NK cells require immune checkpoint receptor LILRB4/gp49B to control neurotropic Zika virus infections in mice
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59 BASIC BIOLOGICAL SCIENCES↗
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Results for “T cell receptor”
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Lectin Receptor-Like Kinases: The Sensor and Mediator at the Plant Cell Surface
Lectin receptor-like kinases (LecRLKs), a plant-specific receptor-like kinase (RLK) sub-family, have been recently found to play crucial roles in plant development and responses to abiotic and biotic stresses. In this review, we first describe the classification and structures of Lectin RLKs. Then we focus on the analysis of functions of LecRLKs in various biological processes and discuss the status of LecRLKs from the ligands they recognize, substrate they target, signaling pathways they are involved in, to the overall regulation of growth-defense tradeoffs. LecRLKs and the signaling components they interact with constitute recognition and protection systems at the plant cell surface contributing to the detection of environmental changes monitoring plant fitness.
59 BASIC BIOLOGICAL SCIENCES↗
Clinical Value of Upfront Cranial Radiation Therapy in Osimertinib-Treated Epidermal Growth Factor Receptor–Mutant Non-Small Cell Lung Cancer With Brain Metastases
As a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), osimertinib has a powerful ability to penetrate the blood-brain barrier and a high potency for controlling brain metastases (BMs) from EGFR-mutant non-small cell lung cancer (NSCLC). The clinical value of cranial radiation therapy in osimertinib-treated NSCLC with BMs remains largely unknown.
62 RADIOLOGY AND NUCLEAR MEDICINE↗
MIP-4 is Induced by Bleomycin and Stimulates Cell Migration Partially via Nir-1 Receptor
Background. CC-chemokine ligand 18 also known as MIP-4 is a chemokine with roles in inflammation and immune responses. It has been shown that MIP-4 is involved in the development of several diseases including lung fibrosis and cancer. How exactly MIP-4 is regulated and exerts its role in lung fibrosis remains unclear. Therefore, in the present study, we examined how MIP-4 is regulated and whether it acts via its potential receptor Nir-1. Materials and Methods. A549 cells were grown and maintained in DMEM : F12 (1 : 1) and supplemented with 10% FBS and 1000 U of penicillin/streptomycin and maintained as recommended by the manufacturer (ATCC). Cell migration and invasion, immunohistochemistry (IHC), Western blot, qPCR, and siRNA Nir-1 were used to determine MIP-4 regulation and its role in cell migration. Results. Cell migration was increased following stimulation of cells with recombinant (r) MIP-4 and bleomycin (BLM), whereas quenching rMIP-4 with its antibody (Ab) or addition of the Ab to BLM or H 2 O 2 diminished rMIP-4-induced cell migration. Along with cell migration, rMIP-4, BLM, and H 2 O 2 induced the formation of actin filaments dynamic structures whereas costimulation with MIP-4 Ab limited BLM- and H 2 O 2 -induced effects. MIP-4 mRNA and protein were increased by BLM and H 2 O 2 , and the addition of its Ab significantly reduced treatments effect. Experiments with siRNA investigating whether Nir-1 is a potential MIR-4 receptor indicated that the inhibition of Nir-1 decreased cell migration/invasion but did not totally inhibit rMIP-4-induced cell migration. Conclusion. Therefore, our data indicate that MIP-4 is regulated by BLM and H 2 O 2 and costimulation with its Ab limits the effects on MIP-4 and that the Nir-1 receptor partially mediates MIP-4’s effects on increased cell migration. These data also evidenced that MIP-4 is regulated by fibrotic and oxidative stimuli and that quenching MIP-4 with its Ab or therapeutically targeting the Nir-1 receptor may partially limit MIP-4 effects under fibrotic or oxidative stimulation.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
In vivo human T cell engineering with enveloped delivery vehicles
Viruses and virally derived particles have the intrinsic capacity to deliver molecules to cells, but the difficulty of readily altering cell-type selectivity has hindered their use for therapeutic delivery. Here, we show that cell surface marker recognition by antibody fragments displayed on membrane-derived particles encapsulating CRISPR–Cas9 protein and guide RNA can deliver genome editing tools to specific cells. Compared to conventional vectors like adeno-associated virus that rely on evolved capsid tropisms to deliver virally encoded cargo, these Cas9-packaging enveloped delivery vehicles (Cas9-EDVs) leverage predictable antibody–antigen interactions to transiently deliver genome editing machinery selectively to cells of interest. Antibody-targeted Cas9-EDVs preferentially confer genome editing in cognate target cells over bystander cells in mixed populations, both ex vivo and in vivo. By using multiplexed targeting molecules to direct delivery to human T cells, Cas9-EDVs enable the generation of genome-edited chimeric antigen receptor T cells in humanized mice, establishing a programmable delivery modality with the potential for widespread therapeutic utility.
