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Systemic lupus erythematosus (SLE) is an autoimmune disease causing significant morbidity and mortality, despite important improvements in its management in the last decades. The objective of this work is to investigate the role of IFN-γ in the pathogenesis of childhood-onset systemic lupus erythematosus (cSLE), evaluating the crosstalk between IFN-α and IFN-γ and the expression of T-bet, a transcription factor induced by IFN-γ, in B cells of patients with cSLE. Expression levels of both IFN-α and IFN-γ-induced genes were upregulated in patients with cSLE. We found increased serum levels of CXCL9 and CXCL10 in patients with cSLE. Type I IFN score decreased with initiation of immunosuppressive treatment; conversely, type II IFN score and levels of CXCL9 were not significantly affected by immunosuppressive treatment. Type II IFN score and CXCL9 were significantly higher in patients with lupus nephritis. We observed the expansion of a population of naïve B cells expressing T-bet in a cluster of patients with cSLE. IFN-γ, but not IFN-α, induced the expression of T-bet in B cells. Our data suggest that IFN-γ is hyperactive in cSLE, especially in patients with lupus nephritis, and it is not modulated by therapy. Our data reinforce the potential of IFN-γ as a therapeutic target in SLE.
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Interferón Tipo I , Lupus Eritematoso Sistémico , Nefritis Lúpica , Humanos , Interferón gamma/metabolismo , Factores de TranscripciónRESUMEN
BACKGROUND: CD47/SIRPα axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer, particularly in hematological malignancies. In preclinical models, CD47/SIRPα blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here, we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS: The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry, transcriptomic analysis, and in vivo immune-cell depletion experiments. RESULTS: We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells, monocytes, dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably, molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation, including IFNγ and IL-12b. Moreover, TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701, highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition, NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro, resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS: The study described the mechanisms afforded by the CD47xCD19 bispecific antibody, NI-1701, at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035).
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The aberrant release of inflammatory mediators often referred to as a cytokine storm or cytokine release syndrome (CRS), is a common and sometimes fatal complication in acute infectious diseases including Ebola, dengue, COVID-19, and influenza. Fatal CRS occurrences have also plagued the development of highly promising cancer therapies based on T-cell engagers and chimeric antigen receptor (CAR) T cells. CRS is intimately linked with dysregulated and excessive cytokine release, including IFN-γ, TNF-α, IL 1, IL-6, and IL-10, resulting in a systemic inflammatory response leading to multiple organ failure. Here, we show that mice intravenously administered the agonistic hamster anti-mouse CD3ε monoclonal antibody 145-2C11 develop clinical and laboratory manifestations seen in patients afflicted with CRS, including body weight loss, hepatosplenomegaly, thrombocytopenia, increased vascular permeability, lung inflammation, and hypercytokinemia. Blood cytokine levels and gene expression analysis from lung, liver, and spleen demonstrated a hierarchy of inflammatory cytokine production and infiltrating immune cells with differentiating organ-dependent kinetics. IL-2, IFN-γ, TNF-α, and IL-6 up-regulation preceded clinical signs of CRS. The co-treatment of mice with a neutralizing anti-cytokine antibody cocktail transiently improved early clinical and laboratory features of CRS. We discuss the predictive use of this model in the context of new anti-cytokine strategies to treat human CRS.
