Cellular Ligands Research Articles

The impact of mineralocorticoid and glucocorticoid receptor interaction on corticosteroid transcriptional outcomes

The mineralocorticoid and glucocorticoid receptors (MR and GR, respectively) are members of the steroid receptor subfamily of nuclear receptors. Their main function is to act as ligand-activated transcription factors, transducing the effects of corticosteroid hormones (aldosterone and glucocorticoids) by modulating gene expression. Corticosteroid signaling is essential for homeostasis and adaptation to different forms of stress. GR responds to glucocorticoids by regulating genes involved in development, metabolism, immunomodulation and brain function. MR is best known for mediating the effects of aldosterone, a key hormone controlling electrolyte and water homeostasis. In addition to aldosterone, MR binds glucocorticoids (cortisol and corticosterone) with equally high affinity. This ligand promiscuity has important repercussions to understand MR function, as well as glucocorticoid signaling. MR and GR share significant sequence and structural similarities, regulate overlapping sets of genes and are able to interact forming heteromeric complexes. However, the precise role of these heteromers in regulating corticosteroid-regulated transcriptional outcomes remains an open question. In this review, we examine the evidence supporting MR-GR heteromerization, the molecular determinants of complex formation and their possible role in differential regulation of transcription in different cellular contexts and ligand availability.

PD-L1 promotes oncolytic virus infection via a metabolic shift that inhibits the type I IFN pathway.

While conventional wisdom initially postulated that PD-L1 serves as the inert ligand for PD-1, an emerging body of literature suggests that PD-L1 has cell-intrinsic functions in immune and cancer cells. In line with these studies, here we show that engagement of PD-L1 via cellular ligands or agonistic antibodies, including those used in the clinic, potently inhibits the type I interferon pathway in cancer cells. Hampered type I interferon responses in PD-L1-expressing cancer cells resulted in enhanced efficacy of oncolytic viruses in vitro and in vivo. Consistently, PD-L1 expression marked tumor explants from cancer patients that were best infected by oncolytic viruses. Mechanistically, PD-L1 promoted a metabolic shift characterized by enhanced glycolysis rate that resulted in increased lactate production. In turn, lactate inhibited type I IFN responses. In addition to adding mechanistic insight into PD-L1 intrinsic function, our results will also help guide the numerous ongoing efforts to combine PD-L1 antibodies with oncolytic virotherapy in clinical trials.

Glutamate Receptor-like (GLR) Family in Brassica napus: Genome-Wide Identification and Functional Analysis in Resistance to Sclerotinia sclerotiorum.

Plant glutamate receptor-like channels (GLRs) are homologs of animal ionotropic glutamate receptors. GLRs are critical in various plant biological functions, yet their genomic features and functions in disease resistance remain largely unknown in many crop species. Here, we report the results on a thorough genome-wide study of the GLR family in oilseed rape (Brassica napus) and their role in resistance to the fungal pathogen Sclerotinia sclerotiorum. A total of 61 GLRs were identified in oilseed rape. They comprised three groups, as in Arabidopsis thaliana. Detailed computational analyses, including prediction of domain and motifs, cellular localization, cis-acting elements, PTM sites, and amino acid ligands and their binding pockets in BnGLR proteins, unveiled a set of group-specific characteristics of the BnGLR family, which included chromosomal distribution, motif composition, intron number and size, and methylation sites. Functional dissection employing virus-induced gene silencing of BnGLRs in oilseed rape and Arabidopsis mutants of BnGLR homologs demonstrated that BnGLR35/AtGLR2.5 positively, while BnGLR12/AtGLR1.2 and BnGLR53/AtGLR3.2 negatively, regulated plant resistance to S. sclerotiorum, indicating that GLR genes were differentially involved in this resistance. Our findings reveal the complex involvement of GLRs in B. napus resistance to S. sclerotiorum and provide clues for further functional characterization of BnGLRs.

Network pharmacology-based strategy combined with molecular docking to explore the potential mechanism of agarwood against recurrent aphthous stomatitis.

