RESUMEN
Despite the remarkable efficacy of tyrosine kinase inhibitors (TKIs) in eliminating differentiated chronic myeloid leukemia (CML) cells, recent evidence suggests that leukemic stem and progenitor cells (LSPCs) persist long term, which may be partly attributable to cytokine-mediated resistance. We evaluated the expression of the interleukin 3 (IL-3) receptor α subunit (CD123), an established marker of acute myeloid leukemia stem cells, on CML LSPCs and the potential of targeting those cells with the humanized anti-CD123 monoclonal antibody CSL362. Compared with normal donors, CD123 expression was higher in CD34(+)/CD38(-) cells of both chronic phase and blast crisis CML patients, with levels increasing upon disease progression. CSL362 effectively targeted CML LSPCs by selective antibody-dependent cell-mediated cytotoxicity (ADCC)-facilitated lysis of CD123(+) cells and reduced leukemic engraftment in mice. Importantly, not only were healthy donor allogeneic natural killer (NK) cells able to mount an effective CSL362-mediated ADCC response, but so were CML patients' autologous NK cells. In addition, CSL362 also neutralized IL-3-mediated rescue of TKI-induced cell death. Notably, combination of TKI- and CSL362-induced ADCC caused even greater reduction of CML progenitors and further augmented their preferential elimination over normal hematopoietic stem and progenitor cells. Thus, our data support the further evaluation of CSL362 therapy in CML.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Subunidad alfa del Receptor de Interleucina-3/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/inmunología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Receptores de Interleucina-3/inmunología , ADP-Ribosil Ciclasa 1/metabolismo , Adulto , Anciano , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/farmacología , Antígenos CD34/metabolismo , Línea Celular , Femenino , Humanos , Subunidad alfa del Receptor de Interleucina-3/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Leucemia Eritroblástica Aguda/inmunología , Leucemia Eritroblástica Aguda/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Células Madre/inmunología , Células Madre/metabolismo , Células Madre/patologíaRESUMEN
The prognosis of older patients with acute myelogenous leukemia is generally poor. The interleukin-3 receptor α-chain (CD123) is highly expressed on the surface of acute leukemia cells compared with normal hematopoietic stem cells. CSL362 is a fully humanized, CD123-neutralizing monoclonal antibody containing a modified Fc structure, which enhances human natural killer cell antibody-dependent cell-mediated cytotoxicity. Six continuous acute myelogenous leukemia xenografts established from patient explants and characterized by cell and molecular criteria, produced progressively lethal disease 42-202 days after transplantation. CSL362 alone reduced engraftment of one of four and three of four acute myelogenous leukemia xenografts in the bone marrow and peripheral organs, respectively. A cytarabine and daunorubicin regimen was optimized using this model to identify potentially synergistic interactions with CSL362. Cytarabine/daunorubicin improved the survival of mice engrafted with four of four acute myelogenous leukemia xenografts by 31-41 days. Moreover, CSL362 extended the survival of cytarabine/daunorubicin-treated mice for two of two acute myelogenous leukemia xenografts, while augmentation of natural killer cell-deficient NSG mice with adoptively transferred human natural killer cells improved survival against a single xenograft. Interestingly, this enhanced CSL362 efficacy was lost in the absence of chemotherapy. This study shows that acute myelogenous leukemia xenografts provide a platform for the evaluation of new therapeutics, simulating complex in vivo interactions, and that the in vivo efficacy of CSL362 supports continued clinical development of this drug.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Huésped Inmunocomprometido , Subunidad alfa del Receptor de Interleucina-3/antagonistas & inhibidores , Leucemia Mieloide Aguda/terapia , Traslado Adoptivo , Animales , Citarabina/farmacología , Daunorrubicina , Femenino , Expresión Génica , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/inmunología , Subunidad alfa del Receptor de Interleucina-3/genética , Subunidad alfa del Receptor de Interleucina-3/inmunología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/mortalidad , Leucemia Mieloide Aguda/patología , Ratones , Análisis de Supervivencia , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
