ATAK receptors, harnessing innate immunity to program myeloid cells to kill cancer

Cytotherapy

2021 May 01

Wang, Y;Diwanji, N;Nicholson, T;Mukherjee, S;Getts, D;
| DOI: 10.1016/S1465324921004217

Background & Aim: T cells therapies have revolutionized cancer treatment for many patients. However, for the majority of patients with advanced solid tumors, sustained clinical benefit has not been achieved. Unlike T cells, myeloid cells readily accumulate in tumors, in some cases contributing up to 50% of the tumor mass. More recently, the potential for engaging innate immune signaling sensors such as Toll-like receptors and STING-cGAS have been investigated as important pathways to drastically upregulate pro-inflammatory anti-tumor immune response and are associated with anti-tumor immunity. These approaches, using synthetic agonists to activate these pathways, can be potent but delivering a localized and tumor specific activation of innate immune signaling is difficult to achieve. Methods, Results & Conclusion: Here we designed and engineered a new class of chimeric antigen receptors that couple tumor recognition with innate immune signaling, referred to as Activate, Target, Attack & Kill (ATAK™) receptors. By combining cancer recognition domains with intracellular signaling domains from innate immune receptors such as Fcg, TLR and cytokine receptors, we show that myeloid cells can be programmed to recognize cancer and elicit a broad and tunable immune response. Our data show the versatility of building ATAK receptors by harnessing innate immune pathways and support their clinical development in cell and direct in vivo therapies.

Spatial Transcriptomics: Molecular Maps of the Mammalian Brain

Annual review of neuroscience

2021 Apr 29

Ortiz, C;Carlén, M;Meletis, K;
PMID: 33914592 | DOI: 10.1146/annurev-neuro-100520-082639

Maps of the nervous system inspire experiments and theories in neuroscience. Advances in molecular biology over the past decades have revolutionized the definition of cell and tissue identity. Spatial transcriptomics has opened up a new era in neuroanatomy, where the unsupervised and unbiased exploration of the molecular signatures of tissue organization will give rise to a new generation of brain maps. We propose that the molecular classification of brain regions on the basis of their gene expression profile can circumvent subjective neuroanatomical definitions and produce common reference frameworks that can incorporate cell types, connectivity, activity, and other modalities. Here we review the technological and conceptual advances made possible by spatial transcriptomics in the context of advancing neuroanatomy and discuss how molecular neuroanatomy can redefine mapping of the nervous system. Expected final online publication date for the Annual Review of Neuroscience, Volume 44 is July 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Pro-Epicardial Cells are Heterogeneous with Specified Smooth Muscle-Like and Pacemaker Progenitor Cells

SSRN Electronic Journal

2021 Apr 29

Miao, L;Li, J;Lu, Y;Yin, C;Sun, D;Lo, E;Song, R;Cai, C;Huang, W;Long, X;McConnell, B;Fan, Z;Singer, H;Schwartz, R;Munshi, N;Wu, M;
| DOI: 10.2139/ssrn.3832987

The heterogeneity and specification of pro-epicardial cells (pro-EpiCs) to fibroblast and smooth muscle cell (SMC) are unknown. We applied single-cell RNA sequencing (scRNA-seq) paired with RNAScope, bioinformatics, and lineage tracing in an unbiased manner to identify the previously uncharacterized molecular heterogeneity of the pro-EpiCs isolated from pro-epicardium (PE). We found that pro-EpiCs labeled by _Tbx18 Cre/+ _ are heterogeneous, with three clusters displaying differential expression profiles and distinct spatial locations. Cluster 1 are mesothelial cells, and Cluster 2 express SMC markers. Surprisingly, Cluster 3 express _Isl1_ and markers of pacemaker progenitor cells (PMPC) but not marker of atrial cardiomyocytes. Our studies conclude that pro-EpiCs are heterogeneous and SMC-like cells are specified in the PE, while fibroblasts are not specified in PE but epicardium. We identified a region in PE that contains PMPCs, which translocate through the inflow tract to the sinoatrial node. The expression profile of Cluster 3 cells unifies previous studies regarding the origins and markers of PMPCs.