To explore the antiinflammatory mechanism of agarwood on recurrent aphthous stomatitis (RAS). RAS is the most common mucosal disease in the oral cavity. The clinical application of traditional Chinese medicine found that agarwood has significant curative effect on peptic ulcer, but the effect and mechanism of agarwood on RAS remain unclear. This study is intended to predict the potential antiinflammatory mechanisms by which agarwood acts on RAS through network pharmacology and molecular docking. TCMSP database was used to screen the active components of agarwood. RAS targets were screened in Genecards, DisGeNET, and OMIM database. Venny, an online tool, screens for interacting genes between the two. Cytoscape software was used to construct the gene regulation map of active compounds target of agarwood. String Database building protein-protein interaction network. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathways were enriched in DAVID database. The key active ingredients and core targets were further verified by molecular docking. There were 9 effective compounds and 186 target genes in agarwood; RAS has 793 target genes. There were 41 interacting genes between agarwood and RAS. Interleukin 6, tumor necrosis factor, interleukin 1 beta, and cellular component motif ligand 2 may be key targets. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses predicted multiple pathways associated with RAS. Molecular docking results showed that the active compounds of agarwood combined well and stably with the target. The Chinese herbal medicine agarwood can relieve the inflammation of RAS through multiple targets and various ways. Its active compounds may be nominated as candidates for antiinflammatory drugs of RAS.

Report from the 29th Meeting on Toxinology, "Toxins: From the Wild to the Lab", Organized by the French Society of Toxinology on 30 November-1 December 2023.

The French Society of Toxinology (SFET), which celebrated its 30th anniversary this year, organized its 29th annual Meeting (RT29), shared by 87 participants, on 30 November-1 December 2023. The RT29 main theme, "Toxins: From the Wild to the Lab", focused on research in the field of animal venoms and animal, bacterial, fungal, or plant toxins, from their discovery in nature to their study in the laboratory. The exploration of the functions of toxins, their structures, their molecular or cellular ligands, their mode of action, and their potential therapeutic applications were emphasized during oral communications and posters through three sessions, of which each was dedicated to a secondary theme. A fourth, "miscellaneous" session allowed participants to present recent out-of-theme works. The abstracts of nine invited and 15 selected lectures, those of 24 posters, and the names of the Best Oral Communication and Best Poster awardees, are presented in this report.

Fluorimetric Detection of Insulin Misfolding by Probes Derived from Functionalized Fluorene Frameworks.

A group of functionalized fluorene derivatives that are structurally similar to the cellular prion protein ligand N,N'-(methylenedi-4,1-phenylene)bis [2-(1-pyrrolidinyl)acetamide] (GN8) have been synthesized. These compounds show remarkable native fluorescence due to the fluorene ring. The substituents introduced at positions 2 and 7 of the fluorene moiety are sufficiently flexible to accommodate the beta-conformational folding that develops in amyloidogenic proteins. Changes in the native fluorescence of these fluorene derivatives provide evidence of transformations in the amyloidogenic aggregation processes of insulin. The increase observed in the fluorescence intensity of the sensors in the presence of native insulin or amyloid aggregates suggest their potential use as fluorescence probes for detecting abnormal conformations; therefore, the compounds can be proposed for use as "turn-on" fluorescence sensors. Protein-sensor dissociation constants are in the 5-10 μM range and an intermolecular charge transfer process between the protein and the sensors can be successfully exploited for the sensitive detection of abnormal insulin conformations. The values obtained for the Stern-Volmer quenching constant for compound 4 as a consequence of the sensor-protein interaction are comparable to those obtained for the reference compound GN8. Fluorene derivatives showed good performance in scavenging reactive oxygen species (ROS), and they show antioxidant capacity according to the FRAP and DPPH assays.

