RESUMEN
Toll-like receptors (TLR) are critical mediators of the immune system with their activation linked to infection, inflammation and the pathogenesis of immune diseases including autoimmunity and cancer. For this reason, over the last 2 decades, TLR and their associated signalling pathways have been targeted therapeutically to enhance innate and adaptive immunity. Several TLR ligands, both endogenous and synthetic are at various phases of clinical testing, and new ligands are continually emerging. Agonists of TLR7 are known immune response modifiers, simultaneously stimulating several cell types, resulting in immune cell activation and cytokine and chemokine release. The immune stimulating properties of the TLR7 agonist Imiquimod has also been exploited for use in the treatment of malignant superficial tumours of the skin. Here, we investigated a novel TLR7 agonist UC-1V150 and demonstrate it activates both human and mouse myeloid cells in vitro and in vivo, to deliver potent FcγR-mediated engulfment of opsonized target cells.
Asunto(s)
Benzaldehídos/química , Benzaldehídos/farmacología , Factores Inmunológicos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Glicoproteínas de Membrana/agonistas , Fagocitosis/inmunología , Purinas/química , Purinas/farmacología , Receptor Toll-Like 7/agonistas , Aminoquinolinas/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Células Cultivadas , Humanos , Imiquimod , Activación de Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Fagocitosis/efectos de los fármacos , Receptores de IgG/biosíntesisRESUMEN
The Fcγ receptor IIIA (FcγRIIIA) has traditionally been known as a positive regulator of immune responses. Consistent with this, mice deficient in FcγRIIIA are protected from various inflammation-associated pathologies including several autoimmune diseases. In contrast to this accepted dogma, we show here that mice lacking FcγRIIIA developed increased rather than reduced both humoral and cellular immune responses to mucosal (sublingual) immunization with ovalbumin (OVA) given together with the strong mucosal adjuvant cholera toxin as well as to parenteral (subcutaneous) immunization with OVA in complete Freund's adjuvant. After either route of immunization, in comparison with concomitantly immunized wild-type mice, FcγRIIIA-/- mice had increased serum anti-OVA IgG (IgG1 but not IgG2) antibody responses as well as augmented cellular responses that included memory B cells and effector T cells. The increments in immune responses in FcγRIIIA-/- mice were similar to those seen in FcγRIIB-/- mice. Furthermore, OVA-pulsed FcγRIIIA-/- DCs, similar to OVA-specific FcγRIIB-/- DCs, had enhanced capacity to activate OVA-specific OT-II T cells, which was even further pronounced when DCs were pulsed with IgG1-complexed OVA. Our data support an inhibitory-regulatory role of FcγRIIIA on vaccine/adjuvant-induced immune responses and demonstrate that lack of FcγRIIIA can promote rather than suppress both humoral and cellular immune responses.
Asunto(s)
Linfocitos B/inmunología , Inmunidad Celular/genética , Inmunidad Humoral/genética , Inmunoglobulina G/inmunología , Receptores de IgG/genética , Linfocitos T/inmunología , Adyuvantes Inmunológicos , Animales , Toxina del Cólera/inmunología , Adyuvante de Freund/inmunología , Inmunidad Celular/inmunología , Inmunidad Humoral/inmunología , Inmunización/métodos , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Membrana Mucosa/inmunología , Ovalbúmina/inmunologíaRESUMEN
Fc gamma receptors (FcγRs) for IgG regulate adaptive immune responses by modulating activating and inhibitory signalling pathways within immune cells. Data from a haemophilia A mouse model demonstrate that genetic deletion or blockade of the inhibitory FcγR (CD32) suppresses the formation of antibody-secreting cells (ASCs) in vitro. Mechanisms preventing the FVIII-specific recall response, however, remain unclear. Here, the potential role of CD32 inhibition was studied by differentially modulating receptor activity with selected anti-CD32 monoclonal antibodies (mAbs). Splenocytes from immunized FVIII-/- mice were restimulated with FVIII in the absence or presence of different anti-CD32 mAbs over 6 days. At day 6, cytokine release was quantified from cell culture supernatant and the formation of FVIII-specific ASCs assessed. Binding of FVIII-containing immune complexes (F8-ICs) to bone marrow-derived dendritic cells (BMdDCs) was also investigated. The antagonistic CD32 mAb AT128 suppressed the formation of FVIII-specific