Immunoglobulin Domain-containing Protein Research Articles

Characterization of a novel anti-PVRIG antibody with Fc-competent function that exerts strong antitumor effects via NK activation in preclinical models

Poliovirus receptor-related immunoglobulin domain-containing protein, or PVRIG, is a newly discovered immune checkpoint that has emerged as a promising target for cancer immunotherapy. It is primarily expressed on activated T and natural killer (NK) cells, and once engaged with its ligand, PVRL2, it induces inhibitory signaling in T cells, thereby promoting the functional exhaustion of tumor-infiltrating lymphocytes (TILs). Here, we characterized IBI352g4a, a novel humanized anti-PVRIG antibody with Fc-competent function, explored the mechanism of its antitumor activity in preclinical models, and systemically evaluated the contribution of FcrR engagement to PVRIG blockade-induced antitumor activity. IBI352g4a binds to the extracellular domain of human PVRIG with high affinity (Kd = 0.53 nM) and specificity, and fully blocks the interaction between PVRIG and its ligand PVRL2. Unlike other immune checkpoints, IBI352g4a significantly induced NK cell activation and degranulation, but had a minimal effect on T-cell activation in in vitro functional assays. IBI352g4a induced strong antitumor effect in several preclinic models, through in vivo mechanism analysis we found that both NK and T cells contribute to the antitumor effect, but NK cells play predominant roles. Specifically, a single dose of IBI352g4a induced significant NK cell activation in TILs, but T-cell activation was observed only after the second dose. Moreover, the Fc effector function is critical for both NK cell activation and treatment efficacy in vitro and in vivo. Our study, for the first time, demonstrates that both NK activation and FcrR engagement are required for antitumor efficacy induced by PVRIG blockade.

Genetic and Epigenetic Regulation in Lingo-1: Effects on Cognitive Function and White Matter Microstructure in a Case-Control Study for Schizophrenia.

Leucine-rich repeat and immunoglobulin domain-containing protein (Lingo-1) plays a vital role in a large number of neuronal processes underlying learning and memory, which are known to be disrupted in schizophrenia. However, Lingo-1 has never been examined in the context of schizophrenia. The genetic association of a single-nucleotide polymorphism (SNP, rs3144) and methylation (CpG sites) in the Lingo-1 3'-UTR region was examined, with the testing of cognitive dysfunction and white matter (WM) integrity in a schizophrenia case-control cohort (n = 268/group). A large subset of subjects (97 control and 161 schizophrenia subjects) underwent structural magnetic resonance imaging (MRI) brain scans to assess WM integrity. Frequency of the rs3144 minor allele was overrepresented in the schizophrenia population (p = 0.03), with an odds ratio of 1.39 (95% CI 1.016-1.901). CpG sites surrounding rs3144 were hypermethylated in the control population (p = 0.032) compared to the schizophrenia group. rs3144 genotype was predictive of membership to a subclass of schizophrenia subjects with generalized cognitive deficits (p < 0.05), in addition to having associations with WM integrity (p = 0.018). This is the first study reporting a potential implication of genetic and epigenetic risk factors in Lingo-1 in schizophrenia. Both of these genetic and epigenetic alterations may also have associations with cognitive dysfunction and WM integrity in the context of the schizophrenia pathophysiology.

Decreased TMIGD1 aggravates colitis and intestinal barrier dysfunction via the BANF1-NF-κB pathway in Crohn’s disease

BackgroundDisrupted intestinal epithelial barrier is one of the major causes of Crohn’s disease (CD). Novel molecular targets for intestinal epithelial barrier are essential to treatment of CD. Transmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is an adhesion molecule that regulates cell adhesion, migration, and enterocyte differentiation. However, the function and mechanism of TMIGD1 in CD and intestinal epithelial barrier has rarely been studied. Furthermore, the association between TMIGD1 and the clinical features of CD remains unclear.MethodsTranscriptome analysis on colonic mucosa from CD patients and healthy individuals were performed to identify dysregulated genes. Multi-omics integration of the 1000IBD cohort including genomics, transcriptomics of intestinal biopsies, and serum proteomics identified the association between genes and characteristics of CD. Inflammation was assessed by cytokine production in cell lines, organoids and intestinal-specific Tmigd1 knockout (Tmigd1INT-KO) mice. Epithelial barrier integrity was evaluated by trans-epithelium electrical resistance (TEER), paracellular permeability, and apical junction complex (AJC) expression. Co-immunoprecipitation, GST pull-down assays, mass spectrometry, proteomics, and transcriptome analysis were used to explore downstream mechanisms.ResultsMulti-omics integration suggested that TMIGD1 was negatively associated with inflammatory characteristics of CD. TMIGD1 was downregulated in inflamed intestinal mucosa of patients with CD and mice colitis models. Tmigd1INT-KO mice were more susceptible to chemically induced colitis. In epithelial cell lines and colonic organoids, TMIGD1 knockdown caused impaired intestinal barrier integrity evidenced by increased paracellular permeability and reduced TEER and AJC expression. TMIGD1 knockdown in intestinal epithelial cells also induced pro-inflammatory cytokine production. Mechanistically, TMIGD1 directly interacted with cytoplasmic BAF nuclear assembly factor 1 (BANF1) to inhibit NF-κB activation. Exogenous expression of TMIGD1 and BANF1 restored intestinal barrier function and inhibited inflammation in vitro and in vivo. TMIGD1 expression predicted response to anti-TNF treatment in patients with CD.ConclusionsOur study demonstrated that TMIGD1 maintained intestinal barrier integrity and inactivated inflammation, and was therefore a potential therapeutic target for CD.

