Inhibitory Signaling Pathways Research Articles

The Dectin-1 and Dectin-2 clusters: C-type lectin receptors with fundamental roles in immunity.

The ability of myeloid cells to recognize and differentiate endogenous or exogenous ligands rely on the presence of different transmembrane protein receptors. C-type lectin receptors (CLRs), defined by the presence of a conserved structural motif called C-type lectin-like domain (CTLD), are a crucial family of receptors involved in this process, being able to recognize a diverse range of ligands from glycans to proteins or lipids and capable of initiating an immune response. The Dectin-1 and Dectin-2 clusters involve two groups of CLRs, with genes genomically linked within the natural killer cluster of genes in both humans and mice, and all characterized by the presence of a single extracellular CTLD. Fundamental immune cell functions such as antimicrobial effector mechanisms as well as internalization and presentation of antigens are induced and/or regulated through activatory, or inhibitory signalling pathways triggered by these receptors after ligand binding. In this review, we will discuss the most recent concepts regarding expression, ligands, signaling pathways and functions of each member of the Dectin clusters of CLRs, highlighting the importance and diversity of their functions.

Relationship of Signaling Pathways between RKIP Expression and the Inhibition of EMT-Inducing Transcription Factors SNAIL1/2, TWIST1/2 and ZEB1/2.

Untreated primary carcinomas often lead to progression, invasion and metastasis, a process that involves the epithelial-to-mesenchymal transition (EMT). Several transcription factors (TFs) mediate the development of EMT, including SNAIL1/SNAIL2, TWIST1/TWIST2 and ZEB1/ZEB2, which are overexpressed in various carcinomas along with the under expression of the metastasis suppressor Raf Kinase Inhibitor Protein (RKIP). Overexpression of RKIP inhibits EMT and the above associated TFs. We, therefore, hypothesized that there are inhibitory cross-talk signaling pathways between RKIP and these TFs. Accordingly, we analyzed the various properties and biomarkers associated with the epithelial and mesenchymal tissues and the various molecular signaling pathways that trigger the EMT phenotype such as the TGF-β, the RTK and the Wnt pathways. We also presented the various functions and the transcriptional, post-transcriptional and epigenetic regulations for the expression of each of the EMT TFs. Likewise, we describe the transcriptional, post-transcriptional and epigenetic regulations of RKIP expression. Various signaling pathways mediated by RKIP, including the Raf/MEK/ERK pathway, inhibit the TFs associated with EMT and the stabilization of epithelial E-Cadherin expression. The inverse relationship between RKIP and the TF expressions and the cross-talks were further analyzed by bioinformatic analysis. High mRNA levels of RKIP correlated negatively with those of SNAIL1, SNAIL2, TWIST1, TWIST2, ZEB1, and ZEB2 in several but not all carcinomas. However, in these carcinomas, high levels of RKIP were associated with good prognosis, whereas high levels of the above transcription factors were associated with poor prognosis. Based on the inverse relationship between RKIP and EMT TFs, it is postulated that the expression level of RKIP in various carcinomas is clinically relevant as both a prognostic and diagnostic biomarker. In addition, targeting RKIP induction by agonists, gene therapy and immunotherapy will result not only in the inhibition of EMT and metastases in carcinomas, but also in the inhibition of tumor growth and reversal of resistance to various therapeutic strategies. However, such targeting strategies must be better investigated as a result of tumor heterogeneities and inherent resistance and should be better adapted as personalized medicine.

Exosomes originating from neural stem cells undergoing necroptosis participate in cellular communication by inducing TSC2 upregulation of recipient cells following spinal cord injury.