ASCs and reduced secretion of IFN-γ and IL-10. In contrast, the agonistic mAbs AT130-2 and AT130-5, and their F(ab')2 fragments, allowed the formation of FVIII-specific ASCs, even though the full IgG of AT130-2 reduced binding of F8-ICs to CD32. Data suggest that an inhibitory signal is transmitted when F8-ICs bind to CD32 and that this signal is required during memory B cell (MBC) activation to support formation of FVIII-specific ASCs. If the inhibitory signal is lacking due to CD32 deletion or blockade with antagonistic anti-CD32 mAbs, FVIII-specific T cell stimulation and ASC formation are suppressed, whereas agonistic stimulation of CD32 restores T cell stimulation and ASC formation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Factor VIII/inmunología , Receptores de IgG/inmunología , Animales , Linfocitos B/metabolismo , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Factor VIII/genética , Factor VIII/metabolismo , Citometría de Flujo , Inmunoglobulina G/inmunología , Memoria Inmunológica/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-10/inmunología , Interleucina-10/metabolismo , Linfocitos/inmunología , Linfocitos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de IgG/genética , Receptores de IgG/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Factores de TiempoRESUMEN
BACKGROUND: Prostate cancer (PCa) is the most common male cancer in the United Kingdom and we aimed to identify clinically relevant biomarkers corresponding to stage progression of the disease. METHODS: We used enhanced proteomic profiling of PCa progression using iTRAQ 3D LC mass spectrometry on high-quality serum samples to identify biomarkers of PCa. RESULTS: We identified >1000 proteins. Following specific inclusion/exclusion criteria we targeted seven proteins of which two were validated by ELISA and six potentially interacted forming an 'interactome' with only a single protein linking each marker. This network also includes accepted cancer markers, such as TNF, STAT3, NF-κB and IL6. CONCLUSIONS: Our linked and interrelated biomarker network highlights the potential utility of six of our seven markers as a panel for diagnosing PCa and, critically, in determining the stage of the disease. Our validation analysis of the MS-identified proteins found that SAA alongside KLK3 may improve categorisation of PCa than by KLK3 alone, and that TSR1, although not significant in this model, might also be a clinically relevant biomarker.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/patología , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Biomarcadores de Tumor/sangre , Análisis Químico de la Sangre/métodos , Cromatografía Liquida , Progresión de la Enfermedad , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Proyectos Piloto , Pronóstico , Neoplasias de la Próstata/diagnóstico , Reproducibilidad de los ResultadosRESUMEN
Rituximab is widely used in the treatment of haematological malignancies, including chronic lymphocytic leukaemia (CLL), the most common leukaemia in adults. However, some patients, especially those with high tumour burden, develop cytokine release syndrome (CRS). It is likely that more patients will develop therapy-linked CRS in the future due to the implementation of other immunotherapies, such as CAR T-cell, for many malignancies. Current methods for CRS risk assessment are limited, hence there is a need to develop new methods. To better recapitulate an in vivo setting, we implemented a unique human whole blood "loop" system to study patient-specific immune responses to rituximab in blood derived from CLL patients. Upon rituximab infusion, both complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) profiles were evident in CLL patient blood, coincident with CLL cell depletion. Whereas B cell depletion is induced in healthy persons in the blood loop, only patients display B cell depletion coupled with CRS. With the exception of one donor who lacked NK cells, all other five patients displayed variable B cell depletion along with CRS profile. Additionally, inhibition of CDC or ADCC via either inhibitors or antibody Fc modification resulted in skewing of the immune killing mechanism consistent with published literature. Herein we have shown that the human whole blood loop model can be applied using blood from a specific indication to build a disease-specific CRS and immune activation profiling ex vivo system. Other therapeutic antibodies used for other indications may benefit from antibody characterization in a similar setting.
Asunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Leucemia Linfocítica Crónica de Células B/inmunología , Rituximab/uso terapéutico , Anciano , Anciano de 80 o más Años , Antirreumáticos , Linfocitos B/inmunología , Recuento de Células Sanguíneas , Activación de Complemento , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/inmunología , Citocinas/sangre , Citotoxicidad Inmunológica , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Células Asesinas Naturales , Leucemia Linfocítica Crónica de Células B/complicaciones , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Recuento de Leucocitos , MasculinoRESUMEN
Over recent decades it has become increasingly apparent that malignant cells, including chronic lymphocytic leukemia (CLL) cells, do not exist in isolation. Rather they coalesce with numerous "normal" cells of the body and, in the case of CLL, inhabit key immunological niches within secondary lymphoid organs (SLO), where a plethora of stromal and immune cells mediate their growth and survival. With the advent and approval of targeted immune therapies such as monoclonal antibodies (mAb), which elicit their efficacy by engaging immune-mediated effector mechanisms, it is important to develop accurate methods to measure their activities. Here, we describe a series of reliable assays capable of measuring important antibody-mediated effector functions: antibody-dependent cellular phagocytosis (ADCP), antibody-dependent cellular cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) that measure these immune activities.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos Inmunológicos/farmacología , Pruebas Inmunológicas de Citotoxicidad/métodos , Ensayos de Selección de Medicamentos Antitumorales/métodos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos/efectos de los fármacos , Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Antineoplásicos Inmunológicos/uso terapéutico , Línea Celular , Técnicas de Cocultivo , Pruebas Inmunológicas de Citotoxicidad/instrumentación , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Macrófagos , Ratones , Monocitos , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Cultivo Primario de Células/instrumentación , Cultivo Primario de Células/métodosRESUMEN
Somatic hypermutation (SHM) is a pivotal process in adaptive immunity that occurs in the germinal centre and allows B cells to change their primary DNA sequence and diversify their antigen receptors. Here, we report that genome binding of Lamin B1, a component of the nuclear envelope involved in epigenetic chromatin regulation, is reduced during B-cell activation and formation of lymphoid germinal centres. Chromatin immunoprecipitation-Seq analysis showed that kappa and heavy variable immunoglobulin domains were released from the Lamin B1 suppressive environment when SHM was induced in B cells. RNA interference-mediated reduction of Lamin B1 resulted in spontaneous SHM as well as kappa-light chain aberrant surface expression. Finally, Lamin B1 expression level correlated with progression-free and overall survival in chronic lymphocytic leukaemia, and was strongly involved in the transformation of follicular lymphoma. In summary, here we report that Lamin B1 is a negative epigenetic regulator of SHM in normal B-cells and a 'mutational gatekeeper', suppressing the aberrant mutations that drive lymphoid malignancy.
Asunto(s)
Linfocitos B/patología , Lamina Tipo B/genética , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Hipermutación Somática de Inmunoglobulina/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina/métodos , Progresión de la Enfermedad , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Linfoma Folicular/genética , Linfoma Folicular/patologíaRESUMEN
Over the past 10-15 years, genetic engineering of monoclonal antibodies has greatly improved their utility in humans and in particular their ability to recruit immunological effectors such as natural killer cells and macrophages. Clinical results now confirm that these new reagents, when directed at the appropriate tumor markers (e.g. CD20 or Her-2), can control disease without untoward side effects. However, despite such success it is still unclear exactly how monoclonal antibodies (mAbs) destroy tumors in vivo. The ability of mAbs to crosslink membrane receptors and generate intracellular signals is part of the mechanism by which they control tumor growth. New data show that such 'signaling' mAbs can be used to sensitize tumors to the action of conventional DNA-damaging drugs.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Neoplasias/terapia , Anticuerpos Monoclonales de Origen Murino , Antígenos CD20/inmunología , Quimioterapia Combinada , Humanos , Linfoma de Células B/terapia , Receptor ErbB-2/inmunología , Rituximab , Transducción de Señal/efectos de los fármacosRESUMEN
