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BACKGROUND: One of the mechanisms regulating the biological activity of tumor necrosis factor (TNF) in cells is the co-expression of TNFR1/TNFR2 receptors. A model with a differential level of receptor expression is required to evaluate the contribution of these mechanisms. AIM: The development of a cellular model to compare the effects of TNF on cells depending on the presence of both receptors and TNFR2 alone. METHODS: TNFR1 absence modifications of ZR-75/1 and K-562 cell lines were obtained by TNFR1 knockout. The presence of deletions was confirmed by Sanger sequencing, and the absence of cell membrane receptor expression was confirmed by flow cytometry. The dose-dependent effect of TNF on intact and knockout cells was comparatively evaluated by the effect on the cell cycle, the type of cell death, and the profile of expressed genes. RESULTS: Knockout of TNFR1 resulted in a redistribution of TNFR2 receptors with an increased proportion of TNFR2+ cells in both lines and a multidirectional change in the density of expression in the lines (increased in K562 and decreased in ZR75/1). The presence of a large number of cells with high TNFR2 density in the absence of TNFR1 in the K562 cells was associated with greater sensitivity to TNF-stimulating doses and increased proliferation but did not result in a significant change in cell death parameters. A twofold increase in TNFR2+ cell distribution in this cell line at a reduced expression density in ZR75/1 cells was associated with a change in sensitivity to low cytokine concentrations in terms of proliferation; an overall increase in cell death, most pronounced at standard stimulating concentrations; and increased expression of the lymphocyte-activation gene groups, host-pathogen interaction, and innate immunity. CONCLUSIONS: The absence of TNFR1 leads to different variants of compensatory redistribution of TNFR2 in cellular models, which affects the type of cell response and the threshold level of sensitivity. The directionality of cytokine action modulation and sensitivity to TNF levels depends not only on the fraction of cells expressing TNFR2 but also on the density of expression.
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This research delves into the intricate landscape of tumor necrosis factor-alpha (TNF-α) signaling, a multi-functional cytokine known for its diverse cellular effects. Specifically, we investigate the roles of two TNF receptors, TNFR1 and TNFR2, in mediating TNF-α-induced transcriptional responses. Using human K562 cell lines with TNFR1 and TNFR2 knockouts, we explore changes in gene expression patterns following TNF-α stimulation. Our findings reveal distinct transcriptional profiles in TNFR1 and TNFR2 knockout cells, shedding light on the unique contributions of these receptors to TNF-α signaling. Notably, several key pathways associated with inflammation, apoptosis, and cell proliferation exhibit altered regulation in the absence of TNFR1 or TNFR2. This study provides valuable insights into the intricate mechanisms governing TNF-α signaling and its diverse cellular effects, with potential implications for targeted therapeutic strategies.
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Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa , Humanos , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Células K562 , Citocinas/metabolismoRESUMEN
Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer.
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TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses.
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Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T , Neoplasias/metabolismo , Inmunoterapia Adoptiva , Citotoxicidad Inmunológica , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
The authors used a method quantitative estimation density of TNFR1/TNFR2 on cells by flow cytometry with calibration particles, which allowed them to estimate the absolute number of receptors on cells regardless of the type of flow cytometer. The TNF receptor expression parameters were used to determine their association with the fact of disease and to build diagnostic models. The proposed methodological approach using a combination of flow cytometry and mathematical modeling techniques represents a promising direction for testing the diagnostic and prognostic significance of the studied biomarkers. The multifactorial regression analysis constructed on the basis of this approach made it possible to refine and supplement diagnostic schemes for determining the probability of rheumatoid arthritis and bronchial asthma in patients.
