RESUMO
Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study, we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells, including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies, the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus, ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity.
Assuntos
Neoplasias , Sialiltransferases , Animais , Camundongos , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/imunologia , Ácido N-Acetilneuramínico/imunologia , Neoplasias/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Sialiltransferases/genética , Sialiltransferases/imunologiaRESUMO
Altered glycosylations, which are associated with expression and activities of glycosyltransferases, can dramatically affect the function of glycoproteins and modify the behavior of tumor cells. ST3GAL1 is a sialyltransferase that adds sialic acid to core 1 glycans, thereby terminating glycan chain extension. In breast carcinomas, overexpression of ST3GAL1 promotes tumorigenesis and correlates with increased tumor grade. In pursuing the role of ST3GAL1 in breast cancer using ST3GAL1-siRNA to knockdown ST3GAL1, we identified CD55 to be one of the potential target proteins of ST3GAL1. CD55 is an important complement regulatory protein, preventing cells from complement-mediated cytotoxicity. CD55 had one N-linked glycosylation site in addition to a Ser/Thr-rich domain, which was expected to be heavily O-glycosylated. Detailed analyses of N- and O-linked oligosaccharides of CD55 released from scramble or ST3GAL1 siRNA-treated breast cancer cells by tandem mass spectrometry revealed that the N-glycan profile was not affected by ST3GAL1 silencing. The O-glycan profile of CD55 demonstrated a shift in abundance to nonsialylated core 1 and monosialylated core 2 at the expense of the disialylated core 2 structure after ST3GAL1 silencing. We also demonstrated that O-linked desialylation of CD55 by ST3GAL1 silencing resulted in increased C3 deposition and complement-mediated lysis of breast cancer cells and enhanced sensitivity to antibody-dependent cell-mediated cytotoxicity. These data demonstrated that ST3GAL1-mediated O-linked sialylation of CD55 acts like an immune checkpoint molecule for cancer cells to evade immune attack and that inhibition of ST3GAL1 is a potential strategy to block CD55-mediated immune evasion.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos/imunologia , Neoplasias da Mama/patologia , Antígenos CD55/imunologia , Evasão da Resposta Imune/imunologia , Sialiltransferases/metabolismo , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glicosilação , Humanos , RNA Interferente Pequeno/metabolismo , Sialiltransferases/genética , Sialiltransferases/imunologia , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Sialic acids (Sias) are the most abundant terminal sugar residues of glycoproteins and glycolipids on the surface of mammalian cells. The nervous tissue is the organ with the highest expression level of Sias. The 'sialylation' of glycoconjugates is performed via sialyltransferases, whereas 'desialylation' is done by sialidases or is a possible consequence of oxidative damage. Sialic acid residues on the neural cell surfaces inhibit complement and microglial activation, as well as phagocytosis of the underlying structures, via binding to (i) complement factor H (CFH) or (ii) sialic acid-binding immunoglobulin-like lectin (SIGLEC) receptors. In contrast, activated microglial cells show sialidase activity that desialylates both microglia and neurons, and further stimulates innate immunity via microglia and complement activation. The desialylation conveys neurons to become susceptible to phagocytosis, as well as triggers a microglial phagocytosis-associated oxidative burst and inflammation. Dysfunctions of the 'Sia-SIGLEC' and/or 'Sia-complement' axes often lead to neurological diseases. Thus, Sias on glycoconjugates of the intact glycocalyx and its desialylation are major regulators of neuroinflammation.
