RESUMO
Blockade of the inhibitory receptor TIM-3 shows efficacy in cancer immunotherapy clinical trials. TIM-3 inhibits production of the chemokine CXCL9 by XCR1+ classical dendritic cells (cDC1), thereby limiting antitumor immunity in mammary carcinomas. We found that increased CXCL9 expression by splenic cDC1s upon TIM-3 blockade required type I interferons and extracellular DNA. Chemokine expression as well as combinatorial efficacy of TIM-3 blockade and paclitaxel chemotherapy were impaired by deletion of Cgas and Sting. TIM-3 blockade increased uptake of extracellular DNA by cDC1 through an endocytic process that resulted in cytoplasmic localization. DNA uptake and efficacy of TIM-3 blockade required DNA binding by HMGB1, while galectin-9-induced cell surface clustering of TIM-3 was necessary for its suppressive function. Human peripheral blood cDC1s also took up extracellular DNA upon TIM-3 blockade. Thus, TIM-3 regulates endocytosis of extracellular DNA and activation of the cytoplasmic DNA sensing cGAS-STING pathway in cDC1s, with implications for understanding the mechanisms underlying TIM-3 immunotherapy.
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DNA/metabolismo , Células Dendríticas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais/fisiologia , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Citoplasma/metabolismo , Endocitose/fisiologia , Feminino , Células HEK293 , Humanos , Imunoterapia/métodos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
An improved understanding of the anti-tumor CD8+ T cell response after checkpoint blockade would enable more informed and effective therapeutic strategies. Here we examined the dynamics of the effector response of CD8+ tumor-infiltrating lymphocytes (TILs) after checkpoint blockade therapy. Bulk and single-cell RNA profiles of CD8+ TILs after combined Tim-3+PD-1 blockade in preclinical models revealed significant changes in the transcriptional profile of PD-1- TILs. These cells could be divided into subsets bearing characterstics of naive-, effector-, and memory-precursor-like cells. Effector- and memory-precursor-like TILs contained tumor-antigen-specific cells, exhibited proliferative and effector capacity, and expanded in response to different checkpoint blockade therapies across different tumor models. The memory-precursor-like subset shared features with CD8+ T cells associated with response to checkpoint blockade in patients and was compromised in the absence of Tcf7. Expression of Tcf7/Tcf1 was requisite for the efficacy of diverse immunotherapies, highlighting the importance of this transcriptional regulator in the development of effective CD8+ T cell responses upon immunotherapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Experimentais/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células , Receptor Celular 2 do Vírus da Hepatite A/antagonistas & inibidores , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Humanos , Memória Imunológica/genética , Imunoterapia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Melanoma Experimental , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/imunologia , TranscriptomaRESUMO
Natural killer (NK) cell immunotherapy has gained attention as a promising strategy for treatment of various malignancies. In this study, we used a genome-wide CRISPR screen to identify genes that provide protection or susceptibility to NK cell cytotoxicity. The screen confirmed the role of several genes in NK cell regulation, such as genes involved in interferon-γ signaling and antigen presentation, as well as genes encoding the NK cell receptor ligands B7-H6 and CD58. Notably, the gene TMEM30A, encoding CDC50A-beta-subunit of the flippase shuttling phospholipids in the plasma membrane, emerged as crucial for NK cell killing. Accordingly, a broad range of TMEM30A knock-out (KO) leukemia and lymphoma cells displayed increased surface levels of phosphatidylserine (PtdSer). TMEM30A KO cells triggered less NK cell degranulation, cytokine production and displayed lower susceptibility to NK cell cytotoxicity. Blockade of PtdSer or the inhibitory receptor TIM-3, restored the NK cell ability to eliminate TMEM30A-mutated cells. The key role of the TIM-3 - PtdSer interaction for NK cell regulation was further substantiated by disruption of the receptor gene in primary NK cells, which significantly reduced the impact of elevated PtdSer in TMEM30A KO leukemic cells. Our study underscores the potential significance of agents targeting the interaction between PtdSer and TIM-3 in the realm of cancer immunotherapy.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Células Matadoras Naturais , Leucemia , Linfoma , Membrana Celular/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Interferon gama/metabolismo , Receptores de Células Matadoras Naturais , Humanos , Leucemia/metabolismo , Linfoma/metabolismo , Proteínas de Membrana/metabolismoRESUMO