A Method to Pre-Screen Rat Mammary Gland Whole-Mounts Prior To RNAscope

Journal of mammary gland biology and neoplasia

2021 Apr 17

Duderstadt, EL;Sanders, MA;Samuelson, DJ;
PMID: 33866475 | DOI: 10.1007/s10911-021-09484-5

RNAscope is a quantitative in situ gene expression measurement technique that preserves the spatial aspect of intact tissue; thus, allowing for comparison of specific cell populations and morphologies. Reliable and accurate measurement of gene expression in tissue is dependent on preserving RNA integrity and the quantitative nature of RNAscope. The purpose of this study was to determine if the quantitative nature of RNAscope was retained following processing and carmine staining of mammary gland whole-mounts, which are commonly used to identify lesions, such as hyperplasia and ductal carcinoma in situ (DCIS). We were concerned that handling and procedures required to visualize microscopic disease lesions might compromise RNA integrity and the robustness of RNAscope. No effect on the quantitative abilities of RNAscope was detected when mammary gland whole-mounts were pre-screened for lesions of interest prior to RNAscope. This was determined in comparison to tissue that had been formalin-fixed and paraffin embedded (FFPE) immediately after collection. The ability to pre-screen whole-mounts allowed unpalpable diseased lesions to be identified without labor-intensive serial sectioning of tissue samples to find diseased tissue. This method is applicable to evaluate mammary gland whole-mounts during normal mammary gland development, function, and disease progression.

Coordination of escape and spatial navigation circuits orchestrates versatile flight from threats

Neuron

2021 Apr 13

Wang, W;Schuette, PJ;Nagai, J;Tobias, BC;Cuccovia V Reis, FM;Ji, S;de Lima, MAX;La-Vu, MQ;Maesta-Pereira, S;Chakerian, M;Leonard, SJ;Lin, L;Severino, AL;Cahill, CM;Canteras, NS;Khakh, BS;Kao, JC;Adhikari, A;
PMID: 33861942 | DOI: 10.1016/j.neuron.2021.03.033

Naturalistic escape requires versatile context-specific flight with rapid evaluation of local geometry to identify and use efficient escape routes. It is unknown how spatial navigation and escape circuits are recruited to produce context-specific flight. Using mice, we show that activity in cholecystokinin-expressing hypothalamic dorsal premammillary nucleus (PMd-cck) cells is sufficient and necessary for context-specific escape that adapts to each environment's layout. In contrast, numerous other nuclei implicated in flight only induced stereotyped panic-related escape. We reasoned the dorsal premammillary nucleus (PMd) can induce context-specific escape because it projects to escape and spatial navigation nuclei. Indeed, activity in PMd-cck projections to thalamic spatial navigation circuits is necessary for context-specific escape induced by moderate threats but not panic-related stereotyped escape caused by perceived asphyxiation. Conversely, the PMd projection to the escape-inducing dorsal periaqueductal gray projection is necessary for all tested escapes. Thus, PMd-cck cells control versatile flight, engaging spatial navigation and escape circuits.

Excitatory cholecystokinin neurons of the midbrain integrate diverse temporal responses and drive auditory thalamic subdomains

Proceedings of the National Academy of Sciences of the United States of America

2021 Mar 09

Kreeger, LJ;Connelly, CJ;Mehta, P;Zemelman, BV;Golding, NL;
PMID: 33658359 | DOI: 10.1073/pnas.2007724118

The central nucleus of the inferior colliculus (ICC) integrates information about different features of sound and then distributes this information to thalamocortical circuits. However, the lack of clear definitions of circuit elements in the ICC has limited our understanding of the nature of these circuit transformations. Here, we combine virus-based genetic access with electrophysiological and optogenetic approaches to identify a large family of excitatory, cholecystokinin-expressing thalamic projection neurons in the ICC of the Mongolian gerbil. We show that these neurons form a distinct cell type, displaying uniform morphology and intrinsic firing features, and provide powerful, spatially restricted excitation exclusively to the ventral auditory thalamus. In vivo, these neurons consistently exhibit V-shaped receptive field properties but strikingly diverse temporal responses to sound. Our results indicate that temporal response diversity is maintained within this population of otherwise uniform cells in the ICC and then relayed to cortex through spatially restricted thalamic subdomains.