Assuntos
Imunidade Inata/genética , Tecido Nervoso/metabolismo , Ácidos Siálicos/genética , Sialiltransferases/genética , Glicoconjugados/genética , Glicoconjugados/imunologia , Humanos , Macrófagos , Microglia/imunologia , Microglia/metabolismo , Tecido Nervoso/imunologia , Neurônios/metabolismo , Neurônios/patologia , Fagocitose/genética , Ácidos Siálicos/imunologia , Ácidos Siálicos/metabolismo , Sialiltransferases/imunologiaRESUMO
The immune system contains a series of checks and balances that maintain tolerance and prevent autoimmunity. Sialic acid-binding Ig-type lectins (Siglecs) are cell surface receptors found on immune cells and inhibit inflammation by recruiting protein tyrosine phosphatases to ITIMs. Islet-resident macrophages express Siglec-E, and Siglec-E expression decreases on islet-resident macrophages as insulitis progresses in the NOD mouse. The sialyltransferase ST8Sia6 generates α-2,8-disialic acids that are ligands for Siglec-E in vivo. We hypothesized that engaging Siglec-E through ST8Sia6-generated ligands may inhibit the development of immune-mediated diabetes. Constitutive overexpression of ST8Sia6 in pancreatic ß cells mitigated hyperglycemia in the multiple low-dose streptozotocin model of diabetes, demonstrating that engagement of this immune receptor facilitates tolerance in the setting of inflammation and autoimmune disease.
Assuntos
Diabetes Mellitus/induzido quimicamente , Diabetes Mellitus/metabolismo , Sialiltransferases/metabolismo , Estreptozocina/farmacologia , Animais , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Autoimunidade/imunologia , Diabetes Mellitus/imunologia , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Hiperglicemia/imunologia , Hiperglicemia/metabolismo , Tolerância Imunológica/imunologia , Inflamação/imunologia , Inflamação/metabolismo , Células Secretoras de Insulina/imunologia , Células Secretoras de Insulina/metabolismo , Ligantes , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/metabolismo , Sialiltransferases/imunologiaRESUMO
Although xenografts are one of the most attractive strategies for overcoming the shortage of organ donors, cellular rejection by macrophages is a substantial impediment to this procedure. It is well known that macrophages mediate robust immune responses in xenografts. Macrophages also express various inhibitory receptors that regulate their immunological function. Recent studies have shown that the overexpression of inhibitory ligands on porcine target cells results in the phosphorylation of tyrosine residues on intracellular immunoreceptor tyrosine-based inhibitory motifs on macrophages, leading to the suppression of xenogenic rejection by macrophages. It has also been reported that myeloid-derived suppressor cells, a heterogeneous population of immature myeloid cells, suppress not only NK and cytotoxic T lymphocyte cytotoxicity but also macrophage-mediated cytotoxicity. This review is focused on the recent findings regarding strategies for inhibiting xenogenic rejection by macrophages.
Assuntos
Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto , Imunidade Celular , Macrófagos/imunologia , Transplante Heterólogo/efeitos adversos , Animais , Antígeno CD47/genética , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Xenoenxertos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Macrófagos/metabolismo , Células Supressoras Mieloides/imunologia , Células Supressoras Mieloides/metabolismo , Células Supressoras Mieloides/transplante , Fagocitose , Proteína D Associada a Surfactante Pulmonar/genética , Proteína D Associada a Surfactante Pulmonar/imunologia , Proteína D Associada a Surfactante Pulmonar/metabolismo , Sialiltransferases/genética , Sialiltransferases/imunologia , Sialiltransferases/metabolismo , Transdução de Sinais , Resultado do Tratamento , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
CD74 is a type II transmembrane glycoprotein that functions as an MHC class II chaperone and displays diverse roles in immune responses. Recently, anti-CD74 immunotherapy has shown promise as an effective treatment strategy for lymphoid neoplasms in preclinical models. Using a human anti-CD74 antibody (SP7219), we defined the expression of CD74 protein in both normal and over 790 neoplastic hematolymphoid tissue samples. We found that CD74 is expressed broadly in normal B-cell compartments including primary and secondary lymphoid follicles and in the thymic medulla. The vast majority of lymphomas expressed CD74, including Hodgkin lymphomas (98%), B-cell lymphomas (96%), extranodal NK/T-cell lymphomas (88%), mature T-cell lymphomas (80%), and plasma cell myeloma (75%). Our findings confirm and expand previous observations regarding the expression of CD74 and suggest that CD74 expression on tumor cells may be directly targeted for immunomodulatory therapy for lymphoid and plasma cell malignancies.
