7 astrocyte immune activation, hyper-responsiveness to glutamate stimulation and altered calcium flux. We further determine that the impacts of Dhcr7 are not astrocyte intrinsic but result from non-cell-autonomous effects of microglia. Our data suggest that astrocyte-microglia crosstalk likely contributes to the neurological phenotypes observed in disorders of cholesterol biosynthesis. Additionally, these data further elucidate a role for cholesterol metabolism within the astrocyte-microglia immune axis, with possible implications in other neurological diseases.

Rapid In-Process Measurement of Live Virus Vaccine Potency Using Laser Force Cytology: Paving the Way for Rapid Vaccine Development.

Vaccinations to prevent infectious diseases are given to target the body's innate and adaptive immune systems. In most cases, the potency of a live virus vaccine (LVV) is the most critical measurement of efficacy, though in some cases the quantity of surface antigen on the virus is an equally critical quality attribute. Existing methods to measure the potency of viruses include plaque and TCID50 assays, both of which have very long lead times and cannot provide real time information on the quality of the vaccine during large-scale manufacturing. Here, we report the evaluation of LumaCyte's Radiance Laser Force Cytology platform as a new way to measure the potency of LVVs in upstream biomanufacturing process in real time and compare this to traditional TCID50 potency. We also assess this new platform as a way to detect adventitious agents, which is a regulatory expectation for the release of commercial vaccines. In both applications, we report the ability to obtain expedited and relevant potency information with strong correlation to release potency methods. Together, our data propose the application of Laser Force Cytology as a valuable process analytical technology (PAT) for the timely measurement of critical quality attributes of LVVs.

Development of process analytical tools for rapid monitoring of live virus vaccines in manufacturing.

In the development of end-to-end large-scale live virus vaccine (LVV) manufacturing, process analytical technology (PAT) tools enable timely monitoring of critical process parameters (CPP) and significantly guide process development and characterization. In a commercial setting, these very same tools can enable real time monitoring of CPPs on the shop floor and inform harvest decisions, predict peak potency, and serve as surrogates for release potency assays. Here we introduce the development of four advanced PAT tools for upstream and downstream process monitoring in LVV manufacturing. The first tool explores the application of capacitance probes for real time monitoring of viable cell density in bioreactors. The second tool utilizes high content imaging to determine optimum time of infection in a microcarrier process. The third tool uses flow virometry (or nanoscale flow cytometry) to monitor total virus particle counts across upstream and downstream process steps and establishes a robust correlation to virus potency. The fourth and final tool explores the use of nucleic acid dye staining to discriminate between "good" and "damaged" virus particles and uses this strategy to also monitor virus aggregates generated sometimes during downstream processing. Collectively, these tools provide a comprehensive monitoring toolbox and represent a significantly enhanced control strategy for the manufacturing of LVVs.

Sox2 Is an Oncogenic Driver of Small-Cell Lung Cancer and Promotes the Classic Neuroendocrine Subtype.

Although many cancer prognoses have improved in the past 50 years due to advancements in treatments, there has been little improvement in therapies for small-cell lung cancer (SCLC). One promising avenue to improve treatment for SCLC is to understand its underlying genetic alterations that drive its formation, growth, and cellular heterogeneity. RB1 loss is one key driver of SCLC, and RB1 loss has been associated with an increase in pluripotency factors such as SOX2. SOX2 is highly expressed and amplified in SCLC and has been associated with SCLC growth. Using a genetically engineered mouse model, we have shown that Sox2 is required for efficient SCLC formation. Furthermore, genome-scale binding assays have indicated that SOX2 can regulate key SCLC pathways such as NEUROD1 and MYC. These data suggest that SOX2 can be associated with the switch of SCLC from an ASCL1 subtype to a NEUROD1 subtype. Understanding this genetic switch is key to understanding such processes as SCLC progression, cellular heterogeneity, and treatment resistance. IMPLICATIONS: Understanding the molecular mechanisms of SCLC initiation and development are key to opening new potential therapeutic options for this devastating disease.