Successful pregnancy is a unique situation requires the maternal immune system to recognize and tolerate a semi-identical fetus and allow normal invasion of trophoblast cells. Although efforts have been made, the deep mechanisms of the maternal-fetal crosstalk have not yet been fully deciphered. Immune checkpoint molecules (ICMs) are a group of negative modulators of the immune response that avoid immune damage. They have been extensively studied in the fields of oncology and transplantation, while the latest evidence suggests that they are closely associated with pregnancy outcomes via multiple inhibitory mechanisms. Although studies have mostly demonstrated the regulatory role of the well-known PD-1, CTLA-4 at the maternal-fetal interface, what is unique about the newly discovered multiple ICMs remains a mystery. Here, we review the latest knowledge on ICMs, focusing on the first generation of checkpoints (PD-1, CTLA-4) and the next generation (Tim-3, Tigit, Lag-3, VISTA) highlighting their immunoregulatory roles in maternal-fetal tolerance and decidual vascular remodeling, and their involvement in pathological pregnancies. The content covers three aspects: the characteristics they possess, the dynamic expression profile of their expression at the maternal-fetal interface, and their involvement in pathological pregnancy. In immunotherapy strategies for pregnancy complications, upregulation of immune checkpoints may play a role. Meanwhile, the impact on pregnancy outcomes when using ICMs in clinical cancer treatment during pregnancy is a topic worth exploring. These may serve as a guide for future basic research and clinical applications of maternal-fetal immunity.
Assuntos
Proteínas de Checkpoint Imunológico , Receptor de Morte Celular Programada 1 , Antígeno CTLA-4 , Feminino , Humanos , Proteínas de Checkpoint Imunológico/genética , Tolerância Imunológica , Imunidade , Gravidez , Receptor de Morte Celular Programada 1/metabolismoRESUMO
Rationale: Unraveling immune-driven vascular pathology in pulmonary arterial hypertension (PAH) requires a comprehensive understanding of the immune cell landscape. Although patients with hereditary (H)PAH and bone morphogenetic protein receptor type 2 (BMPR2) mutations have more severe pulmonary vascular pathology, it is not known whether this is related to specific immune cell subsets. Objectives: This study aims to elucidate immune-driven vascular pathology by identifying immune cell subtypes linked to severity of pulmonary arterial lesions in PAH. Methods: We used cutting-edge multiplexed ion beam imaging by time of flight to compare pulmonary arteries (PAs) and adjacent tissue in PAH lungs (idiopathic [I]PAH and HPAH) with unused donor lungs, as controls. Measurements and Main Results: We quantified immune cells' proximity and abundance, focusing on those features linked to vascular pathology, and evaluated their impact on pulmonary arterial smooth muscle cells (SMCs) and endothelial cells. Distinct immune infiltration patterns emerged between PAH subtypes, with intramural involvement independently linked to PA occlusive changes. Notably, we identified monocyte-derived dendritic cells within PA subendothelial and adventitial regions, influencing vascular remodeling by promoting SMC proliferation and suppressing endothelial gene expression across PAH subtypes. In patients with HPAH, pronounced immune dysregulation encircled PA walls, characterized by heightened perivascular inflammation involving T cell immunoglobulin and mucin domain-3 (TIM-3)+ T cells. This correlated with an expanded DC subset expressing indoleamine 2,3-dioxygenase 1, TIM-3, and SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1, alongside increased neutrophils, SMCs, and alpha-smooth muscle actin (ACTA2)+ endothelial cells, reinforcing the heightened severity of pulmonary vascular lesions. Conclusions: This study presents the first architectural map of PAH lungs, connecting immune subsets not only with specific PA lesions but also with heightened severity in HPAH compared with IPAH. Our findings emphasize the therapeutic potential of targeting monocyte-derived dendritic cells, neutrophils, cellular interactions, and immune responses to alleviate severe vascular pathology in IPAH and HPAH.
Assuntos
Hidralazina/análogos & derivados , Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Humanos , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Células Endoteliais/metabolismo , Hipertensão Pulmonar Primária Familiar/genética , Artéria Pulmonar , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Proliferação de Células , HidrazonasRESUMO
Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.
