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The sialyltransferase ST6GAL1 that adds α2–6 linked sialic acids to N-glycans of cell surface and secreted glycoproteins is prominently associated with many human cancers. Tumor-native ST6GAL1 promotes tumor cell behaviors such as invasion and resistance to cell stress and chemo- and radio-treatments. Canonically, ST6GAL1 resides in the intracellular secretory apparatus and glycosylates nascent glycoproteins in biosynthetic transit. However, ST6GAL1 is also released into the extracellular milieu and extracellularly remodels cell surface and secreted glycans. The impact of this non-canonical extrinsic mechanism of ST6GAL1 on tumor cell pathobiology is not known. We hypothesize that ST6GAL1 action is the combined effect of natively expressed sialyltransferase acting cell-autonomously within the ER-Golgi complex and sialyltransferase from extracellular origins acting extrinsically to remodel cell-surface glycans. We found that shRNA knockdown of intrinsic ST6GAL1 expression resulted in decreased ST6GAL1 cargo in the exosome-like vesicles as well as decreased breast tumor cell growth and invasive behavior in 3D in vitro cultures. Extracellular ST6GAL1, present in cancer exosomes or the freely soluble recombinant sialyltransferase, compensates for insufficient intrinsic ST6GAL1 by boosting cancer cell proliferation and increasing invasiveness. Moreover, we present evidence supporting the existence novel but yet uncharacterized cofactors in the exosome-like particles that potently amplify extrinsic ST6GAL1 action, highlighting a previously unknown mechanism linking this enzyme and cancer pathobiology. Our data indicate that extracellular ST6GAL1 from remote sources can compensate for cellular ST6GAL1-mediated aggressive tumor cell proliferation and invasive behavior and has great clinical potential for extracellular ST6GAL1 as these molecules are in the extracellular space should be easily accessible targets.

Background Tumor-associated macrophages (TAM) are known to facilitate colorectal cancer (CRC) growth. High macrophage infiltration in thymidine phosphorylase (TYMP) expressing CRC may correspond to poor prognosis. The prognostic impact of the expression CD163, a receptor associated with TAM, and TYMP in stroma, respectively, tumor tissue is not yet established. The aim of this study was to identify the potential associations between TYMP and CD163 expression levels and relapse-free survival (RFS) of patients with stage II CRC, and if microdissection is of importance. Methods Stage II CRC patients, radically resected with relapse (n = 104), were matched to patients with a 5-year relapse-free follow-up (n = 206). Gene expression of TYMP and CD163 was analyzed in snap-frozen tumor tissues and in microdissected formalin-fixed tumor tissues separated into tumor epithelium and stroma. Results TYMP expression was high in poorly differentiated tumors, right-sided CRC, and tumors with high microsatellite instability CD163-expressing macrophages near tumor epithelial cells had high expression in poorly differentiated and T4 tumors. High TYMP expression in tumor epithelial cells was in the multivariate analyses associated with shorter relapse-free survival (hazard ratio 1.66; 95% confidence interval: 1.09–2.56; p Conclusions TYMP expression in tumor epithelial cells was associated with RFS and emphasizes the need for tissue microdissection. Additional studies are needed to establish whether TYMP and CD163 could add clinically relevant information to identify high-risk stage II patients that could benefit from adjuvant chemotherapy.

Melanoma is responsible for 90% of skin cancer-related deaths. Major therapeutic advances have led to a considerable improvement in the prognosis of patients, with the development of targeted therapies (BRAF or MEK inhibitors) and immunotherapy (anti-CTLA-4 or -PD-1 antibodies). However, the tumor constitutes an immunosuppressive microenvironment that prevents the therapeutic efficacy and/or promotes the development of secondary resistances. CD160 is an activating NK-cell receptor initially described as delineating the NK and CD8+T-cell cytotoxic populations. Three forms of CD160 have been described: (1) the GPI isoform, constitutively expressed and involved in the initiation of NK-cells' cytotoxic activity, (2) the transmembrane isoform, neo-synthesized upon cell activation, allowing the amplification of NK cells' cytotoxic functions and (3) the soluble form, generated after cleavage of the GPI isoform, which presents an immuno-suppressive activity. By performing immunohistochemistry analyses, we observed a strong expression of CD160 at the primary cutaneous tumor site of melanoma patients. We further demonstrated that melanoma cells express CD160-GPI isoform and constitutively release the soluble form (sCD160) into the tumor environment. sCD160 was shown to inhibit the cytotoxic activity of NK-cells towards their target cells. In addition, it was found in the serum of melanoma patients and associated with increased tumor dissemination. Altogether these results support a role for sCD160 in the mechanisms leading to the inhibition of anti-tumor response and immune surveillance in melanoma.

The innate immune system of mammalian cells is the first line of defense against pathogenic microorganisms. Phagocytes, which play the central role in this system, engulf microorganisms by a mechanism that involves pattern recognition receptors on their own surface and pathogen-associated molecular patterns (PAMPs) expressed by the microorganism. Components of PAMPs include glycans (polysaccharides) and glycoconjugates (carbohydrates covalently linked to other biological molecules). Pathogenic microorganisms display specific binding affinity to various types of glycosphingolipids (sphingosine-containing glycolipids; GSLs), and GSLs are involved in host-pathogen interactions. We observed that lactosylceramide (LacCer), a neutral GSL, binds directly to certain pathogen-specific molecules (e.g., Candida albicans-derived β-glucans, mycobacterial lipoarabinomannan) via carbohydrate-carbohydrate interaction. LacCer is expressed highly on human neutrophils, and forms membrane microdomains. Such LacCer-enriched microdomains mediate several important neutrophil functions, including chemotaxis, phagocytosis, and superoxide generation. Human neutrophils phagocytose pathogenic mycobacteria (including Mycobacterium tuberculosis) through carbohydrate-carbohydrate interaction between LacCer on their own surface and mannose-capped lipoarabinomannan on the bacterium. During recognition of pathogen-specific glycans, direct association of LacCer-containing C24 fatty acid chain with Lyn (a Src family kinase) is necessary for signal transduction from the neutrophil exterior to interior. Pathogenic mycobacteria utilize a similar interaction to avoid killing by neutrophils. We describe here the mechanisms whereby LacCer mediates neutrophil immune systems via carbohydrate-carbohydrate interaction.

Similar to hemophagocytic lymphohistiocytosis (HLH), some patients with SARS-CoV-2 have cytokine storm. Serum soluble interleukin-2 receptor (sCD25) and soluble CD163 (sCD163) are potential diagnostic biomarkers for HLH that help in guiding its treatment. This study was the first to investigate serum sCD25 and sCD163 levels in SARS-CoV-2. Serum sCD25 and sCD163 were measured by ELISA in 29 patients with SARS-CoV-2, aged between 2 months and 16 years (13 had COVID-19 and 16 had multisystem inflammatory syndrome in children (MIS-C)), in comparison to 30 age- and sex-matched healthy control children and 10 patients with HLH. Levels of these markers were re-measured in 21 patients with SARS-CoV-2 who were followed up 3 months after recovery. Patients with SARS-CoV-2 had significantly higher serum sCD25 and sCD163 than healthy control children (P P P Conclusion: Serum sCD25 and sCD163 levels were up-regulated in SARS-CoV-2 patients. Serum sCD25 was a good differentiating marker between SARS-CoV-2 and HLH. This initial report requires further studies, on large scales, to investigate the relationship between SARS-CoV-2 and both sCD25 and sCD163, including the disease severity and outcome. The therapeutic role of sCD25 and sCD163 antagonists should also be studied in SARS-CoV-2 patients. What is Known:• Similar to hemophagocytic lymphohistiocytosis (HLH), some patients with COVID-19 have cytokine storm due to excessive pro-inflammatory host response.• Serum soluble interleukin-2 receptor (sCD25) and soluble CD163 (sCD163) are potential diagnostic biomarkers for HLH. Monitoring of serum sCD25 and sCD163 levels can also help in guiding the treatment. What is New:• Serum sCD25 and sCD163 levels are up-regulated in patients with COVID-19, including patients presenting with multisystem inflammatory syndrome in children (MIS-C).• Serum sCD25 is a good differentiating marker between SARS-CoV-2 and HLH.