Multiple sclerosis risk gene Mertk is required for microglial activation and subsequent remyelination

Cell reports

2021 Mar 09

Shen, K;Reichelt, M;Kyauk, RV;Ngu, H;Shen, YA;Foreman, O;Modrusan, Z;Friedman, BA;Sheng, M;Yuen, TJ;
PMID: 33691116 | DOI: 10.1016/j.celrep.2021.108835

In multiple sclerosis (MS) and other neurological diseases, the failure to repair demyelinated lesions contributes to axonal damage and clinical disability. Here, we provide evidence that Mertk, a gene highly expressed by microglia that alters MS risk, is required for efficient remyelination. Compared to wild-type (WT) mice, Mertk-knockout (KO) mice show impaired clearance of myelin debris and remyelination following demyelination. Using single-cell RNA sequencing, we characterize Mertk-influenced responses to cuprizone-mediated demyelination and remyelination across different cell types. Mertk-KO brains show an attenuated microglial response to demyelination but an elevated proportion of interferon (IFN)-responsive microglia. In addition, we identify a transcriptionally distinct subtype of surviving oligodendrocytes specific to demyelinated lesions. The inhibitory effect of myelin debris on remyelination is mediated in part by IFNγ, which further impedes microglial clearance of myelin debris and inhibits oligodendrocyte differentiation. Together, our work establishes a role for Mertk in microglia activation, phagocytosis, and migration during remyelination.

Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution

Cell reports

2021 Mar 09

Busslinger, GA;Weusten, BLA;Bogte, A;Begthel, H;Brosens, LAA;Clevers, H;
PMID: 33691112 | DOI: 10.1016/j.celrep.2021.108819

The upper gastrointestinal tract, consisting of the esophagus, stomach, and duodenum, controls food transport, digestion, nutrient uptake, and hormone production. By single-cell analysis of healthy epithelia of these human organs, we molecularly define their distinct cell types. We identify a quiescent COL17A1high KRT15high stem/progenitor cell population in the most basal cell layer of the esophagus and detect substantial gene expression differences between identical cell types of the human and mouse stomach. Selective expression of BEST4, CFTR, guanylin, and uroguanylin identifies a rare duodenal cell type, referred to as BCHE cell, which likely mediates high-volume fluid secretion because of continual activation of the CFTR channel by guanylin/uroguanylin-mediated autocrine signaling. Serotonin-producing enterochromaffin cells in the antral stomach significantly differ in gene expression from duodenal enterochromaffin cells. We, furthermore, discover that the histamine-producing enterochromaffin-like cells in the oxyntic stomach express the luteinizing hormone, yet another member of the enteroendocrine hormone family.

Chemokine CCL5 promotes robust optic nerve regeneration and mediates many of the effects of CNTF gene therapy

Proceedings of the National Academy of Sciences of the United States of America

2021 Mar 02

Xie, L;Yin, Y;Benowitz, L;
PMID: 33627402 | DOI: 10.1073/pnas.2017282118

Ciliary neurotrophic factor (CNTF) is a leading therapeutic candidate for several ocular diseases and induces optic nerve regeneration in animal models. Paradoxically, however, although CNTF gene therapy promotes extensive regeneration, recombinant CNTF (rCNTF) has little effect. Because intraocular viral vectors induce inflammation, and because CNTF is an immune modulator, we investigated whether CNTF gene therapy acts indirectly through other immune mediators. The beneficial effects of CNTF gene therapy remained unchanged after deleting CNTF receptor alpha (CNTFRα) in retinal ganglion cells (RGCs), the projection neurons of the retina, but were diminished by depleting neutrophils or by genetically suppressing monocyte infiltration. CNTF gene therapy increased expression of C-C motif chemokine ligand 5 (CCL5) in immune cells and retinal glia, and recombinant CCL5 induced extensive axon regeneration. Conversely, CRISPR-mediated knockdown of the cognate receptor (CCR5) in RGCs or treating wild-type mice with a CCR5 antagonist repressed the effects of CNTF gene therapy. Thus, CCL5 is a previously unrecognized, potent activator of optic nerve regeneration and mediates many of the effects of CNTF gene therapy.