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Anticuerpos Monoclonales/farmacología , Anticuerpos/inmunología , Complejo CD3/antagonistas & inhibidores , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/metabolismo , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Animales , Anticuerpos/efectos adversos , Anticuerpos Monoclonales/uso terapéutico , Síndrome de Liberación de Citoquinas/diagnóstico , Síndrome de Liberación de Citoquinas/tratamiento farmacológico , Citocinas/sangre , Modelos Animales de Enfermedad , Quimioterapia Combinada , Mediadores de Inflamación/sangre , Mediadores de Inflamación/metabolismo , Activación de Linfocitos/inmunología , Ratones , Fenotipo , Índice de Severidad de la Enfermedad , Linfocitos T/inmunología , Linfocitos T/metabolismo , Resultado del TratamientoAsunto(s)
Anticuerpos Biespecíficos/inmunología , Antígeno B7-H1/inmunología , Antígeno CD47/inmunología , Neoplasias/inmunología , Animales , Anticuerpos Biespecíficos/farmacología , Antígeno B7-H1/antagonistas & inhibidores , Antígeno B7-H1/metabolismo , Antígeno CD47/antagonistas & inhibidores , Antígeno CD47/metabolismo , Humanos , Inmunoterapia/métodos , Terapia Molecular Dirigida/métodos , Neoplasias/metabolismo , Neoplasias/terapiaRESUMEN
Oncogenic KRAS mutations are major drivers of lung adenocarcinoma (LAC), yet the direct therapeutic targeting of KRAS has been problematic. Here, we reveal an obligate requirement by oncogenic KRAS for the ADAM17 protease in LAC In genetically engineered and xenograft (human cell line and patient-derived) KrasG12D-driven LAC models, the specific blockade of ADAM17, including with a non-toxic prodomain inhibitor, suppressed tumor burden by reducing cellular proliferation. The pro-tumorigenic activity of ADAM17 was dependent upon its threonine phosphorylation by p38 MAPK, along with the preferential shedding of the ADAM17 substrate, IL-6R, to release soluble IL-6R that drives IL-6 trans-signaling via the ERK1/2 MAPK pathway. The requirement for ADAM17 in KrasG12D-driven LAC was independent of bone marrow-derived immune cells. Furthermore, in KRAS mutant human LAC, there was a significant positive correlation between augmented phospho-ADAM17 levels, observed primarily in epithelial rather than immune cells, and activation of ERK and p38 MAPK pathways. Collectively, these findings identify ADAM17 as a druggable target for oncogenic KRAS-driven LAC and provide the rationale to employ ADAM17-based therapeutic strategies for targeting KRAS mutant cancers.
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Proteína ADAM17/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Receptores de Interleucina-6/metabolismo , Proteína ADAM17/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Genotipo , Humanos , Neoplasias Pulmonares/patología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación , Fosforilación , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Pathophysiology of graft failure (GF) occurring after allogeneic hematopoietic stem cell transplantation (HSCT) still remains elusive. We measured serum levels of several different cytokines/chemokines in 15 children experiencing GF, comparing their values with those of 15 controls who had sustained donor cell engraftment. Already at day +3 after transplantation, patients developing GF had serum levels of interferon (IFN)-γ and CXCL9 (a chemokine specifically induced by IFNγ) significantly higher than those of controls (8859±7502 vs. 0 pg/mL, P=0.03, and 1514.0±773 vs. 233.6±50.1 pg/mlL, P=0.0006, respectively). The role played by IFNγ in HSCT-related GF was further supported by the observation that a rat anti-mouse IFNγ-neutralizing monoclonal antibody promotes donor cell engraftment in Ifngr1-/-mice receiving an allograft. In comparison to controls, analysis of bone marrow-infiltrating T lymphocytes in patients experiencing GF documented a predominance of effector memory CD8+ cells, which showed markers of activation (overexpression of CD95 and downregulation of CD127) and exhaustion (CD57, CD279, CD223 and CD366). Finally, we obtained successful donor engraftment in 2 out of 3 children with primary hemophagocytic lymphohistiocytosis who, after experiencing GF, were re-transplanted from the same HLA-haploidentical donor under the compassionate use coverage of emapalumab, an anti-IFNγ monoclonal antibody recently approved by the US Food and Drug Administration for treatment of patients with primary hemophagocytic lymphohistiocytosis. Altogether, these results suggest that the IFNγ pathway plays a major role in GF occurring after HSCT. Increased serum levels of IFNγ and CXCL9 represent potential biomarkers useful for early diagnosis of GF and provide the rationale for exploring the therapeutic/preventive role of targeted neutralization of IFNγ.