Cancer is one of the diseases with the highest morbidity and mortality rates worldwide, and its therapeutic options are inadequate. The endothelial glycoprotein, also known as CD105, is a type I transmembrane glycoprotein located on the surface of the cell membranes and it is one of the transforming growth factor-β (TGF-β) receptor complexes. It regulates the responses associated with binding to transforming growth factor β1 egg (Activin-A), bone morphogenetic protein 2 (BMP-2), and bone morphogenetic protein 7 (BMP-7). Additionally, it is involved in the regulation of angiogenesis. This glycoprotein is indispensable in the treatment of tumor angiogenesis, and it also plays a leading role in tumor angiogenesis therapy. Therefore, CD105 is considered to be a novel therapeutic target. In this study, we explored the significance of CD105 in the diagnosis, treatment and prognosis of various tumors, and provided evidence for the effect and mechanism of CD105 on tumors.

The immune checkpoint receptor Lymphocyte activation gene-3 (LAG3) inhibits T cell function upon binding to Major Histocompatibility Complex Class II (MHCII) or Fibrinogen-like 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 structures of human and murine LAG3 ectodomains, revealing a dimeric assembly mediated by immunoglobulin domain 2 (D2). Epitope mapping indicates that a potent LAG3 antagonist antibody blocks interactions with MHCII and FGL1 by binding to a flexible “Loop 2” region in LAG3 domain 1 (D1). We also defined the LAG3:FGL1 interface by mapping mutations onto structures of LAG3 and FGL1, and we established that FGL1 crosslinking induces the formation of higher-order LAG3 oligomers. These insights will guide LAG3-based drug development and implicate ligand-mediated LAG3 clustering as a mechanism for disrupting T cell activation.

The insulin receptor (INSR) mediates insulin signaling to modulate cellular functions. Although INSR is expressed at the blood–brain barrier (BBB), its role in the modulation of BBB function is poorly understood. Therefore, in this study, we aimed to analyze the effect of INSR knockdown on the expression levels of functional proteins at the BBB. We established the INSR-knockdown cell line (shINSR) using human cerebral microvascular endothelial cells (hCMEC/D3). The cellular proteome was analyzed using quantitative proteomics. INSR mRNA and protein expressions were decreased in shINSR cells. The suppression of INSR-mediated signaling in shINSR cells was evaluated. The proteins involved in glycolysis and glycogenolysis were suppressed in shINSR cells. As amyloid-β peptide-related proteins, the expressions of presenilin-1 was increased, and those of the insulin-degrading enzyme and neprilysin were decreased. The expressions of BBB transporters, including the ABCB1/MDR1, ABCG2/BCRP, and SLCO2A1/OATP2A1 were significantly decreased by more than 50% in shINSR cells. The efflux activity of ABCB1/MDR1 was also suppressed. The expressions of the low-density lipoprotein receptor-related protein 1 were significantly increased, and those of the transferrin receptor were significantly decreased in shINSR cells. The expression of claudin-5 was also suppressed in shINSR cells. The present study suggests that INSR-mediated signaling is involved in the regulation of functional protein expression at the BBB and contributes to the maintenance of BBB function. Changes in the expressions of amyloid-β peptide-related proteins may contribute to the development of cerebral amyloid angiopathy via the suppression of INSR-mediated signaling.

Leukocyte immunoglobulin-like receptor B (LILRB), a family of immune checkpoint receptors, contributes to acute myeloid leukemia (AML) development, but the specific mechanisms triggered by activation or inhibition of these immune checkpoints in cancer is largely unknown. Here we demonstrate that the intracellular domain of LILRB3 is constitutively associated with the adaptor protein TRAF2. Activated LILRB3 in AML cells leads to recruitment of cFLIP and subsequent NF-κB upregulation, resulting in enhanced leukemic cell survival and inhibition of T-cell-mediated anti-tumor activity. Hyperactivation of NF-κB induces a negative regulatory feedback loop mediated by A20, which disrupts the interaction of LILRB3 and TRAF2; consequently the SHP-1/2-mediated inhibitory activity of LILRB3 becomes dominant. Finally, we show that blockade of LILRB3 signaling with antagonizing antibodies hampers AML progression. LILRB3 thus exerts context-dependent activating and inhibitory functions, and targeting LILRB3 may become a potential therapeutic strategy for AML treatment. Wu et al. show that LILRB3 modulates AML cell survival and antileukemic T-cell responses through regulation of TRAF2–cFLIP–NF-κB signaling and demonstrate the therapeutic efficacy of LILRB3-blocking antibodies in humanized PDX models in vivo.

Abundant evidence has indicated that the prognosis of cutaneous melanoma (CM) patients is highly complicated by the tumour immune microenvironment. We retrieved the clinical data and gene expression data of CM patients in The Cancer Genome Atlas (TCGA) database for modelling and validation analysis. Based on single-sample gene set enrichment analysis (ssGSEA) and consensus clustering analysis, CM patients were classified into three immune level groups, and the differences in the tumour immune microenvironment and clinical characteristics were evaluated. Seven immune-related CM prognostic molecules, including three mRNAs (SUCO, BTN3A1 and TBC1D2), three lncRNAs (HLA-DQB1-AS1, C9orf139 and C22orf34) and one miRNA (hsa-miR-17-5p), were screened by differential expression analysis, ceRNA network analysis, LASSO Cox regression analysis and univariate Cox regression analysis. Their biological functions were mainly concentrated in the phospholipid metabolic process, transcription regulator complex, protein serine/threonine kinase activity and MAPK signalling pathway. We established a novel prognostic model for CM integrating clinical variables and immune molecules that showed promising predictive performance demonstrated by receiver operating characteristic curves (AUC ≥ 0.74), providing a scientific basis for predicting the prognosis and improving the clinical outcomes of CM patients.

Background RNA N6-methyladenosine (m6A) is involved in mammalian spermatogenesis. In both germ cells and Leydig cells, ALKBH5 regulates spermatogenesis and androgen synthesis in an m6A-dependent manner. However, it is unclear whether ALKBH5 plays a role in testicular Sertoli cells, which constitute the blood–testis barrier (BTB) through cell junctions between adjacent Sertoli cells. Methods ALKBH5 expression in the testes of humans and mice was detected by immunohistochemical staining and immunofluorescence staining. BTB integrity was evaluated by BTB assay. m6A-seq was performed to screen for BTB-related molecules regulated by ALKBH5. m6A immunoprecipitation–quantitative real-time polymerase chain reaction (qPCR), RNA immunoprecipitation–qPCR, western blot, coimmunoprecipitation, and polysome fractionation–qPCR analyses were performed to explore the mechanisms of ALKBH5 in BTB. Transmission electron microscopy was applied to observe the BTB ultrastructure. Results ALKBH5 in Sertoli cells is related to the integrity of the BTB. Subsequently, the m6A level on Cdh2 mRNA, encoding a structural protein N-cadherin in the BTB, was found to be regulated by ALKBH5. IGF2BP1/2/3 complexes and YTHDF1 promoted Cdh2 mRNA translation. In addition, we found that basal endoplasmic specialization, in which N-cadherin is a main structural protein, was severely disordered in the testes of Alkbh5-knockout mice. Conclusions Our study revealed that ALKBH5 regulates BTB integrity via basal endoplasmic specialization by affecting Cdh2 mRNA translation.

BackgroundCD6 is one of many cell surface receptors known to regulate signal transduction upon T cell activation. However, whether CD6 mediates costimulatory or inhibitory signals is controversial. When T cells engage with antigen presenting cells (APCs), CD6 interacts with its ligand CD166 at the cell–cell interface while the cytosolic tail assembles a complex signalosome composed of adaptors and effector enzymes, that may either trigger activating signaling cascades, or instead modulate the intensity of signaling. Except for a few cytosolic adaptors that connect different components of the CD6 signalosome, very little is known about the mechanistic effects of the cytosolic effectors that bind CD6.MethodsJurkat model T cells were transfected to express wild-type (WT) CD6, or a cytoplasmic truncation, signaling-disabled mutant, CD6Δcyt. The two resulting cell lines were directly activated by superantigen (sAg)-loaded Raji cells, used as APCs, to assess the net signaling function of CD6. The Jurkat cell lines were further adapted to express a FRET-based unimolecular HRas biosensor that reported the activity of this crucial GTPase at the immunological synapse.ResultsWe show that deletion of the cytosolic tail of CD6 enhances T-cell responses, indicating that CD6 restrains T-cell activation. One component of the CD6-associated inhibitory apparatus was found to be the GTPase activating protein of Ras (RasGAP), that we show to associate with CD6 in a phosphorylation-dependent manner. The FRET HRas biosensor that we developed was demonstrated to be functional and reporting the activation of the T cell lines. This allowed to determine that the presence of the cytosolic tail of CD6 results in the down-regulation of HRas activity at the immunological synapse, implicating this fundamental GTPase as one of the targets inhibited by CD6.ConclusionsThis study provides the first description of a mechanistic sequence of events underlying the CD6-mediated inhibition of T-cell activation, involving the modulation of the MAPK pathway at several steps, starting with the coupling of RasGAP to the CD6 signalosome, the repression of the activity of Ras, and culminating in the reduction of ERK1/2 phosphorylation and of the expression of the T-cell activation markers CD69 and IL-2R α chain.