Assuntos
Antígenos CD/imunologia , Imunoconjugados/uso terapêutico , Linfoma de Células B/tratamento farmacológico , Linfoma Extranodal de Células T-NK/patologia , Mieloma Múltiplo/tratamento farmacológico , Sialiltransferases/imunologia , Antígenos CD/metabolismo , Genes MHC da Classe II/genética , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/patologia , Humanos , Linfoma de Células B/patologia , Linfoma Extranodal de Células T-NK/tratamento farmacológico , Mieloma Múltiplo/patologia , Sialiltransferases/metabolismoRESUMO
ST6Gal1 is a critical sialyltransferase enzyme that controls the addition of α2,6-linked sialic acids to the termini of glycans. Attachment of sialic acids to glycoproteins as a posttranslational modification influences cellular responses, and is a well-known modifier of immune cell behavior. ST6Gal1 activity impacts processes such as: effector functions of immunoglobulin G via Fc sialylation, hematopoietic capacity by hematopoietic stem and progenitor cell surface sialylation, and lymphocyte activation thresholds though CD22 engagement and inhibition of galectins. This review summarizes recent studies that suggest α2,6 sialylation by ST6Gal1 has an immunoregulatory effect on immune reactions.
Assuntos
Imunoglobulina G/imunologia , Fatores Imunológicos/imunologia , Leucócitos/imunologia , Ácidos Siálicos/imunologia , Sialiltransferases/imunologia , Animais , Humanos , Ativação Linfocitária/imunologia , Polissacarídeos/imunologia , Processamento de Proteína Pós-Traducional/imunologiaRESUMO
Germinal centers (GC) are microanatomical niches where B cells proliferate, undergo antibody affinity maturation, and differentiate to long-lived memory B cells and antibody-secreting plasma cells. For decades, GC B cells have been defined by their reactivity to the plant lectin peanut agglutinin (PNA), which binds serine/threonine (O-linked) glycans containing the asialylated disaccharide Gal-ß1,3-GalNAc-Ser/Thr (also called T-antigen). In T cells, acquisition of PNA binding by activated T cells and thymocytes has been linked with altered tissue homing patterns, cell signaling, and survival. Yet, in GC B cells, the glycobiological basis and significance of PNA binding remains surprisingly unresolved. Here, we investigated the basis for PNA reactivity of GC B cells. We found that GC B cell binding to PNA is associated with downregulation of the α2,3 sialyltransferase, ST3GAL1 (ST3Gal1), and overexpression of ST3Gal1 was sufficient to reverse PNA binding in B cell lines. Moreover, we found that the primary scaffold for PNA-reactive O-glycans in B cells is the B cell receptor-associated receptor-type tyrosine phosphatase CD45, suggesting a role for altered O-glycosylation in antigen receptor signaling. Consistent with similar reports in T cells, ST3Gal1 overexpression in B cells in vitro induced drastic shortening in O-glycans, which we confirmed by both antibody staining and mass spectrometric O-glycomic analysis. Unexpectedly, ST3Gal1-induced changes in O-glycan length also correlated with altered binding of two glycosylation-sensitive CD45 antibodies, RA3-6B2 (more commonly called B220) and MEM55, which (in humans) have previously been reported to favor binding to naïve/GC subsets and memory/plasmablast subsets, respectively. Analysis of primary B cell binding to B220, MEM55, and several plant lectins suggested that B cell differentiation is accompanied by significant loss of O-glycan complexity, including loss of extended Core 2 O-glycans. To our surprise, decreased O-glycan length from naïve to post-GC fates best correlated not with ST3Gal1, but rather downregulation of the Core 2 branching enzyme GCNT1. Thus, our data suggest that O-glycan remodeling is a feature of B cell differentiation, dually regulated by ST3Gal1 and GCNT1, that ultimately results in expression of distinct O-glycosylation states/CD45 glycoforms at each stage of B cell differentiation.