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Artritis Reumatoide , Factor de Necrosis Tumoral alfa , Humanos , Factor de Necrosis Tumoral alfa/metabolismo , Artritis Reumatoide/diagnóstico , Biomarcadores , Citometría de FlujoRESUMEN
Alternative splicing is a part of mRNA processing that expands the diversity of proteins encoded by a single gene. Studying the full range of proteins-products of translation of alternatively spliced mRNA is extremely important for understanding the interactions between receptor proteins and ligands since different receptor protein isoforms can provide variation in the activation of signaling pathways. In this study, we investigated the expression of isoforms of TNFR1 and TNFR2 receptors before and after exposure to TNFα in two cell lines that had previously demonstrated diverse effects on cell proliferation under TNFα incubation using RT-qPCR. We found that after incubation with TNFα: (1) expression of isoform 3 of the TNFRSF1A gene was increased in both cell lines; (2) the cell line with increased proliferation, K562, had decreased expression of isoforms 1 and 4 of the TNFRSF1A gene and expression of isoform 2 of TNFRSF1B gene was absent at all; (3) the cell line with decreased proliferation-MCF-7 had significantly increased expression of isoform 2 of TNFRSF1B gene. Thus, we can conclude that TNFα exposure to the K562 and MCF-7 cell lines leads to changes in the expression of TNFα receptor isoforms, which, in turn, can appear via diverse proliferative effects.
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Receptores Tipo II del Factor de Necrosis Tumoral , Receptores Tipo I de Factores de Necrosis Tumoral , Factor de Necrosis Tumoral alfa , Expresión Génica , Isoformas de Proteínas/genética , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Humanos , Células K562 , Células MCF-7 , Receptores Tipo II del Factor de Necrosis Tumoral/genética , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismoRESUMEN
Dendritic cells (DCs) loaded with tumor-associated antigens (TAAs) are known to be crucial for the antitumor response and are still included in various treatment regimens in cancer immunotherapy research. In the present study, a cell-based protocol was evaluated, involving the use of original DNA constructs encoding the wide range of TAA epitopes expressed on different epithelial cancers. The constructs were transfected into in vitro-generated DCs of patients with various types of cancer, including breast, colorectal and non-small cell lung cancer. The direct cytotoxicity assay of effector cells, activated with the transfected DCs, revealed a significant increase in cytotoxicity against autologous tumor cells. The use of DNA constructs encoding a large number of TAAs for insertion into DCs in vitro, aiming to activate a T-cell response may prove to be a reliable and unified approach for immunotherapy and for the prevention of relapse in patients with epithelial cancers.
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BACKGROUND: A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. METHODS: The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). RESULTS: A significant increase in the amounts of FoxP3+ CD4+CD25+ cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. CONCLUSIONS: The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.
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Células Dendríticas , Isoantígenos , Células Dendríticas/metabolismo , Epítopos/genética , Epítopos/metabolismo , Humanos , Tolerancia Inmunológica/genética , Isoantígenos/genética , Isoantígenos/metabolismo , Leucocitos Mononucleares , Linfocitos T ReguladoresRESUMEN
BACKGROUND: The co-expression patterns of type 1 and 2 tumor necrosis factor (TNF)-α membrane receptors (TNFR1/TNFR2) are associated with the presence, stage, and activity of allergic diseases. The aim of this study was to assess the expression levels and dynamics of TNFRs on immune cells and to assess associations between their expression and severity of bronchial asthma (BA). METHODS: Patients with severe (n = 8), moderate (n = 10), and mild (n = 4) BA were enrolled. As a comparison group, data from 46 healthy volunteers (HV) were accessed. Co-expression of TNFR1/2 was evaluated as a percentage of cells and the number of receptors of each type per cell. Multivariate logistic regression analysis was used to identify diagnostic biomarkers of BA. RESULTS: More than 90% of the monocytes in patients with mild BA were TNFR1+TNFR2+ but had significantly lower TNFR1 expression density compared with HV (7.82- to 14.08-fold, depending on disease severity). Lower percentages of the TNFR+ B-lymphocytes were observed in combination with significantly lower receptors density in BA compared with HV (2.59- to 11.64-fold for TNFR1 and 1.72- to 3.4-fold for TNFR2, depending on disease severity). The final multivariate model for predicting the presence of BA included the percentage of double-positive CD5+ B-lymphocytes and average number of TNFR1 molecules expressed on cytotoxic naive T-lymphocytes and T-helper cells (R2 = 0.87). CONCLUSIONS: The co-expression patterns of TNFRs on immune cells in BA differed significantly compared with HV. The expression differences were associated with disease severity. TNFR1 expression changes were key parameters that discriminated patients with BA from those with HV.