Mast cells (MC) are key drivers of allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-6 is an immunoregulatory receptor found on MCs. While it is recognized that engaging Siglecs with antibodies mediates inhibition across immune cells, the mechanisms that govern this agonism are not understood. Here we generated Siglec-6 mAb clones (AK01 to AK18) to better understand Siglec-6-mediated agonism. Siglec-6 mAbs displayed epitope-dependent receptor internalization and inhibitory activity. We identified a Siglec-6 mAb (AK04) that required Fc-mediated interaction for receptor internalization and induced inhibition and antibody-dependent cellular phagocytosis against MCs. AK04-mediated MC inhibition required Siglec-6 immunoreceptor tyrosine-based inhibitory motif (ITIM) and ITIM-like domains and was associated with receptor cluster formation containing inhibitory phosphatases. Treatment of humanized mice with AK04 inhibited systemic anaphylaxis with a single dose and reduced MCs with chronic dosing. Our findings suggest Siglec-6 activity is epitope dependent and highlight an agonistic Siglec-6 mAb as a potential therapeutic approach in allergic disease.

Background Chimeric antigen receptor (CAR) T-cell therapy using brexucabtagene autoleucel (BA) induces remission in many patients with mantle cell lymphoma (MCL), and BA is the only CAR T-cell therapy approved by the FDA for MCL. However, development of relapses to BA is recognized with poor patient outcomes. Multiple CAR T-cell therapies have been approved for other lymphomas and the resistance mechanisms have been investigated. However, the mechanisms underlying BA relapse in MCL have not been investigated and whether any previously reported resistance mechanisms apply to BA-relapsed patients with MCL is unknown. Methods To interrogate BA resistance mechanisms in MCL, we performed single-cell RNA sequencing on 39 longitudinally collected samples from 15 BA-treated patients, and multiplex cytokine profiling on 80 serial samples from 20 patients. Results We demonstrate that after BA relapse, the proportion of T cells, especially cytotoxic T cells (CTLs), decreased among non-tumor cells, while the proportion of myeloid cells correspondingly increased. TIGIT, LAG3, and CD96 were the predominant checkpoint molecules expressed on exhausted T cells and CTLs; only TIGIT was significantly increased after relapse. CTLs expanded during remission, and then contracted during relapse with upregulated TIGIT expression. Tumor cells also acquired TIGIT expression after relapse, leading to the enhanced interaction of tumor cell TIGIT with monocyte CD155/PVR. In myeloid cells, post-relapse HLA-II expression was reduced relative to pretreatment and during remission. Myeloid-derived suppressor cells (MDSCs) were enriched after relapse with elevated expression of activation markers, including CLU (clusterin) and VCAN (versican). Extracellular chemokines (CCL4, CXCL9, CXCL13), soluble checkpoint inhibitors (sPD-L1, sTIM3, s4-1BB), and soluble receptors (sIL-2R, sTNFRII) were decreased during remission but elevated after relapse. Conclusions Our data demonstrate that multiple tumor-intrinsic and -extrinsic factors are associated with T-cell suppression and BA relapse. Among these, TIGIT appears to be the central player given its elevated expression after BA relapse in not only CTLs but also MCL cells. The acquisition of TIGIT expression on tumor cells is MCL-specific and has not been reported in other CAR T-treated diseases. Together, our data suggest that co-targeting TIGIT may prevent CAR T relapses and thus promote long-term progression-free survival in MCL patients.

Background: while improvements in immunoradiotherapy have significantly improved outcomes for cancer patients, this treatment approach has nevertheless proven ineffective at controlling the majority of malignancies. One of the mechanisms of resistance to immunoradiotherapy is that immune cells may be suppressed via the myriad of different immune checkpoint receptors. Therefore, simultaneous blockade of multiple immune checkpoint receptors may enhance the treatment efficacy of immunoradiotherapy. Methods: we combined NBTXR3-enhanced localized radiation with the simultaneous blockade of three different checkpoint receptors: PD1, LAG3, and TIGIT, and tested the treatment efficacy in an anti-PD1-resistant lung cancer model in mice. 129 Sv/Ev mice were inoculated with fifty thousand alpha PD1-resistant 344SQR cells in the right leg on day 0 to establish primary tumors and with the same number of cells in the left leg on day 4 to establish the secondary tumors. NBTXR3 was intratumorally injected into the primary tumors on day 7, which were irradiated with 12 Gy on days 8, 9, and 10. Anti-PD1 (200 mu g), alpha LAG3 (200 mu g), and alpha TIGIT (200 mu g) were given to mice by intraperitoneal injections on days 5, 8, 11, 14, 21, 28, 35, and 42. Results: this nanoparticle-mediated combination therapy is effective at controlling the growth of irradiated and distant unirradiated tumors, enhancing animal survival, and is the only one that led to the destruction of both tumors in approximately 30% of the treated mice. Corresponding with this improved response is robust activation of the immune response, as manifested by increased numbers of immune cells along with a transcriptional signature of both innate and adaptive immunity within the tumor. Furthermore, mice treated with this combinatorial therapy display immunological memory response when rechallenged by the same cancer cells, preventing tumor engraftment. Conclusion: our results strongly attest to the efficacy and validity of combining nanoparticle-enhanced radio-therapy and simultaneous blockade of multiple immune checkpoint receptors and provide a pre-clinical rationale for investigating its translation into human patients., This work was supported by Cancer Center Support (Core) Grant CA016672 to The University of Texas MD Anderson Cancer Center; the Goodwin family research fund; the family of M. Adnan Hamed, and the Orr Family Foundation to MD Anderson Cancer Center's Thoracic Radiation Oncology program; an MD Anderson Knowledge Gap award; Nanobiotix.

Inborn errors of immunity are known to cause not only immunodeficiencies and allergies but also autoimmunity. Leukocyte immunoglobulin-like receptor B1 (LILRB1) is a receptor on leukocytes playing a role in regulating immune responses. No phenotypes have been reported to be caused by germline mutations in LILRB1. We aimed to identify the causative variant in a three-generation family with nine members suffering from one of the three autoimmune diseases—Graves’ disease, Hashimoto's thyroiditis, or systemic lupus erythematosus. Whole-genome linkage study revealed a locus on chromosome 19q13.4 with the maximum LOD score of 2.71. Whole-exome sequencing identified a heterozygous missense variant, c.479G > A (p. G160E) in LILRB1, located within the chromosomal-linked region, in all nine affected members. The variant has never been previously reported. Jurkat cells transfected with the mutant LILRB1, compared with those with the wild-type LILRB1, showed decreased phosphorylation of both LILRB1 and its downstream protein, SHP-1. Flow cytometry was used to study immunophenotype and revealed that LILRB1 was significantly lower on the surface of activated regulatory T lymphocytes (Treg) cells of patients. Single-cell RNA sequencing showed substantially increased M1-like monocytes in peripheral blood mononuclear cells of affected individuals. This study, for the first time, implicates LILRB1 as a new disease gene for autoimmunity.

Background Gastric cancer is a common and lethal human malignancy worldwide and cancer cell metastasis is the leading cause of cancer-related mortality. MICAL2, a flavoprotein monooxygenase, is an important regulator of epithelial-to-mesenchymal transition. The aim of this study was to explore the effects of MICAL2 on gastric cancer cell migration and determine the underlying molecular mechanisms. Methods Cell migration was examined by wound healing and transwell assays. Changes in E-cadherin/β-catenin signaling were determined by qPCR and analysis of cytoplasmic and nuclear protein fractions. E-cadherin/β-catenin binding was determined by co-immunoprecipitation assays. Cdc42 activity was examined by pulldown assay. Results MICAL2 was highly expressed in gastric cancer tissues. The knockdown of MICAL2 significantly attenuated migratory ability and β-catenin nuclear translocation in gastric cancer cells while LiCl treatment, an inhibitor of GSK3β, reversed these MICAL2 knockdown-induced effects. Meanwhile, E-cadherin expression was markedly enhanced in MICAL2-depleted cells. MICAL2 knockdown led to a significant attenuation of E-cadherin ubiquitination and degradation in a Cdc42-dependent manner, then enhanced E-cadherin/β-catenin binding, and reduced β-catenin nuclear translocation. Conclusions Together, our results indicated that MICAL2 promotes E-cadherin ubiquitination and degradation, leading to enhanced β-catenin signaling via the disruption of the E-cadherin/β-catenin complex and, consequently, the promotion of gastric cell migration.

