Uncovering the mode of action of engineered T cells in patient cancer organoids.

Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.

Oncolytic DNX-2401 Virus for Pediatric Diffuse Intrinsic Pontine Glioma.

BACKGROUND: Pediatric patients with diffuse intrinsic pontine glioma (DIPG) have a poor prognosis, with a median survival of less than 1 year. Oncolytic viral therapy has been evaluated in patients with pediatric gliomas elsewhere in the brain, but data regarding oncolytic viral therapy in patients with DIPG are lacking. METHODS: We conducted a single-center, dose-escalation study of DNX-2401, an oncolytic adenovirus that selectively replicates in tumor cells, in patients with newly diagnosed DIPG. The patients received a single virus infusion through a catheter placed in the cerebellar peduncle, followed by radiotherapy. The primary objective was to assess the safety and adverse-event profile of DNX-2401. The secondary objectives were to evaluate the effect of DNX-2401 on overall survival and quality of life, to determine the percentage of patients who have an objective response, and to collect tumor-biopsy and peripheral-blood samples for correlative studies of the molecular features of DIPG and antitumor immune responses. RESULTS: A total of 12 patients, 3 to 18 years of age, with newly diagnosed DIPG received 1×1010 (the first 4 patients) or 5×1010 (the subsequent 8 patients) viral particles of DNX-2401, and 11 received subsequent radiotherapy. Adverse events among the patients included headache, nausea, vomiting, and fatigue. Hemiparesis and tetraparesis developed in 1 patient each. Over a median follow-up of 17.8 months (range, 5.9 to 33.5), a reduction in tumor size, as assessed on magnetic resonance imaging, was reported in 9 patients, a partial response in 3 patients, and stable disease in 8 patients. The median survival was 17.8 months. Two patients were alive at the time of preparation of the current report, 1 of whom was free of tumor progression at 38 months. Examination of a tumor sample obtained during autopsy from 1 patient and peripheral-blood studies revealed alteration of the tumor microenvironment and T-cell repertoire. CONCLUSIONS: Intratumoral infusion of oncolytic virus DNX-2401 followed by radiotherapy in pediatric patients with DIPG resulted in changes in T-cell activity and a reduction in or stabilization of tumor size in some patients but was associated with adverse events. (Funded by the European Research Council under the European Union's Horizon 2020 Research and Innovation Program and others; EudraCT number, 2016-001577-33; ClinicalTrials.gov number, NCT03178032.).

3D spheroid culture to examine adaptive therapy response in invading tumor cells.

3D in vitro culture models of cancer cells in extracellular matrix (ECM) have been developed to investigate drug targeting and resistance or, alternatively, mechanisms of invasion; however, models allowing analysis of shared pathways mediating invasion and therapy resistance are lacking. To evaluate therapy response associated with cancer cell invasion, we here used 3D invasion culture of tumor spheroids in 3D fibrillar collagen and applied Ethanol-Ethyl cinnamate (EtOH-ECi) based optical clearing to detect both spheroid core and invasion zone by subcellular-resolved 3D microscopy. When subjected to a single dose of irradiation (4 Gy), we detected significant cell survival in the invasion zone. By physical separation of the core and invasion zone, we identified differentially regulated genes preferentially engaged in invading cells controlling cell division, repair, and survival. This imaging-based 3D invasion culture may be useful for the analysis of complex therapy-response patterns in cancer cells in drug discovery and invasion-associated resistance development. Supplementary Information: The online version contains supplementary material available at 10.1007/s44164-022-00040-x.

Genes Involved in Energy Metabolism Are Differentially Expressed During the Day-Night Cycle in Murine Retinal Pigment Epithelium.