Assuntos
Glioblastoma , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Prognóstico , Linfócitos T CD8-Positivos , Microambiente TumoralRESUMO
Inflammatory bowel disease (IBD) occurring following allogeneic stem cell transplantation (aSCT) is a very rare condition. The underlying pathogenesis needs to be better defined. There is currently no systematic effort to exclude loss- or gain-of-function mutations in immune-related genes in stem cell donors. This is despite the fact that more than 100 inborn errors of immunity may cause or contribute to IBD. We have molecularly characterized a patient who developed fulminant inflammatory bowel disease following aSCT with stable 100% donor-derived hematopoiesis. A pathogenic c.A291G; p.I97M HAVCR2 mutation encoding the immune checkpoint protein TIM-3 was identified in the patient's blood-derived DNA, while being absent in DNA derived from the skin. TIM-3 expression was much decreased in the patient's serum, and in vitro-activated patient-derived T cells expressed reduced TIM-3 levels. In contrast, T cell-intrinsic CD25 expression and production of inflammatory cytokines were preserved. TIM-3 expression was barely detectable in the immune cells of the patient's intestinal mucosa, while being detected unambiguously in the inflamed and non-inflamed colon from unrelated individuals. In conclusion, we report the first case of acquired, "transplanted" insufficiency of the regulatory TIM-3 checkpoint linked to post-aSCT IBD.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Doenças Inflamatórias Intestinais , Transplante de Células-Tronco , Humanos , Citocinas/metabolismo , Receptor Celular 2 do Vírus da Hepatite A/genética , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/etiologia , Mucosa Intestinal , Transplante de Células-Tronco/efeitos adversosRESUMO
BACKGROUND & AIMS: Carcinoembryonic antigen-related cell adhesion molecule 1 (CC1) acts through homophilic and heterophilic interactions with T cell immunoglobulin domain and mucin domain-containing protein 3 (TIM-3), which regulates innate immune activation in orthotopic liver transplantation (OLT). We investigated whether cluster of differentiation (CD) 4+ T cell-dependent CC1-TIM-3 crosstalk may affect OLT outcomes in mice and humans. METHODS: Wild-type (WT) and CC1-deficient (CC1 knock-out [KO]) mouse livers were transplanted into WT, CC1KO, or T-cell TIM-3 transgenic (TIM-3Tg)/CC1KO double-mutant recipients. CD4+ T cells were adoptively transferred into T/B cell-deficient recombination activating gene 2 protein (Rag2) KO recipients, followed by OLT. The perioperative liver-associated CC1 increase was analyzed in 50 OLT patients. RESULTS: OLT injury in WT livers deteriorated in CC1KO compared with CC1-proficient (WT) recipients. The frequency of TIM-3+CD4+ T cells was higher in WT than CC1KO hosts. Reconstitution of Rag2KO mice with CC1KO-T cells increased nuclear factor (NF)-κB phosphorylation and OLT damage compared with recipients repopulated with WT T cells. T-cell TIM-3 enhancement in CC1KO recipients (WT â TIM3Tg/CC1KO) suppressed NF-κB phosphorylation in Kupffer cells and mitigated OLT injury. However, TIM-3-mediated protection was lost by pharmacologic TIM-3 blockade or an absence of CC1 in the donor liver (CC1KO â TIM-3Tg/CC1KO). The perioperative CC1 increase in human OLT reduced hepatocellular injury, early allograft dysfunction, and the cumulative rejection rate. CONCLUSIONS: This translational study identifies T cell-specific CC1 signaling as a therapeutic means to alleviate OLT injury by promoting T cell-intrinsic TIM-3, which in turn interacts with liver-associated CC1 to suppress NF-κB in Kupffer cells. By suppressing peritransplant liver damage, promoting T-cell homeostasis, and improving OLT outcomes, recipient CC1 signaling serves as a novel cytoprotective sentinel.