We previously demonstrated that inhibiting neural stem cells necroptosis enhances functional recovery after spinal cord injury. While exosomes are recognized as playing a pivotal role in neural stem cells exocrine function, their precise function in spinal cord injury remains unclear. To investigate the role of exosomes generated following neural stem cells necroptosis after spinal cord injury, we conducted single-cell RNA sequencing and validated that neural stem cells originate from ependymal cells and undergo necroptosis in response to spinal cord injury. Subsequently, we established an in vitro necroptosis model using neural stem cells isolated from embryonic mice aged 16-17 days and extracted exosomes. The results showed that necroptosis did not significantly impact the fundamental characteristics or number of exosomes. Transcriptome sequencing of exosomes in necroptosis group identified 108 differentially expressed messenger RNAs, 104 long non-coding RNAs, 720 circular RNAs, and 14 microRNAs compared with the control group. Construction of a competing endogenous RNA network identified the following hub genes: tuberous sclerosis 2 (Tsc2), solute carrier family 16 member 3 (Slc16a3), and forkhead box protein P1 (Foxpl). Notably, a significant elevation in TSC2 expression was observed in spinal cord tissues following spinal cord injury. TSC2-positive cells were localized around SRY-box transcription factor 2-positive cells within the injury zone. Furthermore, in vitro analysis revealed increased TSC2 expression in exosomal receptor cells compared with other cells. Further assessment of cellular communication following spinal cord injury showed that Tsc2 was involved in ependymal cellular communication at 1 and 3 days post-injury through the epidermal growth factor and midkine signaling pathways. In addition, Slc16a3 participated in cellular communication in ependymal cells at 7 days post-injury via the vascular endothelial growth factor and macrophage migration inhibitory factor signaling pathways. Collectively, these findings confirm that exosomes derived from neural stem cells undergoing necroptosis play an important role in cellular communication after spinal cord injury and induce TSC2 upregulation in recipient cells.

Mechanisms for dysregulation of excitatory-inhibitory balance underlying allodynia in dorsal horn neural subcircuits.

Chronic pain is a wide-spread condition that is debilitating and expensive to manage, costing the United States alone around $600 billion in 2010. In a common type of chronic pain called allodynia, non-painful stimuli produce painful responses with highly variable presentations across individuals. While the specific mechanisms remain unclear, allodynia is hypothesized to be caused by the dysregulation of excitatory-inhibitory (E-I) balance in pain-processing neural circuitry in the dorsal horn of the spinal cord. In this work, we analyze biophysically-motivated subcircuit structures that represent common motifs in neural circuits in layers I-II of the dorsal horn. These circuits are hypothesized to be part of the neural pathways that mediate two different types of allodynia: static and dynamic. We use neural firing rate models to describe the activity of populations of excitatory and inhibitory interneurons within each subcircuit. By accounting for experimentally-observed responses under healthy conditions, we specify model parameters defining populations of subcircuits that yield typical behavior under normal conditions. Then, we implement a sensitivity analysis approach to identify the mechanisms most likely to cause allodynia-producing dysregulation of the subcircuit's E-I signaling. We find that disruption of E-I balance generally occurs either due to downregulation of inhibitory signaling so that excitatory neurons are "released" from inhibitory control, or due to upregulation of excitatory neuron responses so that excitatory neurons "escape" their inhibitory control. Which of these mechanisms is most likely to occur, the subcircuit components involved in the mechanism, and the proportion of subcircuits exhibiting the mechanism can vary depending on the subcircuit structure. These results suggest specific hypotheses about diverse mechanisms that may be most likely responsible for allodynia, thus offering predictions for the high interindividual variability observed in allodynia and identifying targets for further experimental studies on the underlying mechanisms of this chronic pain condition.

Human cytomegalovirus UL23 exploits PD-L1 inhibitory signaling pathway to evade T cell-mediated cytotoxicity.