Targeted therapies for extrahepatic cholangiocarcinoma: preclinical and clinical development and prospects for the clinic

Expert opinion on investigational drugs

2021 Feb 23

Cadamuro, M;Lasagni, A;Lamarca, A;Fouassier, L;Guido, M;Sarcognato, S;Gringeri, E;Cillo, U;Strazzabosco, M;Marin, JJ;Banales, JM;Fabris, L;
PMID: 33622120 | DOI: 10.1080/13543784.2021.1880564

Introduction: Until recently, cholangiocarcinoma (CCA) was a largely overlooked disease, and among CCAs, extrahepatic CCA (eCCA) was even more neglected. Despite the growing impact of molecularly targeted therapies and immunotherapy, prognosis of eCCA is dismal. Therefore, unraveling the complex molecular landscape of eCCA has become an urgent need. Deep phenotyping studies have revealed that eCCA is a heterogeneous tumor, harboring specific alterations categorizable into four classes, 'Mesenchymal', 'Proliferation', 'Immune', 'Metabolic'. Molecular alterations convey the activation of several pro-oncogenic pathways, where either actionable drivers or outcome predictors can be identified.Areas covered: We offer insights on perturbed pathways, molecular profiling, and actionable targets in eCCA and present a perspective on the potential stepping-stones to future progress. A systematic literature search in PubMed/ClinicalTrials.gov websites was performed by authors from different disciplines according to their specific topic knowledge to identify the newest and most relevant advances in precision medicine of eCCA.Expert opinion: eCCA is a distinct entity with unique features in terms of molecular classes, oncogenic drivers, and tumor microenvironment. Since more prevalent mutations are currently undruggable, and immunotherapy can be offered only to a minority of patients, international collaborations are instrumental to improve the understanding of the molecular underpins of this disease.

The pulmonary pathology of COVID-19

Virchows Archiv : an international journal of pathology

2021 Feb 19

Bösmüller, H;Matter, M;Fend, F;Tzankov, A;
PMID: 33604758 | DOI: 10.1007/s00428-021-03053-1

The lung is the main affected organ in severe coronavirus disease 2019 (COVID-19) caused by the novel coronavirus SARS-CoV-2, and lung damage is the leading cause of death in the vast majority of patients. Mainly based on results obtained by autopsies, the seminal features of fatal COVID-19 have been described by many groups worldwide. Early changes encompass edema, epithelial damage, and capillaritis/endothelialitis, frequently combined with microthrombosis. Subsequently, patients with manifest respiratory insufficiency exhibit exudative diffuse alveolar damage (DAD) with hyaline membrane formation and pneumocyte type 2 hyperplasia, variably complicated by superinfection, which may progress to organizing/fibrotic stage DAD. These features, however, are not specific for COVID-19 and can be found in other disorders including viral infections. Clinically, the early disease stage of severe COVID-19 is characterized by high viral load, lymphopenia, massive secretion of pro-inflammatory cytokines and hypercoagulability, documented by elevated D-dimers and an increased frequency of thrombotic and thromboembolic events, whereas virus loads and cytokine levels tend to decrease in late disease stages, when tissue repair including angiogenesis prevails. The present review describes the spectrum of lung pathology based on the current literature and the authors' personal experience derived from clinical autopsies, and tries to summarize our current understanding and open questions of the pathophysiology of severe pulmonary COVID-19.

Single-cell transcriptomic analysis of the adult mouse spinal cord reveals molecular diversity of autonomic and skeletal motor neurons

Nature neuroscience

2021 Feb 15

Blum, JA;Klemm, S;Shadrach, JL;Guttenplan, KA;Nakayama, L;Kathiria, A;Hoang, PT;Gautier, O;Kaltschmidt, JA;Greenleaf, WJ;Gitler, AD;
PMID: 33589834 | DOI: 10.1038/s41593-020-00795-0

The spinal cord is a fascinating structure that is responsible for coordinating movement in vertebrates. Spinal motor neurons control muscle activity by transmitting signals from the spinal cord to diverse peripheral targets. In this study, we profiled 43,890 single-nucleus transcriptomes from the adult mouse spinal cord using fluorescence-activated nuclei sorting to enrich for motor neuron nuclei. We identified 16 sympathetic motor neuron clusters, which are distinguishable by spatial localization and expression of neuromodulatory signaling genes. We found surprising skeletal motor neuron heterogeneity in the adult spinal cord, including transcriptional differences that correlate with electrophysiologically and spatially distinct motor pools. We also provide evidence for a novel transcriptional subpopulation of skeletal motor neuron (γ*). Collectively, these data provide a single-cell transcriptional atlas ( http://spinalcordatlas.org ) for investigating the organizing molecular logic of adult motor neuron diversity, as well as the cellular and molecular basis of motor neuron function in health and disease.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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