42 ENGINEERING↗
Ligand-induced transmembrane conformational coupling in monomeric $\mathrm{EGFR}$
Single pass cell surface receptors regulate cellular processes by transmitting ligand-encoded signals across the plasma membrane via changes to their extracellular and intracellular conformations. This transmembrane signaling is generally initiated by ligand binding to the receptors in their monomeric form. While subsequent receptor-receptor interactions are established as key aspects of transmembrane signaling, the contribution of monomeric receptors has been challenging to isolate due to the complexity and ligand-dependence of these interactions. By combining membrane nanodiscs produced with cell-free expression, single-molecule Förster Resonance Energy Transfer measurements, and molecular dynamics simulations, we report that ligand binding induces intracellular conformational changes within monomeric, full-length epidermal growth factor receptor (EGFR). Our observations establish the existence of extracellular/intracellular conformational coupling within a single receptor molecule. We implicate a series of electrostatic interactions in the conformational coupling and find the coupling is inhibited by targeted therapeutics and mutations that also inhibit phosphorylation in cells. Collectively, these results introduce a facile mechanism to link the extracellular and intracellular regions through the single transmembrane helix of monomeric EGFR, and raise the possibility that intramolecular transmembrane conformational changes upon ligand binding are common to single-pass membrane proteins.
59 BASIC BIOLOGICAL SCIENCES↗
Core cysteine residues in the Plasminogen-Apple-Nematode (PAN) domain are critical for HGF/c-MET signaling
The Plasminogen-Apple-Nematode (PAN) domain, with a core of four to six cysteine residues, is found in > 28,000 proteins across 959 genera. Still, its role in protein function is not fully understood. The PAN domain was initially characterized in numerous proteins, including HGF. Dysregulation of HGF-mediated signaling results in multiple deadly cancers. The binding of HGF to its cell surface receptor, c-MET, triggers all biological impacts. Here, we show that mutating four core cysteine residues in the HGF PAN domain reduces c-MET interaction, subsequent c-MET autophosphorylation, and phosphorylation of its downstream targets, perinuclear localization, cellular internalization of HGF, and its receptor, c-MET, and c-MET ubiquitination. Furthermore, transcriptional activation of HGF/c-MET signaling-related genes involved in cancer progression, invasion, metastasis, and cell survival were impaired. Thus, targeting the PAN domain of HGF may represent a mechanism for selectively regulating the binding and activation of the c-MET pathway.
59 BASIC BIOLOGICAL SCIENCES↗
Mitigation of chromosome loss in clinical CRISPR-Cas9-engineered T cells
CRISPR-Cas9 genome editing has enabled advanced T cell therapies, but occasional loss of the targeted chromosome remains a safety concern. To investigate whether Cas9-induced chromosome loss is a universal phenomenon and evaluate its clinical significance, we conducted a systematic analysis in primary human T cells. Arrayed and pooled CRISPR screens revealed that chromosome loss was generalizable across the genome and resulted in partial and entire loss of the targeted chromosome, including in preclinical chimeric antigen receptor T cells. T cells with chromosome loss persisted for weeks in culture, implying the potential to interfere with clinical use. A modified cell manufacturing process, employed in our first-in-human clinical trial of Cas9-engineered T cells (NCT03399448), reduced chromosome loss while largely preserving genome editing efficacy. Expression of p53 correlated with protection from chromosome loss observed in this protocol, suggesting both a mechanism and strategy for T cell engineering that mitigates this genotoxicity in the clinic.