Assuntos
Linfócitos B/imunologia , Diferenciação Celular/imunologia , Polissacarídeos/imunologia , Transdução de Sinais/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Diferenciação Celular/genética , Linhagem Celular Tumoral , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Centro Germinativo/citologia , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Glicosilação , Humanos , Lectinas/imunologia , Lectinas/metabolismo , Aglutinina de Amendoim/imunologia , Aglutinina de Amendoim/metabolismo , Polissacarídeos/metabolismo , Sialiltransferases/genética , Sialiltransferases/imunologia , Sialiltransferases/metabolismo , Transdução de Sinais/genética , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
The modern diagnostic approaches permit to diagnose axial spondylarthrosis (axSpA) at roentgenologic stage corresponding to ankylosing spondylitis (AS). While early diagnostic of non-roentgenologic axSpA (nr-axSpA) is still complicated. This situation conditions a need in searching new laboratory biomarkers for early diagnostic of spondylarthrosis, including auto-antibodies to antigen CD74 described recently. The purpose of study is to evaluate clinical diagnostic significance of auto-antibodies to antigen CD74 in case of axSpA. The technique of quantitative enzyme-linked immunosorbent assay was applied to measure content of auto-antibodies IgA to CD74 in samples of serum from 140 patients with axSpA: 68 with AS, 46 with nr-axSpA, 26 with psoriatic arthritis (PA) and 37 healthy representatives of control group with signs of axSpA totally clinically excluded. The average values of concentration of auto-antibodies IgA to CD74 in patients with axSpA and nr-axSpA made up to 3,5 ± 3,0 and 3,8 ± 2,9 U/ml correspondingly that reliably and significantly differed from patients with PA and healthy individuals - 2,1 ± 1,4 and 1,3 ± 1,4 U/ml correspondingly (p < 0,05). At threshold value of content of auto-antibodies IgA to CD74 higher than 2.0 U/ml in case of axSpA diagnostic sensitivity made up to 64.4%, specificity - 89.2%, risk factor of positive result - 5.9 whereas in patients with nr-axSpA at concentration 1.7 U/ml - 73,1%, 84% and 4,5 correspondingly. The auto-antibodies IgA to antigen CD74 are associated withaxSpA but not with PA that permits to use the given marker for diagnostic of axial spondylarthrosis and also in case of differential diagnostic between axSpA and PA.
Assuntos
Antígenos CD/imunologia , Autoanticorpos/sangue , Sialiltransferases/imunologia , Espondilite Anquilosante/diagnóstico , Artrite Psoriásica , Biomarcadores/sangue , Estudos de Casos e Controles , Humanos , Sensibilidade e Especificidade , Espondilite Anquilosante/sangueRESUMO
BACKGROUND: Polysialic acid (polySia) is a carbohydrate modification of the neural cell adhesion molecule (NCAM), which is implicated in neural differentiation and plays an important role in tumor development and metastasis. Polysialylation of NCAM is mediated by two Golgi-resident polysialyltransferases (polyST) ST8SiaII and ST8SiaIV. Intracellular antibodies (intrabodies; IB) expressed inside the ER and retaining proteins passing the ER such as cell surface receptors or secretory proteins provide an efficient means of protein knockdown. To inhibit the function of ST8SiaII and ST8SiaIV specific ER IBs were generated starting from two corresponding hybridoma clones. Both IBs αST8SiaII-IB and αST8SiaIV-IB were constructed in the scFv format and their functions characterized in vitro and in vivo. RESULTS: IBs directed against the polySTs prevented the translocation of the enzymes from the ER to the Golgi-apparatus. Co-immunoprecipitation of ST8SiaII and ST8SiaIV with the corresponding IBs confirmed the intracellular interaction with their cognate antigens. In CHO cells overexpressing ST8SiaII and ST8SiaIV, respectively, the transfection with αST8SiaII-IB or αST8SiaIV-IB inhibited significantly the cell surface expression of polysialylated NCAM. Furthermore stable expression of ST8SiaII-IB, ST8SiaIV-IB and luciferase in the rhabdomyosarcoma cell line TE671 reduced cell surface expression of polySia and delayed tumor growth if cells were xenografted into C57BL/6 J RAG-2 mice. CONCLUSION: Data obtained strongly indicate that αST8SiaII-IB and αST8SiaIV-IB are promising experimental tools to analyze the individual role of the two enzymes during brain development and during migration and proliferation of tumor cells.
