RESUMEN
TGFß is a potential target in cancer treatment due to its dual role in tumorigenesis and homeostasis. However, the expression of TGFß and its inhibition within the tumor microenvironment has mainly been investigated in stroma-heavy tumors. Using B16 mouse melanoma and CT26 colon carcinoma as models of stroma-poor tumors, we demonstrate that myeloid/dendritic cells are the main sources of TGFß1 and TGFß3. Depending on local expression of TGFß isoforms, isoform specific inhibition of either TGFß1 or TGFß3 may be effective. The TGFß signature of CT26 colon carcinoma is defined by TGFß1 and TGFß1 inhibition results in tumor delay; B16 melanoma has equal expression of both isoforms and inhibition of either TGFß1 or TGFß3 controls tumor growth. Using T cell functional assays, we show that the mechanism of tumor delay is through and dependent on enhanced CD8+ T cell function. To overcome the local immunosuppressive environment, we found that combining TGFß inhibition with immune checkpoint blockade results in improved tumor control. Our data suggest that TGFß inhibition in stroma poor tumors shifts the local immune environment to favor tumor suppression.
Asunto(s)
Antineoplásicos/farmacología , Carcinogénesis , Factor de Crecimiento Transformador beta/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
The dimeric cytokine IL-12 is important in the control of various infections but also contributes to the pathology of certain diseases making it a potential target for therapy. However, its specific inhibition with antibodies is complicated by the fact that its two subunits are present in other cytokines: p40 in IL-23 and p35 in IL-35. This has led to erroneous conclusions like the alleged implication of IL-12 in experimental autoimmune encephalomyelitis (EAE). Here, we report the development of a mouse anti-mouse IL-12 vaccine and the production of monoclonal antibodies (mAbs) that do not react with p40 or p35 (in IL-35) but specifically recognize and functionally inhibit the IL-12 heterodimer. Using one of these mAbs, MM12A1.6, that strongly inhibited IFN-γ production and LPS-induced septic shock after viral infection, we demonstrate the critical role played by IL-12 in the rejection of male skin graft by female C57BL/6 syngeneic recipients and in the clearance of an immunogenic mastocytoma tumor variant by DBA/2 mice, but not in a parent to F1 immune aggression model nor in MOG-induced EAE, which was clearly prevented by anti-p40 mAb C17.8. Given this selective inhibition of IL-12, these mAbs provide new options for reassessing IL-12 function in vivo.
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Anticuerpos Monoclonales/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Rechazo de Injerto/inmunología , Interleucina-12/metabolismo , Mastocitoma/inmunología , Esclerosis Múltiple/inmunología , Infecciones por Nidovirales/inmunología , Nidovirales/fisiología , Subunidades de Proteína/metabolismo , Sepsis/inmunología , Trasplante de Piel , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Modelos Animales de Enfermedad , Epítopos , Humanos , Hibridomas , Interleucina-12/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Neoplasias Experimentales , Subunidades de Proteína/inmunologíaRESUMEN
BACKGROUND: Transforming growth factor-ß (TGFß) is emerging as a promising target for cancer therapy, given its ability to promote progression of advanced tumors and to suppress anti-tumor immune responses. However, TGFß also plays multiple roles in normal tissues, particularly during organogenesis, raising toxicity concerns about TGFß blockade. Dose-limiting cardiovascular toxicity was observed, possibly due to the blockade of all three TGFß isoforms. The dominant isoform in tumors is TGFß1, while TGFß2 and TGFß3 seem to be more involved in cardiovascular development. Recent data indicated that selective targeting of TGFß1 promoted the efficacy of checkpoint inhibitor anti-PD1 in transplanted preclinical tumor models, without cardiovascular toxicity. METHODS: To further explore the therapeutic potential of isoform-specific TGFß blockade, we developed neutralizing mAbs targeting mature TGFß1 or TGFß3, and tested them, in parallel with anti-panTGFß mAb 1D11, in two preclinical models: the transplanted colon cancer model CT26, and the autochthonous melanoma model TiRP. RESULTS: We observed that the blockade of TGFß1, but not that of TGFß3, increased the efficacy of a prophylactic cellular vaccine against colon cancer CT26. This effect was similar to pan-TGFß blockade, and was associated with increased infiltration of activated CD8 T cells in the tumor, and reduced levels of regulatory T cells and myeloid-derived suppressor cells. In contrast, in the autochthonous TiRP melanoma model, we observed therapeutic efficacy of the TGFß1-specific mAb as a single agent, while the TGFß3 mAb was inactive. In this model, the anti-tumor effect of TGFß1 blockade was tumor intrinsic rather than immune mediated, as it was also observed in T-cell depleted mice. Mechanistically, TGFß1 blockade increased mouse survival by delaying the phenotype switch, akin to epithelial-to-mesenchymal transition (EMT), which transforms initially pigmented tumors into highly aggressive unpigmented tumors. CONCLUSIONS: Our results confirm TGFß1 as the relevant isoform to target for cancer therapy, not only in combination with checkpoint inhibitors, but also with other immunotherapies such as cancer vaccines. Moreover, TGFß1 blockade can also act as a monotherapy, through a tumor-intrinsic effect blocking the EMT-like transition. Because human melanomas that resist therapy often express a gene signature that links TGFß1 with EMT-related genes, these results support the clinical development of TGFß1-specific mAbs in melanoma.