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Asma , Linfocitos B , Monocitos , Receptores Tipo II del Factor de Necrosis Tumoral , Receptores Tipo I de Factores de Necrosis Tumoral , Linfocitos B/metabolismo , Humanos , Monocitos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismoRESUMEN
BACKGROUND: Tumor necrosis factor (TNF) plays an important role in immune responses to the causative agent of tuberculosis, Mycobacterium tuberculosis. Additionally, TNF can also mediate many negative disease manifestations. The aim of this study was to assess the contribution of anti-TNF autoantibodies to the pathogenesis of active pulmonary tuberculosis (TB). METHODS: The levels of anti-TNF autoantibody classes and subclasses were determined by applying enzyme-linked immunosorbent assays (ELISAs). The levels of TNF and of its soluble receptors were also evaluated using commercial ELISA kits. RESULTS: The levels of both types of soluble TNF receptors were lower patients with TB than in healthy donors. Patients with TB had higher titers of immunoglobulin (Ig)G class and IgG3 subclass anti-TNF autoantibodies in comparison with healthy donors. Patients who had a disseminated TB infection had higher TNF level and IgG, IgG1 and IgG3 autoantibody titers compared with patients who had a localized TB infection. CONCLUSIONS: Changes in the titers of anti-TNF autoantibody classes and subclasses were noted in patients with TB, suggesting their possible contribution to the disease pathogenesis of TB.
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Tuberculosis Pulmonar , Tuberculosis , Autoanticuerpos/análisis , Humanos , Inmunoglobulina G/análisis , Inhibidores del Factor de Necrosis Tumoral , Factor de Necrosis Tumoral alfaRESUMEN
One of the mechanisms of cellular dysfunction during the chronization of immune-system-mediated inflammatory diseases is a change in the profile of expression and co-expression of receptors on cells. The aim of this study was to compare patterns of redistribution of TNF receptors (TNFRs) among patients with different durations of rheumatoid arthritis (RA) or asthma. Subgroup analysis was performed on RA (n = 41) and asthma (n = 22) patients with disease duration<10 years and >10 years and on 30 comparable healthy individuals. The co-expression profile of TNFR1 and TNFR2 was assessed in T cells, B cells, monocytes, regulatory T cells, T-helper subsets, and cytotoxic T-lymphocyte subsets. Percentages of cells with different co-expression combinations and receptor density per cell were estimated. Longer disease duration was significantly associated with a redistribution of receptors in immunocompetent cell subsets with an increase in the expression of TNFR1 in asthma but did not correlate with significant unidirectional changes in receptor expression in RA. In asthma, a higher proportion of cells with a certain type of TNF receptor (as compared with the healthy group) was correlated with a simultaneous greater density of this receptor type. In RA, an inverse correlation was observed (compensatory lower receptor density). Mechanisms of long-term changes in the expression of TNF receptors differ significantly between the diseases of autoimmune and allergic etiology. The formation of irreversible morphostructural alterations was strongly correlated with changes in the expression of TNFR1 in asthma and with changes in the expression of TNFR2 in RA.
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Artritis Reumatoide , Asma , Humanos , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Expresión GénicaRESUMEN
INTRODUCTION: Modulating specific biological effects through the changes in cytokine receptors' expression level remains poorly understood. This study aimed to investigate the influence of the dose-dependent effect of TNF on the balance between proapoptotic and proliferation response depending on the parameters of TNFR1/2 expression density. METHODS: Tumor cell lines (HEp-2, K-562, MCF-7, ZR-75/1, MOLT-4, IM-9, and Raji) were characterized for TNFR1/2 co-expression using flow cytometry and were studied to reveal the dose-dependent effect of rhTNF on cell cycle and apoptosis parameters. The associations among the studied parameters were estimated by correlation and regression analysis. RESULTS: It was found for ZR-75/1 cells (the cell line characterized by high expression of both types) that a dose-dependent increase in expression of both types of TNF-α receptors on cells reduces the proliferative activity of cells. For MOLT-4 cells (which are characterized by lower expression), an increase in proliferative response of cells was positively associated with the percentage of both TNFR1+ and TNFR2+ cells. However, opposite effects on the cells were shown for the K-562 and MCF-7 lines having a similar expression profile. A similarity (a large percentage of double-positive cells) was revealed for the lines having similar effects (K-562 and ZR-75/1). CONCLUSIONS: High expression of TNF receptor type 1 is not always associated with predominant activation of proapoptotic pathways. However, in the case of simultaneous high expression of both types of receptors, the proportion of double-positive cells is crucial for the activation of either the proapoptotic or proliferation pathways.