59 BASIC BIOLOGICAL SCIENCES↗
IL-10 Signaling Elicited by Nivolumab-Induced Activation of the $\mathrm{MAP}$ Kinase Pathway Does Not Fully Contribute to Nivolumab-Modulated Heterogeneous T Cell Responses
Immune checkpoint inhibitor (ICI) therapy has revolutionized anti-cancer treatment for many late-stage cancer patients. However, ICI therapy has thus far demonstrated limited efficacy for most patients, and it remains unclear why this is so. Interleukin 10 (IL-10) is a cytokine that has been recognized as a central player in cancer biology with its ability to inhibit anti-tumor T cell responses. Recent studies suggest that IL-10 might also exert some intrinsic anti-tumor T cell responses, and clinical studies using recombinant IL-10 alone or in combination with ICI are underway. This paradoxical effect of IL-10 and its underlying mechanisms impacting ICI-modulated T cell responses remain poorly understood. In this study, using an in vitro mixed lymphocyte reaction assay, we found that treatment with ICIs such as the anti-programmed cell death receptor-1 (PD-1) mAb nivolumab elicits a strong expression of IL-10. While neutralization of IL-10 signaling with an anti-IL-10 specific mAb significantly decreases the production of IFN-γ by T cells in a cohort of donor cells, the opposite effect was observed in other donor cells. Similarly, neutralization of IL-10 signaling significantly decreases the expression of T cell activation markers Ki67 and CD25, as well as the production of Granzyme B in a cohort of donor cells, whereas the opposite effect was observed in others. Furthermore, we found that nivolumab and IL-10 differentially modulate the signal transducer and activator of transcription 3 (STAT3) and AKT serine–threonine kinase pathways. Finally, we found that nivolumab activates the mitogen-activated protein kinase (MAPK) pathway, which in turn is responsible for the observed induction of IL-10 production by nivolumab. These findings provide new insights into the mechanisms underlying anti-PD-1-modulated T cell responses by IL-10, which could lead to the discovery of novel combination treatments that target IL-10 and immune checkpoint molecules.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
The HHV-6B U20 glycoprotein binds ULBP1, masking it from recognition by NKG2D and interfering with natural killer cell activation
Human Herpesvirus 6B (HHV-6B) impedes host immune responses by downregulating class I MHC molecules (MHC-I), hindering antigen presentation to CD8+ T cells. Downregulation of MHC-I disengages inhibitory receptors on natural killer (NK) cells, resulting in activation and killing of the target cell if NK cell activating receptors such as NKG2D have engaged stress ligands upregulated on the target cells. Previous work has shown that HHV-6B downregulates three MHC-like stress ligands MICB, ULBP1, and ULBP3, which are recognized by NKG2D. The U20 glycoprotein of the related virus HHV-6A has been implicated in the downregulation of ULBP1, but the precise mechanism remains undetermined. We set out to investigate the role of HHV-6B U20 in modulating NK cell activity. We used HHV-6B U20 expressed as a recombinant protein or transduced into target cells, as well as HHV-6B infection, to investigate binding interactions with NK cell ligands and receptors and to assess effects on NK cell activation. Small-angle X-ray scattering was used to align molecular models derived from machine-learning approaches. We demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity. Transduction of U20 decreases NKG2D binding to ULBP1 at the cell surface but does not decrease ULBP1 protein levels, either at the cell surface or in toto. HHV-6B infection and soluble U20 have the same effect. Transduction of U20 blocks NK cell activation in response to cell-surface ULBP1. Structural modeling of the U20 – ULBP1 complex indicates some similarities to the m152-RAE1γ complex.
60 APPLIED LIFE SCIENCES↗
Preclinical characterization of Pan-NKG2D ligand-binding NKG2D receptor decoys
NKG2D and its ligands are critical regulators of protective immune responses controlling infections and cancer, defining a crucial immune signaling axis. Current therapeutic efforts targeting this axis almost exclusively aim at enhancing NKG2D-mediated effector functions. However, this axis can drive disease processes when dysregulated, in particular, driving stem-like cancer cell reprogramming and tumorigenesis through receptor/ligand self-stimulation on tumor cells. Despite complexities with its structure and biology, we developed multiple novel engineered proteins that functionally serve as axis-blocking NKG2D “decoys” and report biochemical, structural, in vitro , and in vivo evaluation of their functionality.
60 APPLIED LIFE SCIENCES↗
Endocytosis of the non-catalytic ADAM23: Recycling and long half-life properties
A Disintegrin And Metalloprotease 23 (ADAM23) is a member of the ADAMs family of transmembrane proteins, mostly expressed in nervous system, and involved in traffic and stabilization of Kv1-potassium channels, synaptic transmission, neurite outgrowth, neuronal morphology and cell adhesion. Also, ADAM23 has been linked to human pathological conditions, such as epilepsy, cancer metastasis and cardiomyopathy. ADAM23 functionality depends on the molecule presence at the cell surface and along the secretory pathway, as expected for a cell surface receptor. Because endocytosis is an important functional regulatory mechanism of plasma membrane receptors and no information is available about the traffic or turnover of non-catalytic ADAMs, we investigated ADAM23 internalization, recycling and half-life properties. Here, we show that ADAM23 undergoes constitutive internalization from the plasma membrane, a process that depends on lipid raft integrity, and is redistributed to intracellular vesicles, especially early and recycling endosomes. Furthermore, we observed that ADAM23 is recycled from intracellular compartments back to the plasma membrane and thus has longer half-life and higher cell surface stability compared with other ADAMs. Our findings suggest that regulation of ADAM23 endocytosis/stability could be exploited therapeutically in diseases in which ADAM23 is directly involved, such as epilepsy, cancer progression and cardiac hypertrophy.