Asunto(s)
Anticuerpos Neutralizantes/farmacología , Antineoplásicos Inmunológicos/farmacología , Vacunas contra el Cáncer/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Melanoma/tratamiento farmacológico , Neoplasias Cutáneas/tratamiento farmacológico , Factor de Crecimiento Transformador beta1/antagonistas & inhibidores , Animales , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Transición Epitelial-Mesenquimal/efectos de los fármacos , Femenino , Masculino , Melanoma/inmunología , Melanoma/metabolismo , Melanoma/patología , Ratones Endogámicos BALB C , Ratones Transgénicos , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Factor de Crecimiento Transformador beta1/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Crecimiento Transformador beta3/antagonistas & inhibidores , Factor de Crecimiento Transformador beta3/inmunología , Factor de Crecimiento Transformador beta3/metabolismo , Microambiente TumoralRESUMEN
BACKGROUND: Viral infections can reduce early cancer development through enhancement of cancer immunosurveillance. This study was performed to analyse this effect of viral infection in a mouse model of solid tumor. METHODS: The experimental model used was the effect of BALB/c mouse infection by lactate dehydrogenase-elevating virus on AB1 mesothelioma cancer development. RESULTS: Acute infection with lactate dehydrogenase-elevating virus strongly reduced in vivo early AB1 mesothelioma growth and death resulting from cancer development. This effect was not due to a direct cytolytic effect of the virus on AB1 cells, but to an in vivo activation of natural killer cells. Gamma-interferon production rather than cytotoxic activity against AB1 cells mediated this protective effect. This gamma-interferon production by natural killer cells was dependent on interleukin-12 production. CONCLUSIONS: Together with other reported effects of infectious agents on cancer development, this observation may support the hypothesis that enhancement of innate immunosurveillance against tumors may result from infection with common infectious agents through modulation of the host immune microenvironment.
RESUMEN
In spite of considerable therapeutic progress, acute graft-versus-host disease still limits allogeneic hematopoietic cell transplantation. We recently reported that mouse infection with nidovirus lactate dehydrogenase elevating virus impairs disease in non-conditioned B6D2F1 recipients of parental B6 spleen cells. As this virus activates TLR7, we tested a pharmacological TLR7 ligand, R848, in this model and observed complete survival if donor and recipients were treated before transplantation. Mixed lymphocyte culture performed 48 h after R848-treatment of normal mice demonstrated that both T-cell allo-responsiveness and antigen presentation by CD11b+ and CD8α+ dendritic cells were inhibited. These inhibitions were dependent on IFNAR-1 signaling. In the B6 to B6D2F1 transplantation model, R848 decelerated, but did not abrogate, donor T-cell implantation and activation. However, it decreased interferon-gamma, tumor necrosis factor-alpha and interleukin-27 while upregulating active transforming growth factor-beta 1 plasma levels. In addition, donor and recipient Foxp3+ regulatory T-cell numbers were increased in recipient mice and their elimination compromised disease prevention. R848 also strongly improved survival of lethally irradiated BALB/c recipients of B6 hematopoietic cells and this also correlated with an upregulation of CD4 and CD8 Foxp3+ regulatory T cells that could be further increased by inhibition of interleukin-27. The combination of anti-interleukin-27p28 mono -clonal antibody and R848 showed strong synergy in preventing disease in the B6 to B6D2F1 transplantation model when recipients were sublethally irradiated and this also correlated with upregulation of regulatory T cells. We conclude that R848 modulates multiple aspects of graft-versus-host disease and offers potential for safe allogeneic bone marrow transplantation that can be further optimized by inhibition of interleukin-27.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Enfermedad Injerto contra Huésped/prevención & control , Imidazoles/farmacología , Interleucina-27/antagonistas & inhibidores , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Receptor Toll-Like 7/metabolismo , Animales , Citocinas/metabolismo , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Inmunomodulación/efectos de los fármacos , Ligandos , Melanoma Experimental , Ratones , Trasplante de Neoplasias , Linfocitos T Reguladores/inmunologíaRESUMEN