Minimal residual disease (MRD)-negative status in multiple myeloma (MM) is associated with favorable outcomes. Although EuroFlow next-generation flow (NGF) is a global standard for MRD detection, its operating cost is high. Therefore, it is desirable to develop a less expensive method with equivalent sensitivity to that of EuroFlow-NGF. In this study, we compared the analytical ability of our BML 10-color multiparameter flow cytometry (MFC) to that of EuroFlow-NGF. Bone marrow samples collected from 51 patients with MM were subjected to MRD detection using BML 10-color-MFC and EuroFlow-NGF. Our antibody panel consisted of CD38 multiepitope, CD138, CD45, CD56, CD19, CD27, CD81, CD117, cytoplasmic immunoglobulin (cIg) κ, and cIgλ in a single tube. The median percentages of total plasma cells, as per 10-color-MFC and EuroFlow-NGF, were 0.2148% and 0.2200%, respectively, with a good correlation between the methods (r = 0.950). The median percentages of myeloma cells determined via 10-color-MFC and EuroFlow-NGF were 0.0012% and 0.0007%, respectively, with a strong correlation (r = 0.954). Our 10-color-MFC demonstrated high sensitivity to detect MRD; the results showed a good correlation with those obtained using EuroFlow-NGF. Therefore, our cost-effective single-tube MFC (approximately 100 USD/sample) is a promising alternative method for the detection of MRD in patients with MM.

Alloimmunization is a serious complication in β-thalassemia major patients as a result of repeated blood transfusion. The immune checkpoint receptors play an important role in regulating immune system homeostasis and the function of the immune cells. This study aimed to evaluate the expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), and T-cell immunoglobulin and mucin domain-containing protein-3 (TIM-3) immune checkpoint molecules in β-thalassemia major patients with and without alloantibody. For this purpose, 68 β-thalassemia major patients with (34 patients) and without (34 patients) alloantibody as well as 20 healthy controls were enrolled. The expression of these genes was evaluated in different groups of patients by SYBR Green real-time PCR method. Our results showed that the mean expression of LAG-3 was significantly increased in thalassemia patients compared to the control group (*P 0.001). However, there was no significant difference in expression of the CTLA-4 and TIM-3 as well as LAG-3 genes between patients with and without alloantibody (P 0.05). A positive correlation was observed between the level of LAG-3 expression with markers associated with Treg function including FOXP3 and GDF-15 genes in β-thalassemia major patients. Taken together, the LAG-3 molecule might have a more prominent role in the abnormality of the immune system in thalassemia patients especially the function of regulatory T cells (Tregs), prior to the CTLA-4 and TIM-3 genes.

The high cytotoxic activity of Vγ9Vδ2 T lymphocytes against tumor cells makes them useful candidates in anticancer therapies. However, the molecular mechanism of their activation by phosphoantigens (PAgs) is not completely known. Many studies have depicted the mechanism of Vγ9Vδ2 T-cell activation by PAg-sensed accessory cells, such as immune presenting cells or tumor cells. In this study, we demonstrated that pure resting Vγ9Vδ2 T lymphocytes can self-activate through exogenous PAgs, involving their TCR and the butyrophilins BTN3A1 and BTN2A1. This is the first time that these three molecules, concurrently expressed at the plasma membrane of Vγ9Vδ2 T cells, have been shown to be involved together on the same and unique T cell during PAg activation. Moreover, the use of probucol to stimulate the inhibition of this self-activation prompted us to propose that ABCA-1 could be implicated in the transfer of exogenous PAgs inside Vγ9Vδ2 T cells before activating them through membrane clusters formed by γ9TCR, BTN3A1 and BTN2A1. The self-activation of Vγ9Vδ2 T cells, which leads to self-killing, can therefore participate in the failure of γδ T cell-based therapies with exogenous PAgs and should be taken into account.

Integrin CD103 mediates the adhesion and tissue retention of T cells by binding to E-cadherin which is abundant on epithelial cells. Notably, CD103 is highly expressed on CD8 T cells but conspicuously absent on most CD4 T cells. The mechanism controlling such lineage-specific expression of CD103 remains unclear. Using a series of genetically engineered mouse models, here, we demonstrate that the regulatory mechanism of CD103 expression is distinct between CD4 and CD8 T cells, and that the transcription factor Runx3 plays an important but not an essential role in this process. We further found that the availability of integrin β7 which heterodimerizes with CD103 was necessary but also constrained the surface expression of CD103. Notably, the forced surface expression of CD103 did not significantly alter the thymic development of conventional T cells but severely impaired the generation of MHC-II-restricted TCR transgenic T cells, revealing previously unappreciated aspects of CD103 in the selection and maturation of CD4 T cells. Unlike its effect on CD4 T cell development, however, CD103 overexpression did not significantly affect CD4 T cells in peripheral tissues. Moreover, the frequency and number of CD4 T cells in the small intestine epithelium did not increase even though E-cadherin is highly expressed in this tissue. Collectively, these results suggest that most mature CD4 T cells are refractory to the effects of CD103 expression, and that they presumably utilize CD103-independent pathways to control their tissue retention and residency.

The objective of this study is to develop a simple protocol to isolate and characterise small extracellular vesicles (sEVs) from human umbilical cord-derived MSCs (hUC-MSCs). hUC-MSCs were characterised through analysis of morphology, immunophenotyping and multidifferentiation ability. SEVs were successfully isolated by ultrafiltration from the conditioned medium of hUC-MSCs. The sEVs' size distribution, intensity within a specific surface marker population were measured with zetasizer or nanoparticle tracking analysis. The expression of surface and internal markers of sEVs was also assessed by western blotting. Morphology of hUC-MSCs displayed as spindle-shaped, fibroblast-like adherent cells. Phenotypic analysis by flow cytometry revealed that hUC-MSCs expressed MSC surface marker, including CD90, CD73, CD105, CD44 and exhibited the capacity for osteogenic, adipogenic and chondrogenic differentiation. Populations of sEVs with CD9, CD63 and CD81 positive were detected with size distribution in the diameter of 63.2 to 162.5 nm. Typical sEVs biomarkers such as CD9, CD63, CD81, HSP70 and TSG101 were also detected with western blotting. Our study showed that sEVs from hUC-MSCs conditioned medium were successfully isolated and characterised. Downstream application of hUC-MSCs-sEVs will be further explored.

Metastasis is the main cause of poor prognosis in the advanced prostate cancer in clinic. Accumulating evidences have proposed that cell motility greatly contributes to the multiple steps of the metastatic process. MicroRNA-145 (miR-145) has been found to be downregulated in prostate cancer and serve as a putative tumor suppressor via decrease of cell growth and augmentation of cell death; however, the effects and the underlying mechanisms of miR-145 in prostate cancer cell motility have not been completely clarified. In the current study, we first demonstrated that miR-145 exerted inhibitory effects on the aggressive phenotype of the prostate cancer cells. Based on the bioinformatics analysis of the putative target genes of miR-145, we further experimentally identified a novel mechanism of miR-145 suppressing the aggressive phenotype of prostate cancer cells via directly targeting cadherin-2 (CDH2) protein translation. Re-expression of CDH2 could rescue miR-145-triggered cell migration and invasion defects. Our results suggested that miR-145 suppressed the motility of prostate cancer cells via post-transcriptional downregulation of CDH2 expression, and miR-145-CDH2 pair might serve as a potential target for intervention of prostate cancer metastasis.

