ABSTRACT
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 , HumansSubject(s)
Receptors, Chimeric Antigen , Signal Transduction , T-Lymphocytes , fas Receptor , Humans , Signal Transduction/immunology , fas Receptor/immunology , fas Receptor/metabolism , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/genetics , T-Lymphocytes/immunology , Immunotherapy, Adoptive/methods , AnimalsABSTRACT
Autoimmune lymphoproliferative syndrome (ALPS) is a primary immune regulatory disorder characterized by benign or malignant lymphoproliferation and autoimmunity. Classically, ALPS is due to mutations in FAS and other related genes; however, recent research revealed that other genes could be responsible for similar clinical features. Therefore, ALPS classification and diagnostic criteria have changed over time, and several ALPS-like disorders have been recently identified. Moreover, mutations in FAS often show an incomplete penetrance, and certain genotypes have been associated to a dominant or recessive inheritance pattern. FAS mutations may also be acquired or could become pathogenic when associated to variants in other genes, delineating a possible digenic type of inheritance. Intriguingly, variants in FAS and increased TCR αß double-negative T cells (DNTs, a hallmark of ALPS) have been identified in multifactorial autoimmune diseases, while FAS itself could play a potential role in carcinogenesis. These findings suggest that alterations of FAS-mediated apoptosis could trespass the universe of inborn errors of immunity and that somatic mutations leading to ALPS could only be the tip of the iceberg of acquired immunodeficiencies.
Subject(s)
Autoimmune Lymphoproliferative Syndrome/genetics , Mutation , fas Receptor/genetics , Animals , Autoimmune Lymphoproliferative Syndrome/diagnosis , Autoimmune Lymphoproliferative Syndrome/immunology , Autoimmunity , Humans , Neoplasms/diagnosis , Neoplasms/genetics , Neoplasms/immunology , fas Receptor/immunologyABSTRACT
SUMMARY: The aim of our study was to investigate the effect of inflammation in the placenta on the pro-apoptotic development after severe preeclampsia. Placenta tissue samples of 15 HELLP syndrome and 15 healthy 35-38th week-pregnant women were involved in the study. Tissue samples were taken only from the maternal side of the placenta and fixed in 10 % formaldehyde, then blocked in paraffin wax and 4-6 mm-thick sections were cut and stained with Harris Hematoxylene-Eosin. Antigen retrieval was performed for sections, incubated with FAS antibody and anti-IL-6 antibody. After the application of streptavidin peroxidase followed by AEC chromogen solution, sections were counterstained with Harris hematoxylin. Significant thickening of the fibrinoid layer, degeneration and apoptotic change in decidua cells, marked increase in the hyalinized area, degenerative changes in the syncytial regions of the chorionic villus and an increase in syncytial nodes and bridges and IL- expression were observed as positive. FAS expression was positive in the pycnotic nuclei of decidual cells in the maternal region and in the syncytial regions. It was observed that the proapoptotic process increased as a result of severe preeclampsia. It was concluded that the control of cytokine activity and reduction of pro-apoptotic signal during the inflammation process will slow down the development of HELLP syndrome.
RESUMEN: El objetivo de nuestro estudio fue investigar el efecto de la inflamación en la placenta sobre el desarrollo proapoptótico después de la preeclampsia severa. Se recogieron muestras de tejido de placenta de 15 mujeres con síndrome de HELLP y 15 mujeres sanas con un embarazo de 35 a 38 semanas. Se tomaron muestras de tejido solo del lado materno de la placenta y se fijaron en formaldehído al 10 %, luego se bloquearon en parafina y se cortaron secciones de 4-6 mm de espesor y se tiñeron con hematoxilena-eosina de Harris. La recuperación del antígeno se realizó para secciones, incubadas con anticuerpo FAS y anticuerpo anti-IL-6. Después de la aplicación de estreptavidina peroxidasa seguida de solución de cromógeno AEC, las secciones se contrastaron con hematoxilina de Harris. Se observó como positivo un engrosamiento significativo de la capa fibrinoide, degeneración y cambio apoptótico en las células de la decidua, aumento marcado en el área hialinizada, cambios degenerativos en las regiones sincitiales de la vellosidad coriónica y un aumento en los nódulos y puentes sincitiales y la expresión de IL-6. La expresión de FAS fue positiva en los núcleos picnóticos de las células deciduales en la región materna y en las regiones sincitiales. Se observó que el proceso proapoptótico se incrementó como consecuencia de la preeclampsia severa. Se concluyó que el control de la actividad de las citocinas y la reducción de la señal proapoptótica durante el proceso de inflamación ralentizarán el desarrollo del síndrome de HELLP.