The pathogenic role of IL-17 and GM-CSF has been unravelled in experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). However, in most models, EAE is characterised by a monophasic attack which is not representative of the relapsing nature nor the chronicity displayed in MS. Here, we used proteolipid protein peptide (PLP139-151 ) to trigger EAE-relapses (EAE-II) in SJL mice that had recovered from a primary-EAE episode (EAE-I). This procedure resulted in severe and irreversible disease that, unlike EAE-I, was not abolished by anti-IL-17-mAb. In contrast, prophylactic anti-GM-CSF-mAb treatment prevented EAE-I and -II. Strikingly, the expression of T-cell transcription factors and cytokines/chemokines in mice treated with anti-GM-CSF during both EAE episodes was silenced. Anti-GM-CSF-mAb treatment administered only during EAE-II did not completely prevent relapses but mice ultimately reached full recovery. Anti-GM-CSF treatment also strongly impaired and ultimately resolved monophasic MOG35-55 -induced EAE in C57Bl/6 mice. In such protected mice, anti-GM-CSF treatment also prevented a further relapse induced by MOG-revaccination. These results underscore the critical role of GM-CSF on pro-inflammatory mediator production. Furthermore, we observed a strong preventive and curative effect of anti-GM-CSF neutralisation in two EAE models, relapsing and chronic. Altogether these findings are relevant for further MS research.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Interleucina-17/metabolismo , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple/metabolismo , Animales , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Ratones , Ratones Endogámicos C57BL , Glicoproteína Mielina-Oligodendrócito/metabolismo , Recurrencia , Factores de Transcripción/metabolismoRESUMEN
Host directed immunomodulation represents potential new adjuvant therapies in infectious diseases such as tuberculosis. Major cytokines like TNFα exert a multifold role in host control of mycobacterial infections. GM-CSF and its receptor are over-expressed during acute M. tuberculosis infection and we asked how GM-CSF neutralization might affect host response, both in immunocompetent and in immunocompromised TNFα-deficient mice. GM-CSF neutralizing antibodies, at a dose effectively preventing acute lung inflammation, did not affect M. tuberculosis bacterial burden, but increased the number of granuloma in wild-type mice. We next assessed whether GM-CSF neutralization might affect the control of M. tuberculosis by isoniazid/rifampicin chemotherapy. GM-CSF neutralization compromised the bacterial control under sub-optimal isoniazid/rifampicin treatment in TNFα-deficient mice, leading to exacerbated lung inflammation with necrotic granulomatous structures and high numbers of intracellular M. tuberculosis bacilli. In vitro, GM-CSF neutralization promoted M2 anti-inflammatory phenotype in M. bovis BCG infected macrophages, with reduced mycobactericidal NO production and higher intracellular M. bovis BCG burden. Thus, GM-CSF pathway overexpression during acute M. tuberculosis infection contributes to an efficient M1 response, and interfering with GM-CSF pathway in the course of infection may impair the host inflammatory response against M. tuberculosis.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Factores Inmunológicos/metabolismo , Inmunomodulación , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/patología , Animales , Anticuerpos Neutralizantes/administración & dosificación , Antituberculosos/administración & dosificación , Bovinos , Modelos Animales de Enfermedad , Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Factores Inmunológicos/antagonistas & inhibidores , Isoniazida/administración & dosificación , Macrófagos/inmunología , Ratones , Mycobacterium bovis/inmunología , Rifampin/administración & dosificación , Resultado del Tratamiento , Tuberculosis Pulmonar/inmunologíaRESUMEN
Despite impressive clinical success, cancer immunotherapy based on immune checkpoint blockade remains ineffective in many patients due to tumoral resistance. Here we use the autochthonous TiRP melanoma model, which recapitulates the tumoral resistance signature observed in human melanomas. TiRP tumors resist immunotherapy based on checkpoint blockade, cancer vaccines or adoptive T-cell therapy. TiRP tumors recruit and activate tumor-specific CD8+ T cells, but these cells then undergo apoptosis. This does not occur with isogenic transplanted tumors, which are rejected after adoptive T-cell therapy. Apoptosis of tumor-infiltrating lymphocytes can be prevented by interrupting the Fas/Fas-ligand axis, and is triggered by polymorphonuclear-myeloid-derived suppressor cells, which express high levels of Fas-ligand and are enriched in TiRP tumors. Blocking Fas-ligand increases the anti-tumor efficacy of adoptive T-cell therapy in TiRP tumors, and increases the efficacy of checkpoint blockade in transplanted tumors. Therefore, tumor-infiltrating lymphocytes apoptosis is a relevant mechanism of immunotherapy resistance, which could be blocked by interfering with the Fas/Fas-ligand pathway.