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Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Interferón gamma/metabolismo , Adolescente , Animales , Biopsia , Médula Ósea/metabolismo , Médula Ósea/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Niño , Preescolar , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Rechazo de Injerto/diagnóstico , Rechazo de Injerto/etiología , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/metabolismo , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Inmunohistoquímica , Memoria Inmunológica , Lactante , Masculino , Ratones , Ratones Noqueados , Donantes de Tejidos , Trasplante Homólogo , Adulto JovenRESUMEN
CD19 is a B cell-specific receptor that regulates the threshold of B cell receptor (BCR)-mediated cell proliferation. A CD47xCD19 bispecific antibody (biAb) was generated to target and deplete B cells via multiple antibody-mediated mechanisms. Interestingly, the biAb, constructed of a CD19 binding arm and a CD47 binding arm, inhibited BCR-mediated B-cell proliferation with an effect even more potent than a CD19 monoclonal antibody (mAb). The inhibitory effect of the biAb was not attributable to CD47 binding because a monovalent or bivalent anti-CD47 mAb had no effect on B cell proliferation. Fluorescence resonance energy transfer analysis demonstrated that co-engaging CD19 and CD47 prevented CD19 clustering and its migration to BCR clusters, while only engaging CD19 (with a mAb) showed no impact on either CD19 clustering or migration. The lack of association between CD19 and the BCR resulted in decreased phosphorylation of CD19 upon BCR activation. Furthermore, the biAb differentially modulated BCR-induced gene expression compared to a CD19 mAb. Taken together, this unexpected role of CD47xCD19 co-ligation in inhibiting B cell proliferation illuminates a novel approach in which two B cell surface molecules can be tethered, to one another in order, which may provide a therapeutic benefit in settings of autoimmunity and B cell malignancies.
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Anticuerpos Biespecíficos/farmacología , Antígenos CD19/metabolismo , Linfocitos B/metabolismo , Antígeno CD47/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Activación de Linfocitos/efectos de los fármacosRESUMEN
NI-0801 is a fully human monoclonal antibody against chemokine (C-X-C motif) ligand 10 (CXCL10), which is involved in the recruitment of inflammatory T cells into the liver. The safety and efficacy of NI-0801 was assessed in patients with primary biliary cholangitis. In this open-label phase 2a study, patients with primary biliary cholangitis with an inadequate response to ursodeoxycholic acid received six consecutive intravenous administrations of NI-0801 (10 mg/kg) every 2 weeks. Patients were followed up for 3 months after the last infusion. Liver function tests, safety assessments, as well as pharmacokinetic and pharmacodynamic parameters were evaluated at different time points throughout the dosing period and the safety follow-up period. Twenty-nine patients were enrolled in the study and were treated with NI-0801. The most frequently reported adverse events included headaches (52%), pruritus (34%), fatigue (24%), and diarrhea (21%). No study drug-related serious adverse events were reported. NI-0801 administration did not lead to a significant reduction in any of the liver function tests assessed at the end of the treatment period (i.e., 2 weeks after final NI-0801 administration) compared to baseline. Conclusion: Despite clear pharmacologic responses in the blood, no therapeutic benefit of multiple administrations of NI-0801 could be demonstrated. The high production rate of CXCL10 makes it difficult to achieve drug levels that lead to sustained neutralization of the chemokine, thus limiting its targetability. (Hepatology Communications 2018;2:492-503).
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CD47, an ubiquitously expressed innate immune checkpoint receptor that serves as a universal "don't eat me" signal of phagocytosis, is often upregulated by hematologic and solid cancers to evade immune surveillance. Development of CD47-targeted modalities is hindered by the ubiquitous expression of the target, often leading to rapid drug elimination and hemotoxicity including anemia. To overcome such liabilities, we have developed a fully human bispecific antibody, NI-1701, designed to coengage CD47 and CD19 selectively on B cells. NI-1701 demonstrates favorable elimination kinetics with no deleterious effects seen on hematologic parameters following single or multiple administrations to nonhuman primates. Potent in vitro and in vivo activity is induced by NI-1701 to kill cancer cells across a plethora of B-cell malignancies and control tumor growth in xenograft mouse models. The mechanism affording maximal tumor growth inhibition by NI-1701 is dependent on the coengagement of CD47/CD19 on B cells inducing potent antibody-dependent cellular phagocytosis of the targeted cells. NI-1701-induced control of tumor growth in immunodeficient NOD/SCID mice was more effective than that achieved with the anti-CD20 targeted antibody, rituximab. Interestingly, a synergistic effect was seen when tumor-implanted mice were coadministered NI-1701 and rituximab leading to significantly improved tumor growth inhibition and regression in some animals. We describe herein, a novel bispecific antibody approach aimed at sensitizing B cells to become more readily phagocytosed and eliminated thus offering an alternative or adjunct therapeutic option to patients with B-cell malignancies refractory/resistant to anti-CD20-targeted therapy. Mol Cancer Ther; 17(8); 1739-51. ©2018 AACR.
