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T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) expression has been a trending topic in recent years due to its differential expression in a wide range of neoplasms. TIM-3 is one of the key immune checkpoint receptors that interact with GAL-9, PtdSer, HMGB1 and CEACAM1. Initially identified on the surface of T helper 1 (Th1) lymphocytes and later on cytotoxic lymphocytes (CTLs), monocytes, macrophages, natural killer cells (NKs), and dendritic cells (DCs), TIM-3 plays a key role in immunoregulation. Recently, a growing body of evidence has shown that its differential expression in various tumor types indicates a specific prognosis for cancer patients. Here, we discuss which types of cancer TIM-3 can serve as a prognostic factor and the influence of coexpressed immune checkpoint inhibitors, such as LAG-3, PD-1, and CTLA-4 on patients' outcomes. Currently, experimental medicine involving TIM-3 has significantly enhanced the anti-tumor effect and improved patient survival. In this work, we summarized clinical trials incorporating TIM-3 targeting monoclonal and bispecific antibodies in monotherapy and combination therapy and highlighted the emerging role of cell-based therapies.

Immunosuppressive molecules are extremely valuable prognostic biomarkers across different cancer types. However, the diversity of different immunosuppressive molecules makes it very difficult to accurately predict clinical outcomes based only on a single immunosuppressive molecule. Here, we establish a comprehensive immune scoring system (ISS GC ) based on 6 immunosuppressive ligands (NECTIN2, CEACAM1, HMGB1, SIGLEC6, CD44, and CD155) using the LASSO method to improve prognostic accuracy and provide an additional selection strategy for adjuvant chemotherapy of gastric cancer (GC). The results show that ISS GC is an independent prognostic factor and a supplement of TNM stage for GC patients, and it can improve their prognosis prediction accuracy; in addition, it can distinguish GC patients with better prognosis from those with high prognostic nutritional index score; furthermore, ISS GC can also be used as a tool to select GC patients who would benefit from adjuvant chemotherapy independent of their TNM stages, MSI status and EBV status. Expression patterns of immune checkpoints in patients with gastric cancer remain poorly characterized. Here the authors propose an immune scoring system based on the expression of six immunosuppressive ligands to improve the prognostic accuracy in gastric cancer patients and drive the selection of candidates for adjuvant chemotherapy.

Early detection and long‐term monitoring are important for urothelial carcinoma of the bladder (UCB). Urine cytology and existing markers have insufficient diagnostic performance. Here, we examined medium‐sized extracellular vesicles (EVs) in urine to identify specific markers for UCB and evaluated their usefulness as diagnostic material. To identify specific markers in urinary EVs derived from UCB, we undertook shotgun proteomics using urine from four UCB patients and four healthy subjects. Next, 29 healthy specimens, 18 noncancer specimens, and 33 UCB specimens, all from men, were analyzed for urinary EVs by flow cytometry to evaluate the diagnostic performance of UCB‐specific EVs. Nanoparticle‐tracking analysis indicated that the size of EVs extracted from urine was mostly <400 nm. By shotgun proteomics, we detected several proteins characteristic of UCB and found that carcinoembryonic antigen‐related adhesion molecule (CEACAM) proteins were increased in patients. Flow cytometric analysis revealed that the degree of expression of CEACAM1, CEACAM5, and CEACAM6 proteins on the surface of EVs varied among patients. Extracellular vesicles expressing CEACAM proteins also expressed mucin 1, suggesting that they were derived from tumorigenic uroepithelial cells. The number of EVs expressing CEACAM1, 5, and 6 proteins was significantly increased in UCB (mean ± SD, 8.6 ± 13%) compared to non‐UCB (0.69 ± 0.46) and healthy (0.46 ± 0.34) by flow cytometry. The results of receiver operating characteristic (ROC) analysis showed a good score of area under the ROC curve of 0.907. We identified EVs that specifically express CEACAM proteins in urine and have potential for diagnostic applications. These EVs are potential targets in a new liquid biopsy test for UCB patients. ・Increased CEACAM proteins was found in EV fraction of urine from patients with bladder cancer by shotgun proteomics. ・Urinary EVs expressing CEACAM proteins, characterized by flow cytometry, are also accompanied by MUC1, which is increased in patients with bladder cancer. ・EVs expressing CEACAM proteins in urine have good diagnostic performance to detect bladder cancer patients.