PI3Kδ plays pivotal roles in the maintenance, proliferation and survival of malignant B-lymphocytes. Although not curative, PI3Kδ inhibitors (PI3Kδi) demonstrate impressive clinical efficacy and, alongside other signaling inhibitors, are revolutionizing the treatment of hematological malignancies. However, only limited in vivo data are available regarding their mechanism of action. With the rising number of novel treatments, the challenge is to identify combinations that deliver curative regimes. A deeper understanding of the molecular mechanism is required to guide these selections. Currently, immunomodulation, inhibition of B-cell receptor signaling, chemokine/cytokine signaling and apoptosis represent potential therapeutic mechanisms for PI3Kδi. Here we characterize the molecular mechanisms responsible for PI3Kδi-induced apoptosis in an in vivo model of chronic lymphocytic leukemia (CLL). In vitro, PI3Kδi-induced substantive apoptosis and disrupted microenvironment-derived signaling in murine (Eµ-Tcl1) and human (CLL) leukemia cells. Furthermore, PI3Kδi imparted significant therapeutic responses in Eµ-Tcl1-bearing animals and enhanced anti-CD20 monoclonal antibody therapy. Responses correlated with upregulation of the pro-apoptotic BH3-only protein Bim. Accordingly, Bim-/- Eµ-Tcl1 Tg leukemias demonstrated resistance to PI3Kδi-induced apoptosis were refractory to PI3Kδi in vivo and failed to display combination efficacy with anti-CD20 monoclonal antibody therapy. Therefore, Bim-dependent apoptosis represents a key in vivo therapeutic mechanism for PI3Kδi, both alone and in combination therapy regimes.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteína 11 Similar a Bcl2/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Modelos Animales de Enfermedad , Leucemia Linfocítica Crónica de Células B/patología , Animales , Proteína 11 Similar a Bcl2/genética , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/metabolismo , Masculino , Ratones , Ratones SCID , Transducción de Señal/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Genetic recombination during B-cell development regularly results in the generation of autoreactive, potentially pathogenic B-cell receptors (BCRs). Consequently, multiple mechanisms link inappropriate BCR specificity to clonal deletion. Similar pathways remain in malignant B cells, offering the potential for targeting BCR signaling. Recently, small molecule inhibitors have realized this potential and, therefore, a deeper understanding of BCR-induced signaling networks in malignant cells is vital. The BH3-only protein Bim has a key role in BCR-induced apoptosis, but it has long been proposed that additional BH3-only proteins also contribute, although conclusive proof has been lacking. Here, we comprehensively characterized the mechanism of BCR-induced apoptosis in Eµ-Myc murine lymphoma cells. We demonstrate the upregulation of Bim, Bik, and Noxa during BCR signaling in vitro and that intrinsic apoptosis has a prominent role in anti-BCR antibody therapy in vivo. Furthermore, lymphomas deficient in these individual BH3-only proteins display significant protection from BCR-induced cell death, whereas combined loss of Noxa and Bim offers enhanced protection in comparison with loss of Bim alone. Some but not all of these effects were reversed upon inhibition of Syk or MEK. These observations indicate that BCR signaling elicits maximal cell death through upregulation of multiple BH3-only proteins; namely Bim, Bik, and Noxa.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Linfoma de Células B/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcr/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Proteína 11 Similar a Bcl2 , Línea Celular Tumoral , Linfoma de Células B/patología , Proteínas de la Membrana/genética , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Proteínas Mitocondriales/genética , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción de SeñalRESUMEN
The pro-survival Bcl-2 family member Mcl-1 is expressed in chronic lymphocytic leukaemia (CLL), with high expression correlated with progressive disease. The spliceosome inhibitor spliceostatin A (SSA) is known to regulate Mcl-1 and so here we assessed the ability of SSA to elicit apoptosis in CLL. SSA induced apoptosis of CLL cells at low nanomolar concentrations in a dose- and time-dependent manner, but independently of SF3B1 mutational status, IGHV status and CD38 or ZAP70 expression. However, normal B and T cells were less sensitive than CLL cells (P=0.006 and P<0.001, respectively). SSA altered the splicing of anti-apoptotic MCL-1(L) to MCL-1(s) in CLL cells coincident with induction of apoptosis. Overexpression studies in Ramos cells suggested that Mcl-1 was important for SSA-induced killing since its expression inversely correlated with apoptosis (P=0.001). IL4 and CD40L, present in patient lymph nodes, are known to protect tumour cells from apoptosis and significantly inhibited SSA, ABT-263 and ABT-199 induced killing following administration to CLL cells (P=0.008). However, by combining SSA with the Bcl-2/Bcl-x(L) antagonists ABT-263 or ABT-199, we were able to overcome this pro-survival effect. We conclude that SSA combined with Bcl-2/Bcl-x(L) antagonists may have therapeutic utility for CLL.
