RESUMEN
Understanding of how persistent viral infection impacts humoral immunity is incomplete. In this issue of Immunity, Wieland et al. (2015) and Yamada et al. (2015) find that high amounts of IgG-antigen complexes formed during chronic lymphocytic choriomeningitis infection can interfere with Fcγ-receptor-mediated effector activities, potentially contributing to immune dysfunction.
Asunto(s)
Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Evasión Inmune/inmunología , Depleción Linfocítica , Coriomeningitis Linfocítica/inmunología , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/inmunología , AnimalesRESUMEN
Understanding how viruses subvert host immunity and persist is essential for developing strategies to eliminate infection. T cell exhaustion during chronic viral infection is well described, but effects on antibody-mediated effector activity are unclear. Herein, we show that increased amounts of immune complexes generated in mice persistently infected with lymphocytic choriomeningitis virus (LCMV) suppressed multiple Fcγ-receptor (FcγR) functions. The high amounts of immune complexes suppressed antibody-mediated cell depletion, therapeutic antibody-killing of LCMV infected cells and human CD20-expressing tumors, as well as reduced immune complex-mediated cross-presentation to T cells. Suppression of FcγR activity was not due to inhibitory FcγRs or high concentrations of free antibody, and proper FcγR functions were restored when persistently infected mice specifically lacked immune complexes. Thus, we identify a mechanism of immunosuppression during viral persistence with implications for understanding effective antibody activity aimed at pathogen control.
Asunto(s)
Anticuerpos Antivirales/inmunología , Complejo Antígeno-Anticuerpo/inmunología , Evasión Inmune/inmunología , Coriomeningitis Linfocítica/inmunología , Receptores de IgG/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales de Origen Murino/farmacología , Antígenos CD20/biosíntesis , Antígenos CD20/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Tolerancia Inmunológica/inmunología , Factores Inmunológicos/farmacología , Activación de Linfocitos/inmunología , Depleción Linfocítica , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/inmunología , Receptores de IgG/inmunología , RituximabRESUMEN
[Figure: see text].
Asunto(s)
Anticuerpos/farmacología , Antihipertensivos/farmacología , Presión Sanguínea/efectos de los fármacos , Hipertensión/prevención & control , Inmunoglobulina G/sangre , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/efectos de los fármacos , Receptores de IgG/antagonistas & inhibidores , Animales , Estudios de Casos y Controles , Modelos Animales de Enfermedad , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Células HEK293 , Humanos , Hipertensión/genética , Hipertensión/metabolismo , Hipertensión/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Músculo Liso Vascular/fisiopatología , Miocitos del Músculo Liso/metabolismo , Ratas , Receptor de Angiotensina Tipo 1/metabolismo , Receptores de IgG/genética , Receptores de IgG/metabolismo , Transducción de Señal , Remodelación Vascular/efectos de los fármacosRESUMEN
In relapsed/refractory Hodgkin lymphoma (R/R HL), immunotherapies such as the anti-programmed death-1 inhibitor pembrolizumab have demonstrated efficacy as monotherapy and are playing an increasingly prominent role in treatment. The CD30/CD16A-bispecific antibody AFM13 is an innate immune cell engager, a first-in-class, tetravalent antibody, designed to create a bridge between CD30 on HL cells and the CD16A receptor on natural killer cells and macrophages, to induce tumor cell killing. Early studies of AFM13 have demonstrated signs of efficacy as monotherapy for patients with R/R HL and the combination of AFM13 with pembrolizumab represents a rational new treatment modality. Here, we describe a phase 1b, dose-escalation study to assess the safety and preliminary efficacy of AFM13 in combination with pembrolizumab in patients with R/R HL. The primary objective was estimating the maximum tolerated dose; the secondary objectives were to assess safety, tolerability, antitumor efficacy, pharmacokinetics, and pharmacodynamics. In this heavily pretreated patient population, treatment with the combination of AFM13 and pembrolizumab was generally well tolerated, with similar safety profiles compared to the known profiles of each agent alone. The combination of AFM13 with pembrolizumab demonstrated an objective response rate of 88% at the highest treatment dose, with an 83% overall response rate for the overall population. Pharmacokinetic assessment of AFM13 in the combination setting revealed a half-life of up to 20.6 hours. This proof-of-concept study holds promise as a novel immunotherapy combination worthy of further investigation. This phase 1b study was registered at www.clinicaltrials.gov as NCT02665650.