Although CEACAM1 (CC1) glycoprotein resides at the interface of immune liver injury and metabolic homeostasis, its role in orthotopic liver transplantation (OLT) remains elusive. We aimed to determine whether/how CEACAM1 signaling may affect hepatic ischemia-reperfusion injury (IRI) and OLT outcomes. In the mouse, donor liver CC1 null mutation augmented IRI-OLT (CC1-KO→WT) by enhancing ROS expression and HMGB1 translocation during cold storage, data supported by in vitro studies where hepatic flush from CC1-deficient livers enhanced macrophage activation in bone marrow-derived macrophage cultures. Although hepatic CC1 deficiency augmented cold stress-triggered ASK1/p-p38 upregulation, adjunctive ASK1 inhibition alleviated IRI and improved OLT survival by suppressing p-p38 upregulation, ROS induction, and HMGB1 translocation (CC1-KO→WT), whereas ASK1 silencing (siRNA) promoted cytoprotection in cold-stressed and damage-prone CC1-deficient hepatocyte cultures. Consistent with mouse data, CEACAM1 expression in 60 human donor liver biopsies correlated negatively with activation of the ASK1/p-p38 axis, whereas low CC1 levels associated with increased ROS and HMGB1 translocation, enhanced innate and adaptive immune responses, and inferior early OLT function. Notably, reduced donor liver CEACAM1 expression was identified as one of the independent predictors for early allograft dysfunction (EAD) in human OLT patients. Thus, as a checkpoint regulator of IR stress and sterile inflammation, CEACAM1 may be considered as a denominator of donor hepatic tissue quality, and a target for therapeutic modulation in OLT recipients.

To characterize transcriptomic changes in endothelial cells (ECs) infected by coronaviruses, and stimulated by DAMPs, the expressions of 1311 innate immune regulatomic genes (IGs) were examined in 28 EC microarray datasets with 7 monocyte datasets as controls. We made the following findings: The majority of IGs are upregulated in the first 12 hours post-infection (PI), and maintained until 48 hours PI in human microvascular EC infected by middle east respiratory syndrome-coronavirus (MERS-CoV) (an EC model for COVID-19). The expressions of IGs are modulated in 21 human EC transcriptomic datasets by various PAMPs/DAMPs, including LPS, LPC, shear stress, hyperlipidemia and oxLDL. Upregulation of many IGs such as nucleic acid sensors are shared between ECs infected by MERS-CoV and those stimulated by PAMPs and DAMPs. Human heart EC and mouse aortic EC express all four types of coronavirus receptors such as ANPEP, CEACAM1, ACE2, DPP4 and virus entry facilitator TMPRSS2 (heart EC); most of coronavirus replication-transcription protein complexes are expressed in HMEC, which contribute to viremia, thromboembolism, and cardiovascular comorbidities of COVID-19. ECs have novel trained immunity (TI), in which subsequent inflammation is enhanced. Upregulated proinflammatory cytokines such as TNFα, IL6, CSF1 and CSF3 and TI marker IL-32 as well as TI metabolic enzymes and epigenetic enzymes indicate TI function in HMEC infected by MERS-CoV, which may drive cytokine storms. Upregulated CSF1 and CSF3 demonstrate a novel function of ECs in promoting myelopoiesis. Mechanistically, the ER stress and ROS, together with decreased mitochondrial OXPHOS complexes, facilitate a proinflammatory response and TI. Additionally, an increase of the regulators of mitotic catastrophe cell death, apoptosis, ferroptosis, inflammasomes-driven pyroptosis in ECs infected with MERS-CoV and the upregulation of pro-thrombogenic factors increase thromboembolism potential. Finally, NRF2-suppressed ROS regulate innate immune responses, TI, thrombosis, EC inflammation and death. These transcriptomic results provide novel insights on the roles of ECs in coronavirus infections such as COVID-19, cardiovascular diseases (CVD), inflammation, transplantation, autoimmune disease and cancers.

Transfer RNA‐derived fragments are a group of small noncoding single‐stranded RNA that play essential roles in multiple diseases. However, their biological functions in carcinogenesis are not well understood. In this study, 5′tRF‐Gly was found to have significantly high expression in hepatocellular carcinoma (HCC), and the upregulation of 5′tRF‐Gly was positively correlated with tumor size and tumor metastasis. Overexpression of 5′tRF‐Gly induced increased growth rate and metastasis in HCC cells in vitro and in nude mice, while knockdown showed the opposite effect. Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) was confirmed to be a direct target of 5′tRF‐Gly in HCC. In addition, the cytological effect of CEACAM1 knockdown proved to be similar to the overexpression of 5′tRF‐Gly. Moreover, attenuation of CEACAM1 expression rescued the 5′tRF‐Gly‐mediated promoting effects on HCC cells. These data show that 5′tRF‐Gly is a new tumor‐promoting factor and could be a potential diagnostic biomarker or new therapeutic target for HCC. In the present study, the small RNA high‐throughput sequencing results showed that 5’tRF‐Gly was up‐regulated in HCC tissues and was related to poor prognosis. We demonstrated the oncogenic role of 5’tRF‐Gly and its underlying molecular mechanism in HCC. Furthermore, we first verified CEA cell adhesion molecule 1 (CEACAM1) as the direct target of 5’tRF‐Gly and revealed how CEACAM1 inhibits hepatocellular carcinoma growth and metastasis. Our findings not only elucidate the role of 5’tRF‐Gly and CEACAM1 in HCC but also provide new targets for the prognostic evaluation and treatment of HCC.