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Apoptosis , Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Apoptosis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Proteínas Recombinantes/farmacología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
We have previously shown that immunization of C57BL/6 mice, prone to spontaneous development of experimental autoimmune encephalomyelitis (EAE), with three antigens (MOG35-55 , DNA-histone complex or DNA-methylated BSA complex), alters the differentiation profiles of bone marrow haematopoietic stem cells (HSCs). These are associated with the production of autoantibodies (auto-Abs) against these antigens and the formation of abzymes hydrolysing DNA, MOG, myelin basic protein (MBP) and histones. Immunization of mice with antigens accelerates the development of EAE. This work is the first to analyse the ratio of auto-Abs without and with catalytic activities at different stages of EAE development (onset, acute and remission phases) after immunization of mice with the three specific antigens. Prior to immunization and during spontaneous in-time development of EAE, the concentration of auto-Abs against MBP, MOG, histones and DNA and activities of IgG antibodies in the hydrolysis of substrates increased in parallel; correlation coefficients = +0.69-0.94. After immunization with MOG, DNA-histone complex or DNA-met-BSA complex, both positive (from +0.13 to +0.98) and negative correlations (from -0.09 to -0.69) were found between these values. Our study is the first showing that depending on the antigen, the relative amount of harmful auto-Abs without and abzymes with low or high catalytic activities may be produced only at onset and in acute or remission phases of EAE. The antigen governs the EAE development rate, whereby the ratio of auto-Abs without catalytic activity and with enzymatic activities of harmful abzymes hydrolysing MBP, MOG, histones and DNA varies strongly between different disease phases.
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Anticuerpos Catalíticos/inmunología , Antígenos/inmunología , Autoanticuerpos/inmunología , Susceptibilidad a Enfermedades/inmunología , Encefalomielitis Autoinmune Experimental/etiología , Animales , Autoantígenos/inmunología , Diferenciación Celular , Proliferación Celular , ADN/inmunología , Células Madre Hematopoyéticas/metabolismo , Histonas/inmunología , Histonas/metabolismo , Hidrólisis , Inmunización , Inmunoglobulina G/inmunología , RatonesRESUMEN
BACKGROUND: Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. METHODS: IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. RESULTS: We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejection and GVHD but through distinct nonspecific and antigen-specific mechanisms, respectively. Discussion. We provide data that the novel approach for DCs antigen loading using DNA constructs encoding distinct homologous determinants derived from major histocompatibility complex genes is effective in antigen-specific suppression of transplantation reactions. Such an approach eliminates the necessity of donor material use and may be useful in immunosuppressive therapy side effects prevention.
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Células Dendríticas/inmunología , Epítopos/inmunología , Antígenos H-2/inmunología , Tolerancia Inmunológica , Animales , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Epítopos/genética , Femenino , Orden Génico , Rechazo de Injerto/inmunología , Enfermedad Injerto contra Huésped/diagnóstico , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/terapia , Antígenos H-2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Plásmidos/genética , Subgrupos de Linfocitos T , Transfección , Trasplante HomólogoRESUMEN
INTRODUCTION: Density and co-expression of tumor necrosis factor (TNF) receptors may vary among cell populations. However, the role and potential of these changes remain unclear. This study aimed to determine the density of expression and co-expression of TNFR1/2 and the dose-dependent effect of soluble TNF on these parameters. METHODS: Epithelial-like (HEp-2, K-562, MCF-7, ZR-75/1) and lymphoblast-like (MOLT-4, HL-60, Raji, RPMI-8226, IM-9) cell lines were characterized for co-expression of TNFR1/2 using a modified flow cytometry protocol. The dose-dependent effects of rhTNF on TNF receptor expression in these lines were studied. RESULTS: This study reports a protocol for the simultaneous quantitative evaluation of the of TNF receptor number and co-expression of membrane-bound TNFR1/2. Cells within one tumor cell line were found to differ regarding their expression of type 1 and 2 TNFα receptors; simultaneously, cells with all 4 variants of co-expression may be present in culture. CONCLUSION: We demonstrated a dose-dependent effect of TNF on changes in the expression of TNFR1/2 by the percentage of positive cells and by the number of receptors, which may be used to control TNF-mediated processes in target cells.