Assuntos
Hepatopatias , Transplante de Fígado , Humanos , Camundongos , Animais , Receptor Celular 2 do Vírus da Hepatite A/genética , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Linfócitos T , NF-kappa B/metabolismo , Doadores Vivos , Fígado/metabolismo , Camundongos Knockout , Fatores de Transcrição/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis (EBL) in cattle and is widespread in many countries, including Japan. Recent studies have revealed that the expression of immunoinhibitory molecules, such as programmed death-1 (PD-1) and PD-ligand 1, plays a critical role in immunosuppression and disease progression during BLV infection. In addition, a preliminary study has suggested that another immunoinhibitory molecule, T-cell immunoglobulin domain and mucin domain-3 (TIM-3), is involved in immunosuppression during BLV infection. Therefore, this study was designed to further elucidate the immunoinhibitory role of immune checkpoint molecules in BLV infection. TIM-3 expression was upregulated on peripheral CD4+ and CD8+ T cells in BLV-infected cattle. Interestingly, in EBL cattle, CD4+ and CD8+ T cells infiltrating lymphomas expressed TIM-3. TIM-3 and PD-1 were upregulated and coexpressed in peripheral CD4+ and CD8+ T cells from BLV-infected cattle. Blockade by anti-bovine TIM-3 monoclonal antibody increased CD69 expression on T cells and gamma interferon (IFN-γ) production from peripheral blood mononuclear cells from BLV-infected cattle. A syncytium formation assay also demonstrated the antiviral effects of TIM-3 blockade against BLV infection. The combined inhibition of TIM-3 and PD-1 pathways significantly enhanced IFN-γ production and antiviral efficacy compared to inhibition alone. In conclusion, the combined blockade of TIM-3 and PD-1 pathways shows strong immune activation and antiviral effects and has potential as a novel therapeutic method for BLV infection. IMPORTANCE Enzootic bovine leukosis caused by bovine leukemia virus (BLV) is an important viral disease in cattle, causing severe economic losses to the cattle industry worldwide. The molecular mechanisms of BLV-host interactions are complex. Previously, it was found that immune checkpoint molecules, such as PD-1, suppress BLV-specific Th1 responses as the disease progresses. To date, most studies have focused only on how PD-1 facilitates escape from host immunity in BLV-infected cattle and the antiviral effects of the PD-1 blockade. In contrast, how T-cell immunoglobulin domain and mucin domain-3 (TIM-3), another immune checkpoint molecule, regulates anti-BLV immune responses is rarely reported. It is also unclear why PD-1 inhibition alone was insufficient to exert anti-BLV effects in previous clinical studies. In this study, the expression profile of TIM-3 in T cells derived from BLV-infected cattle suggested that TIM-3 upregulation is a cause of immunosuppression in infected cattle. Based on these results, anti-TIM-3 antibody was used to experimentally evaluate its function in influencing immunity against BLV. Results indicated that TIM-3 upregulation induced by BLV infection suppressed T-cell activation and antiviral cytokine production. Some T cells coexpressed PD-1 and TIM-3, indicating that simultaneous inhibition of PD-1 and TIM-3 with their respective antibodies synergistically restored antiviral immunity. This study could open new avenues for treating bovine chronic infections.
Assuntos
Leucose Enzoótica Bovina , Proteínas de Checkpoint Imunológico , Vírus da Leucemia Bovina , Animais , Bovinos , Linfócitos T CD8-Positivos/imunologia , Leucose Enzoótica Bovina/imunologia , Proteínas de Checkpoint Imunológico/imunologia , Interferon gama/imunologia , Vírus da Leucemia Bovina/imunologia , Mucinas/imunologia , Receptor de Morte Celular Programada 1/imunologia , Regulação da Expressão Gênica/imunologiaRESUMO
Members of the T-cell immunoglobulin and mucin (TIM) family, which is crucial for T-cell function, are implicated in autoimmunity. TIM-1 and -3 play distinct roles in autoimmunity, with TIM-1 acting as a costimulatory molecule and TIM-3 regulating Th1 responses. We investigated the therapeutic potential of anti-TIM-1 (RMT1-10) and anti-TIM-3 (RMT3-23) antibodies in an autoimmune arthritis model. Zymosan A was used to induce arthritis in female SKG mice. The arthritis scores, histology, mRNA expression, cytokine levels, micro-CT, and flow cytometry results were obtained. The application of RMT1-10 reduced the arthritis scores, histological damage, and CD4+T cell infiltrations, and it suppressed interleukin (IL)-6 and -17A and reduced TIM-3 mRNA expressions. RMT3-23 also lowered arthritis severity, improved histology, and reduced serum levels of tumor necrosis factor (TNF)-α and IL-17A. RMT3-23 inhibited intracellular TNF-α and IL-6 and early apoptosis. An amelioration of autoimmune arthritis was achieved by blocking the TIM-1 and -3 signaling pathways via RMT1-10 and RMT3-23 administration, leading to a widespread decrease in inflammatory cytokines. Both antibodies exhibited therapeutic effects, suggesting TIM-1 and -3 as potential targets for rheumatoid arthritis.