Purpose: The functional interaction between photoreceptors and retinal pigment epithelium (RPE) cells is essential for vision. Phagocytosis of photoreceptor outer segments (POSs) by the RPE follows a circadian pattern; however, it remains unknown whether other RPE processes follow a daily rhythm. Therefore, our aim was to identify RPE processes following a daily rhythm. Methods: Murine RPE was isolated at Zeitgeber time (ZT) 0, 2, 4, 9, 14, and 19 (n = 5 per time point), after which RNA was isolated and sequenced. Genes with a significant difference in expression between time points (P < 0.05) were subjected to EnrichR pathway analysis to identify daily rhythmic processes. Results: Pathway enrichment revealed 13 significantly enriched KEGG pathways (P < 0.01), including the metabolic pathway (P = 0.002821). Analysis of the metabolic pathway differentially expressed genes revealed that genes involved in adenosine triphosphate production, glycolysis, glycogenolysis, and glycerophospholipid were low at ZT0 (light onset) and high at ZT19 (night). Genes involved in fatty acid degradation and cholesterol synthesis were high at light onset and low at night. Conclusions: Our transcriptome data suggest that the highest energy demand of RPE cells is at night, whereas POS phagocytosis and degradation take place in the morning. Furthermore, we identified genes involved in fatty acid and glycerophospholipid synthesis that are upregulated at night, possibly playing a role in generating building blocks for membrane synthesis.

Differential day-night expression of tight junction components in murine retinal pigment epithelium.

Strong communication and interaction between the retinal pigment epithelium (RPE) and the photoreceptor (PR) cells is essential for vision. RPE cells are essential for supporting and maintaining PR cells by transporting nutrients, waste products and ions, and phagocytosing photoreceptor outer segments (POS). POS phagocytosis follows a circadian pattern, taking place in the morning in human, mice and other organisms. However, it remains unknown whether other RPE processes follow a daily rhythm. To study the daily rhythm of RPE cells, we isolated murine RPE cells at six different time points during a 24 h period, after which RNA was isolated and sequenced. Murine RPE flatmounts were isolated at four different time points to study daily rhythm in protein abundance and localisation. EnrichR pathway analysis resulted in 13 significantly-enriched KEGG pathways (p < 0.01) of which seven showed a large number of overlapping genes. Several genes were involved in intracellular trafficking, possibly playing a role in nutrient transport, POS phagocytosis or membrane protein trafficking, with different expression patterns during the day-night cycle. Other genes were involved in actin cytoskeleton building, remodelling and crosslinking and showed a high expression in the morning, suggesting actin cytoskeleton remodelling at this time point. Finally, tight junction proteins Cldn2 and Cldn4 showed a difference in RNA and protein expression and tight junction localisation over time. Our study suggests that several important processes in the RPE follow a day-night rhythm, including intracellular trafficking, and processes involving the actin cytoskeleton and tight junctions. The differential protein localisation of Cldn2 in the RPE during the day-night cycle suggest that Cldn2 may facilitate paracellular water and sodium transport during the day.

Treatment with Statins Does Not Revert Trained Immunity in Patients with Familial Hypercholesterolemia.

Individuals with elevated LDL-cholesterol levels have an increased risk for cardiovascular disease. Despite lipid lowering strategies, however, a significant cardiovascular risk remains. Bekkering et al. show that monocytes from patients with familial hypercholesterolemia have a trained immunity phenotype and that lipid lowering with statins does not revert this pro-inflammatory phenotype.

The Itaconate Pathway Is a Central Regulatory Node Linking Innate Immune Tolerance and Trained Immunity.

Sepsis involves simultaneous hyperactivation of the immune system and immune paralysis, leading to both organ dysfunction and increased susceptibility to secondary infections. Acute activation of myeloid cells induced itaconate synthesis, which subsequently mediated innate immune tolerance in human monocytes. In contrast, induction of trained immunity by ß-glucan counteracted tolerance induced in a model of human endotoxemia by inhibiting the expression of immune-responsive gene 1 (IRG1), the enzyme that controls itaconate synthesis. ß-Glucan also increased the expression of succinate dehydrogenase (SDH), contributing to the integrity of the TCA cycle and leading to an enhanced innate immune response after secondary stimulation. The role of itaconate was further validated by IRG1 and SDH polymorphisms that modulate induction of tolerance and trained immunity in human monocytes. These data demonstrate the importance of the IRG1-itaconate-SDH axis in the development of immune tolerance and training and highlight the potential of ß-glucan-induced trained immunity to revert immunoparalysis.

A new mouse model of ARX dup24 recapitulates the patients' behavioral and fine motor alterations.