Therapeutic effects of dual-ligated doxorubicin-loaded nanosized metal organic framework decorated with obeticholic acid as a novel nuclear targeting of hepatocellular carcinoma

In this work, we developed a novel and successful hepatocellular carcinoma (HCC) therapeutic approach by constructing doxorubicin (DOX)-loaded biocompatible NH2-UiO-66 NMOF decorated with cellular and nuclear targeting ligands. The design, synthesis and biological evaluations of the NMOF covalently ligated with obeticholic acid (OCA) and lactobionic acid (LA) and loaded with DOX were performed and proved efficient HCC-targeting at both the cellular and nuclear levels and simultaneously for active drug delivery. OCA was exploited as a nuclear targeting agent with affinity towards FXR receptor and also as an anticancer drug that is effective against HCC. NMOF was synthesized and covalently decorated with LA or OCA either alone or as dual-ligated. Comprehensive characterization techniques including XRD, FTIR, DSC and BET were used to demonstrate the success of the ligation procedure. The pristine NMOF, mono-ligated and the dual-ligated NMOFs were loaded with DOX and the cell viability was performed on HepG2 cell line as a representative for HCC. The in-vitro experiments confirmed that the drug-free NMOF was safe and biocompatible for normal cell lines such as human skin fibroblast (HSF). However, compared with either non-ligated or mono-ligated DOX-loaded nanoformulations, the DOX-loaded dual-ligated NMOF affirmed a distinguished activity against HCC. These outputs indicate that the developed DOX@OCA-LA-NMOF nanoformulation has superior killing effects on hepatic tumors because of the concentrated drugs localized in tumor cells and the precise targeting affinity of HepG2 cells.

Ligand field theory, Pauli shields and ultra-covalency in organometallic chemistry.

This paper explores the ligand field picture applied to organometallic compounds. Given the dearth of experimental data, the high-level ab initio ligand field theory (aiLFT) method is deployed as a surrogate for experiment and the necessary d orbital sequences and relative energies are obtained computationally. These are fitted to local cellular ligand field (CLF) σ, π and δ bonding parameters. Results are reported for planar [Cu(CR3)4]-, (R = F, H), octahedral M(CO)6n (M = Fe, Mn, Cr, V, Ti; n = +2, +1, 0, -1, -2), and the sandwich compounds M(Cp)2 (Cp = cyclopentadienyl, M = Fe, Ni, V), [Ni(Cp)2]2+ and Cr(C6H6)2. With respect to the aiLFT framework, these organometallic systems behave just like coordination complexes and most maintain the integrity of their formal dn configurations. Both [Cu(CR3)4]- compounds are formulated as low-spin d8 CuIII species and have normal ligand fields consistent with their planar geometries. The metal carbonyls reveal a new way of counting valence electrons which only requires the CLF d orbital energy level diagram to rationalise the 18-electron rule as well as its many exceptions. The bonding in sandwich compounds shows a remarkable variation. In ferrocene, Cp- behaves as a strong field ligand, comparable to [CN]- in [Fe(CN)6]4-. Fe(Cp)2 is low spin as is Cr(C6H6)2. Cp- in Fe(Cp)2 is a weak σ donor, strong π donor and weak δ acceptor while benzene in Cr(C6H6)2 is also a weak σ and strong π donor but is a much better δ acceptor. In contrast, Cp- is weak field in high spin, 20-electron Ni(Cp)2 but 'ultra-covalent' in [Ni(Cp)2]2+. The formal IV oxidation state is too high for the ligand set and the integrity of the d6 configuration is lost. Similarly, [V(CO)6]- and [Ti(CO)6]2- are ultra-covalent except now the formal metal oxidation states are too negative. Both mechanisms relate to the breaching of the metal's 3s23p6 'Pauli shield' and these ultra-covalent systems lie outside the ab initio ligand field regime. However, within the ligand field regime, the bonding in 'coordination complexes' and 'organometallic compounds' has the same conceptual footing and the nature of the local σ, π and δ interactions can be extracted from analysing the ligand field d orbitals.

Reovirus infection of tumor cells reduces the expression of NKG2D ligands, leading to impaired NK-cell cytotoxicity and functionality.