Background Tumor-associated neutrophils (TANs) have recently been identified as a relevant component of the tumor microenvironment (TME) in solid tumors. Within the TME TANs mediate either tumor-promoting or tumor-inhibiting activities. So far, their prognostic relevance remains to be determined. This study aims to evaluate the prognostic relevance of TANs in different molecular subtypes of gastric and esophageal adenocarcinoma. Methods We analyzed a total of 1118 Caucasian patients divided into gastric adenocarcinoma (n = 458) and esophageal adenocarcinoma cohort (n = 660) of primarily resected and neoadjuvant-treated individuals. The amount of CD66b + TANs in the tumor stroma was determined using quantitative image analysis and correlated to both molecular, as well as clinical data. Results An accumulation of TANs in the tumor stroma of gastric carcinomas was associated to a significant favorable prognosis (p = 0.026). A subgroup analysis showed that this effect was primarily related to the molecular chromosomal instable subtype (CIN) of gastric carcinomas (p = 0.010). This was only observed in female patients (p = 0.014) but not in male patients (p = 0.315). The same sex-specific effect could be confirmed in adenocarcinomas of the esophagus (p = 0.027), as well as in female individuals after receiving neoadjuvant therapy (p = 0.034). Conclusions Together, we show a sex-specific prognostic effect of TANs in gastric cancer within a Caucasian cohort. For the first time, we showed that this sex-specific prognostic effect of TANs can also be seen in esophageal cancer.

Increased circulating intercellular adhesion molecule 3 (ICAM3) is associated with a risk of acute ischemic stroke. This study aims to investigate the association of serum levels of ICAM3 with the severity and short-term outcomes of ischemic stroke. This study recruited 152 stroke patients with supratentorial cerebral infarcts and 133 healthy controls. Patients were followed up for 2 weeks since admission for observation of functional outcomes. Serum ICAM3 concentrations at baseline were determined by Elisa. Serum ICAM3 concentrations were higher in stroke patients than in healthy controls. Serum concentrations of ICAM3 were not associated with stroke severity at baseline, as reflected by NIH Stroke Scale (NIHSS) and infarction volume. However, serum ICAM3 levels were positively associated with mRS scores at 2 weeks since admission. Furthermore, regression analyses found that increased serum ICAM3 levels were associated with worse short-term functional outcomes of stroke. These findings imply that circulating ICAM3 might be a potential short-term prognostic biomarker for acute ischemic stroke.

The clinical performance of the therapeutic monoclonal antibody trastuzumab in the treatment of ErbB2-positive unresectable gastric cancer (GC) is severely hampered by the emergence of molecular resistance. Trastuzumab’s target epitope is localized within the extracellular domain of the oncogenic cell surface receptor tyrosine kinase (RTK) ErbB2, which is known to undergo extensive N-linked glycosylation. However, the site-specific glycan repertoire of ErbB2, as well as the detailed molecular mechanisms through which specific aberrant glycan signatures functionally impact the malignant features of ErbB2-addicted GC cells, including the acquisition of trastuzumab resistance, remain elusive. Here, we demonstrate that ErbB2 is modified with both α2,6- and α2,3-sialylated glycan structures in GC clinical specimens. In-depth mass spectrometry-based glycomic and glycoproteomic analysis of ErbB2’s ectodomain disclosed a site-specific glycosylation profile in GC cells, in which the ST6Gal1 sialyltransferase specifically targets ErbB2 N-glycosylation sites occurring within the receptor’s trastuzumab-binding domain. Abrogation of ST6Gal1 expression reshaped the cellular and ErbB2-specific glycomes, expanded the cellular half-life of the ErbB2 receptor, and sensitized ErbB2-dependent GC cells to trastuzumab-induced cytotoxicity through the stabilization of ErbB dimers at the cell membrane, and the decreased activation of both ErbB2 and EGFR RTKs. Overall, our data demonstrates that ST6Gal1-mediated aberrant α2,6-sialylation actively tunes the resistance of ErbB2-driven GC cells to trastuzumab.

The genetic basis of brain tumor development is poorly understood. Here, leukocyte DNA of 21 patients from 15 families with ≥ 2 glioma cases each was analyzed by whole-genome or targeted sequencing. As a result, we identified two families with rare germline variants, p.(A592T) or p.(A817V), in the E-cadherin gene CDH1 that co-segregate with the tumor phenotype, consisting primarily of oligodendrogliomas, WHO grade II/III, IDH-mutant, 1p/19q-codeleted (ODs). Rare CDH1 variants, previously shown to predispose to gastric and breast cancer, were significantly overrepresented in these glioma families (13.3%) versus controls (1.7%). In 68 individuals from 28 gastric cancer families with pathogenic CDH1 germline variants, brain tumors, including a pituitary adenoma, were observed in three cases (4.4%), a significantly higher prevalence than in the general population (0.2%). Furthermore, rare CDH1 variants were identified in tumor DNA of 6/99 (6%) ODs. CDH1 expression was detected in undifferentiated and differentiating oligodendroglial cells isolated from rat brain. Functional studies using CRISPR/Cas9-mediated knock-in or stably transfected cell models demonstrated that the identified CDH1 germline variants affect cell membrane expression, cell migration and aggregation. E-cadherin ectodomain containing variant p.(A592T) had an increased intramolecular flexibility in a molecular dynamics simulation model. E-cadherin harboring intracellular variant p.(A817V) showed reduced β-catenin binding resulting in increased cytosolic and nuclear β-catenin levels reverted by treatment with the MAPK interacting serine/threonine kinase 1 inhibitor CGP 57380. Our data provide evidence for a role of deactivating CDH1 variants in the risk and tumorigenesis of neuroepithelial and epithelial brain tumors, particularly ODs, possibly via WNT/β-catenin signaling.

BACKGROUND: CDH1 pathogenic variants confer a markedly elevated lifetime risk of developing diffuse gastric cancer (DGC) and lobular breast cancer (LBC). The aim of this study was to evaluate the prevalence and clinical impact of CDH1 pathogenic variants in the unselected and ancestrally diverse BioMe Biobank. METHODS: We evaluated exome sequence data from 30,223 adult BioMe participants to identify CDH1 positive individuals, defined as those harboring a variant previously classified as pathogenic or likely pathogenic (P/LP) or a predicted loss-of-function variant in CDH1. We reviewed electronic health records and BioMe enrollment surveys for personal and family history of malignancy and evidence of prior clinical genetic testing. RESULTS: Using a genomics-first approach, we identified 6 CDH1 positive individuals in BioMe (~1 in 5,000). CDH1 positive individuals had a median age of 42 years (range 35 to 62 years), all were non-European by self-report, and one was female. None had evidence of either a personal or family history of DGC or LBC. CONCLUSION: Our findings suggest a low risk of DGC and LBC in unselected patients harboring a pathogenic variant in CDH1. Knowledge of CDH1-related cancer risk in individuals with no personal or family history may better inform surveillance and prophylactic measures.

Histamine H2 receptor (HRH2) is closely associated with the development of cardiovascular and cerebrovascular diseases. However, systematic Hrh2 knockout mice did not exactly reflect the HRH2 function in specific cell or tissue types. To better understand the physiological and pathophysiological functions of endothelial HRH2, this study constructed a targeting vector that contained loxp sites flanking the ATG start codon located in Hrh2 exon 2 upstream and a neomycin (Neo) resistance gene flanked by self-deletion anchor sites within the mouse Hrh2 allele. The targeting vector was then electroporated into C57BL/6J embryonic stem (ES) cells, and positively targeted ES cell clones were micoinjected into C57BL/6J blastocysts, which were implanted into pseudopregnant females to obtain chimeric mice. The F1 generation of Hrh2flox/+ mice was generated via crossing chimeric mice with wild-type mice to excise Neo. We also successfully generated endothelial cell-specific knockout (ECKO) mice by crossing Hrh2flox/+ mice with Cdh5-Cre mice that specifically express Cre in endothelial cells and identified that Hrh2 deletion was only observed in endothelial cells. Hrh2flox/+ and Hrh2ECKO mice were normal, healthy and fertile and did not display any obvious abnormalities. These novel animal models will create new prospects for exploring roles of HRH2 during the development and treatment of related diseases.