Subject(s)
Humans , Female , Pregnancy , Adult , Young Adult , Placenta/immunology , Interleukin-6/metabolism , HELLP Syndrome/immunology , fas Receptor/metabolism , Immunohistochemistry , Interleukin-6/immunology , HELLP Syndrome/metabolism , fas Receptor/immunology , Fas Ligand Protein , InflammationABSTRACT
Fas-induced apoptosis is a central mechanism of hepatocyte damage during acute and chronic hepatic disorders. Increasing evidence suggests that circadian clock plays critical roles in the regulation of cell fates. In the present study, the potential significance of REV-ERBα, a core ingredient of circadian clock, in Fas-induced acute liver injury has been investigated. The anti-Fas antibody Jo2 was injected intraperitoneally in mice to induce acute liver injury and the REV-ERBα agonist GSK4112 was administered. The results indicated that treatment of GSK4112 decreased the level of plasma ALT and AST, attenuated the liver histological changes, and promoted the survival rate in Jo2-insulted mice. Treatment with GSK4112 also downregulated the activities of caspase-3 and caspase-8, suppressed hepatocyte apoptosis. In addition, treatment with GSK4112 decreased the level of Fas and enhanced the phosphorylation of Akt. In conclusion, treatment with GSK4112 alleviated Fas-induced apoptotic liver damage in mice, suggesting that REV-ERBα agonist might have potential value in pharmacological intervention of Fas-associated liver injury.
Subject(s)
Chemical and Drug Induced Liver Injury/prevention & control , Glycine/analogs & derivatives , Thiophenes/pharmacology , Acute Disease , Animals , Antibodies/adverse effects , Apoptosis/drug effects , Chemical and Drug Induced Liver Injury/pathology , Circadian Rhythm/drug effects , Circadian Rhythm/physiology , Glycine/pharmacology , Liver/drug effects , Liver/pathology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Nuclear Receptor Subfamily 1, Group D, Member 1/agonists , fas Receptor/immunologyABSTRACT
T cell based immunotherapies can be applicable to acute myeloid leukemia (AML). Therefore, the selection of optimal T cells, cell manufacturing, and therapeutic T cell engineering are essential for the development of effective adoptive T cell therapies for AML. Autologous tumor-infiltrating lymphocytes (TILs) have been in clinical trials to treat solid malignancies. Herein, we assessed whether TILs can be isolated from the bone marrow (BM) of AML patients, expanded ex vivo and utilized as a novel therapeutic strategy for AML. To this end, firstly we analyzed the immunophenotypes of a series of primary BM samples from AML patients (N = 10) by flow cytometry. We observed a variable amount of CD3+ TILs (range â¼2.3-â¼32.6% of mononuclear cells) among BM samples. We then developed a novel protocol that produced a three-log ex vivo expansion of TILs isolated from AML patient BM (N = 10) and peripheral blood (PB) (N = 10), including from patients with a low number of CD3+ T cells, within 3, 4 weeks. Further, we identified previously described naïve T cells (CCR7+CD95-/or CD62L+CD45RA+) in AML BM and PB samples, which seemed to be required for a successful TILs ex vivo expansion. Finally, we showed that the expanded TILs could: (1) cause cytotoxicity to autologous AML blasts ex vivo (90.6% in control without T cell treatment vs. 1.89% in experimental groups with PB derived T cells and 1.77% in experimental groups with BM derived TILs, p < 0.01), (2) be genetically engineered to express CYP27B1 gene, and (3) infiltrate the BM and reside in close proximity to pre-injected autologous AML blasts of engrafted immunodeficiency mice. Altogether, these results provide a rationale for further studies of the therapeutic use of TILs in AML.