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Anticuerpos Biespecíficos/genética , Leucemia/genética , Leucemia/terapia , Linfoma de Células B/genética , Linfoma de Células B/terapia , Animales , Antígenos CD19 , Antígeno CD47 , Humanos , Leucemia/patología , Linfoma de Células B/patología , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Bone loss associated with estrogen deficiency occurs due to a high level of bone remodelling, with a greater increase in the level of osteoclast-mediated bone resorption than osteoblast-mediated bone formation. Early studies showed that Interleukin-6 (IL-6) inhibition could prevent the increase in osteoclast numbers associated with ovariectomy. However, IL-6 signals through two possible pathways: classic IL-6 signalling (cis) utilizes a membrane-bound IL-6 receptor (IL-6R), while IL-6 trans-signalling occurs through a soluble IL-6R (sIL-6R). It is not known which of these pathways mediates the bone loss after ovariectomy. We therefore sought to determine whether specific pharmacological inhibition of IL-6 trans-signalling could prevent ovariectomy-induced bone loss in mice. We report that IL-6 trans-signalling inhibition prevented the increase in osteoclasts, and trabecular bone loss, associated with ovariectomy. IL-6 trans-signalling inhibition also reduced bone formation rate, but did not prevent the increase in osteoblast numbers. In contrast, cortical bone loss was not prevented by any IL-6 signalling inhibitor. This suggests that local production of sIL-6R mediates trabecular bone loss in estrogen deficiency, but the increased cortical bone resorption that leads to marrow expansion is independent of IL-6 signalling.
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Resorción Ósea/patología , Hueso Esponjoso/patología , Hueso Cortical/patología , Interleucina-6/metabolismo , Ovariectomía , Transducción de Señal , Animales , Biomarcadores/sangre , Resorción Ósea/sangre , Resorción Ósea/diagnóstico por imagen , Resorción Ósea/metabolismo , Hueso Esponjoso/diagnóstico por imagen , Hueso Esponjoso/metabolismo , Hueso Cortical/diagnóstico por imagen , Hueso Cortical/metabolismo , Femenino , Interleucina-6/sangre , Masculino , Ratones Endogámicos C57BL , Microtomografía por Rayos XRESUMEN
BACKGROUND: The pathogenesis of macrophage activation syndrome (MAS) is not clearly understood: a large body of evidence supports the involvement of mechanisms similar to those implicated in the setting of primary hemophagocytic lymphohistiocytosis. OBJECTIVE: We sought to investigate the pathogenic role of IFN-γ and the therapeutic efficacy of IFN-γ neutralization in an animal model of MAS. METHODS: We used an MAS model established in mice transgenic for human IL-6 (IL-6TG mice) challenged with LPS (MAS mice). Levels of IFN-γ and IFN-γ-inducible chemokines were evaluated by using real-time PCR in the liver and spleen and by means of ELISA in plasma. IFN-γ neutralization was achieved by using the anti-IFN-γ antibody XMG1.2 in vivo. RESULTS: Mice with MAS showed a significant upregulation of the IFN-γ pathway, as demonstrated by increased mRNA levels of Ifng and higher levels of phospho-signal transducer and activator of transcription 1 in the liver and spleen and increased expression of the IFN-γ-inducible chemokines Cxcl9 and Cxcl10 in the liver and spleen, as well as in plasma. A marked increase in Il12a and Il12b expression was also found in livers and spleens of mice with MAS. In addition, mice with MAS had a significant increase in numbers of liver CD68+ macrophages. Mice with MAS treated with an anti-IFN-γ antibody showed a significant improvement in survival and body weight recovery associated with a significant amelioration of ferritin, fibrinogen, and alanine aminotransferase levels. In mice with MAS, treatment with the anti-IFN-γ antibody significantly decreased circulating levels of CXCL9, CXCL10, and downstream proinflammatory cytokines. The decrease in CXCL9 and CXCL10 levels paralleled the decrease in serum levels of proinflammatory cytokines and ferritin. CONCLUSION: These results provide evidence for a pathogenic role of IFN-γ in the setting of MAS.