Circulating proteins and peripheral artery disease risk: observational and Mendelian randomization analyses.

We conducted observational and Mendelian randomization (MR) analyses to explore the associations between blood proteins and risk of peripheral artery disease (PAD). The observational cohort analyses included data on 257 proteins estimated in fasting blood samples from 12 136 Swedish adults aged 55-94 years who were followed up for incident PAD via the Swedish Patient Register. Mendelian randomization analyses were undertaken using cis-genetic variants strongly associated with the proteins as instrumental variables and genetic association summary statistic data for PAD from the FinnGen study (11 924 cases and 288 638 controls) and the Million Veteran Program (31 307 cases and 211 753 controls). The observational analysis, including 86 individuals diagnosed with incident PAD during a median follow-up of 6.6-year, identified 13 proteins [trefoil factor two, matrix metalloproteinase-12 (MMP-12), growth differentiation factor 15, V-set and immunoglobulin domain-containing protein two, N-terminal prohormone brain natriuretic peptide, renin, natriuretic peptides B, phosphoprotein associated with glycosphingolipid-enriched microdomains one, C-C motif chemokine 15, P-selectin, urokinase plasminogen activator surface receptor, angiopoietin-2, and C-type lectin domain family five member A] associated with the risk of PAD after multiple testing correction. Mendelian randomization analysis found associations of T-cell surface glycoprotein CD4, MMP-12, secretoglobin family 3A member 2, and ADM with PAD risk. The observational and MR associations for T-cell surface glycoprotein CD4 and MMP-12 were in opposite directions. This study identified many circulating proteins in relation to the development of incident PAD. Future studies are needed to verify our findings and assess the predictive and therapeutic values of these proteins in PAD.

Abstract 593: Bispecific antibody against TIGIT and PVRIG enhances antitumor immunity

Abstract T cell immunoreceptor with Ig and ITIM domains (TIGIT) and poliovirus receptor-related immunoglobulin domain-containing protein (PVRIG) are two immune checkpoint receptors. They compete with CD226 for binding to CD155 or CD112 and suppress the activity of NK cells and T cells. Here we developed a bispecific antibody KA-1874 targeting TIGIT with an anti-PVRIG scFv fused to the C-terminus. KA-1874 is a fully human IgG4 with high binding affinity to TIGIT (KD=0.08nM) and PVRIG (KD=0.17nM). KA-1874 blocks both bindings of PVRIG-mFc to PVRL2 and TIGIT-mFc to CD155. In cell-based luciferase reporter assays, KA-1874 blocks PVRIG and TIGIT-mediated inhibitory signaling and enhances CD3 signaling with EC50 of 1.1nM and 1.4nM respectively, while COM701 against PVRIG and EOS488 against TIGIT both with EC50 above 2nM. In a CMV recall assay, PBMC increases INF-γ secretion when cultured with KA-1874, suggesting KA-1874 enhances primary T cell function. Immunodeficient mice humanized with PBMC are transplanted with A374 tumor cells. Co-injection of KA-1874 and pembrolizumab, which against PD-1, suppress established tumor growth. In comparison, KA-1874 or pembrolizumab alone fails to inhibit tumor growth. The above results indicate KA-1874 is an excellent candidate therapeutical antibody for cancer immunotherapy. Citation Format: Jiabei Liang, Xiao Gao, Cong Zhang, Hui Zhao, Hao Peng, Feng Hao, Jinying Ning, Guojin Wu. Bispecific antibody against TIGIT and PVRIG enhances antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 593.