Subject(s)
Bone Marrow Cells/immunology , Cell Separation/methods , Leukemia, Myeloid, Acute/immunology , Lymphocytes, Tumor-Infiltrating/immunology , T-Lymphocyte Subsets/immunology , Adoptive Transfer/methods , Adult , Aged , Animals , Bioengineering/methods , Female , Heterografts , Humans , L-Selectin/immunology , Leukocyte Common Antigens/immunology , Lymphocytes, Tumor-Infiltrating/transplantation , Male , Mice , Middle Aged , Receptors, CCR7/immunology , T-Lymphocyte Subsets/transplantation , fas Receptor/immunologyABSTRACT
Colorectal cancer (CRC) is one of the main causes of cancer death in the world. Post-translational modifications (PTMs) have been extensively studied in malignancies due to its relevance in tumor pathogenesis and therapy. This review is focused on the dysregulation of glycosyltransferase expression in CRC and its impact in cell function and in several biological pathways associated with CRC pathogenesis, prognosis and therapeutic approaches. Glycan structures act as interface molecules between cells and their environment and in several cases facilitate molecule function. CRC tissue shows alterations in glycan structures decorating molecules, such as annexin-1, mucins, heat shock protein 90 (Hsp90), ß1 integrin, carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR), insulin-like growth factor-binding protein 3 (IGFBP3), transforming growth factor beta (TGF-ß) receptors, Fas (CD95), PD-L1, decorin, sorbin and SH3 domain-containing protein 1 (SORBS1), CD147 and glycosphingolipids. All of these are described as key molecules in oncogenesis and metastasis. Therefore, glycosylation in CRC can affect cell migration, cell-cell adhesion, actin polymerization, mitosis, cell membrane repair, apoptosis, cell differentiation, stemness regulation, intestinal mucosal barrier integrity, immune system regulation, T cell polarization and gut microbiota composition; all such functions are associated with the prognosis and evolution of the disease. According to these findings, multiple strategies have been evaluated to alter oligosaccharide processing and to modify glycoconjugate structures in order to control CRC progression and prevent metastasis. Additionally, immunotherapy approaches have contemplated the use of neo-antigens, generated by altered glycosylation, as targets for tumor-specific T cells or engineered CAR (Chimeric antigen receptors) T cells.
Subject(s)
Colorectal Neoplasms/genetics , Glycosphingolipids/immunology , Glycosyltransferases/genetics , Mucins/genetics , Neoplasm Proteins/genetics , Protein Processing, Post-Translational , Annexin A1/genetics , Annexin A1/immunology , Colorectal Neoplasms/immunology , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , Decorin/genetics , Decorin/immunology , ErbB Receptors/genetics , ErbB Receptors/immunology , Gene Expression Regulation, Neoplastic , Glycosphingolipids/metabolism , Glycosylation , Glycosyltransferases/immunology , Humans , Immunotherapy, Adoptive/methods , Insulin-Like Growth Factor Binding Protein 3/genetics , Insulin-Like Growth Factor Binding Protein 3/immunology , Integrin beta1/genetics , Integrin beta1/immunology , Microfilament Proteins/genetics , Microfilament Proteins/immunology , Mucins/immunology , Neoplasm Proteins/immunology , fas Receptor/genetics , fas Receptor/immunologyABSTRACT
The diagnosis and management of concussion is hindered by its diverse clinical presentation and assessment tools reliant on subjectively experienced symptoms. The biomechanical threshold of concussion is also not well understood, and asymptomatic concussion or "subconcussive impacts" of variable magnitudes are common in contact sports. Concerns have risen because athletes returning to activity too soon have an increased risk of prolonged recovery or long-term adverse health consequences. To date, little is understood on a molecular level regarding concussion and subconcussive impacts. Recent research suggests that neuroinflammatory mechanisms may serve an important role subsequent to concussion and possibly to subconcussive impacts. These studies suggest that autoantibodies may be a valuable tool for detection of acute concussion and monitoring for changes caused by cumulative exposure to subconcussive impacts. Hence, we aimed to profile the immunoglobulin (Ig)A autoantibody repertoire in saliva by screening a unique sport-related head trauma biobank. Saliva samples (n = 167) were donated by male and female participants enrolled in either the concussion (24-48 h post-injury) or subconcussion (non-concussed