Assuntos
Anticorpos/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Ácidos Siálicos/metabolismo , Sialiltransferases/metabolismo , Animais , Anticorpos/genética , Anticorpos/imunologia , Sequência de Bases , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Moléculas de Adesão de Célula Nervosa/imunologia , Plasmídeos/genética , Plasmídeos/metabolismo , Rabdomiossarcoma/metabolismo , Rabdomiossarcoma/patologia , Sialiltransferases/genética , Sialiltransferases/imunologia , Transplante HeterólogoRESUMO
Responding to systemic demands in producing and replenishing end-effector blood cells is predicated on the appropriate delivery and interpretation of extrinsic signals to the HSPCs. The data presented herein implicate the systemic, extracellular form of the glycosyltransferase ST6Gal-1 in the regulation of late-stage neutrophil development. ST6Gal-1 is typically a membrane-bound enzyme sequestered within the intracellular secretory apparatus, but an extracellular form is released into the blood from the liver. Both human and murine HSPCs, upon exposure to extracellular ST6Gal-1 ex vivo, exhibited decreased proliferation, diminished expression of the neutrophilic primary granule protein MPO, and decreased appearance of CD11b+ cells. HSPC suppression was preceded by decreased STAT-3 phosphorylation and diminished C/EBPα expression, without increased apoptosis, indicating attenuated G-CSF receptor signaling. A murine model to raise systemic ST6Gal-1 level was developed to examine the role of the circulatory enzyme in vivo. Our results show that systemic ST6Gal-1 modified the cell surface of the GMP subset of HSPCs and decreased marrow neutrophil reserves. Acute airway neutrophilic inflammation by LPS challenge was used to drive demand for new neutrophil production. Reduced neutrophil infiltration into the airway was observed in mice with elevated circulatory ST6Gal-1 levels. The blunted transition of GMPs into GPs in vitro is consistent with ST6Gal-1-attenuated granulopoiesis. The data confirm that circulatory ST6Gal-1 is a negative systemic regulator of granulopoiesis and moreover suggest a clinical potential to limit the number of inflammatory cells by manipulating blood ST6Gal-1 levels.
Assuntos
Hematopoese/imunologia , Neutrófilos/citologia , Sialiltransferases/imunologia , Animais , Western Blotting , Diferenciação Celular/imunologia , Citometria de Fluxo , Imunofluorescência , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Sialiltransferases/metabolismo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, ß-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.
Assuntos
Diferenciação Celular/imunologia , Glicoesfingolipídeos/química , Macrófagos/química , Monócitos/química , Polissacarídeos/química , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular , Fucosiltransferases/genética , Fucosiltransferases/imunologia , Gangliosídeo G(M3)/química , Gangliosídeo G(M3)/imunologia , Regulação da Expressão Gênica , Glicoesfingolipídeos/imunologia , Glicosilação , Glicosiltransferases/genética , Glicosiltransferases/imunologia , Humanos , Macrófagos/citologia , Macrófagos/imunologia , Manose/química , Manose/imunologia , Monócitos/citologia , Monócitos/imunologia , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/imunologia , Neuraminidase/genética , Neuraminidase/imunologia , Polissacarídeos/imunologia , Ácidos Siálicos/química , Ácidos Siálicos/imunologia , Sialiltransferases/genética , Sialiltransferases/imunologiaRESUMO
An immunotherapeutic strategy is discussed supporting anti-tumor activity toward malignancies overexpressing ganglioside D3. GD3 can be targeted by NKT cells when derived moieties are presented in the context of CD1d. NKT cells can support anti-tumor responses by secreting inflammatory cytokines and through cytotoxicity toward CD1d+GD3+ tumors. To overexpress GD3, we generated expression vector DNA and an adenoviral vector encoding the enzyme responsible for generating GD3 from its ubiquitous precursor GM3. We show that DNA encoding α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (SIAT8) introduced by gene gun vaccination in vivo leads to overexpression of GD3 and delays tumor growth. Delayed tumor growth is dependent on CD1d expression by host immune cells, as shown in experiments engaging CD1d knockout mice. A trend toward greater NKT cell populations among tumor-infiltrating lymphocytes is associated with SIAT8 vaccination. A single adenoviral vaccination introduces anti-tumor activity similarly to repeated vaccination with naked DNA. Here, greater NKT tumor infiltrates were accompanied by marked overexpression of IL-17 in the tumor, later switching to IL-4. Our results suggest that a single intramuscular adenoviral vaccination introduces overexpression of GD3 by antigen-presenting cells at the injection site, recruiting NKT cells that provide an inflammatory anti-tumor environment. We propose adenoviral SIAT8 (AdV-SIAT8) can slow the growth of GD3 expressing tumors in patients.
