ABSTRACT
Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometrium-like/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain, and susceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorly understood and largely unknown. The prevailing view is that the immune system of endometriosis patients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes are small extracellular vesicles that exhibit immunomodulatory properties. We studied the role of endometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediated mechanisms known to impair the immune response were investigated: 1) downregulation of NKG2D-mediated cytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showed that secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL on their surface, i.e., signature molecules of exosome-mediated immune suppression. Acting as decoys, these exosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity, and induce apoptosis of activated PBMCs and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometriotic exosomes create an immunosuppressive gradient at the ectopic site, forming a "protective shield" around the endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxic attack and creates immunologic privilege by induction of apoptosis in activated immune cells. Taken together, our results provide a plausible, exosome-based mechanistic explanation for the immune dysfunction and the compromised immune surveillance in endometriosis and contribute novel insights into the pathogenesis of this enigmatic disease.
Subject(s)
Apoptosis , Endometriosis , Endometrium , Exosomes , NK Cell Lectin-Like Receptor Subfamily K , TNF-Related Apoptosis-Inducing Ligand , Humans , Endometriosis/immunology , Endometriosis/metabolism , Endometriosis/pathology , Female , Exosomes/metabolism , Exosomes/immunology , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Apoptosis/immunology , Endometrium/immunology , Endometrium/pathology , Endometrium/metabolism , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/immunology , Down-Regulation/immunology , Fas Ligand Protein/metabolism , Fas Ligand Protein/immunology , Cytotoxicity, Immunologic , Adult , Histocompatibility Antigens Class I/metabolism , Histocompatibility Antigens Class I/immunologyABSTRACT
The longevity of grafts remains a major challenge in allogeneic transplantation due to immune rejection. Systemic immunosuppression can impair graft function and can also cause severe adverse effects. Here, we report a local immuno-protective strategy to enhance post-transplant persistence of allografts using a mesenchymal stem cell membrane-derived vesicle (MMV)-crosslinked hydrogel (MMV-Gel). MMVs are engineered to upregulate expression of Fas ligand (FasL) and programmed death ligand 1 (PD-L1). The MMVs are retained within the hydrogel by crosslinking. The immuno-protective microenvironment of the hydrogel protects allografts by presenting FasL and PD-L1. The binding of these ligands to T effector cells, the dominant contributors to graft destruction and rejection, results in apoptosis of T effector cells and generation of regulatory T cells. We demonstrate that implantation with MMV-Gel prolongs the survival and function of grafts in mouse models of allogeneic pancreatic islet cells and skin transplantation.
Subject(s)
Fas Ligand Protein , Hydrogels , Islets of Langerhans Transplantation , Mice, Inbred C57BL , Skin Transplantation , T-Lymphocytes, Regulatory , Transplantation, Homologous , Animals , Hydrogels/chemistry , Mice , Fas Ligand Protein/metabolism , Fas Ligand Protein/immunology , T-Lymphocytes, Regulatory/immunology , Islets of Langerhans Transplantation/methods , Skin Transplantation/methods , B7-H1 Antigen/metabolism , B7-H1 Antigen/immunology , Mesenchymal Stem Cells/immunology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/drug effects , Mice, Inbred BALB C , Graft Survival/drug effects , Graft Survival/immunology , Graft Rejection/prevention & control , Graft Rejection/immunology , Humans , Male , Apoptosis/drug effectsABSTRACT
DNA origami is capable of spatially organizing molecules into sophisticated geometric patterns with nanometric precision. Here we describe a reconfigurable, two-dimensional DNA origami with geometrically patterned CD95 ligands that regulates immune cell signalling to alleviate rheumatoid arthritis. In response to pH changes, the device reversibly transforms from a closed to an open configuration, displaying a hexagonal pattern of CD95 ligands with ~10 nm intermolecular spacing, precisely mirroring the spatial arrangement of CD95 receptor clusters on the surface of immune cells. In a collagen-induced arthritis mouse model, DNA origami elicits robust and selective activation of CD95 death-inducing signalling in activated immune cells located in inflamed synovial tissues. Such localized immune tolerance ameliorates joint damage with no noticeable side effects. This device allows for the precise spatial control of cellular signalling, expanding our understanding of ligand-receptor interactions and is a promising platform for the development of pharmacological interventions targeting these interactions.