60 APPLIED LIFE SCIENCES↗
Delineating the mechanism of anti-Lassa virus GPC-A neutralizing antibodies
Lassa virus (LASV) is the etiologic agent of Lassa Fever, a hemorrhagic disease that is endemic to West Africa. During LASV infection, LASV glycoprotein (GP) engages with multiple host receptors for cell entry. Neutralizing antibodies against GP are rare and principally target quaternary epitopes displayed only on the metastable, pre-fusion conformation of GP. Currently, the structural features of the neutralizing GPC-A antibody competition group are understudied. Structures of two GPC-A antibodies presented here demonstrate that they bind the side of the pre-fusion GP trimer, bridging the GP1 and GP2 subunits. Complementary biochemical analyses indicate that antibody 25.10C, which is broadly specific, neutralizes by inhibiting binding of the endosomal receptor LAMP1 and also by blocking membrane fusion. The other GPC-A antibody, 36.1F, which is lineage-specific, prevents LAMP1 association only. These data illuminate a site of vulnerability on LASV GP and will guide efforts to elicit broadly reactive therapeutics and vaccines.
59 BASIC BIOLOGICAL SCIENCES↗
Pulmonary ACE2 expression in neonatal and adult rats
Children show a distinct presentation of COVID‐19, characterized by a lower incidence and mild phenotype, but the reason for this is still unknown. The angiotensin‐converting enzyme 2 (ACE2) functions as the primary cell entry receptor for Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and is thought to cause distinct clinical features between children and old people. The primary purpose of this study was to determine whether differences exist in the level of expression and distribution of ACE2 between neonatal and adult rat lungs. The lung tissues from rats of various ages were used to investigate the expression patterns of ACE2. Western blot, immunohistochemistry, and immunofluorescence were used to quantify or identify the localization of ACE2 in rat lungs. ACE2 was homogenously expressed in fewer alveolar type II (AT2) cells in the neonatal lung, with no polarization to the alveolar space and additional expression in pulmonary endothelium when compared to adult rat lungs. These findings suggest that the patterns of ACE2 distribution and cellular localization in rat lungs change with age.
Zhao, Depeng↗
Crosstalk between cyclophilins and T lymphocytes in coronary artery disease
Highlights: • CD147 receptor expression is elevated in T lymphocytes from CAD patients. • Intracellular CypA and B expression are augmented in T lymphocytes from CAD patients. • CypA, B and C, and IL-1β and IL-6 serum levels are increased in patients with CAD. • CypA and B levels in T lymphocytes are correlated with CypA and B serum levels, respectively. Cardiovascular diseases and atherosclerosis are currently some of the most widespread diseases of our time. Within cardiovascular disease, coronary artery disease and underlying atherosclerosis were recently linked with systemic and local inflammation. Cyclophilins participate in the initiation and progression of these inflammatory-related diseases. Cyclophilins are released into the extracellular space upon inflammatory stimuli and participate in the pathology of cardiovascular diseases. The cell surface receptor for extracellular cyclophilins, the CD147 receptor, also contributes to coronary artery disease pathogenesis. Nevertheless, the physiological relevance of cyclophilin's family and their receptor in cardiovascular diseases remains unclear. The present study aimed to better understand the role of cyclophilins in cardiovascular artery disease and their relationship with inflammation. Hence, cyclophilins and pro-inflammatory interleukins were measured in the serum of 30 subjects (divided into three groups according to coronary artery disease status: 10 patients with acute coronary syndrome, 10 patients with chronic coronary artery disease, and 10 control volunteers). In addition, cyclophilin levels and CD147 receptor expression were measured in T lymphocytes purified from these subjects. Cyclophilin A, B, and C, pro-inflammatory interleukins, and CD147 membrane expression were significantly elevated in patients with coronary artery disease.