Asunto(s)
Antígenos de Neoplasias/inmunología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Enfermedad de Hodgkin/tratamiento farmacológico , Inmunoterapia , Antígeno Ki-1/antagonistas & inhibidores , Receptores de IgG/antagonistas & inhibidores , Adolescente , Adulto , Anciano , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/efectos adversos , Anticuerpos Biespecíficos/farmacocinética , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Terapia Combinada , Relación Dosis-Respuesta Inmunológica , Femenino , Semivida , Trasplante de Células Madre Hematopoyéticas , Enfermedad de Hodgkin/terapia , Humanos , Inmunidad Innata/efectos de los fármacos , Antígeno Ki-1/inmunología , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Prueba de Estudio Conceptual , Receptores de IgG/inmunología , Recurrencia , Trasplante Autólogo , Adulto JovenRESUMEN
Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (KD: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (KD: â¼6 µM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb-sdAb and sdAb-scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.
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Anticuerpos Biespecíficos/metabolismo , Células Asesinas Naturales/trasplante , Neoplasias/terapia , Proteínas Recombinantes de Fusión/metabolismo , Anticuerpos de Dominio Único/metabolismo , Animales , Anticuerpos Biespecíficos/genética , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos de Neoplasias/metabolismo , Bioensayo , Camélidos del Nuevo Mundo , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/genética , Humanos , Inmunoterapia/métodos , Células Jurkat , Células Asesinas Naturales/metabolismo , Macaca fascicularis , Ratones , Neoplasias/inmunología , Cultivo Primario de Células , Dominios Proteicos/genética , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/genética , Proteínas Recombinantes de Fusión/genética , Anticuerpos de Dominio Único/genéticaRESUMEN
Bispecific antibodies (bsAbs) have emerged as promising therapeutics. A bispecific diabody (bsDb) is a small bsAb consisting of two distinct chimeric single-chain components, with two possible arrangements of the domains. We previously reported the effect of domain order on the function of a humanized bsDb targeting the epidermal growth factor receptor (EGFR) on cancer cells, and CD3 on T cells. Notably, the co-localization of a T-cell receptor (TCR) with CD3 is bulky, potentially affecting the cross-linking ability of bsDbs, due to steric hindrance. Here, we constructed and evaluated humanized bsDbs, with different domain orders, targeting EGFR and CD16 on natural killer (NK) cells (hEx16-Dbs). We predicted minimal effects due to steric hindrance, as CD16 lacks accessory molecules. Interestingly, one domain arrangement displayed superior cytotoxicity in growth inhibition assays, despite similar cross-linking abilities for both domain orders tested. In hEx16-Dbs specifically, domain order might affect the agonistic activity of the anti-CD16 portion, which was supported by a cytokine production test, and likely contributed to the superiority of one of the hEx16-Dbs. Our results indicate that both the target antigen and mode of action of an antibody must be considered in the construction of highly functional bsAbs.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Células Asesinas Naturales/inmunología , Neoplasias/terapia , Receptores de IgG/inmunología , Anticuerpos Biespecíficos/farmacología , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Complejo CD3/inmunología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Humanos , Inmunoterapia , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Receptores de IgG/antagonistas & inhibidores , Linfocitos T/inmunologíaRESUMEN