Subject(s)
Arthritis, Rheumatoid , DNA , Immune Tolerance , Signal Transduction , fas Receptor , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Animals , DNA/chemistry , DNA/immunology , Mice , fas Receptor/metabolism , fas Receptor/immunology , Fas Ligand Protein/metabolism , Fas Ligand Protein/immunology , HumansABSTRACT
Idiopathic severe aplastic anemia (SAA) is a disease of bone marrow failure caused by T-cell-induced destruction of hematopoietic stem and progenitor cells (HSPCs), however the mechanism remains unclear. We performed single-cell RNA sequencing of PBMCs and BMMCs from SAA patients and healthy donors and identified a CD8+ T cell subset with a tissue residency phenotype (Trm) in bone marrow that exhibit high IFN-γ and FasL expression and have a higher ability to induce apoptosis in HSPCs in vitro through FasL expression. CD8+ Trm cells were induced by IL-15 presented by IL-15Rα on monocytes, especially CD16+ monocytes, which were increased in SAA patients. CD16+ monocytes contributed to IL-15-induced CD38+CXCR6+ pre-Trm differentiation into CD8+ Trm cells, which can be inhibited by the CD38 inhibitor 78c. Our results demonstrate that IL-15-induced CD8+ Trm cells are pathogenic cells that mediate HSPC destruction in SAA patients and are therapeutic targets for future treatments.
Subject(s)
Anemia, Aplastic , CD8-Positive T-Lymphocytes , GPI-Linked Proteins , Hematopoietic Stem Cells , Interleukin-15 , Monocytes , Receptors, IgG , Humans , Anemia, Aplastic/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/drug effects , Interleukin-15/pharmacology , Interleukin-15/immunology , Receptors, IgG/metabolism , Receptors, IgG/immunology , Monocytes/immunology , Monocytes/drug effects , Female , Male , Adult , Hematopoietic Stem Cells/immunology , GPI-Linked Proteins/metabolism , GPI-Linked Proteins/immunology , Middle Aged , Fas Ligand Protein/metabolism , Fas Ligand Protein/immunology , Young Adult , Adolescent , Interferon-gamma/immunology , Interferon-gamma/metabolism , Receptors, Interleukin-15/metabolism , Receptors, Interleukin-15/immunology , Apoptosis/drug effects , Cell Differentiation/immunologyABSTRACT
Muscle cell death in polymyositis is induced by CD8+ cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8+ cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8+ cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes.
Subject(s)
HMGB1 Protein/antagonists & inhibitors , Imidazoles/pharmacology , Indoles/pharmacology , Muscle Fibers, Skeletal/drug effects , Myositis/prevention & control , Necroptosis/drug effects , Polymyositis/genetics , Animals , Antibodies, Neutralizing/pharmacology , C-Reactive Protein/administration & dosage , Fas Ligand Protein/genetics , Fas Ligand Protein/immunology , Female , Gene Expression Regulation , Granzymes/genetics , Granzymes/immunology , HMGB1 Protein/genetics , HMGB1 Protein/immunology , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Muscle Fibers, Skeletal/immunology , Muscle Fibers, Skeletal/pathology , Muscle Strength/drug effects , Muscle Strength/immunology , Muscle, Skeletal/drug effects , Muscle, Skeletal/immunology , Muscle, Skeletal/pathology , Myositis/chemically induced , Myositis/genetics , Myositis/immunology , Necroptosis/genetics , Necroptosis/immunology , Perforin/genetics , Perforin/immunology , Polymyositis/immunology , Polymyositis/pathology , Signal Transduction , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/pathologyABSTRACT
BACKGROUNDMultisystem inflammatory syndrome in children (MIS-C) is a rare but potentially severe illness that follows exposure to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Kawasaki disease (KD) shares several clinical features with MIS-C, which prompted the use of intravenous immunoglobulin (IVIG), a mainstay therapy for KD. Both diseases share a robust activation of the innate immune system, including the IL-1 signaling pathway, and IL-1 blockade has been used for the treatment of both MIS-C and KD. The mechanism of action of IVIG in these 2 diseases and the cellular source of IL-1ß have not been defined.METHODSThe effects of IVIG on peripheral blood leukocyte populations from patients with MIS-C and KD were examined using flow cytometry and mass cytometry (CyTOF) and live-cell imaging.RESULTSCirculating neutrophils were highly activated in patients with KD and MIS-C and were a major source of IL-1ß. Following IVIG treatment, activated IL-1ß+ neutrophils were reduced in the circulation. In vitro, IVIG was a potent activator of neutrophil cell death via PI3K and NADPH oxidase, but independently of caspase activation.CONCLUSIONSActivated neutrophils expressing IL-1ß can be targeted by IVIG, supporting its use in both KD and MIS-C to ameliorate inflammation.FUNDINGPatient Centered Outcomes Research Institute; NIH; American Asthma Foundation; American Heart Association; Novo Nordisk Foundation; NIGMS; American Academy of Allergy, Asthma and Immunology Foundation.