Glomeruli and renal tubule injury in chronic kidney disease (CKD) is reported to involve induction of macrophage activation through the CCL2/CCR2 axis. The effects of inhibitors of the CCL2/CCR2 axis, such as anti-CCL2 antibody and CCR2 antagonist, on kidney function in animal models or humans with kidney dysfunction have been demonstrated. The N-terminal glutamine on immature CCL2 is replaced with pyroglutamate (pE) by glutaminyl cyclase (QC) and isoQC. pE-CCL2 is stable and resistant to peptidases. We hypothesized that inhibiting QC/isoQC activity would lead to the degradation of CCL2, thereby ameliorating CKD and reducing kidney inflammation. To test this hypothesis, we investigated the renoprotective properties of the QC/isoQC inhibitor PQ529 in anti-glomerular basement membrane (GBM) antibody–induced glomerulonephritis Wistar Kyoto (WKY) rats. Three-week repeated administration of PQ529 (30 and 100 mg/kg, twice daily) significantly reduced the serum and urine CCL2 and urinary protein excretion in a dose-dependent manner. Correlations between the urinary protein level and serum or urinary CCL2 levels were confirmed in tested animals. Repeated administration of PQ529 significantly reduced the expression of CD68, a macrophage marker, in the kidney cortex and mononuclear infiltration into the tubulointerstitium. In addition, decreased levels of urinary KIM-1, β2 microglobulin, and clusterin were detected, suggesting the inhibition of inflammation in both the proximal and distal tubules. These results suggest that PQ529 suppresses the progression of inflammation-induced renal dysfunction by inhibiting the CCL2/CCR2 axis. Inhibition of QC/isoQC may thus be a viable alternative therapeutic approach for treating glomerulonephritis and CKD patients. Supplementary Information The online version contains supplementary material available at 10.1007/s00210-020-02013-x.

The COVID-19 pandemic poses a major burden on healthcare and economic systems across the globe. Even though a majority of the population develops only minor symptoms upon SARS-CoV-2 infection, a significant number are hospitalized at intensive care units (ICU) requiring critical care. While insights into the early stages of the disease are rapidly expanding, the dynamic immunological processes occurring in critically ill patients throughout their recovery at ICU are far less understood. Here, we have analysed whole blood samples serially collected from 40 surviving COVID-19 patients throughout their recovery in ICU using high-dimensional cytometry by time-of-flight (CyTOF) and cytokine multiplexing. Based on the neutrophil-to-lymphocyte ratio (NLR), we defined four sequential immunotypes during recovery that correlated to various clinical parameters, including the level of respiratory support at concomitant sampling times. We identified classical monocytes as the first immune cell type to recover by restoration of HLA-DR-positivity and the reduction of immunosuppressive CD163 + monocytes, followed by the recovery of CD8 + and CD4 + T cell and non-classical monocyte populations. The identified immunotypes also correlated to aberrant cytokine and acute-phase reactant levels. Finally, integrative analysis of cytokines and immune cell profiles showed a shift from an initially dysregulated immune response to a more coordinated immunogenic interplay, highlighting the importance of longitudinal sampling to understand the pathophysiology underlying recovery from severe COVID-19. Supplementary Information The online version contains supplementary material available at 10.1007/s00018-021-03808-8.

Purpose Immune modulatory therapies including immune checkpoint inhibitors have so far failed to result in clinically meaningful efficacy in glioma. We aimed to investigate lymphocyte activation gene 3 (LAG-3), an inhibitory receptor on immune cells and target of second-generation immune checkpoint inhibitors, in glioma. Methods 97 patients with diffuse glioma (68 with glioblastoma, 29 with WHO grade II-III glioma) were identified from the Neuro-Biobank of the Medical University of Vienna. LAG-3 expression in the inflammatory microenvironment was assessed by immunohistochemistry (monoclonal anti-LAG-3 antibody, clone 17B4) and correlated to CD3+ , CD8+ , CD20+ and PD-1+ tumor-infiltrating lymphocytes (TILs) and PD-L1 expression on tumor cells. Results LAG-3+ TILs could be observed in 10/97 (10.3%) IDH-wildtype samples and in none of the included IDH-mutant glioma samples (p = 0.057). Further, LAG-3+ TILs were only observed in WHO grade IV glioblastoma, while none of the investigated WHO grade II–III glioma presented with LAG-3+ TILs (p = 0.03). No association of O6-methylguanine-DNA-methyltransferase (MGMT) promoter methylation and presence of LAG-3+ TILs was observed (p = 0.726). LAG-3 expression was associated with the presence of CD3+ (p = 0.029), CD8+ (p = 0.001), PD-1+ (p Conclusions LAG-3 is only rarely expressed on TILs in IDH-wildtype glioma and associated with active inflammatory milieu as defined by higher TIL density. Immune microenvironment diversity should be considered in the design of future immunotherapy trials in glioma.

Activated EGFR signalling drives tumorigenicity in 50% of glioblastoma (GBM). However, EGFR-targeting therapy has proven ineffective in treating patients with GBM, indicating that there is redundant EGFR activation. Circular RNAs are covalently closed RNA transcripts that are involved in various physiological and pathological processes. Herein, we report an additional activation mechanism of EGFR signalling in GBM by an undescribed secretory E-cadherin protein variant (C-E-Cad) encoded by a circular E-cadherin (circ-E-Cad) RNA through multiple-round open reading frame translation. C-E-Cad is overexpressed in GBM and promotes glioma stem cell tumorigenicity. C-E-Cad activates EGFR independent of EGF through association with the EGFR CR2 domain using a unique 14-amino-acid carboxy terminus, thereby maintaining glioma stem cell tumorigenicity. Notably, inhibition of C-E-Cad markedly enhances the antitumour activity of therapeutic anti-EGFR strategies in GBM. Our results uncover a critical role of C-E-Cad in stimulating EGFR signalling and provide a promising approach for treating EGFR-driven GBM.

The formation of a functional blood vessel network relies on the ability of endothelial cells (ECs) to dynamically rearrange their adhesive contacts in response to blood flow and guidance cues, such as vascular endothelial growth factor-A (VEGF-A) and class 3 semaphorins (SEMA3s). Neuropilin 1 (NRP1) is essential for blood vessel development, independently of its ligands VEGF-A and SEMA3, through poorly understood mechanisms. Grounding on unbiased proteomic analysis, we report here that NRP1 acts as an endocytic chaperone primarily for adhesion receptors on the surface of unstimulated ECs. NRP1 localizes at adherens junctions (AJs) where, interacting with VE-cadherin, promotes its basal internalization-dependent turnover and favors vascular permeability initiated by histamine in both cultured ECs and mice. We identify a splice variant of tryptophanyl-tRNA synthetase (mini-WARS) as an unconventionally secreted extracellular inhibitory ligand of NRP1 that, by stabilizing it at the AJs, slows down both VE-cadherin turnover and histamine-elicited endothelial leakage. Thus, our work shows a role for NRP1 as a major regulator of AJs plasticity and reveals how mini-WARS acts as a physiological NRP1 inhibitory ligand in the control of VE-cadherin endocytic turnover and vascular permeability. ispartof: NATURE COMMUNICATIONS vol:13 issue:1 ispartof: location:England status: published

Pathological cardiac hypertrophy is an independent risk factor for heart failure and is considered a target for the treatment of heart failure. However, the mechanisms underlying pathological cardiac hypertrophy remain largely unknown. We aimed to investigate the role of angiopoietin-like protein 8 (ANGPTL8) in pathological cardiac hypertrophy. We found that serum ANGPTL8 levels were significantly increased in hypertensive patients with cardiac hypertrophy and in mice with cardiac hypertrophy induced by Ang II or TAC. Furthermore, the secretion of ANGPTL8 from the liver was increased during hypertrophic processes, which were triggered by Ang II. In the Ang II- and transverse aortic constriction (TAC)-induced mouse cardiac hypertrophy model, ANGPTL8 deficiency remarkably accelerated cardiac hypertrophy and fibrosis with deteriorating cardiac dysfunction. Accordingly, both recombinant human full-length ANGPTL8 (rANGPTL8) protein and ANGPTL8 overexpression significantly mitigated Ang II-induced cell enlargement in primary neonatal rat cardiomyocytes (NRCMs) and H9c2 cells. Mechanistically, the antihypertrophic effects of ANGPTL8 depended on inhibiting Akt and GSK-3β activation, and the Akt activator SC-79 abolished the antihypertrophic effects of rANGPTL8 in vitro. Moreover, we demonstrated that ANGPTL8 directly bound to the paired Ig-like receptor PIRB (LILRB3) by RNA-seq and immunoprecipitation-mass screening. Remarkably, the antihypertrophic effects of ANGPTL8 were largely blocked by anti-LILRB3 and siRNA-LILRB3. Our study indicated that ANGPTL8 served as a novel negative regulator of pathological cardiac hypertrophy by binding to LILRB3 (PIRB) and inhibiting Akt/GSK3β activation, suggesting that ANGPTL8 may provide synergistic effects in combination with AT1 blockers and become a therapeutic target for cardiac hypertrophy and heart failure.