T cell immunity is pivotal in controlling primary human cytomegalovirus (HCMV) infection, restricting periodic reactivation, and preventing HCMV-associated diseases. Despite inducing a robust T cell immune response, HCMV has developed sophisticated immune evasion mechanisms that specifically target T cell responses. Although numerous studies have been conducted on HCMV-specific T cells, the primary focus has been on the impact of HCMV on T cell recognition via major histocompatibility complex molecules. Our studies show for the first time that HCMV exploits the programmed death ligand 1 (PD-L1) inhibitory signaling pathway to evade T cell immunity by modulating the activities of T cells and thereby blocking the secretion of IFN-γ, which is directly mediated by HCMV-encoded tegument protein UL23. While PD-L1 has been extensively studied in the context of tumors and viruses, its involvement in HCMV infection and viral immune evasion is rarely reported. We observed an upregulation of PD-L1 in normal cells during HCMV infection and provided strong evidence supporting its critical role in UL23-induced inhibition of T cell-mediated cytotoxicity. The novel strategy employed by HCMV to manipulate the inhibitory signaling pathway of T cell immune activation for viral evasion through its encoded protein offers valuable insights for the understanding of HCMV-mediated T cell immunomodulation and developing innovative antiviral treatment strategies.

Treatment modalities for advanced solid pseudopapillary tumor of the pancreas.

e16327 Background: Solid pseudopapillary tumor (SPT) of the pancreas is a rare low-grade malignant tumor. However, a minority of SPT patients present aggressive behavior and develop recurrent or metastatic diseases. The treatment strategies of advanced SPTs remain unclear. Our study aimed to explore the treatment modalities of aggressive SPTs and the role of endocrine therapy. Methods: The data on clinicopathologic characteristics and treatment of advanced SPT patients from Sun Yat-Sen University Cancer Center and Shen Zhen People's Hospital were collected. The treatment modalities were explored by co-analyzing our local cohort and published literatures. Single sample gene set enrichment analysis (ssGSEA) was used to explore the functional enrichment pathway (GSE43795). Fresh surgical specimens from 10 SPT patients were collected for endocrine drugs sensitive test in vitro by performing tumor histoculture end-point staining computer image analysis (TECIA). Results: A total of 77 advanced SPT patients including 14 cases in our local cohort and 63 cases from published literatures were enrolled. 60 patients received local treatment including cytoreductive surgery, interventional therapy or radiation therapy. 55 patients who had undergone cytoreductive surgery have better outcome compared with others (P = 0.046, HR = 4.12). 31 patients received systemic chemotherapy and 6 patients received targeted therapy. There were 2 patients achieved partial response after chemotherapy. In our local cohort, 5 patients received endocrine therapy with tamoxifen plus megestrol. One patient achieved partial response with duration of response (DOR) of 89 months. 4 patients showed stable disease. ssGSEA for GSE43795 showed that Wnt signaling pathway and its inhibitory pathway-progesterone receptor signaling pathway were simultaneously enriched in SPTs. The results of endocrine drug sensitive test in vitro by TECIA showed response rate of 50% (5/10) in megestrol group, 40% (4/10) in tamoxifen group, and 30% (3/10) in testosterone propionate group. Conclusions: Advanced SPTs did not appear chemosensitive. Cytoreductive surgery may improve survival. Simultaneously enriching Wnt and progesterone receptor signaling pathway may partially explain the low aggressive ability of SPT and their unbalance may lead to the aggressive behavior of SPT. Endocrine therapy with tamoxifen plus megestrol, by enhancing the progesterone signaling pathway, seems to be a promising regimen for advanced SPTs, which is worth to be noted and further studied.

Unveiling mitochondrial and ribosomal gene deregulation and tumor microenvironment dynamics in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a malignant clonal hematopoietic disease with a poor prognosis. Understanding the interaction between leukemic cells and the tumor microenvironment (TME) can help predict the prognosis of leukemia and guide its treatment. Re-analyzing the scRNA-seq data from the CSC and G20 cohorts, using a Python-based pipeline including machine-learning-based scVI-tools, recapitulated the distinct hierarchical structure within the samples of AML patients. Weighted correlation network analysis (WGCNA) was conducted to construct a weighted gene co-expression network and to identify gene modules primarily focusing on hematopoietic stem cells (HSCs), multipotent progenitors (MPPs), and natural killer (NK) cells. The analysis revealed significant deregulation in gene modules associated with aerobic respiration and ribosomal/cytoplasmic translation. Cell-cell communications were elucidated by the CellChat package, revealing an imbalance of activating and inhibitory immune signaling pathways. Interception of genes upregulated in leukemic HSCs & MPPs as well as in NKG2A-high NK cells was used to construct prognostic models. Normal Cox and artificial neural network models based on 10 genes were developed. The study reveals the deregulation of mitochondrial and ribosomal genes in AML patients and suggests the co-occurrence of stimulatory and inhibitory factors in the AML TME.