Subject(s)
COVID-19/complications , Immunoglobulins, Intravenous/therapeutic use , Mucocutaneous Lymph Node Syndrome/immunology , Mucocutaneous Lymph Node Syndrome/therapy , Systemic Inflammatory Response Syndrome/immunology , Systemic Inflammatory Response Syndrome/therapy , COVID-19/blood , COVID-19/immunology , COVID-19/therapy , Case-Control Studies , Cell Death/immunology , Cell Lineage/immunology , Child , Child, Preschool , Fas Ligand Protein/immunology , Female , Humans , Infant , Interleukin-1beta/antagonists & inhibitors , Interleukin-1beta/blood , Leukocyte Count , Male , Mucocutaneous Lymph Node Syndrome/blood , Neutrophil Activation , Neutrophils/classification , Neutrophils/immunology , Neutrophils/pathology , Systemic Inflammatory Response Syndrome/bloodABSTRACT
Differentiation of Ag-specific B cells into class-switched, high-affinity, Ab-secreting cells provides protection against invading pathogens but is undesired when Abs target self-tissues in autoimmunity, beneficial non-self-blood transfusion products, or therapeutic proteins. Essential T cell factors have been uncovered that regulate T cell-dependent B cell differentiation. We performed a screen using a secreted protein library to identify novel factors that promote this process and may be used to combat undesired Ab formation. We tested the differentiating capacity of 756 secreted proteins on human naive or memory B cell differentiation in a setting with suboptimal T cell help in vitro (suboptimal CD40L and IL-21). High-throughput flow cytometry screening and validation revealed that type I IFNs and soluble FAS ligand (sFASL) induce plasmablast differentiation in memory B cells. Furthermore, sFASL induces robust secretion of IgG1 and IgG4 Abs, indicative of functional plasma cell differentiation. Our data suggest a mechanistic connection between elevated sFASL levels and the induction of autoreactive Abs, providing a potential therapeutic target in autoimmunity. Indeed, the modulators identified in this secretome screen are associated with systemic lupus erythematosus and may also be relevant in other autoimmune diseases and allergy.
Subject(s)
Antibody-Producing Cells/immunology , Cell Differentiation/immunology , Fas Ligand Protein/immunology , Immunologic Memory/immunology , Interleukins/immunology , Lupus Erythematosus, Systemic/immunology , Animals , Autoimmunity/immunology , B-Lymphocytes/immunology , CD40 Ligand/immunology , Cell Line , HEK293 Cells , Humans , Lymphocyte Activation/immunology , Mice , NIH 3T3 Cells , Plasma Cells/immunology , T-Lymphocytes/immunologyABSTRACT
Activation of self-reactive CD8+ T cells induces a peripheral tolerance mechanism that involves loss of CD8 expression. Because genetic deficiency of Fas and Fasl causes the accumulation of double-negative (DN; CD3+ TCR-αß+ CD4- CD8-) T cells that have been proposed to derive from CD8+ cells, we decided to explore the role of Fas and FasL in self-antigen-induced CD8 downregulation. To this end, we quantified Fas and FasL induction by different stimuli and analyzed the effects of Fas/FasL deficiency during a protective immune response and after exposure to self-antigens. Our data describes how Fas and FasL upregulation differs depending on the setting of CD8 T cell activation and demonstrates that Fas/FasL signaling maintains CD8 expression during repetitive antigen stimulation and following self-antigen encounter. Together, our results reveal an unexpected role of Fas/FasL signaling and offer a new insight into the role of these molecules in the regulation of immune tolerance.