participants having moderate or high cumulative subconcussive impact exposure) cohorts. Study design included discovery and verification phases. Discovery aimed to identify new candidate autoimmune targets of IgA. Verification tested whether concussion and subconcussion cohorts increased IgA reactivity and whether cohorts showed similarities. The results show a significant increase in the prevalence of IgA toward protein fragments representing 5-hydroxytryptamine receptor 1A (HTR1A), serine/arginine repetitive matrix 4 (SRRM4) and FAS (tumor necrosis factor receptor superfamily member 6) after concussion and subconcussion. These results may suggest that concussion and subconcussion induce similar physiological effects, especially in terms of immune response. Our study demonstrates that saliva is a potential biofluid for autoantibody detection in concussion and subconcussion. After rigorous confirmation in much larger independent study sets, a validated salivary autoantibody assay could provide a non-subjective quantitative means of assessing concussive and subconcussive events.
Subject(s)
Athletic Injuries/metabolism , Autoantibodies/metabolism , Brain Concussion/metabolism , Immunoglobulin A, Secretory/metabolism , Saliva/immunology , Adolescent , Child , Cohort Studies , Female , Humans , Male , Nerve Tissue Proteins/immunology , Receptor, Serotonin, 5-HT1A/immunology , Time Factors , Young Adult , fas Receptor/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
Background: Innate lymphoid cells (ILCs), so far studied mostly in mouse models, are important tissue-resident innate immune cells that play important roles in the colorectal cancer microenvironment and maintain mucosal tissue homeostasis. Plasmacytoid dendritic cells (pDCs) present complexity in various tumor types and are correlated with poor prognosis. pDCs can promote HIV-1-induced group 3 ILC (ILC3) depletion through the CD95 pathway. However, the role of ILC3s in human colon cancer and their correlation with other immune cells, especially pDCs, remain unclear. Methods: We characterized ILCs and pDCs in the tumor microenvironment of 58 colon cancer patients by flow cytometry and selected three patients for RNA sequencing. Results: ILC3s were negatively correlated, and pDCs were positively correlated, with cancer pathological stage. There was a negative correlation between the numbers of ILC3s and pDCs in tumor tissues. RNA sequencing confirmed the correlations between ILC3s and pDCs and highlighted the potential function of many ILC- and pDC-associated differentially expressed genes in the regulation of tumor immunity. pDCs can induce apoptosis of ILC3s through the CD95 pathway in the tumor-like microenvironment. Conclusions: One of the interactions between ILC3s and pDCs is via the CD95 pathway, which may help explain the role of ILC3s in colon cancer.
Subject(s)
Colonic Neoplasms/immunology , Dendritic Cells/immunology , Lymphocytes/immunology , Signal Transduction/immunology , Tumor Microenvironment/immunology , Aged , Colonic Neoplasms/pathology , Dendritic Cells/pathology , Female , Flow Cytometry , Humans , Lymphocytes/pathology , Male , Middle Aged , fas Receptor/immunologyABSTRACT
Cell surface glycosylation has been known as an important modification process that can be targeted and manipulated by malignant cells to escape from host immunosurveillance. We previously showed that the blood group branched I antigen on the leukemia cell surface can regulate the cell susceptibility against natural killer (NK) cell-mediated cytotoxicity through interfering target-NK interaction. In this work, we first identified N-linkage as the major glycosylation linkage type for branched I glycan formation on leukemia cells, and this linkage was responsible for cell sensitivity against therapeutic NK-92MI targeting. Secondly, by examining different leukemia cell surface death receptors, we showed death receptor Fas had highest expressions in both Raji and TF-1a cells. Mutations on two Fas extracellular N-linkage sites (118 and 136) for glycosylation impaired activation of Fas-mediated apoptosis during NK-92MI cytotoxicity. Last, we found that the surface I antigen expression levels enable leukemia cells to respond differently against NK-92MI targeting. In low I antigen expressing K-562 cell, reduction of I antigen presence greatly reduced leukemia cell susceptibility against NK-92MI targeting. But in other high I antigen expressing leukemia cells, similar reduction in I antigen expression did not affect cell susceptibility.