A Systematic Review of the Advances in the Study of T Lymphocyte Suppressor Receptors in HBV Infection: Potential Therapeutic Targets.

Background: HBV-specific T lymphocytes are pivotal in eliminating the hepatitis B virus (HBV) and regulating intrahepatic inflammatory reactions. Effective T cell responses curtail HBV infection; however, compromised immunity can result in persistent infection. Beyond the acute phase, the continued presence of antigens and inflammation leads to the increased expression of various inhibitory receptors, such as PD-1, CTLA-4, Tim-3, LAG3, 2B4, CD160, BTLA, and TIGIT. This escalates the dysfunction of and diminishes the immune and proliferative abilities of T cells. Methods: In this study, we reviewed English-language literature from PubMed, Web of Science, and Scopus up to 9 July 2023. This paper aims to elucidate the inhibitory effects of these receptors on HBV-specific T lymphocytes and how immune function can be rejuvenated by obstructing the inhibitory receptor signaling pathway in chronic HBV patients. We also summarize the latest insights into related anti-HBV immunotherapy. Result: From 66 reviewed reports, we deduced that immunotherapy targeting inhibitory receptors on T cells is a reliable method to rejuvenate T cell immune responses in chronic HBV patients. However, comprehensive combination therapy strategies are essential for a functional cure. Conclusions: Targeting T cell suppressor receptors and combining immunotherapy with antiviral treatments may offer a promising approach towards achieving a functional cure, urging future research to prioritize effective combination therapeutic strategies for chronic HBV infection.

In vitro re-challenge of CAR T cells.

Chimeric antigen receptor (CAR) T cells (CAR T) have emerged as a potential therapy for cancer patients. CAR T cells are capable of recognizing membrane proteins on cancer cells which initiates a downstream signaling in T cells that ends in cancer cell death. Continuous antigen exposure over time, activation of inhibitory signaling pathways and/or chronic inflammation can lead to CAR T cell exhaustion. In this context, the design of CARs can have a great impact on the functionality of CAR T cells, on their potency and exhaustion. Here, using CD19CAR as model, we provide a re-challenge protocol where CAR T cells are cultured weekly with malignant lymphoid cell lines BL-41 and Nalm-6 to simulate them with continuous antigen pressure over a four-week period. This protocol can be value for assessing CAR T cell functionality and for the comparison of different CAR constructs.

Antidiabetic Effect of Standardized Chrysanthemum rubellum Hydroethanolic Extract by Targeting α-Glucosidase and the PTP-1B Signaling Pathway for Alleviating Diabetes in Experimental Model.

The study's goal was to find out whether Chrysanthemum rubellum extract has anti-diabetic properties by concentrating on α-glucosidase and the PTP-1B signaling pathway. C. rubellum flowers were used for extraction using Methanol/water (80/20) as solvent. LC-MS techniques was used to check the presence of phytoconstituents present in C. rubellum extract. In vitro antidiabetic activity was evaluated using α-glucosidase inhibitory activity and PTP-1B signaling pathway. On Streptozotocin (STZ)-induced rats with diabetes, the in vivo antidiabetic efficacy was assessed using a test for oral glucose tolerance. The phytoconstituents identified in the extract of C. rubellum were apigenin, diosmin, myricetin, luteolin, luteolin-7-glucoside, and Quercitrin as compound 1-6, respectively. Results showed that diosmin exhibited highest α-glucosidase inhibitory activity i.e. 90.39%. The protein level of PTP-1B was lowered and the insulin signalling activity was directly increased by compounds 1-6. The maximum blood glucose levels were seen in all groups' OGTT findings at 30 minutes following glucose delivery, followed by gradual drops. In comparison to the control group, the extract's glucose levels were 141 mg/dL at 30 minutes before falling to 104 mg/dL after 120 minutes. The current study has demonstrated, in summary, that extract with phytoconstituents reduce blood sugar levels in rats. This finding suggests that extract may reduce the chance of insulin resistance and shield against disorders like hyperglycemia.