Subject(s)
Autoantigens/metabolism , CD8 Antigens/metabolism , CD8-Positive T-Lymphocytes/metabolism , Fas Ligand Protein/metabolism , Immune Tolerance , Lymphocyte Activation , fas Receptor/metabolism , Adoptive Transfer , Animals , Autoantigens/immunology , CD8 Antigens/immunology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/transplantation , Cells, Cultured , Down-Regulation , Fas Ligand Protein/genetics , Fas Ligand Protein/immunology , Kinetics , Mice, Inbred C57BL , Mice, Transgenic , Phenotype , Signal Transduction , fas Receptor/genetics , fas Receptor/immunologyABSTRACT
By dint of the aging population and further deepened with the Covid-19 pandemic, lung disease has turned out to be a major cause of worldwide morbidity and mortality. The condition is exacerbated when the immune system further attacks the healthy, rather than the diseased, tissue within the lung. Governed by unremittingly proliferating mesenchymal cells and increased collagen deposition, if inflammation persists, as frequently occurs in aging lungs, the tissue develops tumors and/or turns into scars (fibrosis), with limited regenerative capacity and organ failure. Fas ligand (FasL, a ligand of the Fas cell death receptor) is a key factor in the regulation of these processes. FasL is primarily found in two forms: full length (membrane, or mFasL) and cleaved (soluble, or sFasL). We and others found that T-cells expressing the mFasL retain autoimmune surveillance that controls mesenchymal, as well as tumor cell accumulation following an inflammatory response. However, mesenchymal cells from fibrotic lungs, tumor cells, or cells from immune-privileged sites, resist FasL+ T-cell-induced cell death. The mechanisms involved are a counterattack of immune cells by FasL, by releasing a soluble form of FasL that competes with the membrane version, and inhibits their cell death, promoting cell survival. This review focuses on understanding the previously unrecognized role of FasL, and in particular its soluble form, sFasL, in the serum of aged subjects, and its association with the evolution of lung disease, paving the way to new methods of diagnosis and treatment.
Subject(s)
COVID-19/immunology , Fas Ligand Protein/immunology , Lung Diseases/immunology , Lung/immunology , Age Factors , Aged , COVID-19/blood , Cell Death/immunology , Fas Ligand Protein/blood , Humans , Immunity , Lung Diseases/blood , SARS-CoV-2/immunology , SARS-CoV-2/isolation & purification , T-Lymphocytes/immunologyABSTRACT
Exosomes are nanosized extracellular vesicles of endosomal origin that enclose a multitude of functional biomolecules. Exosomes have emerged as key players of intercellular communication in physiological and pathological conditions. In cancer, depending on the context, exosomes can oppose or potentiate the development of an aggressive tumor microenvironment, thereby impacting tumor progression and clinical outcome. Increasing evidence has established exosomes as important mediators of immune regulation in cancer, as they deliver a plethora of signals that can either support or restrain immunosuppression of lymphoid and myeloid cell populations in tumors. Here, we review the current knowledge related to exosome-mediated regulation of lymphoid (T lymphocytes, B lymphocytes, and NK cells) and myeloid (macrophages, dendritic cells, monocytes, myeloid-derived suppressor cells, and neutrophils) cell populations in cancer. We also discuss the translational potential of engineered exosomes as immunomodulatory agents for cancer therapy.
Subject(s)
B7-H1 Antigen/pharmacology , Exosomes/immunology , Fas Ligand Protein/pharmacology , Immunotherapy/methods , Neoplasms/therapy , Tumor Microenvironment/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , B-Lymphocytes/pathology , B7-H1 Antigen/genetics , B7-H1 Antigen/immunology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/pathology , Exosomes/metabolism , Exosomes/transplantation , Fas Ligand Protein/genetics , Fas Ligand Protein/immunology , Gene Expression , Humans , Immunomodulation , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Macrophages/drug effects , Macrophages/immunology , Macrophages/pathology , Monocytes/drug effects , Monocytes/immunology , Monocytes/pathology , Myeloid-Derived Suppressor Cells/drug effects , Myeloid-Derived Suppressor Cells/immunology , Myeloid-Derived Suppressor Cells/pathology , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/pathology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
BACKGROUND: In follicular lymphoma (FL), histologic transformation to high-grade FL and diffuse large B-cell lymphoma (DLBCL) is a critical adverse step in disease progression. Activation of the oncogene c-MYC and tumor microenvironment remodeling account for FL progression. A panel of microRNA (miRNA) was downregulated in transformed FL (tFL). METHODS: Differentially expressed miRNAs were systematically compared in 11 lymph nodes from patients at different stages of disease. Expression of miR-7e-5p was analyzed in 46 B-cell lymphomas, including 30 FL tissues and 16 DLBCL tissues. In FL cells, transcriptional regulation of the oncogene c-MYC on its target miR-7e-5p was revealed by Chromatin Immunoprecipitation (ChIP) assay. Exosome, carrying differentially expressed miR-7e-5p was isolated and visualized by transmission electron microscope and fluorescence tracing. The effect of miR-7e-5p on recipient macrophage was determined by target gene quantification, flow cytometry, and TUNEL method in a cocultured system with miR-7e-5p-mimics or inhibitors treatment. Expression of miR-7e-5p targets, macrophage proportions, and clinical parameters were included for correlation analysis. RESULTS: We determined that downregulation of miR-7e-5p, driven by c-MYC overexpression, was associated with poorer prognosis in FL patients. The decreased expression of miR-7e-5p in lymphoma cells led to a reduced exosomal transfer to surrounding macrophages. As a result, the target gene of miR-7e-5p, Fas ligand (FasL), was upregulated and activated the caspase signaling, which led to the apoptosis of M1 macrophages in tumor stroma. Finally, in transformed FL tissues, overexpression of FasL and activation of caspase proteins was detected in tumor stromal macrophages. Downregulation of miR-7e-5p was associated with poorer clinical outcomes. CONCLUSION: Downregulation of exosomal miR-7e-5p induces stromal M1 macrophage apoptosis, which leads to immunosurveillance and transformation of FL.