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Anticuerpos Neutralizantes/inmunología , Interferón gamma/inmunología , Síndrome de Activación Macrofágica/inmunología , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Alanina Transaminasa/inmunología , Animales , Quimiocina CXCL10/inmunología , Quimiocina CXCL9/inmunología , Citocinas/inmunología , Modelos Animales de Enfermedad , Ferritinas/inmunología , Fibrinógeno/inmunología , Inflamación/inmunología , Linfohistiocitosis Hemofagocítica/inmunología , RatonesRESUMEN
CD47 serves as an anti-phagocytic receptor that is upregulated by cancer to promote immune escape. As such, CD47 is the focus of intense immuno-oncology drug development efforts. However, as CD47 is expressed ubiquitously, clinical development of conventional drugs, e.g., monoclonal antibodies, is confronted with patient safety issues and poor pharmacology due to the widespread CD47 "antigen sink". A potential solution is tumor-directed blockade of CD47, which can be achieved with bispecific antibodies (biAbs). Using mouse CD47-blocking biAbs in a syngeneic tumor model allowed us to evaluate the efficacy of tumor-directed blockade of CD47 in the presence of the CD47 antigen sink and a functional adaptive immune system. We show here that CD47-targeting biAbs inhibited tumor growth in vivo, promoting durable antitumor responses and stimulating CD8+ T cell activation in vitro. In vivo efficacy of the biAbs could be further enhanced when combined with chemotherapy or PD-1/PD-L1 immune checkpoint blockade. We also show that selectivity and pharmacological properties of the biAb are dependent on the affinity of the anti-CD47 arm. Taken together, our study validates the approach to use CD47-blocking biAbs either as a monotherapy or part of a multi-drug approach to enhance antitumor immunity.
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Epstein-Barr virus-induced gene 3 (EBI3) is a subunit of the composite cytokines IL-27 and IL-35. Both have beneficial functions or effects in models of infectious and autoimmune diseases. This suggests that administration of EBI3 could be therapeutically useful by binding free p28 and p35 to generate IL-27 and IL-35. IL-27- and IL-35-independent functions of EBI3 could compromise its therapeutic uses. We therefore assessed the effects of EBI3 on cytokine receptor-expressing cells. We observed that EBI3 activates STAT3 and induces the proliferation of the IL-6-dependent B9 mouse plasmacytoma cell line. Analyses using blocking mAbs and Ba/F3 transfectants expressing gp130 indicate that EBI3 activity was linked to its capacity to mediate IL-6 trans-signaling, albeit less efficiently than soluble IL-6Rα. In line with this interpretation, co-immunoprecipitation and SPR experiments indicated that EBI3 binds IL-6. An important pro-inflammatory function of IL-6 trans-signaling is to activate blood vessel endothelial cells. We observed that EBI3 in combination with IL-6 could induce the expression of chemokines by human venal endothelial cells. Our results indicate that EBI3 can promote pro-inflammatory IL-6 functions by mediating trans-signaling. These unexpected observations suggest that use of EBI3 as a therapeutic biologic for autoimmune diseases will likely require co-administration of soluble gp130 to prevent the side effects associated with IL-6 trans-signaling. Together with previous studies that demonstrated activation of IL-6R by p28 (IL-30), new findings further suggest a complex interrelation between IL-27 and IL-6.