The aristaless-related homeobox (ARX) transcription factor is involved in the development of GABAergic and cholinergic neurons in the forebrain. ARX mutations have been associated with a wide spectrum of neurodevelopmental disorders in humans, among which the most frequent, a 24 bp duplication in the polyalanine tract 2 (c.428_451dup24), gives rise to intellectual disability, fine motor defects with or without epilepsy. To understand the functional consequences of this mutation, we generated a partially humanized mouse model carrying the c.428_451dup24 duplication (Arxdup24/0) that we characterized at the behavior, neurological and molecular level. Arxdup24/0 males presented with hyperactivity, enhanced stereotypies and altered contextual fear memory. In addition, Arxdup24/0 males had fine motor defects with alteration of reaching and grasping abilities. Transcriptome analysis of Arxdup24/0 forebrains at E15.5 showed a down-regulation of genes specific to interneurons and an up-regulation of genes normally not expressed in this cell type, suggesting abnormal interneuron development. Accordingly, interneuron migration was altered in the cortex and striatum between E15.5 and P0 with consequences in adults, illustrated by the defect in the inhibitory/excitatory balance in Arxdup24/0 basolateral amygdala. Altogether, we showed that the c.428_451dup24 mutation disrupts Arx function with a direct consequence on interneuron development, leading to hyperactivity and defects in precise motor movement control and associative memory. Interestingly, we highlighted striking similarities between the mouse phenotype and a cohort of 33 male patients with ARX c.428_451dup24, suggesting that this new mutant mouse line is a good model for understanding the pathophysiology and evaluation of treatment.

Metabolic Induction of Trained Immunity through the Mevalonate Pathway.

Innate immune cells can develop long-term memory after stimulation by microbial products during infections or vaccinations. Here, we report that metabolic signals can induce trained immunity. Pharmacological and genetic experiments reveal that activation of the cholesterol synthesis pathway, but not the synthesis of cholesterol itself, is essential for training of myeloid cells. Rather, the metabolite mevalonate is the mediator of training via activation of IGF1-R and mTOR and subsequent histone modifications in inflammatory pathways. Statins, which block mevalonate generation, prevent trained immunity induction. Furthermore, monocytes of patients with hyper immunoglobulin D syndrome (HIDS), who are mevalonate kinase deficient and accumulate mevalonate, have a constitutive trained immunity phenotype at both immunological and epigenetic levels, which could explain the attacks of sterile inflammation that these patients experience. Unraveling the role of mevalonate in trained immunity contributes to our understanding of the pathophysiology of HIDS and identifies novel therapeutic targets for clinical conditions with excessive activation of trained immunity.

Predicting stimulation-dependent enhancer-promoter interactions from ChIP-Seq time course data.

We have developed a machine learning approach to predict stimulation-dependent enhancer-promoter interactions using evidence from changes in genomic protein occupancy over time. The occupancy of estrogen receptor alpha (ERα), RNA polymerase (Pol II) and histone marks H2AZ and H3K4me3 were measured over time using ChIP-Seq experiments in MCF7 cells stimulated with estrogen. A Bayesian classifier was developed which uses the correlation of temporal binding patterns at enhancers and promoters and genomic proximity as features to predict interactions. This method was trained using experimentally determined interactions from the same system and was shown to achieve much higher precision than predictions based on the genomic proximity of nearest ERα binding. We use the method to identify a genome-wide confident set of ERα target genes and their regulatory enhancers genome-wide. Validation with publicly available GRO-Seq data demonstrates that our predicted targets are much more likely to show early nascent transcription than predictions based on genomic ERα binding proximity alone.

Mouse models of 17q21.31 microdeletion and microduplication syndromes highlight the importance of Kansl1 for cognition.