Subject(s)
Cytotoxicity, Immunologic/immunology , Histocompatibility Antigens Class I/immunology , Killer Cells, Natural/immunology , fas Receptor/immunology , Apoptosis/immunology , Cells, Cultured , Glycosylation , Histocompatibility Antigens Class I/genetics , Humans , Mutation , fas Receptor/geneticsABSTRACT
The identification and characterization of rare immune cell populations in humans can be facilitated by their growth advantage in the context of specific genetic diseases. Here, we use autoimmune lymphoproliferative syndrome to identify a population of FAS-controlled TCRαß+ T cells. They include CD4+, CD8+, and double-negative T cells and can be defined by a CD38+CD45RA+T-BET- expression pattern. These unconventional T cells are present in healthy individuals, are generated before birth, are enriched in lymphoid tissue, and do not expand during acute viral infection. They are characterized by a unique molecular signature that is unambiguously different from other known T cell differentiation subsets and independent of CD4 or CD8 expression. Functionally, FAS-controlled T cells represent highly proliferative, noncytotoxic T cells with an IL-10 cytokine bias. Mechanistically, regulation of this physiological population is mediated by FAS and CTLA4 signaling, and its survival is enhanced by mTOR and STAT3 signals. Genetic alterations in these pathways result in expansion of FAS-controlled T cells, which can cause significant lymphoproliferative disease.
Subject(s)
ADP-ribosyl Cyclase 1/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Leukocyte Common Antigens/metabolism , fas Receptor/immunology , Adolescent , Adult , Aged , Child , Child, Preschool , Female , Humans , Infant , Infant, Newborn , Lymphocyte Activation/immunology , Lymphoproliferative Disorders/immunology , Male , Middle Aged , Signal Transduction/immunology , Young AdultABSTRACT
Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, although it is unclear whether this occurs primarily in a subset of SLE T cells. The process driving the DNA demethylation and the consequences on overall gene expression are also poorly understood and whether this represents a secondary consequence of SLE or a primary contributing factor. Lupus-prone lpr mice accumulate large numbers of T cells with age because of a mutation in Fas (CD95). The accumulating T cells include an unusual population of CD4-CD8-TCR-αß+ (DN) T cells that arise from CD8+ precursors and are also found in human SLE. We have previously observed that T cell accumulation in lpr mice is due to dysregulation of T cell homeostatic proliferation, which parallels an increased expression of numerous genes in the DN subset, including several proinflammatory molecules and checkpoint blockers. We thus determined the DNA methylome in lpr DN T cells compared with their CD8+ precursors. Our findings show that DN T cells manifest discrete sites of extensive demethylation throughout the genome, and these sites correspond to the location of a large proportion of the upregulated genes. Thus, dysregulated homeostatic proliferation in lpr mice and consequent epigenetic alterations may be a contributing factor to lupus pathogenesis.