Asunto(s)
Apoptosis/efectos de los fármacos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/genética , Fosfoproteínas/antagonistas & inhibidores , Piranos/farmacología , Ribonucleoproteína Nuclear Pequeña U2/antagonistas & inhibidores , Compuestos de Espiro/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Humanos , Interleucina-4/farmacología , Leucemia Linfocítica Crónica de Células B/patología , Mutación , Fosfoproteínas/genética , Empalme del ARN , Factores de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U2/genética , Microambiente Tumoral , Proteína bcl-X/antagonistas & inhibidoresRESUMEN
Histone methyltransferases (HMTs) are important epigenetic regulators of gene transcription and are disrupted at the genomic level in a spectrum of human tumours including haematological malignancies. Using high-resolution single nucleotide polymorphism (SNP) arrays, we identified recurrent deletions of the SETD2 locus in 3% (8/261) of chronic lymphocytic leukaemia (CLL) patients. Further validation in two independent cohorts showed that SETD2 deletions were associated with loss of TP53, genomic complexity and chromothripsis. With next-generation sequencing we detected mutations of SETD2 in an additional 3.8% of patients (23/602). In most cases, SETD2 deletions or mutations were often observed as a clonal event and always as a mono-allelic lesion, leading to reduced mRNA expression in SETD2-disrupted cases. Patients with SETD2 abnormalities and wild-type TP53 and ATM from five clinical trials employing chemotherapy or chemo-immunotherapy had reduced progression-free and overall survival compared with cases wild type for all three genes. Consistent with its postulated role as a tumour suppressor, our data highlight SETD2 aberration as a recurrent, early loss-of-function event in CLL pathobiology linked to aggressive disease.
Asunto(s)
Genómica , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Linfocítica Crónica de Células B/genética , Mutación , Proteínas de la Ataxia Telangiectasia Mutada/genética , Supervivencia sin Enfermedad , Femenino , Genes Supresores de Tumor , Histona Metiltransferasas , Humanos , Leucemia Linfocítica Crónica de Células B/enzimología , Leucemia Linfocítica Crónica de Células B/mortalidad , Masculino , Pronóstico , Tasa de Supervivencia , Proteína p53 Supresora de Tumor/genéticaRESUMEN
In this study, we demonstrate for the first time that complement attack of target cells, in the presence of suitably high levels of serum, can induce the oligonucleosomal DNA fragmentation characteristic of apoptosis. This phenomenon requires membrane permeabilisation induced by formation of the complete membrane attack complex and relies on physiologically relevant levels of serum. TUNEL analysis detected complement mediated DNA fragmentation as early as 30 min after the addition of serum and electron microscopy confirmed that chromatin became condensed after complement attack. Various experiments implicate serum DNase I as the mediator of this DNA fragmentation. Intriguingly, membrane permeability induced by melittin gave rise to similar serum dependent DNA fragmentation. The implications of these results for the study of apoptosis in vitro and in vivo are discussed.
Asunto(s)
Apoptosis/inmunología , Proteínas del Sistema Complemento/inmunología , Animales , Cromatina , Fragmentación del ADN , Cobayas , Humanos , Células Tumorales CultivadasRESUMEN
Bfl-1 is a pro-survival Bcl-2 family member overexpressed in a subset of chemoresistant tumours, including melanoma. Here, we characterised the expression and regulation of Bfl-1 in normal and malignant melanocytes and determined its role in protecting these cells from chemotherapy-induced apoptosis. Bfl-1 was mitochondrially resident in both resting and apoptotic cells and experienced regulation by the proteasome and NFκB pathways. siRNA-mediated knockdown enhanced sensitivity towards various relevant drug treatments, with forced overexpression of Bfl-1 protective. These findings identify Bfl-1 as a contributor towards therapeutic resistance in melanoma cells and support the use of NFκB inhibitors alongside current treatment strategies.