Ezetimibe (EZE) and glucuronidated EZE (EZE-Glu) differentially target Niemann-Pick C1-like 1 (NPC1L1) and CD13 (aminopeptidase-N) to inhibit intestinal cholesterol absorption and cholesterol processing in other cells, although the precise molecular mechanisms are not fully elucidated. Cellular effects of EZE, EZE-Glu, and the low-absorbable EZE-analogue S6130 were investigated on human monocyte-derived macrophages upon loading with atherogenic lipoproteins. EZE and S6130, but not EZE-Glu disturbed the colocalization of CD13 and its coreceptor CD64 (Fcγ receptor I) in membrane microdomains, and decreased the presence of both receptors in detergent-resistant membrane fractions. Biotinylated cholesterol absorption inhibitor C-5 (i.e., derivative of EZE) was rapidly internalized to perinuclear tubular structures of cells, resembling endoplasmic reticulum (ER), but CD13 was detected on extracellular sites of the plasma membrane and endolysosomal vesicles. Administration of EZE, but not of EZE-Glu or S6130, was associated with decreased cellular cholesteryl ester content, indicating the sterol-O acyltransferase 1 (SOAT1)-inhibition by EZE. Furthermore, EZE decreased the expression of molecules involved in cholesterol uptake and synthesis, in parallel with increased apolipoprotein A-I-mediated cholesterol efflux and upregulation of efflux-effectors. However, NPC1L1 the other claimed molecular target of EZE, was not detected in macrophages, thereby excluding this protein as target for EZE in macrophages. Thus, EZE is very likely a CD13-linked microdomain-disruptor and SOAT1-inhibitor in macrophages leading to in vitro anti-atherosclerotic effects through a decrease of net cellular cholesterol content. © 2019 International Society for Advancement of Cytometry.
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Antígenos CD13/ultraestructura , Colesterol/aislamiento & purificación , Citometría de Flujo , Proteínas de Transporte de Membrana/genética , Receptores de IgG/ultraestructura , Aterosclerosis/genética , Transporte Biológico/efectos de los fármacos , Antígenos CD13/antagonistas & inhibidores , Colesterol/metabolismo , Ezetimiba/farmacología , Glucuronatos/genética , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/ultraestructura , Proteínas de Transporte de Membrana/metabolismo , Monocitos/metabolismo , Monocitos/ultraestructura , Receptores de IgG/antagonistas & inhibidoresRESUMEN
Immune complexes (IC) containing predominantly malondialdehyde-LDL and the corresponding autoantibodies (MDA-LDL IC) predict acute cardiovascular events, while IC rich in oxidized LDL (oxLDL IC) predict cardiovascular disease progression. Our objective was to determine mechanisms that could explain these prognostic differences. We compared the effects of the interaction of oxLDL, MDA-LDL and the corresponding IC with human macrophages focusing on apoptosis, metalloproteinases, and proinflammatory cytokines. MDA-LDL IC induced higher degrees of apoptosis, higher levels of caspase-3 expression, and increased expression and release of MMP-1 and TNF compared to MDA-LDL, oxLDL, and oxLDL IC. The pro-apoptotic effects of MDA-LDL IC were inhibited by blocking TNFR 1 or FcγRI. Blocking FcγRI abrogated the induction and expression of MMPs and proinflammatory cytokines by MDA-LDL IC. In conclusion, the interaction of MDA-LDL IC with FcγRI triggers macrophage apoptosis and increased expression and release of TNF and MMP-1, which can lead to the rupture of unstable plaques.