Subject(s)
DNA Demethylation , Lupus Erythematosus, Systemic/immunology , Receptors, Antigen, T-Cell, alpha-beta/immunology , T-Lymphocytes/immunology , fas Receptor/immunology , Animals , Cell Proliferation , Gene Expression Regulation , Lupus Erythematosus, Systemic/pathology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
Death receptor Fas-mediated apoptosis not only eliminates nonspecific and autoreactive B cells but also plays a major role in antitumor immunity. However, the possible mechanisms underlying impairment of Fas-mediated induction of apoptosis during lymphomagenesis remain unknown. In this study, we employed our developed syngeneic lymphoma model to demonstrate that downregulation of Fas is required for both lymphoma development and lymphoma cell survival to evade immune cytotoxicity. CD40 signal activation significantly restored Fas expression and thereby induced apoptosis after Fas ligand treatment in both mouse and human lymphoma cells. Nevertheless, certain human lymphoma cell lines were found to be resistant to Fas-mediated apoptosis, with Livin (melanoma inhibitor of apoptosis protein; ML-IAP) identified as a driver of such resistance. High expression of Livin and low expression of Fas were associated with poor prognosis in patients with aggressive non-Hodgkin's lymphoma. Livin expression was tightly driven by bromodomain and extraterminal (BET) proteins BRD4 and BRD2, suggesting that Livin expression is epigenetically regulated in refractory lymphoma cells to protect them from Fas-mediated apoptosis. Accordingly, the combination of CD40-mediated Fas restoration with targeting of the BET proteins-Livin axis may serve as a promising immunotherapeutic strategy for refractory B-cell lymphoma. SIGNIFICANCE: These findings yield insights into identifying risk factors in refractory lymphoma and provide a promising therapy for tumors resistant to Fas-mediated antitumor immunity. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/20/4439/F1.large.jpg.
Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Inhibitor of Apoptosis Proteins/immunology , Lymphoma, Large B-Cell, Diffuse/immunology , Lymphoma, Large B-Cell, Diffuse/pathology , Neoplasm Proteins/immunology , fas Receptor/immunology , Adaptor Proteins, Signal Transducing/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Animals , CD40 Antigens/immunology , CD40 Antigens/metabolism , Cell Line, Tumor , Cell Survival , Child , Child, Preschool , Cytotoxicity, Immunologic , Female , Humans , Inhibitor of Apoptosis Proteins/genetics , Lymphoma, Large B-Cell, Diffuse/genetics , Lymphoma, Large B-Cell, Diffuse/mortality , Male , Mice , Mice, Inbred C57BL , Middle Aged , NIH 3T3 Cells , Neoplasm Proteins/genetics , Neoplasms, Experimental/pathology , Xenograft Model Antitumor Assays , Young Adult , fas Receptor/genetics , fas Receptor/metabolismABSTRACT
Antibodies that target a clinically relevant group of receptors within the tumor necrosis factor receptor superfamily (TNFRSF), including CD40 and CD95 (Fas/Apo-1), also require binding to Fc gamma receptors (FcγRs) to elicit a strong agonistic activity. This FcγR dependency largely relies on the mere cellular anchoring through the antibody's Fc domain and does not involve the engagement of FcγR signaling. The aim of this study was to elicit agonistic activity from αCD40 and αCD95 antibodies in a myeloma cell anchoring-controlled FcγR-independent manner. For this purpose, various antibody variants (IgG1, IgG1N297A, Fab2) against the TNFRSF members CD40 and CD95 were genetically fused to a single-chain-encoded B-cell activating factor (scBaff) trimer as a C-terminal myeloma-specific anchoring domain substituting for Fc domain-mediated FcγR binding. The antibody-scBaff fusion proteins were evaluated in binding studies and functional assays using tumor cell lines expressing one or more of the three receptors of Baff: BaffR, transmembrane activator and CAML interactor (TACI) and B-cell maturation antigen (BCMA). Cellular binding studies showed that the binding properties of the different domains within the fusion proteins remained fully intact in the antibody-scBaff fusion proteins. In co-culture assays of CD40- and CD95-responsive cells with BaffR, BCMA or TACI expressing anchoring cells, the antibody fusion proteins displayed strong agonism while only minor receptor stimulation was observed in co-cultures with cells without expression of Baff-interacting receptors. Thus, our CD40 and CD95 antibody fusion proteins display myeloma cell-dependent activity and promise reduced systemic side effects compared to conventional CD40 and CD95 agonists.