Abstract 1935: Interplay between TIM3+ immune cells and other immune checkpoints in more than 40 different human carcinoma entities using 18+1 BLEACH&amp;STAIN mfIHC

Abstract An increasing number of therapy regimens using a combination of different immune checkpoint inhibitors (ICIs) have shown remarkable results in several different tumor entities. However, the likelihood of a positive response rate to combined ICIs is poor in most tumor entities and depends on several parameters including the tumor microenvironment. Particularly little is known about the spatial orchestration and spatial interplay between different immune checkpoint expressing cells. Given that the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is expressed on both immune cells as well as tumor cells and that several phase I/II studies are currently evaluating anti-TIM3 drugs, the interplay between these immune checkpoints in human cancers is of topical interest. To study the spatial orchestration and interplay between TIM3, CTLA-4, PD-1, and PD-L1 expression on T-cell subsets, macrophage subsets, CD11c+ dendritic cells, CD20+B-cells in relation to panCK+ malignant cells, CD31+ vessels and other structural tumor compartments, a multiplex fluorescence immunohistochemistry approach was used to stain 18 different antibodies on a set of tissue microarrays containing samples from more than 3000 carcinoma samples. In addition, a deep learning-based framework for cell type identification was developed and validated in this study. TIM3, PD-1, PD-L1, and CTLA-4 expression was measured on tumor cells (panCK+), cytotoxic T-cells (CD3+CD8+), T-helper cells (CD3+CD4+), regulatory T-cells (CD3+CD4+FOXP3+), subsets of macrophages (CD68+CD163+/CD68+iNOS+) and dendritic cells (CD11c+). Interestingly, TIM3 as well as CTLA-4 expression on CD3+CD8+ cytotoxic T-cells and CD3+CD4+FOXP3+ regulatory T-cells showed a spatially more diverse expression pattern - particularly inverse expression profile - compared to PD-1 expression on all analyzed T-cells subsets that was consistently accompanied by PD-L1 expression on immune and tumor cells (p&amp;lt;0.001). Combined analysis of cell densities, expression patterns, intensity measurements, interaction and distance analysis between immune cells and tumor cells revealed distinct changes in the immune cell infiltration pattern that was linked to several major immune checkpoint receptor expression profiles. Previously uncharacterized immune cell-composition dynamics in clustered tumor phenotypes, according to the immune checkpoint expression, were detected. This included for instance, a significant inverse association between CTLA-4 expression on T-cells and high expression levels of the PD-1/PD-L1 axis. In conclusion, deep profiling of 18 biomarkers in more than 40 different carcinoma entities revealed complex changes in the spatial orchestration of a wide range of immune cell subsets that was driven by the expression profile and composition of TIM3, PD-1, PD-L1, and CTLA-4. Citation Format: Nicolaus F. Debatin, Elena Bady, Tim Mandelkow, Magalie C. Lurati, Ronald Simon, Claudia Hube-Magg, Maximilian Lennartz, Guido Sauter, Niclas C. Blessin. Interplay between TIM3+ immune cells and other immune checkpoints in more than 40 different human carcinoma entities using 18+1 BLEACH&amp;STAIN mfIHC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1935.

Abstract 1649: Humanized PVRIG mice: A novel tool for evaluating anti-PVRIG immunotherapies

Abstract The use of humanized mouse models provides a promising platform for preclinical studies aimed at developing novel immunotherapies. While current checkpoint inhibitors have improved patient outcomes, combination therapy targeting additional checkpoint receptors may heighten the immune response. Specifically, previous reports have shown that high expression of the immune checkpoint poliovirus receptor-related immunoglobulin domain-containing (PVRIG) protein leads to T-cell exhaustion. Furthermore, in vitro blockade of human PVRIG enabled T-cell activation. To explore the translational potential of inhibiting PVRIG, we evaluated the efficacy of PVRIG antibodies by generating a humanized B-hPVRIG mouse model. In this model, the mouse Pvrig gene was replaced by the human counterpart, and expression of the human PVRIG protein was confirmed in homozygous B-hPVRIG mice. Additionally, flow cytometry analysis showed similar distribution of T-cell subtypes within the blood, spleen, and lymph nodes of B-hPVRIG mice compared to wild-type C57BL/6. Lastly, our in vivo efficacy studies showed that homozygous B-hPVRIG mice that were subcutaneously implanted with murine colon cancer (MC38) cells and treated with 30 mg/kg of the human PVRIG antibody, reached a tumor growth inhibition (TGI) of approximately 40%. This data suggests that human PVRIG antibodies significantly inhibit tumor growth in homozygous B-hPVRIG mice. Altogether, our humanized B-hPVRIG mouse model is an effective tool for in vivo efficacy studies aimed at the development of novel PVRIG immunotherapies. Citation Format: Yifu Zhang, Jiawei Yao, Rebecca Soto, Chonghui Liu, Qingcong Lin. Humanized PVRIG mice: A novel tool for evaluating anti-PVRIG immunotherapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1649.