Chronic inflammation with progressive age, called inflammaging, contributes to the pathogenesis of cardiovascular diseases. Previously, we have shown increased vascular expression of the Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) in aged mice and humans, presumably via mutual upregulation with the pro‐inflammatory cytokine TNF‐α. CEACAM1 is critical for aging‐associated vascular alterations like endothelial dysfunction, fibrosis, oxidative stress, and sustained inflammation and can be regarded as a main contributor to vascular inflammaging. This study was conducted to elucidate the mechanisms underlying endothelial CEACAM1 upregulation by TNF‐α in detail. Using wildtype (WT) and TNF‐α knockout (Tnf−/−) mice, we confirmed that the aging‐related upregulation of endothelial CEACAM1 critically depends on TNF‐α. The underlying mechanisms were analyzed in an endothelial cell culture model. TNF‐α time‐dependently upregulated CEACAM1 in vitro. In pharmacological experiments, we identified an early NF‐κB‐ and a delayed β‐catenin‐mediated response. Involvement of β‐catenin was further substantiated by siRNA‐mediated knockdown of the β‐catenin‐targeted transcription factor TCF4. Both signaling pathways acted independent from each other. Elucidating the delayed response, co‐immunoprecipitation analysis revealed release of β‐catenin from adherens junctions by TNF‐α. Finally, TNF‐α activated Akt kinase by increasing its Ser473 phosphorylation. Consequently, Akt kinase facilitated β‐catenin signaling by inhibiting its degradation via phosphorylation of GSK3β at Ser9 and by increased phosphorylation of β‐catenin at Ser552 that augments its transcriptional activity. Taken together, our study provides novel mechanistic insights into the aging‐related, inflammation‐mediated endothelial upregulation of CEACAM1. Beyond the pathogenesis of cardiovascular diseases, these findings may be significant to all fields of inflammaging. Early response: TNF‐α activates NF‐κB signaling by IKK‐induced degradation of the inhibitor of nuclear factor kappa B (IκB). Released NF‐κB translocates into the nucleus and upregulates CEACAM1 expression detectable after 24 h of TNF‐α stimulation. Delayed response: TNF‐α‐induced adherens junction disassembly releases β‐catenin, promoting its cytosolic and nuclear accumulation. Nuclear β‐catenin binds to transcription factors of the TCF/LEF family to induce CEACAM1 expression. Akt kinase inhibits GSK3β by phosphorylation thereby stabilizing β‐catenin and directly phosphorylates β‐catenin which was reported to increase its transcriptional activity.