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Interleucina-6/metabolismo , Antígenos de Histocompatibilidad Menor/metabolismo , Receptores de Citocinas/metabolismo , Transducción de Señal , Animales , Anticuerpos Monoclonales/química , Línea Celular Tumoral , Proliferación Celular , Quimiocinas/metabolismo , Receptor gp130 de Citocinas/metabolismo , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inflamación , Interleucinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosforilación , Plasmacitoma/metabolismo , Unión Proteica , Receptores de Interleucina-6/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The antiviral restriction factor IFN-induced transmembrane protein 3 (IFITM3) inhibits cell entry of a number of viruses, and genetic diversity within IFITM3 determines susceptibility to viral disease in humans. Here, we used the murine CMV (MCMV) model of infection to determine that IFITM3 limits herpesvirus-associated pathogenesis without directly preventing virus replication. Instead, IFITM3 promoted antiviral cellular immunity through the restriction of virus-induced lymphopenia, apoptosis-independent NK cell death, and loss of T cells. Viral disease in Ifitm3-/- mice was accompanied by elevated production of cytokines, most notably IL-6. IFITM3 inhibited IL-6 production by myeloid cells in response to replicating and nonreplicating virus as well as following stimulation with the TLR ligands Poly(I:C) and CpG. Although IL-6 promoted virus-specific T cell responses, uncontrolled IL-6 expression in Ifitm3-/- mice triggered the loss of NK cells and subsequently impaired control of MCMV replication. Thus, IFITM3 represents a checkpoint regulator of antiviral immunity that controls cytokine production to restrict viral pathogenesis. These data suggest the utility of cytokine-targeting strategies in the treatment of virus-infected individuals with impaired IFITM3 activity.
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Citocinas/fisiología , Infecciones por Herpesviridae/metabolismo , Proteínas de la Membrana/fisiología , Animales , Células Cultivadas , Infecciones por Herpesviridae/inmunología , Inmunidad Celular , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Muromegalovirus/fisiología , Receptores de Interleucina-6/metabolismo , Transducción de Señal , Internalización del Virus , Replicación ViralRESUMEN
CD47 is a ubiquitously expressed immune checkpoint receptor that is often upregulated in cancer. CD47 interacts with its counter-receptor SIRPα on macrophages and other myeloid cells to inhibit cancer cell phagocytosis and drive immune evasion. To overcome tolerability and "antigen sink" issues arising from widespread CD47 expression, we generated dual-targeting bispecific antibodies that selectively block the CD47-SIRPα interaction on malignant cells expressing a specific tumor-associated antigen; e.g., CD19 or mesothelin. These bispecific κλ bodies are fully human, native IgG1 molecules, combining tumor targeting and selective CD47 blockade with immune activating mechanisms mediated by the Fc portion of the antibody. CD47-neutralizing κλ bodies efficiently kill cancer cells in vitro and in vivo but interact only weakly with healthy cells expressing physiological levels of CD47. Accordingly, a κλ body administered to non-human primates showed a typical IgG pharmacokinetic profile and was well tolerated. Importantly, κλ bodies preserve their tumoricidal capabilities in the presence of a CD47 antigen sink. Thus, dual-targeting κλ bodies allow for efficacious yet safe targeting of CD47 in cancer. Such a bispecific design could be applied to limit the extent of neutralization of other ubiquitously expressed therapeutic targets.
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Anticuerpos Biespecíficos/farmacología , Antígeno CD47/metabolismo , Animales , Anticuerpos Biespecíficos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antígenos de Neoplasias/inmunología , Antineoplásicos/farmacología , Antígeno CD47/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Mesotelina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Neoplasias/metabolismo , Fagocitosis/inmunología , Unión Proteica/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
To improve our understanding of properties that confer successful inhibition of chemokines in vivo, we analyzed anti-murine CXCL10 monoclonal antibodies (mAb) having different characteristics. 1B6 displayed potent inhibition of cell recruitment in vitro with an IC50 of 0.5 nm but demonstrated little efficacy in various animal models of human disease. On the contrary, 1F11 showed efficacy in several models of inflammation yet was less potent at inhibiting chemotaxis in vitro with an IC50 of 21 nm Furthermore, we observed that 1B6 displayed a rapid dose-dependent clearance (t½ 10-60 h) in contrast to 1F11, which presented a dose-proportional pharmacokinetic profile and a half-life of 12 days. Moreover, 1B6 recognized glycosaminoglycan (GAG)-bound CXCL10, resulting in target-mediated clearance, which was corroborated using CXCL10-deficient mice. In contrast to 1B6, 1F11 inhibited the interaction of CXCL10 with GAGs, did not recognize GAG-bound CXCL10, and did not display target-mediated drug disposition. Confirming previous animal studies, 1B6 was poor at reversing glycemia in a model of type 1 diabetes, whereas 1F11 induced early and prolonged control of diabetes. Furthermore, when using 1A4, a subsequently generated anti-mCXCL10 mAb that shares the property with 1F11 of being unable to recognize CXCL10 immobilized on GAG, we observed a similar superior control of diabetes as compared with 1B6. We therefore concluded that targeting chemokines with antibodies such as 1B6 that recognize the more abundant GAG-bound form of the chemokine may not be the optimal strategy to achieve disease control.