The Role of TMIGD1 as a Tumor Suppressor in Colorectal Cancer.

Background: Colorectal cancer (CRC) is one of the leading causes of cancer-related death, and up to 50% of individuals will suffer relapse. Although transmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) was found to be a protective factor in several renal and intestinal diseases, the specific role of TMIGD1 in CRC remains unclear. Objective: To determine the tumor suppressor TMIGD1 expression and its function in inhibiting CRC. Methods: We analyzed three Gene Expression Omnibus (GEO) datasets through the GEO2R online tool to obtain the set of differentially expressed genes (DEGs) between CRC and normal tissues, and further analyzed the TMIGD1 gene's expression in databases. Real-time quantitative polymerase chain reaction and western blot assays were used to investigate expression of TMIGD1. Transwell and wound healing assays were performed to detect the migration and invasion ability. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to evaluate cell proliferation. In vivo studies were conducted to illustrate the tumorigenicity. Results: We found that the TMIGD1 gene is one of the highly downregulated genes in CRC through bioinformatic analysis. We also showed that downregulation of TMIGD1 is associated with poor overall survival rate of CRC based on The Cancer Genome Atlas (TCGA) databases. In addition, we showed that overexpression of TMIGD1 protein significantly impaired the metastasis and proliferation ability of the CRC cells. Finally, TMIGD1 also repressed subcutaneous tumorigenesis of CRC cells in vivo. Conclusion: Our findings indicate that downregulation of TMIGD1 may promote CRC progression and invasion. Therefore, TMIGD1 may serve as a biomarker for CRC prognoses.

Implication of the LINGO2 gene in the predisposition to movement disorders

Previous reports on the pathogenesis of age-related movement disorders, such as Parkinson’s disease (PD) and essential tremor (ET), have demonstrated the potential implications of LINGO1 (leucine-rich repeat and immunoglobulin domain-containing protein) gene. Although LINGO2 has a high degree of homology with LINGO1, but it is less characterized and the role of LINGO2 in the development of PD/ET remains unreported. Hence, this meta-analysis was conducted to evaluate the role of LINGO2 in PD/ET pathogenesis. A total of 4 studies, which complied with the Hardy–Weinberg equilibrium, were included in the meta-analysis. Analysis of the pooled odds ratio and confidence interval of the studies were performed for five genetic models, namely: allelic, dominant, recessive, homozygous, and heterozygous. No significant association was observed between the LINGO2 polymorphism and PD/ET, although subgroup analysis through conventional meta-analysis indicated that the recessive models of rs7033345 and rs10812774 are significantly associated with predisposition to ET in the Asian population. However, trial sequential analyses for both polymorphisms were unlikely to reveal any robust effect. Hence, studies with larger samples on this association are needed in the future to corroborate our results.

Myopalladin knockout mice develop cardiac dilation and show a maladaptive response to mechanical pressure overload.

Myopalladin (MYPN) is a striated muscle-specific immunoglobulin domain-containing protein located in the sarcomeric Z-line and I-band. MYPN gene mutations are causative for dilated (DCM), hypertrophic, and restrictive cardiomyopathy. In a yeast two-hybrid screening, MYPN was found to bind to titin in the Z-line, which was confirmed by microscale thermophoresis. Cardiac analyses of MYPN knockout (MKO) mice showed the development of mild cardiac dilation and systolic dysfunction, associated with decreased myofibrillar isometric tension generation and increased resting tension at longer sarcomere lengths. MKO mice exhibited a normal hypertrophic response to transaortic constriction (TAC), but rapidly developed severe cardiac dilation and systolic dysfunction, associated with fibrosis, increased fetal gene expression, higher intercalated disc fold amplitude, decreased calsequestrin-2 protein levels, and increased desmoplakin and SORBS2 protein levels. Cardiomyocyte analyses showed delayed Ca2+ release and reuptake in unstressed MKO mice as well as reduced Ca2+ spark amplitude post-TAC, suggesting that altered Ca2+ handling may contribute to the development of DCM in MKO mice.

Holosteans contextualize the role of the teleost genome duplication in promoting the rise of evolutionary novelties in the ray-finned fish innate immune system.

Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrate species that have diversified across virtually all fresh and saltwater ecosystems. This ecological breadth raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.