In recent years, reoviruses have been of major interest in immunotherapy because of their oncolytic properties. Preclinical and clinical trials, in which reovirus was used for the treatment of melanoma and glioblastoma, have paved the way for future clinical use of reovirus. However, little is known about how reovirus infection affects the tumor microenvironment and immune response towards infected tumor cells. Studies have shown that reovirus can directly stimulate natural killer (NK) cells, but how reovirus affects cellular ligands on tumor cells, which are ultimately key to tumor recognition and elimination by NK cells, has not been investigated. We tested how reovirus infection affects the binding of the NK Group-2 member D (NKG2D) receptor, which is a dominant mediator of NK cell anti-tumor activity. Using models of human-derived melanoma and glioblastoma tumors, we demonstrated that NKG2D ligands are downregulated in tumor cells post-reovirus-infection due to the impaired translation of these ligands in reovirus-infected cells. Moreover, we showed that downregulation of NKG2D ligands significantly impaired the binding of NKG2D to infected tumor cells. We further demonstrated that reduced recognition of NKG2D ligands significantly alters NK cell anti-tumor cytotoxicity in human primary NK cells and in the NK cell line NK-92. Thus, this study provides novel insights into reovirus-host interactions and could lead to the development of novel reovirus-based therapeutics that enhance the anti-tumor immune response.

Arachidonic acid reverses cholesterol and zinc inhibition of human voltage-gated proton channels

Unlike other members of the voltage-gated ion channel superfamily, voltage-gated proton (Hv) channels are solely composed of voltage sensor domains without separate ion-conducting pores. Due to their unique dependence on both voltage and transmembrane pH gradients, Hv channels normally open to mediate proton efflux. Multiple cellular ligands were also found to regulate the function of Hv channels, including Zn2+, cholesterol, polyunsaturated arachidonic acid, and albumin. Our previous work showed that Zn2+ and cholesterol inhibit the human voltage-gated proton channel hHv1 by stabilizing its S4 segment at resting state conformations. Released from phospholipids by phospholipase A2 in cells upon infection or injury, arachidonic acid regulates the function of many ion channels, including hHv1. In the present work, we examined the effects of arachidonic acid on purified hHv1 channels using liposome flux assays and revealed underlying structural mechanisms using single-molecule Fluorescence Resonance Energy Transfer (smFRET). Our data indicated that arachidonic acid strongly activates hHv1 channels by promoting transitions of the S4 segment towards opening or 'pre-opening' conformations. Moreover, we found that arachidonic acid even activates hHv1 channels inhibited by Zn2+ and cholesterol, providing a biophysical mechanism to activate hHv1 channels in non-excitable cells upon infection or injury.

Hematopoietic stem cells and betaherpesvirus latency.

The human betaherpesviruses including human cytomegalovirus (HCMV), human herpesvirus (HHV)-6a and HHV-6b, and HHV-7 infect and establish latency in CD34+ hematopoietic stem and progenitor cells (HPCs). The diverse repertoire of HPCs in humans and the complex interactions between these viruses and host HPCs regulate the viral lifecycle, including latency. Precise manipulation of host and viral factors contribute to preferential maintenance of the viral genome, increased host cell survival, and specific manipulation of the cellular environment including suppression of neighboring cells and immune control. The dynamic control of these processes by the virus regulate inter- and intra-host signals critical to the establishment of chronic infection. Regulation occurs through direct viral protein interactions and cellular signaling, miRNA regulation, and viral mimics of cellular receptors and ligands, all leading to control of cell proliferation, survival, and differentiation. Hematopoietic stem cells have unique biological properties and the tandem control of virus and host make this a unique environment for chronic herpesvirus infection in the bone marrow. This review highlights the elegant complexities of the betaherpesvirus latency and HPC virus-host interactions.