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Complejo Antígeno-Anticuerpo/inmunología , Apoptosis/inmunología , Aterosclerosis/inmunología , Autoanticuerpos/inmunología , Lipoproteínas LDL/inmunología , Macrófagos/inmunología , Malondialdehído/análogos & derivados , Placa Aterosclerótica/inmunología , Complejo Antígeno-Anticuerpo/metabolismo , Aterosclerosis/metabolismo , Autoanticuerpos/metabolismo , Enfermedades Cardiovasculares/inmunología , Enfermedades Cardiovasculares/metabolismo , Caspasa 3/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Citocinas/inmunología , Progresión de la Enfermedad , Expresión Génica , Humanos , Interleucina-6/genética , Interleucina-6/inmunología , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Malondialdehído/inmunología , Malondialdehído/metabolismo , Metaloproteinasa 1 de la Matriz/genética , Metaloproteinasa 1 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Placa Aterosclerótica/metabolismo , Receptores de IgG/antagonistas & inhibidores , Receptores Tipo I de Factores de Necrosis Tumoral/antagonistas & inhibidores , Inhibidor Tisular de Metaloproteinasa-1/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
In this issue of Blood, Yu et al describe a novel antiFcγ receptor III (FcγRIII)-albumin fusion protein that inhibits the development of thrombocytopenia in a murine model of immune thrombocytopenia (ITP).1 The unique aspect of this protein is that it blocks FcγRIII-mediated uptake of antibody-coated platelets without activating FcγRIII and the associated inflammatory response.
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Albúminas/inmunología , Anticuerpos Monoclonales/farmacología , Fragmentos Fc de Inmunoglobulinas/inmunología , Púrpura Trombocitopénica Idiopática/terapia , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/inmunología , Animales , Femenino , HumanosRESUMEN
Patients with immune thrombocytopenia (ITP) commonly have antiplatelet antibodies that cause thrombocytopenia through Fcγ receptors (FcγRs). Antibodies specific for FcγRs, designed to inhibit antibody-FcγR interaction, had been shown to improve ITP in refractory human patients. However, the development of such FcγR-specific antibodies has stalled because of adverse events, a phenomenon recapitulated in mouse models. One hypothesis behind these adverse events involved the function of the Fc region of the antibody, which engages FcγRs, leading to inflammatory responses. Unfortunately, inhibition of Fc function by deglycosylation failed to prevent this inflammatory response. In this work, we hypothesize that the bivalent antigen-binding fragment regions of immunoglobulin G are sufficient to trigger adverse events and have reasoned that designing a monovalent targeting strategy could circumvent the inflammatory response. To this end, we generated a fusion protein comprising a monovalent human FcγRIIIA-specific antibody linked in tandem to human serum albumin, which retained FcγR-binding activity in vitro. To evaluate clinically relevant in vivo FcγR-blocking function and inflammatory effects, we generated a murine version targeting the murine FcγRIII linked to murine albumin in a passive murine ITP model. Monovalent blocking of FcγR function dramatically inhibited antibody-dependent murine ITP and successfully circumvented the inflammatory response as assessed by changes in body temperature, basophil activation, and basophil depletion. Consistent with our hypothesis, in vivo cross-linking of the fusion protein induced these inflammatory effects, recapitulating the adverse events of the parent antibody. Thus, monovalent blocking of FcγR function demonstrates a proof of concept to successfully treat FcγR-mediated autoimmune diseases.