CircRNA LRIG3 knockdown inhibits hepatocellular carcinoma progression by regulating miR-223-3p and MAPK/ERK pathway

IntroductionEmerging evidence suggests that circular RNAs (circRNAs) play critical roles in tumorigenesis. However, the roles and molecular mechanisms of circRNA leucine-rich repeat immunoglobulin domain-containing protein 3 (circ_LRIG3) in hepatocellular carcinoma (HCC) has not been investigated.Material and methodsThe expression levels of circ_LRIG3, miR-223-3p, and mitogen-activated protein kinase kinase 6 (MAP2K6) were determined by qRT-PCR. Flow cytometry was applied to determine the cell cycle distribution and apoptosis. Cell proliferation, migration and invasion were assessed by MTT, colony formation, and transwell assays. Western blot assay was employed to measure the protein levels of the snail, E-cadherin, MAP2K6, mitogen-activated protein kinase (MAPK), phospho-MAPK (p-MAPK), extracellular signal-regulated kinases (ERKs), and phospho-ERKs (p- ERKs). The relationship between miR-223-3p and circ_LRIG3 or MAP2K6 was predicted by bioinformatics tools and verified by dual-luciferase reporter assay. A xenograft tumor model was established to confirm the functions of circ_LRIG3 in vivo.ResultsCirc_LRIG3 and MAP2K6 expression were enhanced while miR-223-3p abundance was reduced in HCC tissues and cells. Knockdown of circ_LRIG3 inhibited cell proliferation, metastasis, and increasing apoptosis. MiR-223-3p was a target of circ_LRIG3, and its downregulation reversed the inhibitory effect of circ_LRIG3 knockdown on the progression of HCC cells. Moreover, MAP2K6 could bind to miR-223-3p, and MAP2K6 upregulation also abolished the suppressive impact of circ_LRIG3 interference on progression of HCC cells. Additionally, the silence of circ_LRIG3 suppressed the activation of the MAPK/ERK pathway and tumor growth by upregulating miR-223-3p and downregulating MAP2K6.ConclusionsCirc_LRIG3 knockdown inhibited HCC progression through regulating miR-223-3p/MAP2K6 axis and inactivating MAPK/ERK pathway.

TIM-3 Genetic Variants Are Associated with Altered Clinical Outcome and Susceptibility to Gram-Positive Infections in Patients with Sepsis.

Background: Previous studies have reported the fundamental role of immunoregulatory proteins in the clinical phenotype and outcome of sepsis. This study investigated two functional single nucleotide polymorphisms (SNPs) of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), which has a negative stimulatory function in the T cell immune response. Methods: Patients with sepsis (n = 712) were prospectively enrolled from three intensive care units (ICUs) at the University Medical Center Goettingen since 2012. All patients were genotyped for the TIM-3 SNPs rs1036199 and rs10515746. The primary outcome was 28-day mortality. Disease severity and microbiological findings were secondary endpoints. Results: Kaplan–Meier survival analysis demonstrated a significantly lower 28-day mortality for TIM-3 rs1036199 AA homozygous patients compared to C-allele carriers (18% vs. 27%, p = 0.0099) and TIM-3 rs10515746 CC homozygous patients compared to A-allele carriers (18% vs. 26%, p = 0.0202). The TIM-3 rs1036199 AA genotype and rs10515746 CC genotype remained significant predictors for 28-day mortality in the multivariate Cox regression analysis after adjustment for relevant confounders (adjusted hazard ratios: 0.67 and 0.70). Additionally, patients carrying the rs1036199 AA genotype presented more Gram-positive and Staphylococcus epidermidis infections, and rs10515746 CC homozygotes presented more Staphylococcus epidermidis infections. Conclusion: The studied TIM-3 genetic variants are associated with altered 28-day mortality and susceptibility to Gram-positive infections in sepsis.

Decrease of T-cells exhaustion markers programmed cell death-1 and T-cell immunoglobulin and mucin domain-containing protein 3 and plasma IL-10 levels after successful treatment of chronic hepatitis C