Assuntos
Gangliosídeos/biossíntese , Melanoma Experimental/imunologia , Melanoma/imunologia , Sialiltransferases/imunologia , Animais , Biolística , Linhagem Celular Tumoral , Gangliosídeos/imunologia , Células HEK293 , Humanos , Melanoma/enzimologia , Melanoma/terapia , Melanoma Experimental/enzimologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sialiltransferases/genética , Vacinas de DNA/imunologiaRESUMO
Sialic acid sugars are vital regulators of the immune system through binding to immunosuppressive sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on immune cells. Aberrant sialic acid-Siglec interactions are associated with an increasing number of pathologies including infection, autoimmunity, and cancer. Therefore, the sialic acid-Siglec axis is an emerging target to prevent or affect the course of several diseases. Chemical modifications of the natural sialic acid ligands have led to sialic acid mimetics (SAMs) with improved binding affinity and selectivity towards Siglecs. Recent progress in glycobiotechnology allows the presentation of these SAMs on nanoparticles, polymers, and living cells via bioorthogonal synthesis. These developments now enable the detailed study of the sialic acid-Siglec axis including its therapeutic potential as an immune modulator.
Assuntos
Envelhecimento/imunologia , Materiais Biomiméticos/uso terapêutico , Doenças do Sistema Imunitário/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Ácidos Siálicos/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Materiais Biomiméticos/química , Sequência de Carboidratos , Portadores de Fármacos , Expressão Gênica , Humanos , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/imunologia , Doenças do Sistema Imunitário/patologia , Fatores Imunológicos/química , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/patologia , Terapia de Alvo Molecular , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/patologia , Nanopartículas/uso terapêutico , Ligação Proteica , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/antagonistas & inibidores , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/genética , Ácidos Siálicos/antagonistas & inibidores , Ácidos Siálicos/química , Sialiltransferases/antagonistas & inibidores , Sialiltransferases/genética , Sialiltransferases/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Acute myeloid leukemia (AML) is a heterogeneous group of malignancies that may be sensitive to the NK cell antitumor response. However, NK cells are frequently defective in AML. In this study, we found in an exploratory cohort (n = 46) that NK cell status at diagnosis of AML separated patients in two groups with a different clinical outcome. Patients with a deficient NK cell profile, including reduced expression of some activating NK receptors (e.g., DNAX accessory molecule-1, NKp46, and NKG2D) and decreased IFN-γ production, had a significantly higher risk of relapse (p = 0.03) independently of cytogenetic classification in multivariate analysis. Patients with defective NK cells showed a profound gene expression decrease in AML blasts for cytokine and chemokine signaling (e.g., IL15, IFNGR1, IFNGR2, and CXCR4), Ag processing (e.g., HLA-DRA, HLA-DRB1, and CD74) and adhesion molecule pathways (e.g., PVR and ICAM1). A set of 388 leukemic classifier genes defined in the exploratory cohort was independently validated in a multicentric cohort of 194 AML patients. In total, these data evidenced the interplay between NK cells and AML blasts at diagnosis allowing an immune-based stratification of AML patients independently of clinical classifications.