EWI2 is a transmembrane immunoglobulin superfamily (IgSF) protein that physically associates with tetraspanins and integrins. It inhibits cancer cells by influencing the interactions among membrane molecules including the tetraspanins and integrins. The present study revealed that, upon EWI2 silencing or ablation, the elevated movement and proliferation of cancer cells in vitro and increased cancer metastatic potential and malignancy in vivo are associated with (i) increases in clustering, endocytosis, and then activation of EGFR and (ii) enhancement of Erk MAP kinase signaling. These changes in signaling make cancer cells (i) undergo partial epithelial-to-mesenchymal (EMT) for more tumor progression and (ii) proliferate faster for better tumor formation. Inhibition of EGFR or Erk kinase can abrogate the cancer cell phenotypes resulting from EWI2 removal. Thus, to inhibit cancer cells, EWI2 prevents EGFR from clustering and endocytosis to restrain its activation and signaling.

Tumor endothelial marker 1 (TEM1) is a transmembrane glycoprotein that appears on mesenchymal lineage-derived cells during embryogenesis, but its expression greatly reduces after birth. Re-upregulation of TEM1 is found in tumor angiogenesis, organ fibrosis and wound healing indicating its potential role in tissue remodeling and repair. The expression level and function of TEM1 in adult heart are unknown. In explanted hearts from heart failure (HF) patients received cardiac transplantation, immunofluorescence staining showed TEM1 was expressed in cardiomyocytes (CMs) and cardiac fibroblasts. Bioinformatics analysis showed TEM1 upregulation in mouse heart after coronary ligation. Cardiac TEM1 expression was reconfirmed in mouse HF induced by coronary ligation or doxorubicin injection. TEM1 expression increased in cultured CMs stimulated with mechanical stretch, doxorubicin and hypoxia. Further studies showed recombinant TEM1 (rTEM1) was a functional protein that influenced cell behaviors of CMs. It directly activated Erk and Akt through interaction with PDGF receptor. TEM1lacZ/lacZ mice had less collagen deposition and worse cardiac function than wild type mice. These results indicate that TEM1 expression increases in the heart after cardiac injury and works as a functional protein that participates in cardiac remodeling.

SummaryNatural killer (NK) cells are innate cytotoxic lymphoid cells (ILCs) involved in the killing of infected and tumor cells. Among human and mouse NK cells from the spleen and blood, we previously identified by single-cell RNA sequencing (scRNAseq) two similar major subsets, NK1 and NK2. Using the same technology, we report here the identification, by single-cell RNA sequencing (scRNAseq), of three NK cell subpopulations in human bone marrow. Pseudotime analysis identified a subset of resident CD56bright NK cells, NK0 cells, as the precursor of both circulating CD56dim NK1-like NK cells and CD56bright NK2-like NK cells in human bone marrow and spleen under physiological conditions. Transcriptomic profiles of bone marrow NK cells from patients with acute myeloid leukemia (AML) exhibited stress-induced repression of NK cell effector functions, highlighting the profound impact of this disease on NK cell heterogeneity. Bone marrow NK cells from AML patients exhibited reduced levels of CD160, but the CD160high group had a significantly higher survival rate.

Parkinson’s disease (PD) and restless legs syndrome/Willis-Ekbom disease (RLS/WED) are both common movement disorders. Based on their clinical overlap, association studies of PD and RLS/WED have been conducted for many years. To investigate whether or not the genetic risk factor of PD was also associated with RLS/WED. We included 102 idiopathic RLS/WED patients and 189 matched controls from southeast China. The clinical data included the International Restless Legs Syndrome Study Group Rating Scale, the subtypes of RLS/WED symptoms (painful or other discomfort), the comorbidities, the pregnancy history of female patients, the Hamilton Depression Scale (HAMD), and the Pittsburgh Sleep Quality Index (PSQI) questionnaire. Risk gene analysis between RLS/WED and control groups including 21 SNPs (single nucleotide polymorphisms) was conducted. Genotyping was done by Sanger sequencing. We found that rs4273468 polymorphism of BST1 gene increased the risk of idiopathic RLS/WED patients in southeastern Chinese population (P =

Background Immunotherapy targeting PD-1 provides a limited survival benefit in patients with unresectable advanced or recurrent gastric cancer (GC). Beside PD-L1, the expression of inhibitory ligands such as CEACAM-1 and LSECtin on GC cells account for this limitation. Here we assessed their expression and immune suppressive effect in GC patients. Methods Using multiplexed immunohistochemistry staining, we evaluated the distribution of different inhibitory ligands, including PD-L1, CEACAM-1, LSECtin, and MHC class II, in 365 GC patients. We analyzed their correlations and overall survival (OS) based on the expression of each inhibitory ligand and the independent prognostic factors that affect OS. Subsequently, we evaluated the additive effect of anti-PD-1 mAb or anti-PD-L1 mAb with/without anti-Lag-3 mAb with/without anti-Tim-3 mAb in cytotoxic assay using tumor-antigen specific CTL clones against GC cell lines. Results Co-expression of the inhibitory ligands for PD-1, Tim-3, and Lag-3 was observed in the largest proportion (34.7%). CEACAM-1, LSECtin, and MHC class II expression showed significant correlation with PD-L1 expression and OS. Multivariable analysis demonstrated that CEACAM-1 low is an independent prognostic factor. Furthermore, combining dual and triple ICIs yielded additive effect on cytotoxicity of CTL clones against each immune inhibitory ligand positive GC cell lines. Conclusions Our findings suggested that the expression of inhibitory ligands for Tim-3 and Lag-3 on GC cells serve as potential biomarkers to predict the response to anti-PD-1 therapy and the combinatorial immunotherapy with ICIs targeting for PD-1, Tim-3, and Lag-3 has a therapeutic potential for GC patients.

Human ovarian follicular fluid (HOFF) contains proteins, extracellular matrixes necessary for growth and maturation of oocytes as well as granulosa cells. Epithelial cells and stem cells can be isolated from HOFF. However, information regarding stem cells derived from HOFF is still lacking. The objectives of the present study were to isolate, characterize, and differentiate cells derived from HOFF. HOFF was collected during the routine aspiration of oocytes in an assisted fertilization program and subjected to cell isolation, characterization, and in vitro culture. After 24 h of culture, different cell morphologies including epithelial-like-, neural-like- and fibroblast-like cells were observed. Immunocytochemistry reveals the expression of pluripotent stem cell markers (OCT4, NANOG, SSEA4), epithelial marker (CK18), FSH- and LH-receptor. For in vitro culture, the isolated cells were continuously cultured in a growth medium; alpha MEM containing 10% FBS and epidermal growth factor (EGF). After 2 weeks of in vitro culture, cells with fibroblast-like morphology dominantly grow in the culture vessels and resemble mesenchymal stem cells (MSCs). HOFF-derived cells exhibited MSC expression of CD44, CD73, CD90, CD105, CD146, and STRO-1, and were capable of differentiation into osteoblasts, chondrocytes, and adipocytes. After induction of neural differentiation, HOFF-derived cells formed spheroidal structures and expressed neural stem cell markers including Nestin, β-tubulin III, and O4. Besides, the oocyte-like structure was observed after prolonged culture of HOFF. In conclusion, cells derived from follicular fluid exhibited stem cell characteristics, which could be useful for regenerative medicine applications and cell-based therapies.

Most ovarian cancers are infiltrated by prognostically relevant activated T cells1–3, yet exhibit low response rates to immune checkpoint inhibitors4. Memory B cell and plasma cell infiltrates have previously been associated with better outcomes in ovarian cancer5,6, but the nature and functional relevance of these responses are controversial. Here, using 3 independent cohorts that in total comprise 534 patients with high-grade serous ovarian cancer, we show that robust, protective humoral responses are dominated by the production of polyclonal IgA, which binds to polymeric IgA receptors that are universally expressed on ovarian cancer cells. Notably, tumour B-cell-derived IgA redirects myeloid cells against extracellular oncogenic drivers, which causes tumour cell death. In addition, IgA transcytosis through malignant epithelial cells elicits transcriptional changes that antagonize the RAS pathway and sensitize tumour cells to cytolytic killing by T cells, which also contributes to hindering malignant progression. Thus, tumour-antigen-specific and -antigen-independent IgA responses antagonize the growth of ovarian cancer by governing coordinated tumour cell, T cell and B cell responses. These findings provide a platform for identifying targets that are spontaneously recognized by intratumoural B-cell-derived antibodies, and suggest that immunotherapies that augment B cell responses may be more effective than approaches that focus on T cells, particularly for malignancies that are resistant to checkpoint inhibitors. In patients with high-grade serous ovarian cancer, robust and protective humoral responses are dominated by B-cell-derived polyclonal IgA that binds to polymeric IgA receptors that are universally expressed on ovarian cancer cells.