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Albúminas/inmunología , Anticuerpos Monoclonales/farmacología , Fragmentos Fc de Inmunoglobulinas/inmunología , Púrpura Trombocitopénica Idiopática/terapia , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/inmunología , Proteínas Recombinantes de Fusión/inmunología , Albúminas/metabolismo , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Células Cultivadas , Femenino , Humanos , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Púrpura Trombocitopénica Idiopática/inmunología , Receptores de IgG/metabolismoRESUMEN
Patients with acute myeloid leukemia (AML) often relapse after initial therapy because of persistence of leukemic stem cells that frequently express the IL-3 receptor alpha chain CD123. Natural killer (NK) cell-based therapeutic strategies for AML show promise and we explore the NK cell lines, NK-92 and CD16+ NK-92, as a treatment for AML. NK-92 has been tested in phase I clinical trials with minimal toxicity; irradiation prior to infusion prevents risk of engraftment. The CD16 negative NK-92 parental line was genetically modified to express the high affinity Fc gamma receptor, enabling antibody-dependent cell-mediated cytotoxicity, which we utilized in combination with an anti-CD123 antibody to target leukemic stem cells. NK-92 was preferentially cytotoxic against leukemic stem and progenitor cells compared with bulk leukemia in in vitro assays, while CD16+ NK-92 in combination with an anti-CD123 mAb mediated antibody-dependent cell-mediated cytotoxicity against CD123+ leukemic targets. Furthermore, NK-92 infusions (with or without prior irradiation) improved survival in a primary AML xenograft model. Mice xenografted with primary human AML cells had a superior survival when treated with irradiated CD16+NK-92 cells and an anti-CD123 monoclonal antibody (7G3) versus treatment with irradiated CD16+NK-92 cells combined with an isotype control antibody. In this proof-of-principle study, we show for the first time that a CD16+NK-92 cell line combined with an antibody that targets a leukemic stem cell antigen can lead to improved survival in a relevant pre-clinical model of AML.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Subunidad alfa del Receptor de Interleucina-3/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Proteínas de Neoplasias/antagonistas & inhibidores , Receptores de IgG/antagonistas & inhibidores , Animales , Proteínas Ligadas a GPI/antagonistas & inhibidores , Proteínas Ligadas a GPI/metabolismo , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Células K562 , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/metabolismo , Receptores de IgG/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Eosinophils contribute to the pathogenesis of multiple diseases, including asthma. Treatment with antibodies targeting IL-5 or IL-5 receptor α reduces the frequency of asthma exacerbations. Eosinophil receptors for IL-5 share a common ß-chain with IL-3 and GM-CSF receptors. We recently reported that IL-3 is more potent than IL-5 or GM-CSF in maintaining the ERK/p90S6K/RPS6 ribosome-directed signaling pathway, leading to increased protein translation. OBJECTIVE: We aimed to determine disease-relevant consequences of prolonged eosinophil stimulation with IL-3. RESULTS: Human blood eosinophils were used to establish the impact of activation with IL-3 on IgG-driven eosinophil degranulation. When compared to IL-5, continuing exposure to IL-3 further induced degranulation of eosinophils on aggregated IgG via increased production and activation of both CD32 (low affinity IgG receptor) and αMß2 integrin. In addition, unlike IL-5 or GM-CSF, IL-3 induced expression of CD32B/C (FCGRIIB/C) subtype proteins, without changing CD32A (FCGRIIA) protein and CD32B/C mRNA expression levels. Importantly, these in vitro IL-3-induced modifications were recapitulated in vivo on airway eosinophils. CONCLUSIONS AND CLINICAL RELEVANCE: We observed for the first time upregulation of CD32B/C on eosinophils, and identified IL-3 as a potent inducer of CD32- and αMß2-mediated eosinophil degranulation.
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Degranulación de la Célula/inmunología , Eosinófilos/inmunología , Eosinófilos/metabolismo , Interleucina-3/metabolismo , Antígeno de Macrófago-1/metabolismo , Receptores de IgG/metabolismo , Anticuerpos Monoclonales/farmacología , Biomarcadores , Degranulación de la Célula/efectos de los fármacos , Células Cultivadas , Eosinófilos/efectos de los fármacos , Humanos , Interleucina-3/farmacología , Receptores de IgG/antagonistas & inhibidoresRESUMEN
The CD32a immunoglobulin G (IgG) receptor (Fcγ receptor IIa) is a potential therapeutic target for diseases in which IgG immune complexes (ICs) mediate inflammation, such as heparin-induced thrombocytopenia, rheumatoid arthritis, and systemic lupus erythematosus. Monoclonal antibodies (mAbs) are a promising strategy for treating such diseases. However, IV.3, perhaps the best characterized CD32a-blocking mAb, was recently shown to induce anaphylaxis in immunocompromised "3KO" mice. This anaphylactic reaction required a human CD32a transgene because mice lack an equivalent of this gene. The finding that IV.3 induces anaphylaxis in CD32a-transgenic mice was surprising because IV.3 had long been thought to lack the intrinsic capacity to trigger cellular activation via CD32a. Such an anaphylactic reaction would also limit potential therapeutic applications of IV.3. In the present study, we examine the molecular mechanisms by which IV.3 induces anaphylaxis. We now report that IV.3 induces anaphylaxis in immunocompetent CD32a-transgenic "FCGR2A" mice, along with the novel finding that IV.3 and 2 other well-characterized CD32a-blocking mAbs, AT-10 and MDE-8, also induce severe thrombocytopenia in FCGR2A mice. Using recombinant variants of these same mAbs, we show that IgG "Fc" effector function is necessary for the induction of anaphylaxis and thrombocytopenia in FCGR2A mice. Variants of these mAbs lacking the capacity to activate mouse IgG receptors not only failed to induce anaphylaxis or thrombocytopenia, but also very potently protected FCGR2A mice from near lethal doses of IgG ICs. Our findings show that effector-deficient IV.3, AT-10, and MDE-8 are promising candidates for developing therapeutic mAbs to treat CD32a-mediated diseases.