BackgroundA recent EULAR Task force identified critical variability in interferon (IFN) assay methods and defined a research agenda to improve and harmonise assays. IFN scores A and B, developed using IFN stimulated genes (ISG) measured in peripheral blood mononuclear cells (PBMC) differentiate between clinical subsets of patients with autoimmune disease [1]. Whole blood (WB) samples are more commonly used in clinical practice and trials. Regardless of sample type, real time polymerase chain reaction (RT-PCR) experiments require a suitable reference gene (RG) which is uniformly expressed across different experimental conditions in order to normalise target gene expression quantities [2].Objectives(i) To determine whether sample type affects the previously defined IFN scores; (ii) if so, to develop revised IFN scores in WB; (iii) to identify the best RG for future RT-PCR experiments.MethodsSamples were collected from participants enrolled in 5 studies; DEFINITION, CONVAS, USEFUL, MASTERPLANs and BRAGGSS. PBMCs were separated from blood using leucosep tubes and WB was collected using Tempus™ Blood RNA Tubes. Undetected delta Ct values were singly imputed.IFN scores A and B were calculated in paired WB and PBMC samples and agreement between the sample types was assessed via the Bland-Altman (BA) method. New IFN scores for WB were derived via exploratory factor analysis (EFA) of 31 ISG as per the original work [1]. Separation of clinical subgroups was visualized using violin plots and effect sizes calculated using Hedges’ g. RG identification from 16 candidates used online software.ResultsWe used samples from 342 participants with systemic lupus erythematosus (SLE), 6 ANA positive at risk progressors, 7 at risk non-progressors, 30 with rheumatoid arthritis (RA) and 10 healthy controls (HC). Subsets were used for sample comparison (n=45) and RG work (n=22). Comparing score A in PBMC versus WB matched samples resulted in bias of -1.44, limits of agreement (LOA 0.08, -2.96), the spread of data on BA plot showed a systematic difference. The same comparison in score B gave bias of -1.23 (LOA 0.26, 2.73), data on BA plot suggested differences were not systematic. EFA in WB produced a two-factor solution which explained 97% of the variation. The first (score C) contained 3 ISG and the second (score D) contained 4 (Table 1), compared with score A (12 ISG) and score B (14 ISG) [1]. Both scores showed potential to differentiate between HC and those with SLE or RA, but neither separated SLE from RA as the original PBMC scores A and B had done (Figure 1). YWHAZ, PGK1 and GUSB were the most stable RG. Others such as ACTB performed poorly as RG.ConclusionPBMC and WB may be used interchangeably to calculate IFN score A, but not score B. This suggests an influence of neutrophils, including the demethylation of neutrophil ISG cytosine-guanine sites, on Score B genes [3]. In WB we derived a further two scores which differentiated SLE or RA from HC, although neither score distinguished between SLE and RA. Further work is needed to assess the performance of scores A-D in WB samples. The Minimum Information for Publication of Quantitative Real-Time PCR Experiments recommend using ≥3 RG to avoid bias[4]; we found that YWHAZ, PGK1 and GUSB were the most stable genes across a range of experimental conditions.References[1]El-Sherbiny, Y.M. et al., Sci Rep, 2018. 8(1): p. 5793.[2]Schmittgen, T.D. and K.J. Livak, Nat Protoc, 2008. 3(6): p. 1101-8.[3]Coit, P., et al., J Autoimmun, 2015. 58: p. 59-66.[4]Bustin, S.A., et al., BMC Molecular Biology, 2010. 11(1): p. 74.Table 1.Interferon stimulated genes selected in two-factor solution derived via exploratory factor analysis in whole blood.VariableScore CScore DCCL81.01IFI270.84CXCL100.80LAMP30.73ISG150.66CASP10.93CEACAM10.89TRIM380.73SOCS10.58Figure 1.Violin plots (bold dashed line = median, dotted lines = upper and lower quartiles) and effect sizes presented as top value and lower and upper 95% CI presented underneath.Acknowledgements:NIL.Disclosure of InterestsNone Declared.

The production of adenosine by CD73 on cancer cells in the tumor microenvironment is a recognized immunosuppressive mechanism contributing to immune evasion in many solid tumors. While NK cells have been purported to overexpress CD73 under certain conditions, this phenomenon has remained elusive and unclear. We have found that while NK cells are able to upregulate expression of CD73 on their surface when exposed to CD73+ cancer cells, this upregulation is not universal, nor is it often substantial. Rather, our data point to the extent of CD73 expression on NK cells to be both cancer-specific and environmentally-driven, and largely limited in intensity. We found that NK cell overexpression of CD73 responds to the level of CD73 on cancer cells and is enhanced in hypoxia. Interestingly, human CD73+ NK cells appear hyperfunctional in vitro compared to CD73− NK cells, suggesting that CD73 expression could be a bystander of NK cell activation. In addition, glioblastoma patient data show that tumor-infiltrating NK cells express CD73 variably, depending on donor, and present lower expression of CD16, alongside patient-specific changes in CEACAM1, CXCR3 and TIM-3, suggesting some functional changes in NK cell responses associated with expression of CD73 on NK cells in vivo. Taken together, our study is the first to show that while NK cells are largely resistant to the upregulation of CD73, CD73 expression is inducible on NK cells in response to CD73 on cancer cells, and these cells are associated with distinct functional signatures.

F. nucleatum is an anaerobic bacterium that is associated with several tumor entities and promotes tumorigenesis. Recent evidence suggests that F. nucleatum binds the inhibitory receptor carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) via the trimeric autotransporter adhesin CbpF. However, whether this binding is functional or whether other fusobacterial trimeric autotransporter adhesins are involved in CEACAM1 activation is unknown. In this study, using F. nucleatum mutants lacking the type 5c trimeric autotransporter adhesins fvcA (CbpF), fvcB, fvcC, and fvcD, we show that F. nucleatum CbpF binds and activates CEACAM1 and also binds carcinoembryonic antigen (CEA), a tumor-associated protein. We further find that CEACAM antibodies directed against the CEACAM N-terminal domain block the CbpF-CEACAM1 interaction. In functional assays, we demonstrate CbpF-dependent inhibition of CD4 + T cell response. Thus, we characterize an immune evasion mechanism in which F. nucleatum uses its surface protein CbpF to inhibit T cell function by activating CEACAM1.