Flow virometry for process monitoring of live virus vaccines-lessons learned from ERVEBO.

Direct at line monitoring of live virus particles in commercial manufacturing of vaccines is challenging due to their small size. Detection of malformed or damaged virions with reduced potency is rate-limited by release potency assays with long turnaround times. Thus, preempting batch failures caused by out of specification potency results is almost impossible. Much needed are in-process tools that can monitor and detect compromised viral particles in live-virus vaccines (LVVs) manufacturing based on changes in their biophysical properties to provide timely measures to rectify process stresses leading to such damage. Using ERVEBO, MSD's Ebola virus vaccine as an example, here we describe a flow virometry assay that can quickly detect damaged virus particles and provide mechanistic insight into process parameters contributing to the damage. Furthermore, we describe a 24-h high throughput infectivity assay that can be used to correlate damaged particles directly to loss in viral infectivity (potency) in-process. Collectively, we provide a set of innovative tools to enable rapid process development, process monitoring, and control strategy implementation in large scale LVV manufacturing.

Human NK cell deficiency as a result of biallelic mutations in MCM10.

Human natural killer cell deficiency (NKD) arises from inborn errors of immunity that lead to impaired NK cell development, function, or both. Through the understanding of the biological perturbations in individuals with NKD, requirements for the generation of terminally mature functional innate effector cells can be elucidated. Here, we report a cause of NKD resulting from compound heterozygous mutations in minichromosomal maintenance complex member 10 (MCM10) that impaired NK cell maturation in a child with fatal susceptibility to CMV. MCM10 has not been previously associated with monogenic disease and plays a critical role in the activation and function of the eukaryotic DNA replisome. Through evaluation of patient primary fibroblasts, modeling patient mutations in fibroblast cell lines, and MCM10 knockdown in human NK cell lines, we have shown that loss of MCM10 function leads to impaired cell cycle progression and induction of DNA damage-response pathways. By modeling MCM10 deficiency in primary NK cell precursors, including patient-derived induced pluripotent stem cells, we further demonstrated that MCM10 is required for NK cell terminal maturation and acquisition of immunological system function. Together, these data define MCM10 as an NKD gene and provide biological insight into the requirement for the DNA replisome in human NK cell maturation and function.

Foxi1 inactivation rescues loss of principal cell fate selection in Hes1-deficient kidneys but does not ensure maintenance of principal cell gene expression.

The distal nephron and collecting duct segments of the mammalian kidney consist of intercalated cell types intermingled among principal cell types. Notch signaling ensures that a sufficient number of cells select a principal instead of an intercalated cell fate. However, the precise mechanisms by which Notch signaling patterns the distal nephron and collecting duct cell fates is unknown. Here we observed that Hes1, a direct target of Notch signaling pathway, is required within the mouse developing collecting ducts for repression of Foxi1 expression, an essential intercalated cell specific transcription factor. Interestingly, inactivation of Foxi1 in Hes1-deficient collecting ducts rescues the deficiency in principal cell fate selection, overall urine concentrating deficiency, and reduces the occurrence of hydronephrosis. However, Foxi1 inactivation does not rescue the reduction in expression of all principal cell genes in the Hes1-deficient kidney collecting duct cells that select the principal cell fate. Additionally, suppression of Notch/Hes1 signaling in mature principal cells reduces principal cell gene expression without activating Foxi1. We conclude that Hes1 is a Notch signaling target that is essential for normal patterning of the collecting ducts with intermingled cell types by repressing Foxi1, and for maintenance of principal cell gene expression independent of repressing Foxi1.

Notch signaling regulates Akap12 expression and primary cilia length during renal tubule morphogenesis.