Transcriptomic analysis of B cells suggests that CD70 and LY9 may be novel features in patients with systemic lupus erythematosus

Dysfunction of B-cell subsets is critical in the development of systemic lupus erythematosus (SLE). There is a great diversity of B-lineage cells, and their features and functions in SLE need to be clarified. In this study, we analyzed single-cell RNA sequencing (scRNA-seq) data from peripheral blood mononuclear cells (PBMCs) and bulk transcriptomic data of isolated B-cell subsets from patients with SLE and healthy controls (HCs). We preformed scRNA-seq analysis focused on the diversity of B-cell subsets and identified a subset of antigen-presenting B cells in SLE patients that highly expressed ITGAX. A list of marker genes of each B-cell subset in patients with SLE was also identified. Comparison of bulk transcriptomic data of isolated B-cell subpopulations between SLE patients and HCs revealed the upregulated differentially expressed genes (DEGs) for each B-cell subpopulation in SLE. Common genes identified using these two methods were considered to be upregulated marker genes of B cells in SLE. The scRNA-seq data of SLE patients and HCs revealed that CD70 and LY9 were overexpressed in B cells vs. other cell types from SLE patients, and this pattern was validated by RT‒qPCR. Because CD70 is the cellular ligand of CD27, previous studies on CD70 have focused mainly on T cells from SLE patients. LY9 appears to have different functions in mice and humans: its expression is decreased in lupus-prone mice but is increased in T cells and some B-cell subpopulations in SLE patients. Here, we describe the overexpression of two costimulatory molecules, CD70 and LY9, which may be a novel feature of B cells in SLE patients.

In silico characterization of cysteine-stabilized αβ defensins from neglected unicellular microeukaryotes

BackgroundThe emergence of multi-resistant pathogens have increased dramatically in recent years, becoming a major public-health concern. Among other promising antimicrobial molecules with potential to assist in this worldwide struggle, cysteine-stabilized αβ (CS-αβ) defensins are attracting attention due their efficacy, stability, and broad spectrum against viruses, bacteria, fungi, and protists, including many known human pathogens.ResultsHere, 23 genomes of ciliated protists were screened and two CS-αβ defensins with a likely antifungal activity were identified and characterized, using bioinformatics, from a culturable freshwater species, Laurentiella sp. (LsAMP-1 and LsAMP-2). Although any potential cellular ligand could be predicted for LsAMP-2; evidences from structural, molecular dynamics, and docking analyses suggest that LsAMP-1 may form stably associations with phosphatidylinositol 4,5-bisphosphates (PIP2), a phospholipid found on many eukaryotic cells, which could, in turn, represent an anchorage mechanism within plasma membrane of targeted cells.ConclusionThese data stress that more biotechnology-oriented studies should be conducted on neglected protists, such ciliates, which could become valuable sources of novel bioactive molecules for therapeutic uses.

Single-cell sequencing reveals that endothelial cells, EndMT cells and mural cells contribute to the pathogenesis of cavernous malformations

Cavernous malformations (CMs) invading the central nervous system occur in ~0.16–0.4% of the general population, often resulting in hemorrhages and focal neurological deficits. Further understanding of disease mechanisms and therapeutic strategies requires a deeper knowledge of CMs in humans. Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from twelve human CM samples and three control samples. A total of 112,670 high-quality cells were clustered into 11 major cell types, which shared a number of common features in CMs harboring different genetic mutations. A new EC subpopulation marked with PLVAP was uniquely identified in lesions. The cellular ligand‒receptor network revealed that the PLVAP-positive EC subcluster was the strongest contributor to the ANGPT and VEGF signaling pathways in all cell types. The PI3K/AKT/mTOR pathway was strongly activated in the PLVAP-positive subcluster even in non-PIK3CA mutation carriers. Moreover, endothelial-to-mesenchymal transition (EndMT) cells were identified for the first time in CMs at the single-cell level, which was accompanied by strong immune activation. The transcription factor SPI1 was predicted to be a novel key driver of EndMT, which was confirmed by in vitro and in vivo studies. A specific fibroblast-like phenotype was more prevalent in lesion smooth muscle cells, hinting at the role of vessel reconstructions and repairs in CMs, and we also confirmed that TWIST1 could induce SMC phenotypic switching in vitro and in vivo. Our results provide novel insights into the pathomechanism decryption and further precise therapy of CMs.