RESUMO
BACKGROUND: Overexpression of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3) is related to the exhaustion of CD8+ tumor-infiltrating lymphocytes (TILs) in diffuse large B-cell lymphoma (DLBCL). However, the mechanism of TIM3-mediated CD8+TILs exhaustion in DLBCL remains poorly understood. Therefore, we aimed to clarify the potential pathway involved in TIM3-mediated CD8+TILs exhaustion and its significance in DLBCL. METHODS: The expression of TIM3 and its correlation with CD8+TILs exhaustion, the key ligand of TIM3, and the potential pathway of TIM3-mediated CD8+TILs exhaustion in DLBCL were analyzed using single-cell RNA sequencing and validated by RNA sequencing. The biological significance of TIM3-related pathway in DLBCL was investigated based on RNA sequencing, immunohistochemistry, and reverse transcription-quantitative polymerase chain reaction data. Finally, the possible regulatory mechanism of TIM3-related pathway in DLBCL was explored using single-cell RNA sequencing and RNA sequencing. RESULTS: Our results demonstrated that CD8+TILs, especially the terminally exhausted state, were the major clusters that expressed TIM3 in DLBCL. Galectin-9, mainly expressed in M2 macrophages, is the key ligand of TIM3 and can induce the exhaustion of CD8+TILs through TIM3/Galectin-9 pathway. Meanwhile, high TIM3/Galectin-9 enrichment is related to immunosuppressive tumor microenvironment, severe clinical manifestations, inferior prognosis, and poor response to CHOP-based chemotherapy, and can predict the clinical efficacy of immune checkpoint blockade therapy in DLBCL. Furthermore, the TIM3/Galectin-9 enrichment in DLBCL may be regulated by the IFN-γ signaling pathway. CONCLUSIONS: Our study highlights that TIM3/Galectin-9 pathway plays a crucial role in CD8+TILs exhaustion and the immune escape of DLBCL, which facilitates further functional studies and could provide a theoretical basis for the development of novel immunotherapy in DLBCL.
Assuntos
Linfócitos T CD8-Positivos , Galectinas , Receptor Celular 2 do Vírus da Hepatite A , Linfoma Difuso de Grandes Células B , Humanos , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Ligantes , Linfócitos do Interstício Tumoral , Linfoma Difuso de Grandes Células B/patologia , Microambiente Tumoral , Galectinas/metabolismoRESUMO
We investigated expressions of PD-L1, LAG-3, TIM-3, and OX40L as immune checkpoint proteins, and MSI (repetitive short-DNA-sequences due to defective DNA-repair system) status were analyzed with immunohistochemistry from tissue blocks. Of 83 patients, PD-L1 expression was observed in 18.1% (n = 15) of the patients. None of the patients exhibited LAG-3 expression. TIM-3 expression was 4.9% (n = 4), OX40L was 22.9% (n = 19), and 8.4% (n = 7) of the patients had MSI tumor. A low-to-intermediate positive correlation was observed between PD-L1 and TIM-3 expressions (rho: 0.333, p < 0.01). Although PD-L1 expression was higher in grade 3 NET/NEC, MSI status was prominent in grade 1/2 NET.
Assuntos
Antígeno B7-H1 , Neoplasias Gastrointestinais , Receptor Celular 2 do Vírus da Hepatite A , Proteínas de Checkpoint Imunológico , Tumores Neuroendócrinos , Neoplasias Pancreáticas , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Antígeno B7-H1/análise , Antígeno B7-H1/metabolismo , Reparo do DNA , Neoplasias Gastrointestinais/química , Neoplasias Gastrointestinais/patologia , Receptor Celular 2 do Vírus da Hepatite A/análise , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Proteínas de Checkpoint Imunológico/análise , Proteínas de Checkpoint Imunológico/metabolismo , Proteína do Gene 3 de Ativação de Linfócitos/análise , Proteína do Gene 3 de Ativação de Linfócitos/metabolismo , Tumores Neuroendócrinos/química , Tumores Neuroendócrinos/patologia , Ligante OX40/análise , Ligante OX40/metabolismo , Neoplasias Pancreáticas/química , Neoplasias Pancreáticas/patologia , Estudos Retrospectivos , Imuno-Histoquímica , Gradação de TumoresRESUMO
BACKGROUND: Galectin-9 (Gal-9) has been implicated in allergic and autoimmune diseases, but its role and relevance in chronic spontaneous urticaria (CSU) are unclear. OBJECTIVES: To characterize the role and relevance of Gal-9 in the pathogenesis of CSU. METHODS: We assessed 60 CSU patients for their expression of Gal-9 on circulating eosinophils and basophils as well as T cell expression of the Gal-9 receptor TIM-3, compared them with 26 healthy controls (HCs), and explored possible links with disease features including disease activity (urticaria activity score, UAS), total IgE, basophil activation test (BAT), and response to omalizumab treatment. We also investigated potential drivers of Gal-9 expression by eosinophils and basophils. RESULTS: Our CSU patients had markedly increased rates of circulating Gal-9+ eosinophils and basophils and high numbers of lesional Gal-9+ cells. High rates of blood Gal-9+ eosinophils/basophils were linked to high disease activity, IgE levels, and BAT negativity. Serum levels of TNF-α were positively correlated with circulating Gal-9+ eosinophils/basophils, and TNF-α markedly upregulated Gal-9 on eosinophils. CSU patients who responded to omalizumab treatment had more Gal-9+ eosinophils/basophils than non-responders, and omalizumab reduced blood levels of Gal-9+ eosinophils/basophils in responders. Gal-9+ eosinophils/basophils were negatively correlated with TIM-3+TH17 cells. CONCLUSION: Our findings demonstrate a previously unrecognized involvement of the Gal-9/TIM-3 pathway in the pathogenesis CSU and call for studies that explore its relevance.