Alagille syndrome patients present with loss of function mutations in either JAG1 or NOTCH2. About 40%-50% of patients have kidney abnormalities, and frequently display multicystic, dysplastic kidneys. Additionally, gain-of-function mutations in NOTCH2 are associated with cystic kidneys in Hajdu-Cheney syndrome patients. How perturbations in Notch signaling cause renal tubular cysts remains unclear. Here, we have determined that reduced Notch signaling mediated transcription by ectopic expression of dominant-negative mastermind-like (dnMaml) peptide in the nephrogenic epithelia from after the s-shaped body formation and in the developing collecting ducts results in proximal tubular and collecting duct cysts, respectively. An acute inhibition of Notch signaling for two days during kidney development is sufficient to disrupt tubule formation, and significantly increases Akap12 expression. Ectopic expression of Akap12 in renal epithelia results in abnormally long primary cilia similar to that observed in Notch-signaling-deficient epithelia. Both loss of Notch signaling and elevated Akap12 expression disrupt the ability of renal epithelial cells to form spherical structures with a single lumen when grown embedded in matrix. Interestingly, Akap12 can inhibit Notch signaling mediated transcription, which likely explains how both loss of Notch signaling and ectopic expression of Akap12 result in similar renal epithelial abnormalities. We conclude that Notch signaling regulates Akap12 expression while also ensuring normal primary cilia length and renal epithelial morphogenesis, and suggest that one aspect of diseases associated with defective Notch signaling, such as Alagille syndrome, maybe mechanistically related to ciliopathies.

Notch Signaling in Kidney Development, Maintenance, and Disease.

Kidney development involves formation of nephrons intricately aligned with the vasculature and connected to a branched network of collecting ducts. Notch signaling plays multiple roles during kidney development involving the formation of nephrons composed of diverse epithelial cell types arranged into tubular segments, all the while maintaining a nephron progenitor niche. Here, we review the roles of Notch signaling identified from rodent kidney development and injury studies, while discussing human kidney diseases associated with aberrant Notch signaling. We also review Notch signaling requirement in maintenance of mature kidney epithelial cell states and speculate that Notch activity regulation mediates certain renal physiologic adaptations.

Publisher Correction: Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

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

Human DEF6 deficiency underlies an immunodeficiency syndrome with systemic autoimmunity and aberrant CTLA-4 homeostasis.

Immune responses need to be controlled tightly to prevent autoimmune diseases, yet underlying molecular mechanisms remain partially understood. Here, we identify biallelic mutations in three patients from two unrelated families in differentially expressed in FDCP6 homolog (DEF6) as the molecular cause of an inborn error of immunity with systemic autoimmunity. Patient T cells exhibit impaired regulation of CTLA-4 surface trafficking associated with reduced functional CTLA-4 availability, which is replicated in DEF6-knockout Jurkat cells. Mechanistically, we identify the small GTPase RAB11 as an interactor of the guanine nucleotide exchange factor DEF6, and find disrupted binding of mutant DEF6 to RAB11 as well as reduced RAB11+CTLA-4+ vesicles in DEF6-mutated cells. One of the patients has been treated with CTLA-4-Ig and achieved sustained remission. Collectively, we uncover DEF6 as player in immune homeostasis ensuring availability of the checkpoint protein CTLA-4 at T-cell surface, identifying a potential target for autoimmune and/or cancer therapy.

Retraction Note: A homing system targets therapeutic T cells to brain cancer.

This Article has been retracted; see accompanying Retraction.

Endogenous Notch Signaling in Adult Kidneys Maintains Segment-Specific Epithelial Cell Types of the Distal Tubules and Collecting Ducts to Ensure Water Homeostasis.