Assuntos
Citotoxicidade Imunológica/imunologia , Células Matadoras Naturais/imunologia , Leucemia Mieloide Aguda/imunologia , Receptores de Células Matadoras Naturais/metabolismo , Evasão Tumoral/imunologia , Adulto , Idoso , Antígenos CD/imunologia , Moléculas de Adesão Celular/imunologia , Feminino , Cadeias alfa de HLA-DR/imunologia , Cadeias HLA-DRB1/imunologia , Humanos , Interferon gama/biossíntese , Interleucina-15/biossíntese , Leucemia Mieloide Aguda/diagnóstico , Masculino , Pessoa de Meia-Idade , Receptores CXCR4/biossíntese , Receptores de Interferon/biossíntese , Sialiltransferases/imunologia , Evasão Tumoral/genética , Adulto Jovem , Receptor de Interferon gamaRESUMO
Myxoma virus (MYXV) induces a lethal disease called Myxomatosis in European rabbits. MYXV is one of the rare viruses that encodes an α2,3-sialyltransferase through its M138L gene. In this study, we showed that although the absence of the enzyme was not associated with any in vitro deficit, the M138L deficient strains are highly attenuated in vivo. Indeed, while all rabbits infected with the parental and the revertant strains died within 9 days post-infection from severe myxomatosis, all but one rabbit inoculated with the M138L deficient strains survived the infection. In primary lesions, this resistance to the infection was associated with an increased ability of innate immune cells, mostly neutrophils, to migrate to the site of virus replication at 4 days post-infection. This was followed by the development of a better specific immune response against MYXV. Indeed, at day 9 post-infection, we observed an important proliferation of lymphocytes and an intense congestion of blood vessels in lymph nodes after M138L knockouts infection. Accordingly, in these rabbits, we observed an intense mononuclear cell infiltration throughout the dermis in primary lesions and higher titers of neutralizing antibodies. Finally, this adaptive immune response provided protection to these surviving rabbits against a challenge with the MYXV WT strain. Altogether, these results show that expression of the M138L gene contributes directly or indirectly to immune evasion by MYXV. In the future, these results could help us to better understand the pathogenesis of myxomatosis but also the importance of glycans in regulation of immune responses.
Assuntos
Tolerância Imunológica/imunologia , Myxoma virus/imunologia , Mixomatose Infecciosa/imunologia , Sialiltransferases/imunologia , Proteínas Virais/imunologia , Imunidade Adaptativa/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , DNA Viral/sangue , DNA Viral/genética , DNA Viral/imunologia , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Masculino , Myxoma virus/patogenicidade , Myxoma virus/fisiologia , Mixomatose Infecciosa/sangue , Mixomatose Infecciosa/virologia , Coelhos , Sialiltransferases/genética , Sialiltransferases/metabolismo , Análise de Sobrevida , Fatores de Tempo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência/genética , Virulência/imunologia , Fatores de Virulência/genética , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismoRESUMO
Macrophages play an important role in xenogenic rejection and therefore may represent a major obstacle in clinical application of xenograft. CD33-related sialic acid-binding immunoglobulin-like lectins (Siglecs) belong to the immunoglobulin superfamily and contain a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) that is able to inhibit cytokine production. Because human macrophages express various CD33-related Siglecs, we hypothesized that overexpression of α-2,6-sialyltransferase (2,6-ST) in swine endothelial cells (SECs) might prevent the cytotoxicity mediated by macrophages. To confirm our hypothesis, the cytotoxicity of macrophages against 2,6-ST-overexpressing SECs was determined with the use of in vitro-generated macrophages as an effector and naïve or 2,6-ST-overexpressing SECs as a target. The 2,6-ST-overexpressing SECs were established by transfection with the genes for 2,6-ST. Transfection of 2,6-ST led to significant reduction in cytotoxicity compared with naïve SECs. These findings indicate that the sialylated ligands against CD33-related Siglecs may provide an effective therapeutic strategy to inhibit macrophage-mediated xenograft rejection in xenotransplantation.
Assuntos
Células Endoteliais/imunologia , Ativação de Macrófagos , Macrófagos/imunologia , Sialiltransferases/imunologia , Animais , Linhagem Celular , Técnicas de Cocultura , Células Endoteliais/enzimologia , Indução Enzimática , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Humanos , Ligantes , Macrófagos/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/imunologia , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Sialiltransferases/biossíntese , Sialiltransferases/genética , Suínos , Transfecção , Transplante Heterólogo , beta-D-Galactosídeo alfa 2-6-SialiltransferaseRESUMO
Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas from melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.