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Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Neutralizantes/administración & dosificación , Quimiocina CXCL10/antagonistas & inhibidores , Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Tipo 1/prevención & control , Endotelio Vascular/metabolismo , Glicosaminoglicanos/metabolismo , Animales , Anticuerpos Monoclonales/farmacocinética , Células Cultivadas , Quimiocina CXCL10/inmunología , Quimiocina CXCL10/metabolismo , Quimiotaxis de Leucocito/fisiología , Cricetinae , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/patología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/inmunología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Distribución TisularRESUMEN
OBJECTIVES: Interferon-γ (IFNγ) is the pivotal mediator in murine models of primary haemophagocytic lymphohistiocytosis (pHLH). Given the similarities between primary and secondary HLH (sec-HLH), including macrophage activation syndrome (MAS), we investigate the involvement of the IFNγ pathway in MAS by evaluating levels of IFNγ and of the induced chemokines, and their relation with laboratory parameters of MAS in systemic juvenile idiopathic arthritis (sJIA) patients with MAS and in a murine MAS model. METHODS: The Luminex multiplexing assay was used to assess serum levels of interleukin (IL)-1ß, IL-6, IFNγ and of the IFNγ-induced chemokines CXCL9, CXCL10 and CXCL11 in patients with sec-HLH (n=11) and in patients with sJIA (n=54), of whom 20 had active MAS at sampling. Expression of IFNγ-induced chemokines was assessed in IL-6 transgenic mice in which MAS is induced by TLR4 stimulation with lipopolysaccharide. RESULTS: Levels of IFNγ and of IFNγ-induced chemokines were markedly elevated during active MAS and sec-HLH and were significantly higher in patients with MAS compared with active sJIA without MAS. Levels in patients with active sJIA without MAS were comparable to those of patients with clinically inactive sJIA. During MAS, ferritin and alanine transferase levels and neutrophil and platelet counts were significantly correlated with serum levels of IFNγ and CXCL9. In murine MAS, serum levels of ferritin were significantly correlated with mRNA levels of Cxcl9 in liver and spleen. CONCLUSIONS: The high levels of IFNγ and of IFNγ-induced chemokines and their correlation with the severity of laboratory abnormalities of MAS suggest a pivotal role of IFNγ in MAS.
Asunto(s)
Artritis Juvenil/complicaciones , Quimiocinas/sangre , Interferón gamma/sangre , Síndrome de Activación Macrofágica/etiología , Adolescente , Animales , Artritis Juvenil/inmunología , Biomarcadores/sangre , Niño , Preescolar , Modelos Animales de Enfermedad , Femenino , Humanos , Interferón gamma/inmunología , Interleucina-1beta/sangre , Interleucina-6/sangre , Síndrome de Activación Macrofágica/inmunología , Masculino , Ratones Transgénicos , Índice de Severidad de la EnfermedadRESUMEN
Life-threatening cytokine release syndromes include primary (p) and secondary (s) forms of hemophagocytic lymphohistiocytosis (HLH). Below detection in healthy individuals, interferon γ (IFNγ) levels are elevated to measurable concentrations in these afflictions suggesting a central role for this cytokine in the development and maintenance of HLH. Mimicking an infection-driven model of sHLH in mice, we observed that the tissue-derived levels of IFNγ are actually 500- to 2000-fold higher than those measured in the blood. To identify a blood biomarker, we postulated that the IFNγ gene products, CXCL9 and CXCL10 would correlate with disease parameters in the mouse model. To translate this into a disease relevant biomarker, we investigated whether CXCL9 and CXCL10 levels correlated with disease activity in pediatric sHLH patients. Our data demonstrate that disease control in mice correlates with neutralization of IFNγ activity in tissues and that the 2 chemokines serve as serum biomarkers to reflect disease status. Importantly, CXCL9 and CXCL10 levels in pediatric sHLH were shown to correlate with key disease parameters and severity in these patients. Thus, the translatability of the IFNγ-biomarker correlates from mouse to human, advocating the use of serum CXCL9 or CXCL10 as a means to monitor total IFNγ activity in patients with sHLH.