Subject(s)
Fas Ligand Protein/immunology , Lymphoma, Follicular/immunology , Macrophages/metabolism , MicroRNAs/immunology , Down-Regulation , Fas Ligand Protein/metabolism , Female , Humans , Lymphoma, Follicular/genetics , Lymphoma, Follicular/pathology , Macrophages/pathology , Male , MicroRNAs/genetics , MicroRNAs/metabolism , Middle Aged , Neoplasm Grading , Stromal Cells/immunology , Stromal Cells/pathology , Transfection , Up-RegulationABSTRACT
Neutralization of FasL is linked to suppression of hypertension, placental inflammation, and endothelin system activation in an animal model of hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. During HELLP syndrome the placenta has been reported to serve as the primary source of Fas ligand (FasL), which has an impact on inflammation and hypertension during pregnancy and is dysregulated in women with severe preeclampsia and HELLP syndrome. We hypothesize that neutralization of FasL during pregnancy in an animal model of HELLP syndrome decreases inflammation and placental apoptosis, improves endothelial damage, and improves hypertension. On gestational day (GD) 12, rats were chronically infused with placental antiangiogenic factors sFlt-1 and sEng to induce HELLP syndrome. To neutralize FasL, MFL4 or FasL antibody was infused into a subset of HELLP or normal pregnant rats on GD13. IgG infusion into another group of NP and HELLP rats on GD13 was used as a control for FasL antibody, and all rats were euthanized on GD19 after blood pressure measurement. Plasma and placentas were collected to assess inflammation, apoptosis, and the degree of placental debris activation of endothelial cells. Administration of MFL4 to HELLP rats significantly decreased blood pressure compared with untreated HELLP rats and HELLP rats infused with IgG and improved the biochemistry of HELLP syndrome. Both circulating and placental FasL were significantly attenuated in response to MFL4 infusion, as were levels of placental and circulating TNFα when compared with untreated HELLP rats and HELLP rats infused with IgG. Endothelial cells exposed to placental debris and media from HP + MFL4 rats secreted significantly less endothelin-1 compared with stimulated endothelial cells from HELLP placentas. Neutralization of FasL is associated with decreased MAP and improvement in placental inflammation and endothelial damage in an animal model of HELLP syndrome.
Subject(s)
Antibodies, Neutralizing/therapeutic use , Endothelin-1/blood , Fas Ligand Protein/immunology , HELLP Syndrome/drug therapy , Placenta/physiopathology , Animals , Disease Models, Animal , Fas Ligand Protein/blood , Female , HELLP Syndrome/blood , HELLP Syndrome/immunology , HELLP Syndrome/physiopathology , Immunoglobulin G , Placenta/immunology , Pregnancy , Rats , Rats, Sprague-Dawley , Treatment Outcome , Tumor Necrosis Factor-alpha/blood , Vascular Endothelial Growth Factor Receptor-1/bloodABSTRACT
OBJECTIVE: To date, little is known about the roles of FasL and TILs in cervical cancer. This study aims to determine the correlation between FasL expression and TILs presence in cervical cancer. METHODS: In this study, we analysed the FasL and TIL presence in 32 squamous cell carcinoma or adenocarcinoma that were obtained from early stage (≤ IIA2) cervical cancer patients using immunohistochemistry. The level of FasL and TIL was assessed qualitatively, and then quantified with the H-Score system. RESULTS: Most of the patients were between 30 to 50 years old (59,4%), and had never taken pap smear examination before (96,9%). Based on the Pearson analysis of FasL and TIL presence, we found that FasL was inversely correlated with CD45 or TIL number when the level of FasL is above 140 and the CD45 is below 160. Based on Chi-Square test of FasL and TIL classification, there was a nine-fold odds ratio (OR) of lower TILs classification in high expression of FasL classification (OR 9, p=0.01). CONCLUSION: An inverse correlation between FasL expression and TILs level, that might indicate FasL-induced TILs apoptosis in tumor tissue, was observed. The strong inverse correlation between FasL and TILs presence showed some insight about the interactions between cancer cells and its surroundings inside of the cervical cancer tissue. This might also be further developed to tailor a prognostic marker that can predict the outcome of therapy in patients, not only in cervical cancer, but generally in all cancer.