LIGHT (TNFSF14), a tumor necrosis factor superfamily member expressed by activated T cells, binds to herpes virus entry mediator (HVEM) which is constitutively expressed by T cells and costimulates T cell activation in a CD28-independent manner. Given interest in regulating the effector functions of T cells in vivo, we examined the role of LIGHT-HVEM costimulation in a murine cardiac allograft rejection model. Normal hearts lacked LIGHT or HVEM mRNA expression, but allografts showed strong expression of both genes from day 3 after transplant, and in situ hybridization and immunohistology-localized LIGHT and HVEM to infiltrating leukocytes. To test the importance of LIGHT expression on allograft survival, we generated LIGHT-/- mice by homologous recombination. The mean survival of fully major histocompatibility complex-mismatched vascularized cardiac allografts in LIGHT-/- mice (10 days, P < 0.05) or cyclosporine A (CsA)-treated LIGHT+/+ mice (10 days, P < 0.05) was only slightly prolonged compared with LIGHT+/+ mice (7 days). However, mean allograft survival in CsA-treated LIGHT-/- allograft recipients (30 days) was considerably enhanced (P < 0.001) compared with the 10 days of mean survival in either untreated LIGHT-/- mice or CsA-treated LIGHT+/+ controls. Molecular analyzes showed that the beneficial effects of targeting of LIGHT in CsA-treated recipients were accompanied by decreased intragraft expression of interferon (IFN)-gamma, plus IFN-gamma-induced chemokine, inducible protein-10, and its receptor, CXCR3. Treatment of LIGHT+/+ allograft recipients with HVEM-Ig plus CsA also enhanced mean allograft survival (21 days) versus wild-type controls receiving HVEM-Ig (mean of 7 days) or CsA alone (P < 0.001). Our data suggest that T cell to T cell-mediated LIGHT/HVEM-dependent costimulation is a significant component of the host response leading to cardiac allograft rejection.
Asunto(s)
Supervivencia de Injerto/inmunología , Trasplante de Corazón , Proteínas de la Membrana/inmunología , Linfocitos T/inmunología , Inmunología del Trasplante , Factor de Necrosis Tumoral alfa/inmunología , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Trasplante Homólogo , Miembro 14 de la Superfamilia de Ligandos de Factores de Necrosis TumoralRESUMEN
The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. A specialized glial cell within the olfactory nerve, called olfactory ensheathing cell (OEC), and their associated extracellular matrix are thought to play an important role during regenerative events in this system. To gain insight into novel molecules that could mediate the OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments were conducted which revealed high expression of the glycoprotein fibulin-3 in OECs. This observation was confirmed with quantitative PCR. In vivo, the distribution of all members of the fibulin family, fibulin-3 included, was localized to the lamina propria underneath the olfactory epithelium, in close association within olfactory nerve bundles. To manipulate fibulin-3 gene expression in cultured OECs, lentiviral vector constructs were designed to either transgenically express or knock-down fibulin-3. Experimental data showed that increased levels of fibulin-3 induced profound morphological changes in cultured OECs, impeded with their migratory abilities and also suppressed OEC-mediated neurite outgrowth. Knock-down of fibulin-3 levels resulted in reduced OEC proliferation. In conclusion, the data provide novel insights into a putative role for fibulin-3 in the regulation of cell migration and neurite outgrowth within the primary olfactory pathway.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Movimiento Celular/fisiología , Tamaño de la Célula , Expresión Génica/fisiología , Neuroglía/fisiología , Bulbo Olfatorio/citología , Animales , Bromodesoxiuridina/metabolismo , Proteínas de Unión al Calcio/genética , Células Cultivadas , Análisis por Conglomerados , Técnicas de Cocultivo/métodos , Perfilación de la Expresión Génica/métodos , Técnicas de Transferencia de Gen , Neuritas/fisiología , Neuroglía/citología , Vías Olfatorias/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Endogámicas F344 , Células de Schwann/fisiología , Estadísticas no Paramétricas , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/metabolismoRESUMEN
The neuregulins (NRGs) are a family of four structurally related growth factors that are expressed in the developing and adult brain. NRG-1 is essential for normal heart formation and has been implicated in the development and maintenance of both neurons and glia. NRG-2 was identified on the basis of its homology to NRG-1 and, like NRG-1, is expressed predominantly by neurons in the central nervous system. We have generated mice with the active domain of NRG-2 deleted in an effort to characterize the biological function of NRG-2 in vivo. In contrast to the NRG-1 knockout animals, NRG-2 knockouts have no apparent heart defects and survive embryogenesis. Mutant mice display early growth retardation and reduced reproductive capacity. No obvious histological differences were observed in the major sites of NRG-2 expression. Our results indicate that in vivo NRG-2 activity differs substantially from that of NRG-1 and that it is not essential for normal development in utero.