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Anafilaxia/inducido químicamente , Anticuerpos Monoclonales de Origen Murino/efectos adversos , Anticuerpos Neutralizantes/efectos adversos , Receptores de IgG/antagonistas & inhibidores , Trombocitopenia/inducido químicamente , Anafilaxia/inmunología , Anafilaxia/patología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Humanos , Ratones , Ratones Transgénicos , Receptores de IgG/genética , Receptores de IgG/inmunología , Trombocitopenia/genética , Trombocitopenia/inmunología , Trombocitopenia/patologíaRESUMEN
Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.
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Proteínas Portadoras/metabolismo , Citomegalovirus/inmunología , Glicoproteínas/metabolismo , Fragmentos Fc de Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de IgG/antagonistas & inhibidores , Proteínas Virales/metabolismo , Animales , Células Cultivadas , Chlorocebus aethiops , Citomegalovirus/fisiología , Células HEK293 , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno/inmunología , Humanos , Unión Proteica , Receptores de IgG/metabolismo , Replicación ViralRESUMEN
The autoantibodies (auto-Abs) that are a hallmark of neonatally thymectomized (NTx) mice with autoimmune gastritis (AIG) have been poorly explored. We investigated their immune significance using B cell-deficient (B(-)) mice and found that B(-) mice are totally resistant to AIG but become susceptible to AIG after receiving bone marrow cells from B(+) mice. This susceptibility is most likely caused by the production of auto-Abs by B cells because B(-) pups also became susceptible to AIG when nourished by an AIG dam producing auto-Abs of the IgG class during the suckling period. NTx B(-) mice receiving purified IgG auto-Abs at this developmental stage similarly developed AIG. Auto-Abs probably act on antigen handling for antigen presentation because the treatment of NTx B(+) mice with anti-FcγR Abs prevented the development of AIG. Auto-Abs are indispensable for AIG development but are not sufficient because auto-Ab treatment did not increase AIG incidence in NTx B(+) mice above the baseline.
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Anticuerpos Antiidiotipos/inmunología , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Gastritis/inmunología , Receptores de IgG/inmunología , Traslado Adoptivo , Animales , Animales Recién Nacidos , Linfocitos B , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Receptores de IgG/antagonistas & inhibidores , TimectomíaRESUMEN
Invasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.