Koolen-de Vries syndrome (KdVS) is a multi-system disorder characterized by intellectual disability, friendly behavior, and congenital malformations. The syndrome is caused either by microdeletions in the 17q21.31 chromosomal region or by variants in the KANSL1 gene. The reciprocal 17q21.31 microduplication syndrome is associated with psychomotor delay, and reduced social interaction. To investigate the pathophysiology of 17q21.31 microdeletion and microduplication syndromes, we generated three mouse models: 1) the deletion (Del/+); or 2) the reciprocal duplication (Dup/+) of the 17q21.31 syntenic region; and 3) a heterozygous Kansl1 (Kans1+/-) model. We found altered weight, general activity, social behaviors, object recognition, and fear conditioning memory associated with craniofacial and brain structural changes observed in both Del/+ and Dup/+ animals. By investigating hippocampus function, we showed synaptic transmission defects in Del/+ and Dup/+ mice. Mutant mice with a heterozygous loss-of-function mutation in Kansl1 displayed similar behavioral and anatomical phenotypes compared to Del/+ mice with the exception of sociability phenotypes. Genes controlling chromatin organization, synaptic transmission and neurogenesis were upregulated in the hippocampus of Del/+ and Kansl1+/- animals. Our results demonstrate the implication of KANSL1 in the manifestation of KdVS phenotypes and extend substantially our knowledge about biological processes affected by these mutations. Clear differences in social behavior and gene expression profiles between Del/+ and Kansl1+/- mice suggested potential roles of other genes affected by the 17q21.31 deletion. Together, these novel mouse models provide new genetic tools valuable for the development of therapeutic approaches.

Somatic mutations reveal asymmetric cellular dynamics in the early human embryo.

Somatic cells acquire mutations throughout the course of an individual's life. Mutations occurring early in embryogenesis are often present in a substantial proportion of, but not all, cells in postnatal humans and thus have particular characteristics and effects. Depending on their location in the genome and the proportion of cells they are present in, these mosaic mutations can cause a wide range of genetic disease syndromes and predispose carriers to cancer. They have a high chance of being transmitted to offspring as de novo germline mutations and, in principle, can provide insights into early human embryonic cell lineages and their contributions to adult tissues. Although it is known that gross chromosomal abnormalities are remarkably common in early human embryos, our understanding of early embryonic somatic mutations is very limited. Here we use whole-genome sequences of normal blood from 241 adults to identify 163 early embryonic mutations. We estimate that approximately three base substitution mutations occur per cell per cell-doubling event in early human embryogenesis and these are mainly attributable to two known mutational signatures. We used the mutations to reconstruct developmental lineages of adult cells and demonstrate that the two daughter cells of many early embryonic cell-doubling events contribute asymmetrically to adult blood at an approximately 2:1 ratio. This study therefore provides insights into the mutation rates, mutational processes and developmental outcomes of cell dynamics that operate during early human embryogenesis.

Histone Methylation by the Kleefstra Syndrome Protein EHMT1 Mediates Homeostatic Synaptic Scaling.

Homeostatic plasticity, a form of synaptic plasticity, maintains the fine balance between overall excitation and inhibition in developing and mature neuronal networks. Although the synaptic mechanisms of homeostatic plasticity are well characterized, the associated transcriptional program remains poorly understood. We show that the Kleefstra-syndrome-associated protein EHMT1 plays a critical and cell-autonomous role in synaptic scaling by responding to attenuated neuronal firing or sensory drive. Chronic activity deprivation increased the amount of neuronal dimethylated H3 at lysine 9 (H3K9me2), the catalytic product of EHMT1 and an epigenetic marker for gene repression. Genetic knockdown and pharmacological blockade of EHMT1 or EHMT2 prevented the increase of H3K9me2 and synaptic scaling up. Furthermore, BDNF repression was preceded by EHMT1/2-mediated H3K9me2 deposition at the Bdnf promoter during synaptic scaling up, both in vitro and in vivo. Our findings suggest that H3K9me2-mediated changes in chromatin structure govern a repressive program that controls synaptic scaling.

Conditional depletion of intellectual disability and Parkinsonism candidate gene ATP6AP2 in fly and mouse induces cognitive impairment and neurodegeneration.