B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic malignancy of B-type lymphoid precursor cells. Minimal/measurable residual disease (MRD) is an important prognostic factor for B-ALL relapse. Traditional flow cytometry detection mainly relies on CD19-based gating strategies. However, relapse of CD19-negative B-ALL frequently occurs in patients who receive cellular and targeted therapy. This review will summarize the technical aspects of standard MRD assessment in B-ALL by flow cytometry, and then discuss the challenges of MRD strategies to deal with the scenario of CD19 negative or dim B-ALL relapse.

One of the main challenges in liver cell therapy is the replacement of damaged cells and the induction of a tolerogenic microenvironment to promote graft acceptance by the recipient. Adult-derived human liver stem/progenitor cells (ADHLSCs) are currently evaluated at the clinical levels as a promising pro-regenerative and immune-modulatory tool. The expression profile of several immunological molecules may influence the local immune-inflammatory response and, therefore, modulate the tissue healing process. To increase the quality and safety of ADHLSCs before transplantation requires an appropriate analysis and characterization of their pattern expression of immune-inflammatory-associated molecules., info:eu-repo/semantics/published

Endometriosis is a chronic inflammatory disease. Endometriotic cysts contain hemoglobin (Hb) and infiltrated macrophages, indicating that the metabolism of Hb by macrophages may play an important role in the inflammation of endometriotic cysts. In this study, we investigated the distribution of immune cells and CD163 (Hb receptor)-positive cells in the endometriotic cyst wall using immunohistochemistry. We also examined the role of macrophage activation by Hb on the pathogenesis of endometriotic cysts by measuring the cytokine concentration in the cystic fluids and macrophage-culture supernatant using ELISA. Macrophages were the most prominent immune cells observed in the endometriotic cysts and were differentially distributed in the different histological areas of the cyst wall. The localization of CD163-positive macrophages was restricted to the hemorrhagic and outer areas in the cyst wall. High concentrations of IL-6 and CCL2 were found in the cystic fluids, and inflammatory cytokines (IL-6, TNF-α, and CCL2) were secreted from macrophages on stimulation by Hb. IL-6 is a promotional factor for endometriotic stromal cells and ovarian clear cell carcinoma, the most common histological subtype of endometriosis-related ovarian cancer, hence, the continuous activation of macrophages by Hb could be a potential mechanism underlying endometriosis development and carcinogenesis.

Recent studies have indicated that CD47, interacting with SIRP-α, conveys “don’t eat me” signal in evasion of tumor cells and serves as a potential target for cancer immunotherapy. The purpose of this study was to investigate the clinical correlation of CD47 and uncover prognostic implications of CD47 and CD68 in non-small cell lung cancer (NSCLC). The specimens from 384 patients with completely resected NSCLC were collected for immunohistochemical assays of CD47 and CD68. Cox multivariate proportion hazard analyses were conducted to confirm the independent prognostic value of CD47 and CD68. TCGA database and GSE37745 were used to identify the association between CD47 and immune cells. In 186 pairs of lung cancer and adjacent tissues, the RNA of CD47 was overexpressed in lung cancer tissues (P

Non-small-cell lung cancer (NSCLC) remains the leading cause of cancer-related death worldwide. Accumulating research has highlighted the ability of exosome-encapsulated microRNAs (miRNAs or miRs) as potential circulating biomarkers for lung cancer. The current study aimed to evaluate the clinical significance of serum-derived exosomal miR-let-7e as a biomarker in the metastasis of NSCLC. Initially, the expression of miR-let-7e, SUV39H2, and CDH1 in human NSCLC tissues and exosomes isolated from the serum of NSCLC patients was determined by RT-qPCR, demonstrating that miR-let-7e was downregulated in NSCLC tissues and serum-derived exosomes, while SUV39H2 was upregulated in NSCLC tissues. Kaplan-Meier method revealed that both lower miR-let-7e expression and higher SUV39H2 expression were correlated with a lower survival rate of NSCLC patients. Next, SUV39H2 was predicted and validated to be a target of miR-let-7e using dual-luciferase reporter assay. NSCLC H1299 cells following ectopic expression and depletion experiments of miR-let-7e and SUV39H2 were treated with serum-derived exosomes, after which the viability, migration, and invasion of H1299 cells were detected using CCK-8 and Transwell assays. Further, in vivo experiments were conducted to elucidate the effect of exosomal miR-let-7e on tumorigenesis. Results revealed that miR-let-7e overexpression in serum-derived exosomes inhibited SUV39H2, resulting in impaired cell viability, migration, and invasion in vitro as well as delayed tumor growth in vivo. In conclusion, the key findings of the current study demonstrate that exosomal miR-let-7e from serum possesses anticarcinogenic properties against NSCLC via the SUV39H2/LSD1/CDH1 axis.

BACKGROUND: Germline inactivating variants in the CDH1 tumor suppressor gene impart an elevated lifetime risk of diffuse gastric cancer. The current endoscopic surveillance method depends upon random gastric biopsies for early cancer detection. METHODS: Asymptomatic adults with pathogenic or likely pathogenic CDH1 variants referred for endoscopic gastric cancer surveillance were included in this retrospective cohort. Upper gastrointestinal endoscopy was performed according to the consensus Cambridge method, in the early period, or a systematic (Bethesda) protocol as part of an ongoing natural history study. The primary outcome measure was cancer detection. RESULTS: Collectively, 135 endoscopic surveillance procedures were performed in 120 patients. Twenty-six (19%, 26/135) procedures were performed using Cambridge method and 109 (81%) using the Bethesda protocol. Gastric signet ring cell carcinomas were detected in 15% (4/26) using the Cambridge method and 36% (40/109) using the Bethesda protocol (p

We studied the effect of erythropoietin on the morphofunctional status of bone marrow mesenchymal stem cells in patients with coronary heart disease. It was shown that the duration of cell exposure with erythropoietin had different effects on the expression levels of adhesion molecules, erythropoietin receptors, and co-expression of the erythropoietin receptor and common β-chain of cytokines, apoptosis/necrosis, and the cell cycle. In most cases, erythropoietin increased proliferation, migration, and NO production by "aged" mesenchymal stem cells (passage 8) and passage 4 mesenchymal stem cells grown during the previous 3 passages in the presence of 33.4 U/ml erythropoietin. Erythropoietin increased the expression of the autophagy marker LC3B in mesenchymal stem cells grown in the presence of erythropoietin in the culture medium. Thus, long-term culturing of mesenchymal stem cells in the presence of erythropoietin in the culture medium increased their resistance to adverse microenvironment factors - oxidative stress and hyperglycemia.

Minimal residual disease of leukemia may reside in the bone marrow (BM) microenvironment and escape the effects of chemotherapeutic agents. This study investigated interactions between B cell precursor (BCP)-acute lymphoblastic leukemia (ALL) cells and BM mesenchymal stromal cells (BM-MSCs) in vitro. Five BCP-ALL cell lines established from pediatric patients and primary samples from a BCP-ALL patient were examined by flow cytometry and immunocytochemistry for expression of specific cell surface markers and cell adhesion proteins. The cell lines developed chemoresistance to commonly used anti-leukemic agents through adhesion to MSC-TERT cells in long-term culture. The change in chemosensitivity after adhering to BM-MSCs was associated with the expression of CD34, CD133, P-glycoprotein and BCRP/ABCG2, and downregulation of CD38. Similar phenotypic changes were observed in primary samples obtained by marrow aspiration or biopsy from a BCP-ALL patient. BM-MSC-adhering leukemia cells also showed deceleration of cell proliferation and expressed proteins in the Cadherin and Integrin pathways. These results suggest that BCP-ALL cells residing in the BM microenvironment may acquire chemoresistance by altering their phenotype to resemble that of cancer stem cells. Our results indicate that cell adhesion could be potentially targeted to improve the chemosensitivity of residual BCP-ALL cells in the BM microenvironment.