Wilforine inhibits rheumatoid arthritis pathology through the Wnt11/β-catenin signaling pathway axis

BackgroundWilforine (WFR) is a monomeric compound of the anti-RA plant Tripterygium wilfordii Hook. f. (TwHF). Whether WFR has anti-RA effect, its molecular mechanism has not been elucidated.Aim of the studyOur study aims to clarify how WFR inhibits fibroblast-like synovial cells (FLS) activation and improves RA through Wnt11 action on the Wnt11/β-catenin signaling pathway.MethodsThe therapeutic effect of WFR on collagen-induced arthritis (CIA) rats was evaluated using methods such as rat arthritis score. The inhibitory effects and signaling pathways of WFR on the proliferation and inflammatory response of CIA FLS and RA FLS were studied using ELISA, CCK-8, RT-qPCR, Western blot, and immunofluorescence methods.ResultsWFR could effectively alleviate the arthritis symptoms of CIA rats; reduce the levels of IL-6, IL-1β, and TNF-α in the peripheral blood of CIA rats; and inhibit the expression of MMP3 and fibronectin. The data showed that WFR has a significant inhibitory effect on FLS proliferation. Furthermore, WFR inhibited the activation of Wnt/β-catenin signaling pathway and decreased the expression of Wnt11, β-catenin, CCND1, GSK-3β, and c-Myc, while the effects of WFR were reversed after overexpression of Wnt11.ConclusionsWFR improves RA by inhibiting the Wnt11/β-catenin signaling pathway, and Wnt11 is the direct target of WFR. This study provides a new molecular mechanism for WFR to improve RA and contributes to the clinical promotion of WFR.

Platelets: Physiology and Biochemistry.

This article represents a republication of an article originally published in STH in 2005. This republication is to help celebrate 50 years of publishing for STH. The original abstract follows.Platelets are specialized blood cells that play central roles in physiologic and pathologic processes of hemostasis, inflammation, tumor metastasis, wound healing, and host defense. Activation of platelets is crucial for platelet function that includes a complex interplay of adhesion and signaling molecules. This article gives an overview of the activation processes involved in primary and secondary hemostasis, for example, platelet adhesion, platelet secretion, platelet aggregation, microvesicle formation, and clot retraction/stabilization. In addition, activated platelets are predominantly involved in cross-talk to other blood and vascular cells. Stimulated "sticky" platelets enable recruitment of leukocytes at sites of vascular injury under high shear conditions. Platelet-derived microparticles as well as soluble adhesion molecules, sP-selectin and sCD40L, shed from the surface of activated platelets, are capable of activating, in turn, leukocytes and endothelial cells. This article focuses further on the new view of receptor-mediated thrombin generation of human platelets, necessary for the formation of a stable platelet-fibrin clot during secondary hemostasis. Finally, special emphasis is placed on important stimulatory and inhibitory signaling pathways that modulate platelet function.

A high-throughput two-cell assay for interrogating inhibitory signaling pathways in T cells

The recent success of immunotherapies relying on manipulation of T-cell activation highlights the value of characterising the mediators of immune checkpoint signaling. CRISPR/Cas9 is a popular approach for interrogating signaling pathways; however, the lack of appropriate assays for studying inhibitory signaling in T cells is limiting the use of large-scale perturbation-based approaches. Here, we adapted an existing Jurkat cell-based transcriptional reporter assay to study both activatory and inhibitory (PD-1-mediated) T-cell signaling using CRISPR-based genome screening in arrayed and pooled formats. We targeted 64 SH2 domain-containing proteins expressed by Jurkat T cells in an arrayed screen, in which individual targets could be assessed independently, showing that arrays can be used to study mediators of both activatory and inhibitory signaling. Pooled screens succeeded in simultaneously identifying many of the known mediators of proximal activating and inhibitory T-cell signaling, including SHP2 and PD-1, confirming the utility of the method. Altogether, the data suggested that SHP2 is the major PD-1-specific, SH2 family mediator of inhibitory signaling. These approaches should allow the systematic analysis of signaling pathways in T cells.