Asunto(s)
Factores de Crecimiento Nervioso/deficiencia , Factores de Crecimiento Nervioso/genética , Animales , Secuencia de Bases , Encéfalo/crecimiento & desarrollo , ADN Complementario/genética , Femenino , Marcación de Gen , Genes erbB , Corazón/crecimiento & desarrollo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Crecimiento Nervioso/fisiología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Neurregulina-1 , FenotipoRESUMEN
Injured neurons in the mammalian central nervous system (CNS) do not normally regenerate their axons after injury. Neurotrauma to the CNS usually results in axonal damage and subsequent loss of communication between neuronal networks, causing long-term functional deficits. For CNS regeneration, repair strategies need to be developed that promote regrowth of lesioned axon projections and restoration of neuronal connectivity. After spinal cord injury (SCI), cystic cavitations are often found, particularly in the later stages, due to the loss of neural tissue at the original impact site. Ultimately, for the promotion of axonal regrowth in these situations, some form of transplantation will be required to provide lesioned axons with a supportive substrate along which they can extend. Here, we review the use of olfactory ensheathing cells: their location and role in the olfactory system, their use as cellular transplants in SCI paradigms, alone or in combination with gene therapy, and the unique properties of these cells that may give them a potential advantage over other cellular transplants.
Asunto(s)
Trasplante de Células , Vías Olfatorias/citología , Animales , Terapia Combinada , Ingeniería Genética , Terapia Genética , Humanos , Vías Olfatorias/fisiologíaRESUMEN
The SOCS family of genes are negative regulators of cytokine signalling with SOCS-1 displaying tumor suppressor activity. SOCS-1, CIS and SOCS-3 have been implicated in the regulation of red blood cell production. In this study, a detailed examination was conducted on the expression patterns of these three SOCS family members in normal erythroid progenitors and a panel of erythroleukemic cell lines. Unexpectedly, differences in SOCS gene expression were observed during maturation of normal red cell progenitors, viz changes to CIS were inversely related to the alterations of SOCS-1 and SOCS-3. Similarly, these SOCS genes were differentially expressed in transformed erythoid cells - erythroleukemic cells immortalized at an immature stage of differentiation expressed SOCS-1 and SOCS-3 mRNA constitutively, whereas in more mature cell lines SOCS-1 and CIS were induced only after exposure to erythropoietin (Epo). Significantly, when ectopic expression of the tyrosine kinase Lyn was used to promote differentiation of immature cell lines, constitutive expression of SOCS-1 and SOCS-3 was completely suppressed. Modulation of intracellular signalling via mutated Epo receptors in mature erythroleukemic lines also highlighted different responses by the three SOCS family members. Close scrutiny of SOCS-1 revealed that, despite large increases in mRNA levels, the activity of the promoter did not alter after erythropoietin stimulation; in addition, erythroid cells from SOCS-1-/- mice displayed increased sensitivity to Epo. These observations indicate complex, stage-specific regulation of SOCS genes during normal erythroid maturation and in erythroleukemic cells.