Asunto(s)
Interferón gamma/sangre , Linfohistiocitosis Hemofagocítica/sangre , Animales , Biomarcadores/sangre , Quimiocina CXCL10/sangre , Quimiocina CXCL9/sangre , Niño , Modelos Animales de Enfermedad , Femenino , Humanos , Linfohistiocitosis Hemofagocítica/patología , Ratones Endogámicos C57BL , Pruebas de Neutralización , Oligodesoxirribonucleótidos/administración & dosificación , Oligodesoxirribonucleótidos/farmacología , SíndromeRESUMEN
BACKGROUND: Increased expression of toll-like receptor 4 (TLR4) and its endogenous ligands, is characteristic of rheumatoid arthritis (RA) synovitis. In this study, we evaluated how these TLR4 ligands may drive pathogenic processes and whether the fine profiling of anti-citrullinated protein antibodies (ACPA) based on their target specificity might provide a simple means to predict therapeutic benefit when neutralizing TLR4 in this disease. METHODS: The capacity of RA synovial fluids (RASF) to stimulate cytokine production in monocytes from patients with RA was analyzed by ELISA. The presence of TLR4 activators in RASF was determined by measuring the levels of ACPA, ACPA subtypes with reactivity to specific citrullinated peptides and other TLR4 ligands. Neutralization of TLR4 signaling was investigated using NI-0101, a therapeutic antibody that targets TLR4. RESULTS: RASF exhibited a heterogeneous capacity to induce production of proinflammatory cytokines by monocytes isolated from patients with RA. Such cytokine responses were significantly modified by TLR4 blockade achieved using NI-0101. The analysis of the content of RASF and matched sera demonstrated that ACPA fine specificities in patient samples predict cellular response to anti-TLR4 exposure in vitro. CONCLUSION: TLR4 represents a possible therapeutic target in RA. Our study demonstrates that TLR4 inhibition in an ex vivo model of RA pathogenesis can significantly modulate cytokine release and does so in specific subgroups of RA patient-derived samples. It also suggests that ACPA fine profiling has the potential to identify RA patients with a predominantly TLR4-driven pathotype that could be used to predict preferential response to TLR4 antagonism.
Asunto(s)
Artritis Reumatoide/inmunología , Autoanticuerpos/inmunología , Líquido Sinovial/inmunología , Receptor Toll-Like 4/inmunología , Anciano , Anticuerpos Monoclonales Humanizados/farmacología , Autoantígenos/inmunología , Citocinas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptidos Cíclicos/inmunología , Receptor Toll-Like 4/antagonistas & inhibidoresRESUMEN
Increased expression of endogenous Toll-like receptor 4 (TLR4) ligands (e.g., Tenascin-C, S100A8/A9, citrullinated fibrinogen (cFb) immune complexes) has been observed in patients with rheumatoid arthritis (RA). However, their roles in RA pathogenesis are not well understood. Here, we investigated the expression kinetics and role of endogenous TLR4 ligands in the murine model of collagen-induced arthritis (CIA). Tenascin-C was upregulated in blood early in CIA, and correlated positively with the clinical score at day 56. Levels of S100A8/A9 increased starting from day 28, peaking at day 42, and correlated positively with joint inflammation. Levels of anti-cFb antibodies increased during the late phase of CIA and correlated positively with both joint inflammation and cartilage damage. Blockade of TLR4 activation at the time of the first TLR4 ligand upregulation prevented clinical and histological signs of arthritis. A TLR4-dependent role was also observed for Tenascin-C and cFb immune complexes in osteoclast differentiation in vitro. Taken together, our data suggests that the pathogenic contribution of TLR4 in promoting joint inflammation and bone erosion during CIA occurs via various TLR4 ligands arising at different stages of disease. The data also suggests that Blockade of TLR4 with monoclonal antibodies is a promising strategy in RA treatment.