During chronic hepatitis C virus (HCV) infection, both CD4+ and CD8+ T-cells become functionally exhausted, which is reflected by increased expression of programmed cell death-1 (PD-1) and T-cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), and elevated anti-inflammatory interleukin 10 (IL-10) plasma levels. We studied 76 DAA-treated HCV-positive patients and 18 non-infected controls. Flow cytometry measured pretreatment frequencies of CD4+PD-1+, CD4+PD-1+Tim-3+ and CD8+PD-1+Tim-3+ T-cells and IL-10 levels measured by ELISA were significantly higher and CD4+PD-1−Tim-3− and CD8+PD-1−Tim-3− T-cells were significantly lower in patients than in controls. Treatment resulted in significant decrease of CD4+Tim-3+, CD8+Tim-3+, CD4+PD-1+Tim-3+ and CD8+PD-1+Tim-3+ T-cell frequencies as well as IL-10 levels and increase in CD4+PD-1−Tim-3− and CD8+PD-1−Tim-3− T-cells. There were no significant changes in the frequencies of CD4+PD-1+ T-cells, while CD8+PD-1+ T-cells increased. Patients with advanced liver fibrosis had higher PD-1 and lower Tim-3 expression on CD4+T-cells and treatment had little or no effect on the exhaustion markers. HCV-specific CD8+T-cells frequency has declined significantly after treatment, but their PD-1 and Tim-3 expression did not change. Successful treatment of chronic hepatitis C with DAA is associated with reversal of immune exhaustion phenotype, but this effect is absent in patients with advanced liver fibrosis.

Abstract 4970: Prognostic role of CD112R, PD-1 and Ki67 expression in CD8+cytotoxic T cells in colorectal cancer

Abstract Expression of inhibitory immune checkpoint receptors such as CD112R (Poliovirus Receptor-Related Immunoglobulin Domain-Containing Protein, PVRIG) and PD-1 on CD8+ lymphocytes are considered to indicate an “exhausted” stage of T cell activation while Ki67 protein expression in these cells is viewed as sign of activity or expansion. To assess the prognostic impact of the clinical impact of CD112R, PD-1, and Ki67 expression of CD8+ lymphocytes, expression of these proteins was analyzed by fluorescent multiplex immunohistochemistry on a tissue microarray containing 384 colorectal cancers with clinical follow-up data. An elevated fraction of CD8+Ki67+ cells was linked to the absence of nodal metastasis and longer survival in these tumors (p≤0.0039 each). CD112R and PD-1 expression was detected at variable intensity levels and were separately quantitated in both ki67+ (expanding) and Ki67 negative (non-expanding) CD8+ cytotoxic lymphocytes. Elevated PD-1 expression in CD8+ T cells was associated with an absence of nodal metastasis (p=0.004) and longer overall survival in all CD8+ cells (p=0.005). This association was more evident in the subset of expanding (p&amp;lt;0.0001) than in non-expanding (p=0.034) cytotoxic T cells. Elevated CD112R expression level in CD8+ T cells were linked to a low tumor grade (p=0.048), absence of nodal metastasis (p=0.023) and a prolonged overall survival (p=0.041) but these associations were not retained in subgroups of either expanding or non-expanding CD8+ cells. Patients with an elevated fraction of CD8+ki67+CD112R+PD-1+cells exhibited a particularly good prognosis. In conclusion, our data reveal that “exhausted” CD112R+/ PD-1+CD8+ cytotoxic T lymphocytes are able to expand (ki67+). This observation in combination with the good prognosis of cancers with high number of expanding exhausted cells argues for retained cytotoxic activity of CD112R+/ PD-1+ CD8+ T lymphocytes. Citation Format: Ronald Simon, Niclas Christian Blessin, Tim Mandelkow, Elena Bady, Claudia Hube-Magg, Guido Sauter, Maximilian Lennartz, Christoph Fraune, Sören A. Weidemann, Katharina Möller, Franziska Büscheck. Prognostic role of CD112R, PD-1 and Ki67 expression in CD8+cytotoxic T cells in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4970.

The modulatory activity of T cell immunoglobulin and mucin domain-containing protein 3 on T lymphocytes in patients with chronic heart failure