Plant-derived Durvalumab variants show efficient PD-1/PD-L1 blockade and therapeutically favourable FcR binding.

Immune checkpoint blocking therapy targeting the PD-1/PD-L1 inhibitory signalling pathway has produced encouraging results in the treatment of a variety of cancers. Durvalumab (Imfinzi®) targeting PD-L1 is currently used for immunotherapy of several tumour malignancies. The Fc region of this IgG1 antibody has been engineered to reduce FcγR interactions with the aim of enhancing blockade of PD-1/PD-L1 interactions without the depletion of PD-L1-expressing immune cells. Here, we used Nicotiana benthamiana to produce four variants of Durvalumab (DL): wild-type IgG1 and its 'Fc-effector-silent' variant (LALAPG) carrying further modifications to increase antibody half-life (YTE); IgG4S228P and its variant (PVA) with Fc mutations to decrease binding to FcγRI. In addition, DL variants were produced with two distinct glycosylation profiles: afucosylated and decorated with α1,6-core fucose. Plant-derived DL variants were compared to the therapeutic antibody regarding their ability to (i) bind to PD-L1, (ii) block PD-1/PD-L1 inhibitory signalling and (iii) engage with the neonatal Fc receptor (FcRn) and various Fcγ receptors. It was found that plant-derived DL variants bind to recombinant PD-L1 and to PD-L1 expressed in gastrointestinal cancer cells and are able to effectively block its interaction with PD-1 on T cells, thereby enhancing their activation. Furthermore, we show a positive impact of Fc amino acid mutations and core fucosylation on DL's therapeutic potential. Compared to Imfinzi®, DL-IgG1 (LALAPG) and DL-IgG4 (PVA)S228P show lower affinity to CD32B inhibitory receptor which can be therapeutically favourable. Importantly, DL-IgG1 (LALAPG) also shows enhanced binding to FcRn, a key determinant of serum half-life of IgGs.

Single-cell transcriptomics reveals ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia exerts antitumor effects by alleviating macrophage suppression in breast cancer

The potential of Ferrimagnetic vortex iron oxide nanoring-mediated mild magnetic hyperthermia (FVIO-MHT) in solid tumor therapy has been demonstrated. However, the impact of FVIO-MHT on the tumor microenvironment (TME) remains unclear. This study utilized single-cell transcriptome sequencing to examine the alterations in the TME in response to FVIO-MHT in breast cancer. The results revealed the cellular composition within the tumor microenvironment (TME) was primarily modified due to a decrease in tumor cells and an increased infiltration of myeloid cells. Subsequently, an enhancement in active oxygen (ROS) metabolism was observed, indicating oxidative damage to tumor cells. Interestingly, FVIO-MHT reprogrammed the macrophages’ phenotypes, as evidenced by alterations in the transcriptome characteristics associated with both classic and alternative activated phenotypes. And an elevated level of ROS generation and oxidative phosphorylation suggested that activated phagocytosis and inflammation occurred in macrophages. Additionally, cell–cell communication analysis revealed that FVIO-MHT attenuated the suppression between tumor cells and macrophages by inhibiting phagocytic checkpoint and macrophage migration inhibitory factor signaling pathways. Inhibition of B2m, an anti-phagocytosis checkpoint, could promote macrophage-mediated phagocytosis and significantly inhibit tumor growth. These data emphasize FVIO-MHT may promote the antitumor capabilities of macrophages by alleviating the suppression between tumor cells and macrophages.

CRISPR-Cas9-Based Gene Knockout of Immune Checkpoints in Expanded NK Cells.