Assuntos
Antialérgicos , Basófilos , Biomarcadores , Urticária Crônica , Eosinófilos , Galectinas , Omalizumab , Humanos , Basófilos/metabolismo , Basófilos/imunologia , Galectinas/metabolismo , Omalizumab/uso terapêutico , Omalizumab/farmacologia , Urticária Crônica/tratamento farmacológico , Masculino , Feminino , Eosinófilos/metabolismo , Eosinófilos/imunologia , Adulto , Pessoa de Meia-Idade , Antialérgicos/uso terapêutico , Antialérgicos/farmacologia , Resultado do Tratamento , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Índice de Gravidade de Doença , Estudos de Casos e Controles , Idoso , Adulto JovemRESUMO
T cell transmembrane, Immunoglobulin, and Mucin (TIM) are important immune system proteins which are especially present in T-cells and regulated the immune system by sensing cell engulfment and apoptotic processes. Their role is exerted by the capacity to detect the presence of phosphatidyl-serine lipid polar head in the outer leaflet of cellular membranes (correlated with apoptosis). In this contribution by using equilibrium and enhanced sampling molecular dynamics simulation we unravel the molecular bases and the thermodynamics of TIM, and in particular TIM-3, interaction with phosphatidyl serine in a lipid bilayer. Since TIM-3 deregulation is an important factor of pro-oncogenic tumor micro-environment understanding its functioning at a molecular level may pave the way to the development of original immunotherapeutic approaches.
Assuntos
Receptor Celular 2 do Vírus da Hepatite A , Proteínas de Membrana , Proteínas de Membrana/metabolismo , Mucina-3 , Fosfatidilserinas , Lipídeos de Membrana , Linfócitos T , Mucinas , SerinaRESUMO
BACKGROUND: T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1. METHODS: Cell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively. RESULTS: The most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001). CONCLUSION: Taken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.
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Glutamina , Leucemia Mieloide Aguda , Humanos , Ácido Glutâmico , Receptor Celular 2 do Vírus da Hepatite A , Células HL-60RESUMO
T cell immunoglobulin and mucin domain-3 (TIM3; HAVCR2) is a transmembrane protein that exerts negative regulatory control over T cell responses. Studies have demonstrated an upregulation of TIM3 expression in tumor-infiltrating lymphocytes (TILs) in cancer patients. In this investigation, a series of monoclonal antibodies targeting TIM3 were produced by hybridoma technology. Among them, C23 exhibited favorable biological properties. To enable specific binding, we developed a 124I/125I-C23 radio-tracer via N-bromosuccinimide (NBS)-mediated labeling of the monoclonal antibody C23. Binding affinity and specificity were assessed using the 293T-TIM3 cell line, which overexpresses TIM3, and the parent 293T cells. Furthermore, biodistribution and in vivo imaging of 124I/125I-C23 were examined in HEK293TIM3 xenograft models and allograft models of 4T1 (mouse breast cancer cells) and CT26 (mouse colon cancer cells). Micro-PET/CT imaging was conducted at intervals of 4, 24, 48, 72, and/or 96 h post intravenous administration of 3.7-7.4 MBq 124I-C23 in the respective model mice. Additionally, immunohistochemistry (IHC) staining of TIM3 expression in dissected tumor organs was performed, along with an assessment of the corresponding expression of Programmed Death 1 (PD1), CD3, and CD8 in the tumors. The C23 monoclonal antibody (mAb) specifically binds to TIM3 protein with a dissociation constant of 23.28 nM. The 124I-C23 and 125I-C23 radio-tracer were successfully