Objective: To investigate the expression of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) in patients with chronic heart failure and its modulatory activity on T lymphocytes. Methods: Eighty-six patients with chronic heart failure (CHF group) who were hospitalized in Department of Cardiology, the First Affiliated Hospital of Zhengzhou University between January and October 2018 were enrolled in the study. Meanwhile, thirty-two healthy controls (HC group) who received healthy examination were also selected. Peripheral blood mononuclear cells were isolated, and TIM-3 expression of CD4(+)and CD8(+)T cells was investigated by flow cytometry. CD4(+)and CD8(+)T cells were purified, and were stimulated by anti-TIM-3 antibody. Interferon-γ (IFN-γ), interleukin (IL)-4, IL-10, IL-35, IL-17, and IL-22 expressions in the supernatants of cultured CD4(+)T cells and tumor necrosis factor-α (TNF-α) and IFN-γ expressions in the supernatants of cultured CD8(+) T cells were measured by enzyme-linked immunosorbent assay. mRNA expressions of T-bet, GATA-3, FoxP3, and RORγt in CD4(+)T cells and perforin and granzyme B in CD8(+)T cells were semi-quantified by real-time PCR. Student t test or paired t test was used for comparisons between the two groups. Results: TIM-3(+) CD4(+) T cell percentage significantly increased in CHF group than that of HC group (3.47%±1.06% vs 0.92%±0.27%, P<0.001). TIM-3(+)CD8(+)T cell percentage also notably elevated in CHF group compared to HC group (6.12%±1.91% vs 1.77%±0.63%, P<0.001). CD4(+)T and CD8(+)T cells were dysfunctional in chronic heart failure. The levels of IFN-γ, IL-17, and IL-22 secreted by purified CD4(+) T cells significantly reduced in CHF group, while IL-10 and IL-35 expressions elevated in CHF group (all P<0.05). The relative mRNA expression levels of T-bet and RORγt in CD4(+) T cells remarkably decreased in CHF group than those of HC group (all P<0.01), while relative expression level of FoxP3 mRNA increased in CHF group (1.93±0.88 vs 0.97±0.28, P=0.031). The levels of TNF-α and IFN-γ produced by purified CD8(+)T cells notably reduced in CHF group than those of HC group (all P<0.05), and relative mRNA expression levels of perforin and granzyme B also decreased in CHF group (all P<0.05). The levels of anti-TIM-3 antibody stimulation-produced IFN-γ, IL-17, and IL-22 increased (all P<0.05) but IL-35 secretion reduced [(61±13) ng/ml vs (72±17) ng/ml, P=0.029] by CD4(+)T cells in CHF group. The relative mRNA expression levels of T-bet and RORγt also elevated in response to anti-TIM-3 antibody stimulation (all P<0.05). Anti-TIM-3 antibody stimulation promoted TNF-α and IFN-γ production by CD8(+)T cells in CHF group (all P<0.01). The relative mRNA expression levels of perforin and granzyme B also increased (all P<0.05). Conclusion: TIM-3 was increasingly expressed in T cells from patients with chronic heart failure, and might take part in the regulation of T cell dysfunction in chronic heart failure.

The mitochondrial outer membrane protein SYNJ2BP interacts with the cell adhesion molecule TMIGD1 and can recruit it to mitochondria

BackgroundTransmembrane and immunoglobulin domain-containing protein 1 (TMIGD1) is a recently identified cell adhesion molecule which is predominantly expressed by epithelial cells of the intestine and the kidney. Its expression is downregulated in both colon and renal cancer suggesting a tumor suppressive activity. The function of TMIGD1 at the cellular level is largely unclear. Published work suggests a protective role of TMIGD1 during oxidative stress in kidney epithelial cells, but the underlying molecular mechanisms are unknown.ResultsIn this study, we address the subcellular localization of TMIGD1 in renal epithelial cells and identify a cytoplasmic scaffold protein as interaction partner of TMIGD1. We find that TMIGD1 localizes to different compartments in renal epithelial cells and that this localization is regulated by cell confluency. Whereas it localizes to mitochondria in subconfluent cells it is localized at cell-cell contacts in confluent cells. We find that cell-cell contact localization is regulated by N-glycosylation and that both the extracellular and the cytoplasmic domain contribute to this localization. We identify Synaptojanin 2-binding protein (SYNJ2BP), a PDZ domain-containing cytoplasmic protein, which localizes to both mitochondria and the plasma membrane, as interaction partner of TMIGD1. The interaction of TMIGD1 and SYNJ2BP is mediated by the PDZ domain of SYNJ2BP and the C-terminal PDZ domain-binding motif of TMIGD1. We also find that SYNJ2BP can actively recruit TMIGD1 to mitochondria providing a potential mechanism for the localization of TMIGD1 at mitochondria.ConclusionsThis study describes TMIGD1 as an adhesion receptor that can localize to both mitochondria and cell-cell junctions in renal epithelial cells. It identifies SYNJ2BP as an interaction partner of TMIGD1 providing a potential mechanism underlying the localization of TMIGD1 at mitochondria. The study thus lays the basis for a better understanding of the molecular function of TMIGD1 during oxidative stress regulation.

An Ancient BCR-like Signaling Promotes ICP Production and Hemocyte Phagocytosis in Oyster.