Natural killer (NK) cell immunotherapy has emerged as a novel treatment modality for various cancer types, including leukemia. The modulation of inhibitory signaling pathways in T cells and NK cells has been the subject of extensive investigation in both preclinical and clinical settings in recent years. Nonetheless, further research is imperative to optimize antileukemic activities, especially regarding NK-cell-based immunotherapies. The central scientific question of this study pertains to the potential for boosting cytotoxicity in expanded and activated NK cells through the inhibition of inhibitory receptors. To address this question, we employed the CRISPR-Cas9 system to target three distinct inhibitory signaling pathways in NK cells. Specifically, we examined the roles of A2AR within the metabolic purinergic signaling pathway, CBLB as an intracellular regulator in NK cells, and the surface receptors NKG2A and CD96 in enhancing the antileukemic efficacy of NK cells. Following the successful expansion of NK cells, they were transfected with Cas9+sgRNA RNP to knockout A2AR, CBLB, NKG2A, and CD96. The analysis of indel frequencies for all four targets revealed good knockout efficiencies in expanded NK cells, resulting in diminished protein expression as confirmed by flow cytometry and Western blot analysis. Our in vitro killing assays demonstrated that NKG2A and CBLB knockout led to only a marginal improvement in the cytotoxicity of NK cells against AML and B-ALL cells. Furthermore, the antileukemic activity of CD96 knockout NK cells did not yield significant enhancements, and the blockade of A2AR did not result in significant improvement in killing efficiency. In conclusion, our findings suggest that CRISPR-Cas9-based knockout strategies for immune checkpoints might not be sufficient to efficiently boost the antileukemic functions of expanded (and activated) NK cells and, at the same time, point to the need for strong cellular activating signals, as this can be achieved, for example, via transgenic chimeric antigen receptor expression.

Molecular and structural basis of TIGIT: Nectin-4 interaction, a recently discovered pathway crucial for cancer immunotherapy

TIGIT (T cell immunoglobulin and ITIM domain) is an inhibitory receptor expressed on T and NK cells that interact with cell surface glycoprotein belonging to the nectin and nectin-like family of cell adhesion molecules, particularly nectin-2 and nectin-like 5 (PVR). Nectin-4 has been recently identified as a novel ligand for TIGIT and the interaction among them inhibits NK cell cytotoxicity. In this study, biophysical experiments were conducted to decipher the mechanism of this novel interaction, followed by structure-guided mutagenesis studies to map the nectin-4 binding interface on TIGIT. Using surface plasmon resonance, we deduced that TIGIT recognizes the membrane distal ectodomain of nectin-4 and the interaction is weaker than the well-characterized TIGIT: nectin-2 interaction. Deciphering the molecular basis of this newly identified interaction between TIGIT and nectin-4 will provide us important insight into the manipulation of this inhibitory signaling pathway, especially targeting cancer cells overexpressing nectin-4 that evade the immune surveillance of the body.

Restraint stress promotes monobenzone-induced depigmentation in mice via the activation of glucocorticoid receptor/macrophage migration inhibitory factor signaling pathway

Psychological stress triggers onset and development of vitiligo in humans. However, the mechanism of psychological stress on vitiligo remains unclear. The study aims to investigate whether psychological stress promotes vitiligo and explore the underlying mechanism. A depigmentation mouse model induced by applying a skin-bleaching reagent monobenzone to dorsal skin and an in vitro HaCaT keratinocyte death model induced by monobenzone were employed to explore the effect of restraint stress, which mimics psychological stress, on depigmentation. The results indicated that restraint stress promoted vitiligo-related depigmentation, vacuolisation, spongiosis, CD8+ T lymphocyte infiltration, and loss of melanocytes in the skin. Restraint stress activated cutaneous NLR family containing pyrin domain protein 3 (NLRP3) inflammasome. In addition, restraint stress aggravated anxiety-like behaviors and increased levels of macrophage migration inhibitory factor (MIF) and corticosterone in the circulation, accompanied with decreasing the expression of cutaneous 8-oxoguanine DNA glycosylase (OGG1) in depigmentation mice. In vitro experiments demonstrated that activation of glucocorticoid receptor (GR) by cortisol upregulated NLRP3 expression dependent on MIF, and directly decreased the transcription of OGG1. Blockade of MIF reversed the NLRP3 signal in restraint stress-induced depigmentation mice. In conclusion, restraint stress promotes vitiligo-related depigmentation in mice via the activation of GR/MIF signaling pathway. The findings provide a theoretical basis for prevention and treatments of vitiligo with therapies of targeting GR, MIF, and OGG1.