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Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Línea Celular Tumoral , Células HL-60 , Humanos , Células K562 , Células MCF-7RESUMEN
INTRODUCTION: Dendritic cells (DCs) control immune responses by modulating T and B cells towards effector or tolerogenic responses. In this study, we evaluated the effects of different immunosuppressive molecules on the phenotypic and functional characteristics of primary dendritic cells from C57BL/6 and CBA mice. METHODS: DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4. DCs were then treated with different types of immunosuppressive molecules (rmIL-10, rmTGF-ß, and BAY 11-7082) and cocultured with syngeneic splenocytes. The amount of CD4+CD25hiFoxP3+ Tregs, IL-10 expression, and proliferation were evaluated. RESULTS: Tolerogenic factors were found to have different effects on DCs C57Bl/6 mice. In C57Bl/6 mice, BAY 11-7082 alone had no effect on the expression of DC maturation molecules (CD80, CD86). Transforming growth factor beta (TGF-ß), alone and in combination with BAY 11-7082, reduced the expression of these molecules. Cocultivation of DCs with splenocytes in the presence of TGF-ß and BAY 11-7082 favored regulatory T cell (CD4+CD25hiFoxP3+) differentiation and disfavored differentiation of CD4+ T cells producing IL-10. In CBA mice, we found that rmIL-10 and rmTGF-ß have a weak effect on maturation of DCs and their functional properties to induce Treg cells and IL-10 production. CONCLUSION: These results indicate that TGF-ß and IL-10 have different effects on the phenotypic and functional characteristics of DCs and that the NF-κB inhibitor, BAY 11-7082, has no synergistic effect on these treatments. In mice with an opposite nature of the immune response, the effects of immunoregulatory cytokines (IL-10 and TGF-b) differ on maturation of dendritic cells.
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Células Dendríticas/inmunología , Inmunosupresores/farmacología , Linfocitos T Reguladores/inmunología , Animales , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Sinergismo Farmacológico , Quimioterapia Combinada , Interleucina-10/farmacología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , FN-kappa B/antagonistas & inhibidores , Nitrilos/farmacología , Cultivo Primario de Células , Sulfonas/farmacología , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
Tolerogenic dendritic cells (tolDCs) and T-regulatory cells (Tregs) are involved in maintaining tolerance to self-antigens and foreign antigens. The cells are used as therapeutic tools for inducing tolerance to transplanted organs or tissues. We investigated the possibility of inducing Tregs in splenocyte cultures using DCs transfected with a DNA construct encoding mouse interleukin-10 (DCpIL-10). DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4 and electroporated with a plasmid encoding mouse IL-10. Furthermore, DCpIL-10 was cocultured with syngeneic splenocytes. The CD4+CD25hiFoxP3+ Treg frequency, IL-10 expression, and inhibition of the mixed lymphocyte reaction were evaluated. C57Bl/6 and CBA mice differ in their initial frequency of CD4+CD25hiFoxP3+ Tregs and baseline IL-10 production. Also, the effectiveness of CD4+CD25hiFoxP3+ Treg upregulation by tolDCpIL-10 was different. In this study, DCpIL-10 from C57Bl/6 mice induced CD4+CD25hiFoxP3+ Tregs in syngenic splenocytes, which was accompanied by an increase in the IL-10 production and a decrease in the proliferation of splenocytes in response to the alloantigen. DCpIL-10 may be used to induce CD4+CD25hiFoxP3+ Tregs and the regulatory potential of splenocytes.