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Candida albicans/inmunología , Candidiasis/prevención & control , Inmunidad Innata/inmunología , Neutrófilos/inmunología , Candida albicans/crecimiento & desarrollo , Candidiasis/inmunología , Candidiasis/microbiología , Células Cultivadas , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lectinas Tipo C/antagonistas & inhibidores , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Fagocitosis , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/genética , Receptores de IgG/metabolismo , Quinasa SykRESUMEN
Canonical neutrophil antimicrobial effector mechanisms, such as degranulation, production of reactive oxygen species, and release of neutrophil extracellular traps (NETs), can result in severe pathology. Activation of neutrophils through immune complexes (ICs) plays a central role in the pathogenesis of many autoimmune inflammatory diseases. In this study, we report that immobilized ICs (iICs), which are hallmarks of several autoimmune diseases, induce the release of NETs from primary human neutrophils. The iIC-induced NET formation was found to require production of reactive oxygen species by NADPH oxidase and myeloperoxidase and to be mediated by FcγRIIIb. Blocking of the ß2 integrin macrophage-1 Ag but not lymphocyte function-associated Ag-1 abolished iIC-induced NET formation. This suggests that FcγRIIIb signals in association with macrophage-1 Ag. As intracellular signaling pathways involved in iIC-induced NET formation we identified the tyrosine kinase Src/Syk pathway, which downstream regulates the PI3K/Akt, p38 MAPK, and ERK1/2 pathways. To our knowledge, the present study shows for the first time that iICs induce NET formation. Thus, we conclude that NETs contribute to pathology in autoimmune inflammatory disorders associated with surface-bound ICs.
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Complejo Antígeno-Anticuerpo/inmunología , Antígeno de Macrófago-1/inmunología , Activación Neutrófila/inmunología , Neutrófilos/inmunología , Receptores de IgG/inmunología , Aminopirina/farmacología , Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Enfermedades Autoinmunes/inmunología , Butadienos/farmacología , Antígeno CD11a/metabolismo , Antígenos CD18/metabolismo , Degranulación de la Célula , Células Cultivadas , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Proteínas Ligadas a GPI/inmunología , Humanos , Imidazoles/farmacología , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/inmunología , Antígeno-1 Asociado a Función de Linfocito/inmunología , Antígeno de Macrófago-1/metabolismo , Mesalamina/farmacología , Nitrilos/farmacología , Compuestos Onio/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/inmunología , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Piridinas/farmacología , Pirimidinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Receptores de IgG/antagonistas & inhibidores , Quinasa Syk , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/inmunologíaRESUMEN
BACKGROUND: Clinical observations suggest that anaphylaxis is more common in adult women compared with adult men, although the mechanistic basis for this sex bias is not well understood. OBJECTIVES: We sought to document sex-dependent differences in a mouse model of anaphylaxis and explore the role of female sex hormones and the mechanisms responsible. METHODS: Passive systemic anaphylaxis was induced in female and male mice by using histamine, as well as IgE or IgG receptor aggregation. Anaphylaxis was assessed by monitoring body temperature, release of mast cell mediators and/or hematocrit, and lung weight as a measure of vascular permeability. A combination of ovariectomy, estrogen receptor antagonism, and estrogen administration techniques were used to establish estrogen involvement. RESULTS: Anaphylactic responses were more pronounced in female than male mice. The enhanced severity of anaphylaxis in female mice was eliminated after pretreatment with an estrogen receptor antagonist or ovariectomy but restored after administration of estradiol in ovariectomized mice, demonstrating that the sex-specific differences are due to the female steroid estradiol. Estrogen did not affect mast cell responsiveness or anaphylaxis onset. Instead, it increased tissue expression of endothelial nitric oxide synthase (eNOS). Blockage of NOS activity with the inhibitor L-NG-nitroarginine methyl ester or genetic eNOS deficiency abolished the sex-related differences. CONCLUSION: Our study defines a contribution of estrogen through its regulation of eNOS expression and nitric oxide production to vascular hyperpermeability and intensified anaphylactic responses in female mice, providing additional mechanistic insights into risk factors and possible implications for clinical management in the further exploration of human anaphylaxis.