SummaryBCR/TCR-based adaptive immune systems arise in the jawed vertebrates, and B cell receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. The existence of BCR-like molecule and the activation mechanism of the downstream response are still not clear in invertebrates. In this study, an ancient BCR-like molecule (designated as CgIgR) with an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic tail was identified from the Pacific oyster Crassostrea gigas to investigate its involvement in immune response. CgIgR could bind different bacteria through five extracellular Ig domains and formed dimers. The activated CgIgR recruited CgSyk to promote CgERK phosphorylation. The CgIgR-mediated signaling promoted the production of immunoglobulin domain-containing proteins (CgICP-2 and CgLRRIG-1) through inducing CgH3K4me2. The produced CgICPs eventually facilitated hemocytes to phagocytize and eliminate V. splendidus. This study proposed that there was an ancient BCR-like molecule and BCR-like signaling in molluscs.

&lt;i&gt;LINGO2&lt;/i&gt; and Movement Disorder Predisposition: Results of a Meta-Analysis

Previous reports on the pathogenesis of age-related movement disorders such as Parkinson’s disease (PD) and essential tremor (ET) have shown the potential implication of the LINGO1 (leucine-rich repeat and immunoglobulin domain-containing protein) gene. Although LINGO2 has a high-degree of homology with LINGO1, it is less characterized, and the role of LINGO2 in the development of PD/ET remains elusive. This meta-analysis was conducted to evaluate the role of LINGO2 in PD/ET pathogenesis. A literature search was conducted until 30 March 2020 in accordance to the PRISMA guidelines. The keywords “LINGO2”, “PD”, and “ET” were used in combination to obtain case-control studies evaluating LINGO2 gene polymorphism and PD/ET predisposition. A total of 4 studies were included, all of which complied with the Hardy-Weinberg Equilibrium. Analysis on the pooled odds ratio and CI of the studies were performed for five genetic models, including allelic (mutant type (M) vs. wild type (W)), dominant (MM+WM vs. WW), recessive (MM vs. WM+WW), homozygous (MM vs. WW), and heterozygous (WM vs. WW) models. No significant association was observed between the LINGO2 polymorphism with PD/ET, although subgroup analysis indicated that the recessive models of rs7033345 and rs10812774 are significantly associated with ET predisposition in the Asian population. Larger studies on this association is needed in the future to warrant our results.

T cell immunoglobulin and mucin domain-containing protein 3 inhibits interleukin-12 secretion by CD14+ monocytes from patients with psoriasis vulgaris

Objective To assess the role of T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) in regulation of interleukin (IL) -12 secretion by CD14+ peripheral blood monocytes from patients with psoriasis vulgaris. Methods From November 2017 to March 2018, a total of 47 patients with psoriasis vulgaris (psoriasis group) and 19 healthy volunteers (control group) were enrolled from Shanxi Provincial People′s Hospital. Peripheral blood mononuclear cells (PBMC) were isolated, and stimulated with Toll-like receptor (TLR) ligands. TIM-3 expression and IL-12 secretion by CD14+ monocytes were measured by flow cytometry. After blockade of TIM-3 pathway by anti-TIM-3 neutralizing antibody, changes in the downstream signaling pathway molecules in and IL-12 secretion by CD14+ monocytes were investigated. Two independent samples t-test was used for comparison between two groups, and Pearson correlation test for correlation analysis. Results Under the unstimulated condition, the level of IL-12 secreted by CD14+ monocytes was very low, and the proportion of CD14+ TIM-3+ cells was significantly higher in the psoriasis group (12.20% ± 2.83%) than in the control group (9.91% ± 1.77%, t = 3.270, P = 0.001 7) . After the stimulation with TLR ligands, the proportion of CD14+ IL-12+ cells was significantly lower in the psoriasis group (13.49% ± 2.80%) than in the control group (28.97% ± 8.97%, t = 10.71, P < 0.000 1) , but the proportion of CD14+TIM-3+ cells was still higher in the psoriasis group (6.80% ± 1.11%) than in the control group (4.85% ± 1.37%, t = 6.064, P < 0.000 1) . The proportion of CD14+TIM-3+ cells was negatively correlated with that of CD14+ IL-12+ cells in both the control group and psoriasis group (r = -0.473, -0.371 respectively, both P < 0.05) . The TIM-3 pathway blockade could induce the decrease of suppressor of cytokine signaling 1 in CD14+ monocytes, increase the phosphorylation of signaling transducers and activators of transcription 1, and promote IL-12 secretion by CD14+ monocytes induced by keratinocytes isolated from psoriatic skin lesions. Conclusion TIM-3 plays a crucial role in the negative regulation of CD14+ monocyte-induced innate immune response in psoriasis. Key words: Psoriasis; Monocytes; Interleukin-12; Antigens, CD14; T cell immunoglobulin and mucin domain-containing protein 3