Subject(s)
Fas Ligand Protein/immunology , Lymphocytes, Tumor-Infiltrating/immunology , Uterine Cervical Neoplasms/immunology , Adult , Biopsy , Female , Humans , Middle Aged , Neoplasm Staging , Uterine Cervical Neoplasms/pathologyABSTRACT
CD5 is expressed on T cells and a subset of B cells (B1a). It can attenuate TCR signalling and impair CTL activation and is a therapeutic targetable tumour antigen expressed on leukemic T and B cells. However, the potential therapeutic effect of functionally blocking CD5 to increase T cell anti-tumour activity against tumours (including solid tumours) has not been explored. CD5 knockout mice show increased anti-tumour immunity: reducing CD5 on CTLs may be therapeutically beneficial to enhance the anti-tumour response. Here, we show that ex vivo administration of a function-blocking anti-CD5 MAb to primary mouse CTLs of both tumour-naïve mice and mice bearing murine 4T1 breast tumour homografts enhanced their capacity to respond to activation by treatment with anti-CD3/anti-CD28 MAbs or 4T1 tumour cell lysates. Furthermore, it enhanced TCR signalling (ERK activation) and increased markers of T cell activation, including proliferation, CD69 levels, IFN-γ production, apoptosis and Fas receptor and Fas ligand levels. Finally, CD5 function-blocking MAb treatment enhanced the capacity of CD8+ T cells to kill 4T1-mouse tumour cells in an ex vivo assay. These data support the potential of blockade of CD5 function to enhance T cell-mediated anti-tumour immunity.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , CD28 Antigens/immunology , CD5 Antigens/immunology , Mammary Neoplasms, Experimental/drug therapy , T-Lymphocytes, Cytotoxic/drug effects , Animals , Antineoplastic Agents, Immunological , CD28 Antigens/antagonists & inhibitors , CD28 Antigens/genetics , CD5 Antigens/antagonists & inhibitors , CD5 Antigens/genetics , Cell Extracts/pharmacology , Cell Line, Tumor , Cell Proliferation , Fas Ligand Protein/genetics , Fas Ligand Protein/immunology , Female , Gene Expression Regulation , Interferon-gamma/genetics , Interferon-gamma/immunology , Lymphocyte Activation/drug effects , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/immunology , Mammary Glands, Animal/pathology , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/immunology , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Knockout , Signal Transduction , T-Lymphocytes, Cytotoxic/immunology , fas Receptor/genetics , fas Receptor/immunologyABSTRACT
Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.
Subject(s)
CTLA-4 Antigen/administration & dosage , Fas Ligand Protein/administration & dosage , Recombinant Fusion Proteins/pharmacology , Amino Acid Sequence , Animals , CTLA-4 Antigen/immunology , Fas Ligand Protein/adverse effects , Fas Ligand Protein/immunology , Fas Ligand Protein/pharmacokinetics , Female , Humans , Jurkat Cells , Macaca fascicularis , Mice , Mice, Inbred BALB C , Mice, Nude , Primates , Random Allocation , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/pharmacokinetics , Xenograft Model Antitumor AssaysABSTRACT
The progression of AIDS depends on the complex host and virus interactions. The most important disease progression hallmarks are immune activation and apoptosis. In this study, we address the prevalence of polymorphisms related to proinflammatory and apoptotic genes, such as IFNG (+874T/A), TNF (308G/A), IL6 (-174G/C), IL8 (-251A/T), FAS (-670A/G), and FASL (-124A/G) in 160 ethnically mixed HIV-1-infected patients from multicentre cohorts with different clinical outcomes (13 elite controllers [EC], 66 slow long-term non-progressors [LTNPs], and 81 progressors [P]). The genotyping was accomplished by TaqMan-qPCR. Among all the polymorphisms analyzed in the cytokines, the IL6 -174G/C polymorphism showed a higher frequency of GG genotype in the LTNP and LTNP+EC groups as compared to the P group. Moreover, there was a significantly higher frequency of the G allele in the LTNP and LTNP+EC groups as compared to the P group. On the other hand, the levels of CD4+ T lymphocytes were higher among individuals showing the AA and AG genotypes for the FASL -124A/G polymorphism as compared to the GG genotype. Furthermore, the AG and AA genotypes were more frequent, as compared to the GG genotype, in individuals showing a lower viral load. In contrast, for the FAS -670A/G polymorphism, a significantly higher viral load was observed in individuals with the AG genotype as compared to the GG genotype. In conclusion, we found three genetic allelic variants of the IL6 -174G/C, FASL -124A/G, and FAS -670A/G polymorphisms that were related to disease progression and immunological and virological markers in cohorts of HIV-1-positive ethnically mixed patients.