Tackling of Immunorefractory Tumors by Targeting Alternative Immune Checkpoints.

Physiologically, well known or traditional immune checkpoints (ICs), such as CTLA-4 and PD-1, are in place to promote tolerance to self-antigens and prevent generation of autoimmunity. In cancer, the ICs are effectively engaged by the tumor cells or stromal ells from the tumor microenvironment through expression of cognate ligands for the ICs present on the cell surface of CD8+ T lymphocytes. The ligation of ICs on CD8+ T lymphocytes triggers inhibitory signaling pathways, leading to quiescence or an exhaustion of CD8+ T lymphocytes. This results in failure of immunotherapy. To overcome this, several FDA-approved therapeutic antibodies are available, but the clinical outcome is quite variable due to the resistance encountered through upregulated expression of alternate ICs such as VISTA, LAG-3, TIGIT and TIM-3. This review focuses on the roles played by the traditional as well as alternate ICs and the contribution of associated signaling pathways in generating such resistance to immunotherapy. Combinatorial targeting of traditional and alternate ICs might be beneficial for immune-refractory tumors.

The HDAC inhibitor domatinostat induces type I interferon α in Merkel cell carcinoma by HES1 repression

BackgroundClass I selective histone deacetylase inhibitors (HDACi) have been previously demonstrated to not only increase major histocompatibility complex class I surface expression in Merkel cell carcinoma (MCC) cells by restoring the antigen processing and presentation machinery, but also exert anti-tumoral effect by inducing apoptosis. Both phenomena could be due to induction of type I interferons (IFN), as has been described for HDACi. However, the mechanism of IFN induction under HDACi is not fully understood because the expression of IFNs is regulated by both activating and inhibitory signaling pathways. Our own preliminary observations suggest that this may be caused by suppression of HES1.MethodsThe effect of the class I selective HDACi domatinostat and IFNα on cell viability and the apoptosis of MCPyV-positive (WaGa, MKL-1) and -negative (UM-MCC 34) MCC cell lines, as well as, primary fibroblasts were assessed by colorimetric methods or measuring mitochondrial membrane potential and intracellular caspase-3/7, respectively. Next, the impact of domatinostat on IFNA and HES1 mRNA expression was measured by RT-qPCR; intracellular IFNα production was detected by flow cytometry. To confirm that the expression of IFNα induced by HDACi was due to the suppression of HES1, it was silenced by RNA interference and then mRNA expression of IFNA and IFN-stimulated genes was assessed.ResultsOur studies show that the previously reported reduction in viability of MCC cell lines after inhibition of HDAC by domatinostat is accompanied by an increase in IFNα expression, both of mRNA and at the protein level. We confirmed that treatment of MCC cells with external IFNα inhibited their proliferation and induced apoptosis. Re-analysis of existing single-cell RNA sequencing data indicated that induction of IFNα by domatinostat occurs through repression of HES1, a transcriptional inhibitor of IFNA; this was confirmed by RT-qPCR. Finally, siRNA-mediated silencing of HES1 in the MCC cell line WaGa not only increased mRNA expression of IFNA and IFN-stimulated genes but also decreased cell viability.ConclusionOur results demonstrate that the direct anti-tumor effect of HDACi domatinostat on MCC cells is at least in part mediated via decreased HES1 expression allowing the induction of IFNα, which in turn causes apoptosis.