Mechanisms of ligand regulation in human voltage-gated proton channel.

Voltage-gated proton (Hv) channels are standalone voltage sensors without separate pore-forming domains in canonical voltage-gated ion channels. They normally function as acid extruders for their unique voltage- and pH-dependent gating. Aside from voltage and pH, Hv channels are also gated by multiple cellular ligands, including Zn2+, polyunsaturated arachidonic acid, albumin, and membrane cholesterol, identified in our recent work. Our previous results have established the S4 segment as the central gating machinery in hHv1 channels by showing that voltage activation enriched its opening conformations while inhibitory ligands, including proton, Zn2+ and cholesterol, stabilized it at resting state conformations. How is the function of hHv1 channels regulated in cells? Our functional characterizations on purified hHv1 channels and those expressed in cells indicated that arachidonic acid has very strong activating effects on hHv1 channels, even being able to reverse both Zn2+ and cholesterol inhibition at physiological levels. Examining the structural effects of arachidonic acid on hHv1 channels using single-molecule FRET further revealed that it activates hHv1 channels by promoting ‘pre-opening’ rather than the opening conformations. In many cells, hHv1 channels are closely associated with NADPH oxidases to compensate for charge/pH imbalances, and both were found in cholesterol-rich lipid rafts. Our results propose that cholesterol perhaps determines the basal activities of the hHv1 channel in cells, while releases of arachidonic acid following irritation or injury may serve as a major mechanism to activate hHv1 channels to promote respiration burst mediating by NADPH oxidases.

Hydrogel-based microenvironment engineering of haematopoietic stem cells.

Haematopoietic Stem cells (HSCs) have the potential for self-renewal and multilineage differentiation, and their behaviours are finely tuned by the microenvironment. HSC transplantation (HSCT) is widely used in the treatment of haematologic malignancies while limited by the quantity of available HSCs. With the development of tissue engineering, hydrogels have been deployed to mimic the HSC microenvironment in vitro. Engineered hydrogels influence HSC behaviour by regulating mechanical strength, extracellular matrix microstructure, cellular ligands and cytokines, cell-cell interaction, and oxygen concentration, which ultimately facilitate the acquisition of sufficient HSCs. Here, we review recent advances in the application of hydrogel-based microenvironment engineering of HSCs, and provide future perspectives on challenges in basic research and clinical practice.

How tobacco (Nicotiana tabacum) BY-2 cells cope with Eu(III) - a microspectroscopic study.

The extensive use of lanthanides in science, industry and high-technology products is accompanied by an anthropogenic input of rare earth elements into the environment. Knowledge of a metal's environmental fate is essential for reasonable risk assessment and remediation approaches. In the present study, Eu(III) was representatively used as a luminescent probe to study the chemical environment and to elucidate the molecular interactions of lanthanides with a suspension cell culture of Nicotiana tabacum BY-2. Biochemical methods were combined with luminescence spectroscopy, two-dimensional microspectroscopic mappings, and data deconvolution methods to resolve the bioassociation behavior and spatial distribution of Eu(III) in plant cells. BY-2 cells were found to gradually take up the metal after exposure to 100 μM Eu(III) without significant loss of viability. Time-resolved luminescence measurements were used to specify the occurrence of Eu(III) species as a function of time, revealing the transformation of an initial Eu(III) species into another after 24 h exposure. Chemical microscopy and subsequent iterative factor analysis reveal the presence of four distinct Eu(III) species located at different cellular compartments, e.g., the cell nucleus, nucleolus and cell walls, which could be assigned to intracellular binding motifs. In addition, a special type of bioaccumulation occurs through the formation of a Eu(III)-containing oxalate biomineral, which is already formed within the first 24 hours after metal exposure. Oxalate crystals were also obtained in analogous experiments with Gd and Sm. These results indicate that tobacco BY-2 cells induce the precipitation of metal oxalate biominerals for detoxification of lanthanides, although they also bind to other cellular ligands at the same time.