prepared with a labeling yield of 83.59 ± 0.35% and 92.35 ± 0.20%, respectively, and over 95.00% radiochemical purity. Stability results indicated that the radiochemical purity of 124I/125I-C23 in phosphate-buffered saline (PBS) and 5% human serum albumin (HSA) was still >80% after 96 h. 125I-C23 uptake in 293T-TIM3 cells was 2.80 ± 0.12%, which was significantly higher than that in 293T cells (1.08 ± 0.08%), and 125I-C23 uptake by 293T-TIM3 cells was significantly blocked at 60 and 120 min in the blocking groups. Pharmacokinetics analysis in vivo revealed an elimination time of 14.62 h and a distribution time of 0.4672 h for 125I-C23. Micro-PET/CT imaging showed that the 124I-C23 probe uptake in the 293T-TIM3 model significantly differed from that of the negative control group and blocking group. In the humanized mouse model, the 124I-C23 probe had obvious specific uptake in the 4T1 and CT26 models and maximum uptake at 24 h in tumor tissues (SUVmax (the maximum standardized uptake value) in 4T1 and CT26 humanized TIM3 murine tumor models: 0.59 ± 0.01 and 0.76 ± 0.02, respectively). Immunohistochemistry of tumor tissues from these mouse models showed comparable TIM3 expression. CD3 and CD8 cells and PD-1 expression were also observed in TIM3-expressing tumor tissues. The TIM3-targeting antibody C23 showed good affinity and specificity. The 124I/125I-C23 probe has obvious targeting specificity for TIM3 in vitro and in vivo. Our results suggest that 124I/125I-C23 is a promising tracer for TIM3 imaging and may have great potential in monitoring immune checkpoint drug efficacy.
Assuntos
Anticorpos Monoclonais , Neoplasias , Animais , Humanos , Camundongos , Anticorpos Monoclonais/química , Linhagem Celular Tumoral , Receptor Celular 2 do Vírus da Hepatite A/metabolismo , Radioisótopos do Iodo , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
PURPOSE: Immune checkpoint inhibitors (ICI) are then backbone in the therapy of metastatic renal cell carcinoma (RCC). The aim of this analysis was to explore the different expression of the ICI PD-L1, BTLA, and TIM-3 at the different tumor locations of the invasion front and the tumor center. METHODS: Large-area sections of the tumor center and invasion front of 44 stage pT1-4 clear cell RCCs were examined immunohistochemically using antibodies against BTLA, TIM-3, and PD-L1 and subsequently correlated with clinicopathologic data. RESULTS: TIM-3 was most strongly expressed at the invasion front (mean ± SD: 84.1 ± 46.6, p = 0.094). BTLA expression was highest in normal tissue, with weak staining in the tumor center and at the invasion front [110.2 vs. 18.6 (p < 0.001) vs. 32.2 (p = 0.248)]. PD-L1 was weakly expressed at the tumor center (n = 5/44) and at the invasion front (n = 5/44). Correlation with clinicopathological parameters revealed significantly higher BTLA expression in ≥ T3 tumors compared to T1/2 tumors (tumor center p = 0.009; invasion front p = 0.005). BTLA-positive tumors at the tumor center correlated with worse CSS (median 48.46 vs. 68.91 months, HR 4.43, p = 0.061). PD-L1 expression was associated with worse CSS (median 1.66 vs. 4.5 years, HR 1.63, p = 0.652). For TIM-3, there were no significant associations with clinicopathological parameters and survival. CONCLUSION: The present results show heterogeneous intratumoral and intertumoral expression of the investigated checkpoint receptors PD-L1, BTLA, and TIM-3. In the clinical practice tumor sampling should include different tumor locations, and multiple inhibition of different checkpoint receptors seems reasonable to increase the therapeutic success.