60 APPLIED LIFE SCIENCES↗
Phytosulfokine downregulates defense‐related WRKY transcription factors and attenuates pathogen‐associated molecular pattern‐triggered immunity
SUMMARY Phytosulfokine (PSK) is a plant growth‐promoting peptide hormone that is perceived by its cell surface receptors PSKR1 and PSKR2 in Arabidopsis. Plants lacking the PSK receptors show phenotypes consistent with PSK signaling repressing some plant defenses. To gain further insight into the PSK signaling mechanism, comprehensive transcriptional profiling of Arabidopsis treated with PSK was performed, and the effects of PSK treatment on plant defense readouts were monitored. Our study indicates that PSK's major effect is to downregulate defense‐related genes; it has a more modest effect on the induction of growth‐related genes. WRKY transcription factors (TFs) emerged as key regulators of PSK‐responsive genes, sharing commonality with a pathogen‐associated molecular pattern (PAMP) responses, flagellin 22 (flg22), but exhibiting opposite regulatory directions. These PSK‐induced transcriptional changes were accompanied by biochemical and physiological changes that reduced PAMP responses, notably mitogen‐activated protein kinase (MPK) phosphorylation (previously implicated in WRKY activation) and the cell wall modification of callose deposition. Comparison with previous studies using other growth stimuli (the sulfated plant peptide containing sulfated tyrosine [PSY] and Pseudomonas simiae strain WCS417) also reveals WRKY TFs' overrepresentations in these pathways, suggesting a possible shared mechanism involving WRKY TFs for plant growth–defense trade‐off.
Liu, Dian [Biochemistry and Molecular Biophysics T↗
Peptoid-Based Nanosheets Exhibiting Broad Antiviral Activity Against Enveloped RNA Viruses
Enveloped RNA viruses, such as Influenza A (H1N1) and Sindbis virus, pose persistent global health threats due to their high mutation rates, efficient transmission, and frequent drug resistance. By mimicking host cell membrane receptors, multivalent virus inhibitors can block viral attachment, making them promising broad-spectrum antiviral agents. However, most of existing antivirals are often limited by strain specificity, short-lived efficacy, and toxicity. Here, we introduce a broad-spectrum antiviral platform based on highly tunable and biocompatible two-dimensional nanomembranes (2DNMs) self-assembled from amphiphilic peptoids, operating via a non-genomic, mutation-insensitive mechanism. By varying peptoid sequence, we design and synthesize over twenty different 2DNMs with various surface charge and high density of viral-attachment ligands (VALs). The self-assembled architecture of these stable 2DNMs provides cooperative noncovalent multivalent binding to virus particles that result in effective inhibition of viral infection. Screening of variants identified three leads that potently suppressed Influenza A (H1N1) and Sindbis virus infection across median tissue culture infectious dose (TCID50), plaque, RT–qPCR, and immunofluorescence assays, while maintaining >90% cell viability. These nanosheets significantly reduced infectious titers, viral RNA replication, and intracellular viral protein expression, indicating inhibition at early stages of viral entry and propagation. The sequence programmability, chemical robustness, and mutation-insensitive antiviral activity distinguish 2DNMs from traditional antivirals and positions them as a versatile materials platform for antiviral coatings, protective barriers, and prophylactic biomedical applications.
Influenza A virus↗
Molecular pathways for learning in the single-cell Stentor coeruleus
The single-cell Stentor coeruleus contracts in response to mechanical taps but habituates and learns to ignore the taps after repeated stimulation. Here, we explored the molecular changes that occur during the formation of this cellular memory in order to improve our understanding of non-synaptic learning. We impaired cellular protein synthesis with cycloheximide and puromycin and found that, contrary to the effects of such treatments on metazoa, these drugs accelerate habituation and prolong memory retention in Stentor . Exploratory proteomic and transcriptomic analyses identified candidate proteins and genes that changed over the course of habituation and response recovery, pointing toward the regulation of Stentor learning by calcium signaling and protein phosphorylation. Building on these results, we found that using RNA interference to knock down the calcium-binding, EF-hand domain-containing protein SteCoe_6763 accelerated habituation. Furthermore, increased extracellular calcium improved Stentor learning, while treatment with kinase and phosphatase inhibitors impaired learning. In particular, KN-93, a drug known to inhibit calcium/calmodulin-dependent kinase II and voltage-gated calcium channels, decreased both the rate and extent of habituation in Stentor , similar to its effects on learning in metazoa. We also discovered that habituation memory can be maintained in progeny following cell division. Taken together, these results suggest that response recovery in Stentor requires new protein synthesis and that memory formation involves the modification of delocalized mechanoreceptors by phosphorylation and calcium signaling. This is consistent with our previous model of Stentor learning, in which habituation occurs through the inactivation of cell-surface receptors.
CaMKII↗