ATP6AP2, an essential accessory component of the vacuolar H+ ATPase (V-ATPase), has been associated with intellectual disability (ID) and Parkinsonism. ATP6AP2 has been implicated in several signalling pathways; however, little is known regarding its role in the nervous system. To decipher its function in behaviour and cognition, we generated and characterized conditional knockdowns of ATP6AP2 in the nervous system of Drosophila and mouse models. In Drosophila, ATP6AP2 knockdown induced defective phototaxis and vacuolated photoreceptor neurons and pigment cells when depleted in eyes and altered short- and long-term memory when depleted in the mushroom body. In mouse, conditional Atp6ap2 deletion in glutamatergic neurons (Atp6ap2(Camk2aCre/0) mice) caused increased spontaneous locomotor activity and altered fear memory. Both Drosophila ATP6AP2 knockdown and Atp6ap2(Camk2aCre/0) mice presented with presynaptic transmission defects, and with an abnormal number and morphology of synapses. In addition, Atp6ap2(Camk2aCre/0) mice showed autophagy defects that led to axonal and neuronal degeneration in the cortex and hippocampus. Surprisingly, axon myelination was affected in our mutant mice, and axonal transport alterations were observed in Drosophila. In accordance with the identified phenotypes across species, genome-wide transcriptome profiling of Atp6ap2(Camk2aCre/0) mouse hippocampi revealed dysregulation of genes involved in myelination, action potential, membrane-bound vesicles and motor behaviour. In summary, ATP6AP2 disruption in mouse and fly leads to cognitive impairment and neurodegeneration, mimicking aspects of the neuropathology associated with ATP6AP2 mutations in humans. Our results identify ATP6AP2 as an essential gene for the nervous system.

Epigenome data release: a participant-centered approach to privacy protection.

Large-scale epigenome mapping by the NIH Roadmap Epigenomics Project, the ENCODE Consortium and the International Human Epigenome Consortium (IHEC) produces genome-wide DNA methylation data at one base-pair resolution. We examine how such data can be made open-access while balancing appropriate interpretation and genomic privacy. We propose guidelines for data release that both reduce ambiguity in the interpretation of open-access data and limit immediate access to genetic variation data that are made available through controlled access.

Stable methylation at promoters distinguishes epiblast stem cells from embryonic stem cells and the in vivo epiblasts.

Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representatives of naïve and primed pluripotency, respectively. It is currently unclear how their epigenomes underpin the phenotypic and molecular characteristics of these distinct pluripotent states. Here, we performed a genome-wide comparison of DNA methylation between ESCs and EpiSCs by MethylCap-Seq. We observe that promoters are preferential targets for methylation in EpiSC compared to ESCs, in particular high CpG island promoters. This is in line with upregulation of the de novo methyltransferases Dnmt3a1 and Dnmt3b in EpiSC, and downregulation of the demethylases Tet1 and Tet2. Remarkably, the observed DNA methylation signature is specific to EpiSCs and differs from that of their in vivo counterpart, the postimplantation epiblast. Using a subset of promoters that are differentially methylated, we show that DNA methylation is established within a few days during in vitro outgrowth of the epiblast, and also occurs when ESCs are converted to EpiSCs in vitro. Once established, this methylation is stable, as ES-like cells obtained by in vitro reversion of EpiSCs display an epigenetic memory that only extensive passaging and sub-cloning are able to almost completely erase.

The PinkThing for analysing ChIP profiling data in their genomic context.

BACKGROUND: Current epigenetic research makes frequent use of whole-genome ChIP profiling for determining the in vivo binding of proteins, e.g. transcription factors and histones, to DNA. Two important and recurrent questions for these large scale analyses are: 1) What is the genomic distribution of a set of binding sites? and 2) Does this genomic distribution differ significantly from another set of sites? FINDINGS: We exemplify the functionality of the PinkThing by analysing a ChIP profiling dataset of cohesin binding sites. We show the subset of cohesin sites with no CTCF binding have a characteristic genomic distribution different from the set of all cohesin sites. CONCLUSIONS: The PinkThing is a web application for fast and easy analysis of the context of genomic loci, such as peaks from ChIP profiling experiments. The output of the PinkThing analysis includes: categorisation of position relative to genes (intronic, exonic, 5' near, 3' near 5' far, 3' far and distant), distance to the closest annotated 3' and 5' end of genes, direction of transcription of the nearest gene, and the option to include other genomic elements like ESTs and CpG islands. The PinkThing enables easy statistical comparison between experiments, i.e. experimental versus background sets, reporting over- and underrepresentation as well as p-values for all comparisons. Access and use of the PinkThing is free and open (without registration) to all users via the website: http://pinkthing.cmbi.ru.nl