BACKGROUND: Notch signaling is required during kidney development for nephron formation and principal cell fate selection within the collecting ducts. Whether Notch signaling is required in the adult kidney to maintain epithelial diversity, or whether its loss can trigger principal cell transdifferentiation (which could explain acquired diabetes insipidus in patients receiving lithium) is unclear. METHODS: To investigate whether loss of Notch signaling can trigger principal cells to lose their identity, we genetically inactivated Notch1 and Notch2, inactivated the Notch signaling target Hes1, or induced expression of a Notch signaling inhibitor in all of the nephron segments and collecting ducts in mice after kidney development. We examined renal function and cell type composition of control littermates and mice with conditional Notch signaling inactivation in adult renal epithelia. In addition, we traced the fate of genetically labeled adult kidney collecting duct principal cells after Hes1 inactivation or lithium treatment. RESULTS: Notch signaling was required for maintenance of Aqp2-expressing cells in distal nephron and collecting duct segments in adult kidneys. Fate tracing revealed mature principal cells in the inner stripe of the outer medulla converted to intercalated cells after genetic inactivation of Hes1 and, to a lesser extent, lithium treatment. Hes1 ensured repression of Foxi1 to prevent the intercalated cell program from turning on in mature Aqp2+ cell types. CONCLUSIONS: Notch signaling viaHes1 regulates maintenance of mature renal epithelial cell states. Loss of Notch signaling or use of lithium can trigger transdifferentiation of mature principal cells to intercalated cells in adult kidneys.

A homing system targets therapeutic T cells to brain cancer.

Successful T cell immunotherapy for brain cancer requires that the T cells can access tumour tissues, but this has been difficult to achieve. Here we show that, in contrast to inflammatory brain diseases such as multiple sclerosis, where endothelial cells upregulate ICAM1 and VCAM1 to guide the extravasation of pro-inflammatory cells, cancer endothelium downregulates these molecules to evade immune recognition. By contrast, we found that cancer endothelium upregulates activated leukocyte cell adhesion molecule (ALCAM), which allowed us to overcome this immune-evasion mechanism by creating an ALCAM-restricted homing system (HS). We re-engineered the natural ligand of ALCAM, CD6, in a manner that triggers initial anchorage of T cells to ALCAM and conditionally mediates a secondary wave of adhesion by sensitizing T cells to low-level ICAM1 on the cancer endothelium, thereby creating the adhesion forces necessary to capture T cells from the bloodstream. Cytotoxic HS T cells robustly infiltrated brain cancers after intravenous injection and exhibited potent antitumour activity. We have therefore developed a molecule that targets the delivery of T cells to brain cancer.

Chimeric Antigen Receptor Signaling Domains Differentially Regulate Proliferation and Native T Cell Receptor Function in Virus-Specific T Cells.

The efficacy of T cells expressing chimeric antigen receptors (CARs) for solid tumors has been limited by insufficient CAR T cell expansion and persistence. The use of virus-specific T cells (VSTs) as carriers for CARs may overcome this limitation since CAR-VSTs can be boosted by viral vaccines or oncolytic viruses. However, there is limited understanding of the optimal combination of endodomains and their influence on the native T cell receptor (TCR) in VSTs. We therefore compared the function of GD2.CARs expressing the TCR zeta chain (ζ) alone or combined with endodomains from CD28 and 4-1BB in varicella zoster virus-specific (VZV) T cells. VZVSTs expressing GD2-CARs recognized VZV-derived peptides and killed GD2-expressing tumor cells. However, after repeated stimulation through their native TCR, the expansion of GD2-CAR.CD28ζ-VZVSTs was 3.3-fold greater (p < 0.001) than non-transduced VZVSTs, whereas GD2-CARζ- and GD2-CAR.41BBζ inhibited VZVST expansion (p < 0.01). Compared to control VZVSTs, GD2-CAR.ζ VZVSTs showed a greater frequency of apoptotic (p < 0.01) T cells, whereas prolonged downregulation of the native αß TCR was observed in GD2-CAR.41BBζ VZVSTs (p < 0.001). We confirmed that CD28ζ can best maintain TCR function by expressing GD2.CARs in Epstein-Barr virus-specific T cells and CD19-CARs in VZVSTs. In response to CAR stimulation VSTs with CD28ζ endodomains also showed the greatest expansion (6 fold > GD2-CAR.41BBζ VZVSTs (p < 0.001), however anti-tumor efficacy was superior in GD2-CAR.41BBζ-VZVSTs. These findings demonstrate that CAR signaling domains can enhance or diminish the function of the native TCR and indicate that only CD28ζ may preserve the function of the native TCR in tonically signaling CAR-VSTs.