Subject(s)
Antibodies, Monoclonal/immunology , B-Cell Activating Factor/immunology , B-Cell Activation Factor Receptor/agonists , B-Cell Maturation Antigen/agonists , CD40 Antigens/immunology , Immunoglobulin Fab Fragments/immunology , Recombinant Fusion Proteins/immunology , Transmembrane Activator and CAML Interactor Protein/agonists , fas Receptor/immunology , Antibodies, Monoclonal/genetics , B-Cell Activating Factor/genetics , B-Cell Activation Factor Receptor/genetics , B-Cell Activation Factor Receptor/immunology , B-Cell Maturation Antigen/genetics , B-Cell Maturation Antigen/immunology , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/genetics , Jurkat Cells , Recombinant Fusion Proteins/genetics , Transmembrane Activator and CAML Interactor Protein/genetics , Transmembrane Activator and CAML Interactor Protein/immunologyABSTRACT
PURPOSE: Esophageal cancer (EC) is one of the most lethal gastrointestinal malignancies. Immunotherapy is a promising treatment modality for this disease. However, broader implementation of EC immunotherapy has been discouraged because of insufficient understanding of tumor interactions with the immune system. As with other malignancies, the current research on EC focuses on deciphering the immune cell signatures within the tumor microenvironment. However, the disease-elicited immune cell profiles in the paratumoral compartments are largely unknown. METHODS: We examined the immune cell signatures in 62 tissue samples from 16 EC patients in different esophageal tissue compartments: tumor tissue, peritumoral tissue, healthy esophageal tissue, and adjacent lymph nodes. We analyzed the proportions and distribution patterns of NK cells and CD4+ and CD8+ T cells as well as their death receptor (FasR, FasR/DR3)-expressing subpopulations. The analyzed data were then compared and correlated with the patients' clinicopathological data. RESULTS: We found that the FasR+ NK cells, CD4+ and CD8+ T cells infiltrated lymph nodes at the lowest levels and that the FasR+DR3+ CD4+ T cells were enhanced in tumors. The comparisons with the clinicopathological data revealed a major impact of active smoking on the reduction in paratumoral NK cells and the upregulation of FasR in tumor-infiltrating NK and CD8+ T cells. The lymph node metastatic stage, tumor stage, and Mandard grade correlated with the compartmental proportions of the evaluated immune cells. CONCLUSION: The novel association of the disease state with tumoral and paratumoral immune cell signatures suggests new possibilities for personalized immunotherapy for EC patients.
Subject(s)
Esophageal Neoplasms/immunology , Esophageal Neoplasms/therapy , Lymphocytes/immunology , Adenocarcinoma/immunology , Adenocarcinoma/pathology , Adenocarcinoma/therapy , Aged , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/pathology , Case-Control Studies , Esophageal Neoplasms/pathology , Esophageal Squamous Cell Carcinoma/immunology , Esophageal Squamous Cell Carcinoma/pathology , Esophageal Squamous Cell Carcinoma/therapy , Female , Humans , Immunotherapy/methods , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Lymph Nodes/immunology , Lymph Nodes/pathology , Lymphocytes/pathology , Lymphocytes, Tumor-Infiltrating/immunology , Lymphocytes, Tumor-Infiltrating/pathology , Male , Middle Aged , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/pathology , fas Receptor/immunologyABSTRACT
Under lymphopenic conditions, the rapid spontaneous proliferation produces cells that robustly differentiate into effector memory T (TEM) cells, and the aberrant expansion is preferentially driven by self-antigens. The pool size of effector memory T-cell is governed by a complex homeostatic balance between proliferation and death. Perp is a critical effector involved in the p53-dependent apoptotic pathway and widely expressed in mammalian tissues. We have previously shown that Perp has a prominent role in activation-induced cell death of peripheral Th17 cells. Here, we show that Peripheral Perp-/-CD4+ TEM cells outcompete wild type TEM cells for access to splenic niches in vivo. The skewing of the Perp-/- TEM cells compartment was not the result of a difference in lymphopenia-induced proliferation, but the resistance to apoptosis, particularly after anti-Fas treatment. Data presented in this work indicate that Perp mediates the persistence of CD4+ TEM cells in irradiation-induced lymphopenic settings.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Lymphopenia/immunology , Membrane Proteins/metabolism , Animals , Antibodies, Monoclonal/metabolism , Apoptosis , Cell Proliferation , Cells, Cultured , Immunologic Memory , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Radiation, Ionizing , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , fas Receptor/immunologyABSTRACT