Subject(s)
Fas Ligand Protein/genetics , HIV Seropositivity/genetics , HIV Seropositivity/immunology , Interleukin-6/genetics , fas Receptor/genetics , Adult , Disease Progression , Ethnicity , Fas Ligand Protein/immunology , Female , Genetic Predisposition to Disease , Genotype , HIV Seropositivity/ethnology , HIV-1/genetics , HIV-1/immunology , Humans , Interleukin-6/immunology , Male , Middle Aged , Polymorphism, Single Nucleotide , Young Adult , fas Receptor/immunologyABSTRACT
BACKGROUND: The increase in CD4+ T cell infiltration and overproduction of CD4+ T cell-associated cytokines have been observed in the inflamed colon mucosa of patients with ulcerative colitis (UC); the underlying mechanisms are not fully understood. Survivin plays a critical role in the interference with apoptotic machinery. This study aims to elucidate the role of survivin in the interference with the apoptotic machinery in CD4+ T cells of UC patients. METHODS: Peripheral blood samples were collected from UC patients (UC group) and healthy subjects (healthy group). The apoptotic status in CD4+ T cells was analyzed by flow cytometry. RESULTS: We observed that the expression of survivin was significantly higher in CD4+ T cells of UC patients than in healthy subjects. UC CD4+ T cells were resistant to apoptosis induction. A complex of survivin and c-Myc, the transcription factor of FasL, was detected in CD4+ T cells in UC patients, which prevented the binding of c-Myc to the FasL promoter and interfered with the expression of FasL. Increased expression of survivin prevented the activation-induced CD4+ T cells from apoptosis. CONCLUSIONS: The data indicate that UC CD4+ T cells express high levels of survivin, which impairs the apoptotic machinery in CD4+ T cells and prevents the activation-induced CD4+ T cell apoptosis. Therefore, target therapy against survivin has translational potential in the treatment of UC patients.
Subject(s)
Apoptosis/immunology , CD4-Positive T-Lymphocytes/immunology , Colitis, Ulcerative/immunology , Survivin/immunology , Adult , Colitis, Ulcerative/pathology , Fas Ligand Protein/immunology , Female , Humans , Male , Proto-Oncogene Proteins c-myc/immunologyABSTRACT
Soluble CD95L (s-CD95L) is a chemoattractant for certain lymphocyte subpopulations. We examined whether this ligand is a prognostic marker for high-grade serous ovarian cancer (HGSOC) and whether it is associated with accumulation of immune cells in the tumor. Serum s-CD95L levels in 51 patients with advanced ovarian cancer were tested by ELISA. IHC staining of CD3, CD4, CD8, CD20, CD163, CD31, FoxP3, CCR6, IL-17, Granzyme B, PD-L1, and membrane CD95L was used to assess tumor-infiltrating immune cells. Although the intensity of CD3, CD8, CD4, CD20, and CD163 in tumor tissues remained constant regardless of membrane CD95L expression, tumors in patients with HGSOC with s-CD95L levels ≥516 pg/mL showed increased infiltration by CD3+ T cells (P = 0.001), comprising both cytotoxic CD8+ (P = 0.01) and CD4+ (P = 0.0062) cells including FoxP3+ regulatory T cells (P = 0.0044). Also, the number of tumor-infiltrating CD20+ B cells (P = 0.0094) increased in these patients. Multivariate analyses revealed that low s-CD95L concentrations [<516 pg/mL, HR, 3.54; 95% confidence interval (CI), 1.13-11.11), and <1,200 activated CD8+ (Granzyme B+) cells (HR, 2.63; 95% CI, 1.16-5.95) were independent poor prognostic factors for recurrence, whereas >6,000 CD3+ cells (HR, 0.34; 95% CI, 0.15-0.79) was a good prognostic factor. Thus, low levels of s-CD95L (<516 pg/mL) are correlated with lower numbers of tumor-infiltrating lymphocytes (CD3+ and CD8+, and also CD4 and FoxP3 T cells) in advanced HGSOC and are a poor prognostic marker. IMPLICATIONS: Serum s-CD95L is correlated with a number of tumor-infiltrating immune cells in HGSOC and could be used as a noninvasive marker of tumor immune infiltration to select patients referred for immunotherapy trials that evaluate checkpoint inhibitor treatment.