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Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/patologia , Receptor Celular 2 do Vírus da Hepatite A , Antígeno B7-H1 , Neoplasias Renais/patologia , Biomarcadores Tumorais , Prognóstico , Receptores Imunológicos/metabolismoRESUMO
BACKGROUND: The immune system is recognized to have therapeutic potential to destroy cancer cells. Soluble T-cell immunoglobulin mucin domain-3 (sTIM-3) and its ligand galectin 9 (Gal-9) cause suppression of cytokine production, cell cycle arrest and cell death. sTIM-3 and Gal-9 levels may have prognostic implications in non-small-cell lung cancer (NSCLC) patients. METHODS: This prospective cohort study was performed at Instituto de Medicina Integral Prof. Fernando Figueira, Recife, Pernambuco, Brazil. Fifty-eight patients were diagnosed with advanced NSCLC from January 2019 to January 2020. RESULTS: The age median was of 64.0 years. Soluble galectin-9 (sGal-9) levels in the smokers compared to nonsmoker patients (p < 0.0001). By using the receiver operating characteristic curve, we found that a baseline of 1694 pg/mL (cutoff). sGAL9 with specificity (72.2%), sensitivity (83.2%) and area under the curve = 0.8497 (p < 0.0004). Until 18.2 months, 46.8% and 72.9% were alive in the sGAL9low and sGAL9high groups, respectively (log-rank test; p = 0.02). The median survival was 15.9 months for sGAL9low (≤1694 pg/mL). CONCLUSION: This study indicated an association of tobacco with the release of circulating sGal-9 levels and the accuracy of sGal-9 as a potential biomarker predictive of survival time in advanced NSCLC patients. Furthermore, sGal-9 has may be a potential therapeutic target in the advanced NSCLC.
RESUMO
BACKGROUND: Exploring predictive biomarkers and therapeutic strategies of ICBs has become an urgent need in clinical practice. Increasing evidence has shown that ARID1A deficiency might play a critical role in sculpting tumor environments in various tumors and might be used as pan-cancer biomarkers for immunotherapy outcomes. The current study aims to explored the immune-modulating role of ARID1A deficiency in Hepatitis B virus (HBV) related hepatocellular carcinoma (HBV-HCC) and its potential immunotherapeutic implications. METHODS: In the current study, we performed a comprehensive analysis using bioinformatics approaches and pre-clinical experiments to evaluate the ARID1A regulatory role on the biological behavior, and immune landscape of Hepatitis B virus (HBV) related hepatocellular carcinoma (HBV-HCC). A total of 425 HBV-related hepatocellular carcinoma patients from TCGA-LIHC, AMC and CHCC-HBV cohort were enrolled in bioinformatics analysis. Immunohistochemical staining of HBV-HCC specimens and ARID1A deficiency cellular models were used to validate the results of the analysis. RESULTS: Our results have shown that ARID1A deficiency promoted tumor proliferation and metastasis. More importantly, ARID1A deficiency in HBV-HCC was associated with the higher TMB, elevated immune activity, and up-regulated expression of immune checkpoint proteins, especially TIM-3 in HBV-HCC. Further, the expression of Galectin-9, which is the ligand of TIM-3, was elevated in the ARID1A knockout HBV positive cell line. CONCLUSION: To conclude, we have shown that the ARID1A deficiency was correlated with more active immune signatures and higher expression of immune checkpoints in HBV-HCC. Additionally, the present study provides insights to explore the possibility of the predictive role of ARID1A in HBV-HCC patients responsive to immunotherapy.
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Carcinoma Hepatocelular , Hepatite B , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Vírus da Hepatite B/genética , Neoplasias Hepáticas/patologia , Receptor Celular 2 do Vírus da Hepatite A , Biomarcadores Tumorais , Hepatite B/complicações , Proteínas de Ligação a DNA , Fatores de TranscriçãoRESUMO
Leukemia is a malignancy of the bone marrow and blood originating from self-renewing cancerous immature blast cells or transformed leukocytes. Despite improvements in treatments, leukemia remains still a serious disease with poor prognosis because of disease heterogeneity, drug resistance and relapse. There is emerging evidence that differentially expression of co-signaling molecules play a critical role in tumor immune evasion. Galectin-9 (Gal-9) is one of the key proteins that leukemic cells express, secrete, and use to proliferate, self-renew, and survive. It also suppresses host immune responses controlled by T and NK cells, enabling leukemic cells to evade immune surveillance. The present review provides the molecular mechanisms of Gal-9-induced immune evasion in leukemia. Understanding the complex immune evasion machinery driven by Gal-9 expressing leukemic cells will enable the identification of novel therapeutic strategies for efficient immunotherapy in leukemic patients. Combined treatment approaches targeting T-cell immunoglobulin and mucin domain-3 (Tim-3)/Gal-9 and other immune checkpoint pathways can be considered, which may enhance the efficacy of host effector cells to attack leukemic cells.