Assuntos
Interleucina-6/imunologia , Queratinócitos/efeitos da radiação , Melanócitos/enzimologia , Melanoma/enzimologia , Sialiltransferases/genética , Fator de Necrose Tumoral alfa/imunologia , Linhagem Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Queratinócitos/imunologia , Melanócitos/imunologia , Melanócitos/efeitos da radiação , Melanoma/genética , Melanoma/imunologia , Sialiltransferases/imunologia , Raios Ultravioleta , Regulação para CimaRESUMO
Breast milk oligosaccharides shape the intestinal environment by affecting mucosal immunity and bacterial colonization. To clarify the role of milk oligosaccharide sialyl(α2,3)lactose (3SL) in intestinal physiology and disease, we investigated colitis development in Il10(-/-) mice exposed to normal or 3SL-deficient milk during lactation. Onset and progression of intestinal inflammation were delayed in Il10(-/-) mice deficient for the α2,3 sialyltransferase 4 (ST3GAL4) responsible for 3SL biosynthesis. The proinflammatory role of 3SL was confirmed by showing that oral supplementation of newborn Il10(-/-);St3gal4(-/-) mice with 3SL increased colitis severity. Conversely, fostering of newborn Il10(-/-) mice to lactating St3gal4(-/-) mothers reduced colitis severity. 3SL directly stimulated mesenteric lymph node CD11c(+) dendritic cells and induced production of cytokines required for expansion of TH1 and TH17 T cells. The stimulatory effect of 3SL was attenuated in Tlr4-deficient CD11c(+) cells, demonstrating that 3SL induces inflammation through Toll-like receptor 4 (TLR4) signaling. Thus, 3SL directly modulates mucosal immunity, which increases susceptibility to colitis.
Assuntos
Antígeno CD11c/imunologia , Intestinos/imunologia , Lactose/análogos & derivados , Ácidos Siálicos/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Animais Recém-Nascidos , Bactérias/classificação , Bactérias/genética , Bactérias/imunologia , Antígeno CD11c/metabolismo , Células Cultivadas , Colite/genética , Colite/imunologia , Colite/metabolismo , Citocinas/genética , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Trato Gastrointestinal/patologia , Expressão Gênica/imunologia , Imunidade nas Mucosas/genética , Imunidade nas Mucosas/imunologia , Interleucina-10/deficiência , Interleucina-10/genética , Interleucina-10/imunologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Lactação/imunologia , Lactose/imunologia , Lactose/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Leite/química , Leite/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ácidos Siálicos/metabolismo , Sialiltransferases/deficiência , Sialiltransferases/genética , Sialiltransferases/imunologia , Receptor 4 Toll-Like/metabolismo , beta-Galactosídeo alfa-2,3-SialiltransferaseRESUMO
Sialic acid-binding, Ig-like lectin (Siglec)-F is highly expressed on mouse eosinophils and plays an important role in regulating levels of eosinophilic lung inflammation. In this study we investigated the mechanism of constitutive and inducible Siglec-F ligand expression by lung airway epithelial cells and inflammatory cells in wild-type (WT) and genetically altered mice (ST3Gal-III heterozygotes, Fuc-TIV/VII double null, STAT6 null). Flow cytometry demonstrated that Siglec-F ligands are constitutively expressed in vitro and in vivo in selected lung cell types (epithelial cells, eosinophils, macrophages, and mast cells, but not CD4, CD8, or B cells) and are induced in response to divergent stimuli, including innate stimuli (TLR ligands, Alternaria), Th2 cytokines (IL-4, IL-13), and adaptive immune stimuli (OVA allergen). Furthermore, studies of deficient mice demonstrated the greater importance of the sialyltransferase ST3Gal-III compared with fucosyltransferases Fuc-TIV/VII in the synthesis of the constitutive and inducible Siglec-F ligands by lung epithelial and nonepithelial cells. In keeping with this, ST3Gal-III heterozygote mice (deficient in expression of Siglec-F ligands) also had significantly enhanced OVA-induced eosinophilic airway inflammation associated with reduced eosinophil apoptosis. Reduced eosinophil apoptosis in the lung of ST3Gal-III-deficient mice is likely mediated by reduced epithelial expression of Siglec-F ligands as WT eosinophils (which highly express Siglec-F) cultured with ST3Gal-III-deficient epithelial cells (which do not express Siglec-F ligand) showed reduced eosinophil apoptosis compared with WT eosinophils cultured with WT epithelial cells. Overall, these studies demonstrate that ST3Gal-III plays an important role in Siglec-F ligand formation and eosinophil apoptosis with resultant effects on eosinophilic inflammation in the lung.