Subject(s)
Biomarkers, Tumor/blood , Cystadenocarcinoma, Serous/blood , Fas Ligand Protein/blood , Ovarian Neoplasms/blood , Apoptosis/genetics , B-Lymphocytes/immunology , Cell Movement/genetics , Cell Proliferation/genetics , Cystadenocarcinoma, Serous/immunology , Cystadenocarcinoma, Serous/pathology , Fas Ligand Protein/immunology , Female , Forkhead Transcription Factors/genetics , Humans , Lymphocytes, Tumor-Infiltrating/immunology , Middle Aged , Neoplasm Staging , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Prognosis , T-Lymphocytes, Regulatory/immunologyABSTRACT
Influenza A virus (IAV)-specific T cell responses are important correlates of protection during primary and subsequent infections. Generation and maintenance of robust IAV-specific T cell responses relies on T cell interactions with dendritic cells (DCs). In this study, we explore the role of nucleotide-binding domain leucine-rich repeat containing receptor family member NLRC4 in modulating the DC phenotype during IAV infection. Nlrc4-/- mice had worsened survival and increased viral titers during infection, normal innate immune cell recruitment and IAV-specific CD8 T cell responses, but severely blunted IAV-specific CD4 T cell responses compared to wild-type mice. The defect in the pulmonary IAV-specific CD4 T cell response was not a result of defective priming or migration of these cells in Nlrc4-/- mice but was instead due to an increase in FasL+ DCs, resulting in IAV-specific CD4 T cell death. Together, our data support a novel role for NLRC4 in regulating the phenotype of lung DCs during a respiratory viral infection, and thereby influencing the magnitude of protective T cell responses.
Subject(s)
Apoptosis Regulatory Proteins/immunology , CD4-Positive T-Lymphocytes/immunology , Calcium-Binding Proteins/immunology , Dendritic Cells/immunology , Fas Ligand Protein/immunology , Gene Expression Regulation/immunology , Influenza A virus/immunology , Lung/immunology , Orthomyxoviridae Infections/immunology , Animals , Apoptosis Regulatory Proteins/genetics , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , Calcium-Binding Proteins/genetics , Dendritic Cells/pathology , Fas Ligand Protein/genetics , Lung/pathology , Mice , Mice, Knockout , Orthomyxoviridae Infections/pathologyABSTRACT
The objective of this article is to evaluate whether the tumoricidal activity of mouse IFN R-/- nature killer (NK) cells is induced by Newcastle disease virus hemagglutinin-neuraminidase (NDV-HN) stimulation, and to investigate what is the mechanism of the HN-stimulated NK cells to kill mouse hepatoma cell line in vitro. The mouse IFN R-/- NK cells were stimulated for 16 hr with 500 ng/mL NDV-HN in 1640 medium. Quantify the cytotoxic activities of NK cells against mouse hepatoma cells (Hepa1-6) by flow cytometry. Granzymes B (GrB) and Fas/FasL concentrations in the supernatants of IFN R-/- NK cells medium were determined by specific ELISA assay. The expression of cell surface GrB and Fas was determined by Western blot. NDV-HN stimulation enhanced tumoricidal activity of IFN R-/- NK cells toward Hepa1-6 in vitro. Treating with anti-HN neutralizing mAb induced significant decline in the cytotoxicity of IFN R-/- NK cells toward Hepa1-6 cell line (P < 0.05). After treating with anti-HN protein (1 µL/mL), Syk-specific inhibitor Herbimycin A(250 ng/mL) and NF-κB inhibitor PDTC (500 ng/mL) downregulated the tumoricidal activity of HN-stimulated IFN R-/- NK cells (P < 0.05). Moreover, significant suppressions in the production of GrB and Fas/FasL were observed in HN-stimulated IFN R-/- NK cells (P < 0.05). Thus, we concluded that killer activation receptors pathway is involved in the IFN-γ-independent GrB and Fas/FasL expression of NDV-HN-stimulated IFN R-/- NK cells, and these are activated by Syk and NF-κB. Anat Rec, 302:1718-1725, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association for Anatomy.