Asunto(s)
Melanoma/metabolismo , Melanoma/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Apoptosis/genética , Línea Celular Tumoral , Supervivencia Celular , Células Cultivadas , Citoprotección , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Melanocitos/metabolismo , Melanoma/genética , Antígenos de Histocompatibilidad Menor , Mitocondrias/metabolismo , Mutación/genética , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Neoplasias Cutáneas/genéticaRESUMEN
The naturally occurring oncolytic virus (OV), reovirus, replicates in cancer cells causing direct cytotoxicity, and can activate innate and adaptive immune responses to facilitate tumour clearance. Reovirus is safe, well tolerated and currently in clinical testing for the treatment of multiple myeloma, in combination with dexamethasone/carfilzomib. Activation of natural killer (NK) cells has been observed after systemic delivery of reovirus to cancer patients; however, the ability of OV to potentiate NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) is unexplored. This study elucidates the potential of oncolytic reovirus for the treatment of chronic lymphocytic leukaemia (CLL), both as a direct cytotoxic agent and as an immunomodulator. We demonstrate that reovirus: (i) is directly cytotoxic against CLL, which requires replication-competent virus; (ii) phenotypically and functionally activates patient NK cells via a monocyte-derived interferon-α (IFNα)-dependent mechanism; and (iii) enhances ADCC-mediated killing of CLL in combination with anti-CD20 antibodies. Our data provide strong preclinical evidence to support the use of reovirus in combination with anti-CD20 immunotherapy for the treatment of CLL.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos/inmunología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Leucemia Linfocítica Crónica de Células B/inmunología , Orthoreovirus Mamífero 3/inmunología , Virus Oncolíticos/inmunología , Rituximab/inmunología , Rituximab/uso terapéutico , Anciano , Anciano de 80 o más Años , Antineoplásicos/inmunología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Efecto Citopatogénico Viral , Femenino , Humanos , Inmunidad Innata , Factores Inmunológicos/inmunología , Factores Inmunológicos/uso terapéutico , Inmunofenotipificación , Inmunoterapia , Células Asesinas Naturales/inmunología , Leucemia Linfocítica Crónica de Células B/diagnóstico , Activación de Linfocitos/inmunología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Replicación ViralRESUMEN
Mitotic death is a delayed response of p53 mutant tumours that are resistant to genotoxic damage. Questions surround why this response is so delayed and how its mechanisms serve a survival function. After uncoupling apoptosis from G1 and S phase arrests and adapting these checkpoints, p53 mutated tumour cells arrive at the G2 compartment where decisions regarding survival and death are made. Missed or insufficient DNA repair in G1 and S phases after severe genotoxic damage results in cells arriving in G2 with an accumulation of point mutations and chromosome breaks. Double strand breaks can be repaired by homologous recombination during G2 arrest. However, cells with excessive chromosome lesions either directly bypass the G2/M checkpoint, starting endocycles from G2 arrest, or are subsequently detected by the spindle checkpoint and present with the features of mitotic death. These complex features include apoptosis from metaphase and mitosis restitution, the latter of which can also facilitate transient endocycles, producing endopolyploid cells. The ability of cells to initiate endocycles during G2 arrest and mitosis restitution most likely reflects their similar molecular environments, with down-regulated mitosis promoting factor activity. Resulting endocycling cells have the ability to repair damaged DNA, and although mostly reproductively dead, in some cases give rise to mitotic progeny. We conclude that the features of mitotic death do not simply represent aberrations of dying cells but are indicative of a switch to amitotic modes of cell survival that may provide additional mechanisms of genotoxic resistance.
RESUMEN
We report the in vivo radioimmunotherapy (RIT) of a new variant of the BCL1 syngeneic mouse B-cell lymphoma model, pi-BCL1, using a panel of monoclonal antibodies (MoAb) reactive with B cell-associated antigens (CD19, CD22, CD40, MHC II, and idiotype). MoAb were radiolabeled with 131I or used in conjunction with external beam irradiation. When administered early in disease (day 4) 131I-anti-MHC II MoAb produced long term disease free survivors as a result of targeted irradiation alone; the equivalent unlabelled MoAb was non-therapeutic. In contrast, 131I-anti-CD40, and 131I-anti-Idiotype (Id) MoAb administered at day 4 despite targeting irradiation and having intrinsic therapeutic activity as unconjugated antibodies, protected mice for approximately 30 days. The 131I-anti-CD19 and anti-CD22 were therapeutically inactive. Treating later in the disease (day 14, after tumor inoculation) permitted study of the efficacy in the presence of an increased tumor load. An increased tumor burden brought about an expected reduction in therapeutic activity with 131I-anti-MHC II, but surprisingly, 131I anti-CD40 and 131I-anti-Id were able to produce prolonged disease free survival in most mice. This unexpected potency of 131I-anti-CD40 and 131I-anti-Id late in disease appeared to result from the direct cytotoxic action induced by these MoAb. Mechanisms by which these two MoAb operate, in producing long-term disease free survival in animals with advanced disease appear different. Our therapeutic results have important implications for the selection of reagents for RIT and demonstrate that successful treatment with such agents may involve more than simple targeting of irradiation.