AIMS/HYPOTHESIS: The aim of this study was to characterise islet autoantibody profiles and immune cell phenotypes in slow progressors to type 1 diabetes. METHODS: Immunological variables were compared across peripheral blood samples obtained from slow progressors to type 1 diabetes, individuals with newly diagnosed or long-standing type 1 diabetes, and healthy individuals. Polychromatic flow cytometry was used to characterise the phenotypic attributes of B and T cells. Islet autoantigen-specific B cells were quantified using an enzyme-linked immunospot (ELISpot) assay and islet autoantigen-specific CD8+ T cells were quantified using peptide-HLA class I tetramers. Radioimmunoassays were used to detect islet autoantibodies. Sera were assayed for various chemokines, cytokines and soluble receptors via ELISAs. RESULTS: Islet autoantibodies were lost over time in slow progressors. Various B cell subsets expressed higher levels of CD95 in slow progressors, especially after polyclonal stimulation, compared with the corresponding B cell subsets in healthy donors (p < 0.05). The phenotypic characteristics of CD4+ and CD8+ T cells were similar in slow progressors and healthy donors. Lower frequencies of CD4+ T cells with a central memory phenotype (CD27int, CD127+, CD95int) were observed in slow progressors compared with healthy donors (mean percentage of total CD4+ T cells was 3.00% in slow progressors vs 4.67% in healthy donors, p < 0.05). Autoreactive B cell responses to proinsulin were detected at higher frequencies in slow progressors compared with healthy donors (median no. of spots was 0 in healthy donors vs 24.34 in slow progressors, p < 0.05) in an ELISpot assay. Islet autoantigen-specific CD8+ T cell responses were largely absent in slow progressors and healthy donors. Serum levels of DcR3, the decoy receptor for CD95L, were elevated in slow progressors compared with healthy donors (median was 1087 pg/ml in slow progressors vs 651 pg/ml in healthy donors, p = 0.06). CONCLUSIONS/INTERPRETATION: In this study, we found that slow progression to type 1 diabetes was associated with a loss of islet autoantibodies and a distinct B cell phenotype, consistent with enhanced apoptotic regulation of peripheral autoreactivity via CD95. These phenotypic changes warrant further studies in larger cohorts to determine their functional implications.
Subject(s)
Autoantibodies/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Diabetes Mellitus, Type 1/immunology , fas Receptor/immunology , Autoantibodies/metabolism , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Diabetes Mellitus, Type 1/metabolism , Flow Cytometry , Humans , Proinsulin/immunology , Proinsulin/metabolism , fas Receptor/metabolismABSTRACT
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
Aberrant T cell differentiation and lymphopenia are hallmarks of severe COVID-19 disease. Since T cells must race to cull infected cells, they are quick to differentiate and achieve cytotoxic function. With this responsiveness, comes hastened apoptosis, due to a coupled mechanism of death and differentiation in both CD4+ and CD8+ lymphocytes via CD95 (Fas) and serine-threonine kinase (Akt). T cell lymphopenia in severe cases may represent cell death or peripheral migration. These facets depict SARS-Cov-2 as a lympho-manipulative pathogen; it distorts T cell function, numbers, and death, and creates a dysfunctional immune response. Whether preservation of T cells, prevention of their aberrant differentiation, and expansion of their population may alter disease course is unknown. Its investigation requires experimental interrogation of the linked differentiation and death pathway by agents known to uncouple T cell proliferation and differentiation in both CD4+ and CD8+ T cells.