Single-cell discovery of the scene and potential immunotherapeutic target in hypopharyngeal tumor environment

Hypopharyngeal carcinoma is a cancer with the worst prognosis. We constructed the first single-cell transcriptome map for hypopharyngeal carcinoma and explored its underlying mechanisms. We systematically studied single-cell transcriptome data of 17,599 cells from hypopharyngeal carcinoma and paracancerous tissues. We identified categories of cells by dimensionality reduction and performed further subgroup analysis. Focusing on the potential mechanism in the cellular communication of hypopharyngeal carcinoma, we predicted ligand-receptor interactions and verified them via immunohistochemical and cellular experiments. In total, seven cell types were identified, including epithelial and myeloid cells. Subsequently, subgroup analysis showed significant tumor heterogeneity. Based on the pathological type of squamous cell carcinoma, we focused on intercellular communication between epithelial cells and various cells. We predicted the crosstalk and inferred the regulatory effect of cellular active ligands on the surface receptor of epithelial cells. From the top potential pairs, we focused on the BMPR2 receptor for further research, as it showed significantly higher expression in epithelial cancer tissue than in adjacent tissue. Further bioinformatics analysis, immunohistochemical staining, and cell experiments also confirmed its cancer-promoting effects. Overall, the single-cell perspective revealed complex crosstalk in hypopharyngeal cancer, in which BMPR2 promotes its proliferation and migration, providing a rationale for further study and treatment of this carcinoma.

NKp44-based chimeric antigen receptor effectively redirects primary T cells against synovial sarcoma

BackgroundT-cell receptor-engineered T-cell therapies have achieved promising response rates against synovial sarcoma in clinical trials, but their applicability is limited owing to the HLA matching requirement. Chimeric antigen receptor (CAR) can redirect primary T cells to tumor-associated antigens without requiring HLA matching. However, various obstacles, including the paucity of targetable antigens, must be addressed for synovial sarcoma. Ligands for natural killer (NK) cell-activating receptors are highly expressed by tumor cells. MethodsThe surface expression of ligands for NK cell-activating receptors in synovial sarcoma cell lines was analyzed. We analyzed RNA sequencing data deposited in a public database to evaluate NKp44-ligand expression. Primary T cells retrovirally transduced with CAR targeting NKp44 ligands were evaluated for their functions in synovial sarcoma cells. Alterations induced by various stimuli, including a histone deacetylase inhibitor, a hypomethylating agent, inflammatory cytokines, and ionizing radiation, in the expression levels of NKp44 ligands were investigated. Results: Ligands for NKp44 and NKp30 were expressed in all cell lines. NKG2D ligands were barely expressed in a single cell line. None of the cell lines expressed NKp46 ligand. Primary synovial sarcoma cells expressed the mRNA of the truncated isoform of MLL5, a known cellular ligand for NKp44. NKp44-based CAR T cells specifically recognize synovial sarcoma cells, secrete interferon-γ, and exert suppressive effects on tumor cell growth. No stimulus altered the expression of NKp44 ligands. ConclusionNKp44-based CAR T cells can redirect primary human T cells to synovial sarcoma cells. CAR-based cell therapies may be an option for treating synovial sarcomas.

LAG3 ectodomain structure reveals functional interfaces for ligand and antibody recognition.

The immune checkpoint receptor lymphocyte activation gene 3 protein (LAG3) inhibits T cell function upon binding to major histocompatibility complex class II (MHC class II) or fibrinogen-like protein 1 (FGL1). Despite the emergence of LAG3 as a target for next-generation immunotherapies, we have little information describing the molecular structure of the LAG3 protein or how it engages cellular ligands. Here we determined the structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by Ig domain 2. Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHC class II and FGL1 by binding to a flexible 'loop 2' region in LAG3 domain 1. We also defined the LAG3-FGL1 interface by mapping mutations onto structures of LAG3 and FGL1 and established that FGL1 cross-linking induces the formation of higher-order LAG3 oligomers. These insights can guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.