Asunto(s)
Moléculas de Adhesión Celular , Lectinas , Linfoma de Células B/radioterapia , Radioinmunoterapia , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/inmunología , Antígenos CD/análisis , Antígenos CD19/análisis , Antígenos de Diferenciación de Linfocitos B/análisis , Apoptosis/efectos de la radiación , Antígenos CD40/análisis , ADN de Neoplasias/análisis , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Genes MHC Clase I/genética , Genes MHC Clase II/genética , Técnicas In Vitro , Radioisótopos de Yodo/uso terapéutico , Ratones , Ratones Endogámicos BALB C , Lectina 2 Similar a Ig de Unión al Ácido Siálico , Análisis de Supervivencia , Factores de Tiempo , Células Tumorales Cultivadas/citologíaRESUMEN
We report a new variant of the BCL1 syngeneic mouse B-cell lymphoma model, which we have called pi-BCL1. pi-BCL1 can be established as a syngeneic tumor in BALB/c mice. Tumors can be removed, prepared and easily grown in liquid culture and subsequently transferred back successfully as syngeneic tumors. As a syngeneic tumor pi-BCL1 behave more like a lymphoma with solid tumor masses, than a chronic lymphocytic leukaemia of the original BCL1 model. The immunophenotype and the growth characteristics of the pi-BCL1 and BCL1 tumors appear very similar. Cytologically, pi-BCL1 appears to be a transformation from a small lymphocytic lymphoma to a more diffuse large cell centroblast-like higher-grade lymphoma. We are not aware of any previous reports of such transformation events in a syngeneic animal model of B cell lymphoma. We believe pi-BCL1 provides a useful new tool for the study of B cell lymphoma in vitro and in vivo and enables reduced numbers of tumor passage in mice.
Asunto(s)
Linfoma de Células B/patología , Animales , Antígenos de Superficie/análisis , ADN de Neoplasias/análisis , Modelos Animales de Enfermedad , Femenino , Técnica del Anticuerpo Fluorescente , Inmunofenotipificación , Técnicas In Vitro , Cariotipificación , Hígado/citología , Hígado/patología , Linfoma Folicular/patología , Linfoma Folicular/ultraestructura , Ratones , Ratones Endogámicos BALB C , Bazo/citología , Bazo/patología , Células Tumorales Cultivadas/citologíaRESUMEN
After DNA or spindle damage, p53-defective tumor cells undergo a complex cycle of reversible polyploidy. How this process occurs and more importantly, why, has recently become the focus of several research groups, prompting this review in which we discuss two related phenomena that accompany the reversible polyploidy of tumor cells: the induction of meiosis genes such as MOS and the decrease in genomic instability observed during the reversion from polyploidy to para-diploidy. The reversible polyploidy likely provides the means through which the balance between increased chromosome instability (CIN), driving genetic variation and decreased CIN, necessary for perpetuating these malignant clones, is maintained. These concepts are integrated with recent findings that many meiotic and self-renewal genes become activated during reversible polyploidy and lead us to the hypothesis that tumor cell immortality may be achieved through germline-like transmission.
Asunto(s)
Aneuploidia , Regulación Neoplásica de la Expresión Génica , Genes mos/genética , Neoplasias/genética , Ploidias , Animales , HumanosRESUMEN
Rituximab is a chimeric anti-CD20 monoclonal antibody that has revolutionised the treatment of many B-cell malignancies, and is now increasingly being used in non-malignant conditions such as auto-immune disorders. Serum rituximab levels are highly variable in patients receiving similar 'standard' approved doses. Little is known regarding the factors that affect serum rituximab concentration and that in turn may influence clinical outcome. In order to provide a tool that may ultimately enable patient specific dosing of rituximab therapy, we have validated a reliable, robust ELISA for the quantitation of serum rituximab levels to provide accurate pharmacokinetic (PK) data that will guide the optimisation of rituximab dosing regimes. Extensive validation of the assay was performed in order to utilise the assay for clinical applications. The within and between day plate coating reproducibility was tested and proved a robust starting platform for the assay. The within day precision for the assay was determined using spiked serum samples and was shown to have a coefficient of variation (CV) of <10% with an accuracy between 91 and 125%. The between day precision (CV) was <25% with an accuracy between 95 and 109%. Dilution linearity and parallelism were demonstrated. Spike recovery for all concentrations and donors was shown to be within +/-15% on average, with a CV below 10%. This assay is highly accurate and reproducible in determining the levels of rituximab in spiked serum samples. It meets stringent acceptance criteria, is fit for purpose, and is currently being applied to several clinical trials incorporating rituximab in the treatment of lymphoma. This assay represents a useful tool for clinical application of this widely used therapeutic.