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Anafilaxia/genética , Anafilaxia/fisiopatología , Estradiol/metabolismo , Pulmón/enzimología , Óxido Nítrico Sintasa de Tipo III/inmunología , Óxido Nítrico/biosíntesis , Anafilaxia/enzimología , Anafilaxia/inmunología , Animales , Temperatura Corporal , Permeabilidad Capilar , Modelos Animales de Enfermedad , Estradiol/farmacología , Antagonistas del Receptor de Estrógeno/farmacología , Femenino , Expresión Génica , Histamina/inmunología , Histamina/farmacología , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/fisiopatología , Masculino , Mastocitos/efectos de los fármacos , Mastocitos/inmunología , Mastocitos/patología , Ratones , Ratones Noqueados , Óxido Nítrico Sintasa de Tipo III/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo III/genética , Ovariectomía , Agregado de Proteínas , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/química , Receptores de Estrógenos/inmunología , Receptores de IgE/antagonistas & inhibidores , Receptores de IgE/química , Receptores de IgE/inmunología , Receptores de IgG/antagonistas & inhibidores , Receptores de IgG/química , Receptores de IgG/inmunología , omega-N-Metilarginina/farmacologíaRESUMEN
To evade opsonophagocytosis, Staphylococcus aureus secretes various immunomodulatory molecules that interfere with effective opsonization by complement and/or IgG. Immune-evasion molecules targeting the phagocyte receptors for these opsonins have not been described. In this study, we demonstrate that S. aureus escapes from FcγR-mediated immunity by secreting a potent FcγR antagonist, FLIPr, or its homolog FLIPr-like. Both proteins were previously reported to function as formyl peptide receptor inhibitors. Binding of FLIPr was mainly restricted to FcγRII receptors, whereas FLIPr-like bound to different FcγR subclasses, and both competitively blocked IgG-ligand binding. They fully inhibited FcγR-mediated effector functions, including opsonophagocytosis and subsequent intracellular killing of S. aureus by neutrophils and Ab-dependent cellular cytotoxicity of tumor cells by both neutrophils and NK cells. In vivo, treatment of mice with FLIPr-like prevented the development of an immune complex-mediated FcγR-dependent Arthus reaction. This study reveals a novel immune-escape function for S. aureus-secreted proteins that may lead to the development of new therapeutic agents in FcγR-mediated diseases.
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Proteínas Bacterianas/fisiología , Receptores de IgG/antagonistas & inhibidores , Staphylococcus aureus/inmunología , Animales , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión de Anticuerpos/inmunología , Humanos , Evasión Inmune/inmunología , Leucemia P388/inmunología , Leucemia P388/microbiología , Ratones , Ratones Endogámicos BALB C , Fagocitosis/inmunología , Unión Proteica/inmunología , Receptores de IgG/química , Receptores de IgG/fisiología , Homología de Secuencia de Aminoácido , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/prevención & control , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/patogenicidadRESUMEN
Cholinergic neural output has been shown to modulate innate immune responses to infection, injury and ischemia through stimulation of α7 nicotinic acetylcholine receptors (α7nAChR) on mononuclear phagocytes. We tested the hypothesis that cholinergic neurotransmitters, similar to those released through activation of a neural reflex, regulate responses to products of the adaptive immune system, specifically immune complex (IC)-mediated activation of effector cells. In this study, we show that stimulation of α7nAChR on human polymorphonuclear neutrophils (PMNs) and blood mononuclear phagocytes in vitro attenuates C5aR- and FcγR-triggered generation of reactive oxygen species, expression of leukocyte markers involved in cell recruitment and adhesion, and release of TNF-α and other proinflammatory cytokines. We show that this pathway is operative in vivo. Ligation of cholinergic receptors blunts IC-triggered responses in the reverse peritoneal Arthus reaction in mice. The selective 7nAChR agonist GTS21 decreased PMN accumulation and release of cytokines and chemokines at sites of IC deposition. In addition, mice lacking α7nAChR had exaggerated responses to reverse peritoneal Arthus reaction characterized by increased infiltration of PMNs and elevated of levels of TNF-α and CXCL1 in peritoneal fluid compared with wild-type mice. Taken together, these findings suggest that cholinergic output has the potential to exert tonic inhibitory activity that dampens responses to ICs and C5a and thus may be a target to minimize tissue damage in autoimmune diseases.