Asunto(s)
Proteínas Portadoras/genética , Células Precursoras Eritroides/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas/genética , Proteínas Represoras , Factores de Transcripción , Animales , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/fisiología , Diferenciación Celular , Línea Celular Transformada , Células Cultivadas , Células Precursoras Eritroides/citología , Células Precursoras Eritroides/efectos de los fármacos , Eritropoyetina/farmacología , Regulación de la Expresión Génica , Proteínas Inmediatas-Precoces/biosíntesis , Ratones , Mutación , Regiones Promotoras Genéticas , Biosíntesis de Proteínas , ARN Mensajero/biosíntesis , Receptores de Eritropoyetina/genética , Proteína 1 Supresora de la Señalización de Citocinas , Proteína 3 Supresora de la Señalización de Citocinas , Proteínas Supresoras de la Señalización de Citocinas , Activación Transcripcional , Familia-src Quinasas/metabolismoRESUMEN
Interleukin-13 (IL-13) is a cytokine implicated in airway diseases such as asthma and idiopathic pulmonary fibrosis. IL-13 signals through a heterodimeric receptor complex consisting of IL-13Rα1 and IL-4Rα, known as the type II IL-4R. IL-4 also signals through this receptor and as such many of the biological effects of IL-13 and IL-4 are similar. Here we describe the development of two sensitive bioassays to determine the potency of antagonists of the mouse type II IL-4R. Both IL-13 and IL-4 dose-dependently induce CCL17 production from J774 mouse monocytic cells and CCL11 production from NIH3T3 mouse fibroblasts in the presence of TNFα. The assays were optimized to minimize TNFα concentration, cell number and incubation time whilst retaining a suitable signal-to-background ratio. Anti-cytokine antibodies or recombinant soluble receptors completely neutralized IL-13 or IL-4 activity in these bioassays. The J774 assay was used to screen a panel of anti-mIL-13Rα1 antibodies for neutralizing activity against this receptor. We report the identification of the first monoclonal antibodies that bind mouse IL-13Rα1 and neutralize both IL-13-induced and IL-4-induced cellular function. These antibodies should prove useful for determining the effects of neutralizing IL-13Rα1 in mouse models of disease. In addition, these bioassays may be used for measuring the bioactivity of mouse IL-13 and IL-4 and for the discovery of additional antagonists of the mouse IL-13Rα1/IL-4Rα complex.
Asunto(s)
Anticuerpos Neutralizantes/análisis , Subunidad alfa1 del Receptor de Interleucina-13/antagonistas & inhibidores , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Quimiocina CCL11/metabolismo , Citocinas/metabolismo , Fibroblastos/inmunología , Inmunoensayo , Interleucina-13/metabolismo , Subunidad alfa1 del Receptor de Interleucina-13/inmunología , Interleucina-4/metabolismo , Ratones , Monocitos/inmunología , Células 3T3 NIH , Receptores de Superficie Celular/inmunología , Transducción de SeñalRESUMEN
Interleukin-3 (IL-3) is an activated T cell product that bridges innate and adaptive immunity and contributes to several immunopathologies. Here, we report the crystal structure of the IL-3 receptor α chain (IL3Rα) in complex with the anti-leukemia antibody CSL362 that reveals the N-terminal domain (NTD), a domain also present in the granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-5, and IL-13 receptors, adopting unique "open" and classical "closed" conformations. Although extensive mutational analyses of the NTD epitope of CSL362 show minor overlap with the IL-3 binding site, CSL362 only inhibits IL-3 binding to the closed conformation, indicating alternative mechanisms for blocking IL-3 signaling. Significantly, whereas "open-like" IL3Rα mutants can simultaneously bind IL-3 and CSL362, CSL362 still prevents the assembly of a higher-order IL-3 receptor-signaling complex. The discovery of open forms of cytokine receptors provides the framework for development of potent antibodies that can achieve a "double hit" cytokine receptor blockade.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Antineoplásicos/química , Subunidad alfa del Receptor de Interleucina-3/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales Humanizados/inmunología , Antineoplásicos/metabolismo , Sitios de Unión de Anticuerpos , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Subunidad alfa del Receptor de Interleucina-3/inmunología , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor alpha chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing to bone marrow (BM) and activating innate immunity of nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival. Mice with pre-established disease showed reduced AML burden in the BM and periphery and impaired secondary transplantation upon treatment, establishing that AML-LSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34(+)CD38(-) cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.