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Antígenos CD4/inmunología , Células Dendríticas/inmunología , Factores de Transcripción Forkhead/inmunología , Interleucina-10/inmunología , Subunidad alfa del Receptor de Interleucina-2/inmunología , Linfocitos T Reguladores/inmunología , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBARESUMEN
Minimal residual disease remaining after resection of primary tumors can lead to tumor recurrence and metastasis, increasing mortality and morbidity rates among cancer patients. Thus, there is a need for new technologies for recognition and elimination of single cancer cells remaining in a patient's body after radiation therapy, chemotherapy, or surgical resection. Effector CD8+ T cells, also commonly known as cytotoxic T lymphocytes (CTLs), play a key role in antitumor cellular immunity and, when properly activated, are able to effectively destroy tumor cells. The aims of this study were to obtain CD8+ CTLs specific for the HER2/neu epitopes E75 and E88 and to assess the cytotoxic activity and composition of these cells in terms of the distribution of memory T-cell subsets. We obtained HER2-specific CD8+ T cells and assessed T cell subset distribution among them including naive T cells (TN), central memory T cells (TCM), effector memory T cells (TEM), stem cell-like memory T cells (TSCM) and terminally-differentiated T cells (TEMRA) via eight-color flow cytometry. HER2-specific CTLs were largely (~40-50%) represented by TSCM cells, a population capable of mounting pronounced antitumor immune responses due to a combination of effector function and self-maintenance. In comparison with activated peripheral blood mononuclear cells (PBMCs) and bulk CD8+ T cells, HER2-specific CTLs exhibited greater cytotoxicity against the HER2-expressing human breast adenocarcinoma cell line MCF-7 and produced higher levels of IFN-γ in response to tumor cells. We also showed the presence of HER2-specific CTLs in healthy individuals and increase in them in HER2-positive breast cancer patients. Collectively, our results suggest that HER2-specific CD8+ T cells isolated using this approach could be used for adoptive T-cell transfer to eliminate tumor cells and prevent metastasis and relapse in patients with HER2-overexpressing cancers.
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Neoplasias de la Mama/terapia , Linfocitos T CD8-positivos/inmunología , Linfocitos T Citotóxicos/inmunología , Adulto , Anciano , Neoplasias de la Mama/inmunología , Células Cultivadas , Citotoxicidad Inmunológica , Epítopos/inmunología , Femenino , Antígeno HLA-A2/metabolismo , Humanos , Memoria Inmunológica , Masculino , Persona de Mediana Edad , Receptor ErbB-2/inmunologíaRESUMEN
The immunoregulatory cytokine tumor necrosis factor α plays crucial roles in the pathogenesis of a broad spectrum of disorders. However, its effect may depend on the expression and co-expression of receptors on the target cell. The aim of the present study was to evaluate the expression levels of type 1 and 2 tumor necrosis factor α receptors (TNFR1/2) on individual cell subsets from peripheral blood of healthy volunteers. Flow cytometry analysis was used to study whole populations as well as subsets (T regulatory cells, T memory cells, cytotoxic T cells, T helper cells). Significant differences in the co-expression of TNFR1/2 were seen within subsets and total pools. Further studies are necessary to explore the implications of the observed differences in the modulation of tumor necrosis factor α function in health and pathology.
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Receptores Tipo II del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Subgrupos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adolescente , Adulto , Anciano , Circulación Sanguínea , Separación Celular , Femenino , Citometría de Flujo , Regulación de la Expresión Génica , Voluntarios Sanos , Humanos , Inmunidad Celular , Masculino , Persona de Mediana Edad , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Transcriptoma , Adulto JovenRESUMEN
The expression of cytokine receptors has a crucial role in many cellular processes. Recent studies reported that changes of receptor expression could control the action of mediators on target cells. The initiation of different signaling pathways and, therefore, specific effects on cells, depends on certain components forming the cytokine-receptor complex. These mechanisms control the immune response and affect both the course of diseases (oncological, autoimmune, inflammatory) and the effectiveness of therapy. This review describes the potential of immune mediator receptors to regulate the efficiency of cytokine activity during pathologic processes and ensure the variability of their biological effects. Our aim was to investigate the spectrum of potential roles of changes in mediator receptor expression for main classes of pathologies. For all major types of immune mediators (cytokines, interleukins, chemokines, growth factors, and tumor necrosis factors), it has been shown that changes in their receptor expression are associated with impaired functioning of the organism in chronic diseases.