Asunto(s)
Inmunoterapia , Linfocitos Infiltrantes de Tumor/inmunología , Melanoma Experimental/inmunología , Melanoma Experimental/terapia , Animales , Apoptosis/inmunología , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Proteína Ligando Fas/antagonistas & inhibidores , Proteína Ligando Fas/genética , Proteína Ligando Fas/inmunología , Femenino , Humanos , Inmunoterapia/métodos , Inmunoterapia Adoptiva , Linfocitos Infiltrantes de Tumor/patología , Masculino , Melanoma Experimental/patología , Ratones , Ratones Transgénicos , Microambiente Tumoral/inmunología , Receptor fas/antagonistas & inhibidores , Receptor fas/inmunologíaRESUMEN
INTRODUCTION: Viruses have developed multiple mechanisms to alter immune reactions. In 1969, it was reported that lactate dehydrogenase-elevating virus (LDV), a single stranded positive sense mouse nidovirus, delays skin allograft rejection and inhibits spleen alterations in graft versus host disease (GVHD). As the underlying mechanisms have remained unresolved and given the need for new therapies of this disease, we reassessed the effects of the virus on GVHD and tried to uncover its mode of action. METHODS: GVHD was induced by transfer of parent (B6) spleen cells to non-infected or LDV-infected B6D2F1 recipients. In vitro mixed-lymhocyte culture (MLC) reactions were used to test the effects of the virus on antigen-presenting cells (APC) and responder T cells. RESULTS: LDV infection resulted in a threefold increase in survival rate with reduced weight loss and liver inflammation but with the establishment of permanent chimerism that correlated with decreased interleukine (IL)-27 and interferon (IFN)γ plasma levels. Infected mice showed a transient elimination of splenic CD11b+ and CD8α+ conventional dendritic cells (cDCs) required for allogeneic CD4 and CD8 T cell responses in vitro. This drop of APC numbers was not observed with APCs derived from toll-like receptor (TLR)7-deficient mice. A second effect of the virus was a decreased T cell proliferation and IFNγ production during MLC without detectable changes in Foxp3+ regulatory T cell (Tregs) numbers. Both cDC and responder T cell inhibition were type I IFN dependent. Although the suppressive effects were very transient, the GVHD inhibition was long-lasting. CONCLUSION: A type I IFN-dependent suppression of DC and T cells just after donor spleen cell transplantation induces permanent chimerism and donor cell implantation in a parent to F1 spleen cell transplantation model. If this procedure can be extended to full allogeneic bone marrow transplantation, it could open new therapeutic perspectives for hematopoietic stem cell transplantation (HSCT).
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Enfermedad Injerto contra Huésped/inmunología , Interferón Tipo I/inmunología , Infecciones por Nidovirales/inmunología , Nidovirales/inmunología , Aloinjertos , Animales , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/patología , Células Dendríticas/patología , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/patología , Interferón Tipo I/genética , Ratones , Ratones NoqueadosRESUMEN
The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases.
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Sistemas de Liberación de Medicamentos/métodos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Interleucina-17/metabolismo , Pulmón/metabolismo , Polietilenglicoles/metabolismo , Animales , Autoanticuerpos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Pulmón/efectos de los fármacos , Masculino , Ratones , Células 3T3 NIH , Polietilenglicoles/administración & dosificación , Conejos , Ratas , Ratas Sprague-Dawley , Especificidad de la EspecieRESUMEN
Periostin (POSTN), a secreted homodimeric protein that binds integrins αvß3, αvß5, and α6ß4, was originally found to be expressed in fetal tissues and in the adult upon injury particularly bone fractures due to its role in remodelling and repair. Recently it was found to be over-expressed in human breast cancer and a variety of other tumour types including head and neck squamous cell carcinoma, where its overexpression correlates with increased tumour invasion. Progress in studying its functional role in tumour pathogenesis has been hampered by the paucity of antibodies for its specific and sensitive detection. It has proven very difficult to obtain monoclonal antibodies (mAbs) against this highly conserved protein but we report here that combining infection of mice with lactate dehydrogenase elevating virus (LDV), a B cell activating arterivirus, with conjugation of human POSTN to ovalbumin as an immunogenic carrier, enabled us to develop six mAbs recognizing both human and mouse POSTN and inhibiting its binding to αvß3 integrin. Two of the mAbs, MPB4B1 and MPC5B4, were tested and found to inhibit POSTN-induced migration of human endothelial colony forming cells. All six mAbs recognized amino acids 136-51 (APSNEAWDNLDSDIRR) within the POSTN fascilin (FAS) 1-1 domain revealing the functional importance of this motif; this was further highlighted by the ability of aa 136-151 peptide to inhibit integrin-mediated cell migration. Immunohistochemistry using MPC5B4, indicated that breast tumour cell POSTN expression was a strong prognostic indicator, along with tumour size, lymph node, and human epidermal growth factor receptor 2 (HER2) status.
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Anticuerpos Monoclonales , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/patología , Moléculas de Adhesión Celular/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Secuencias de Aminoácidos , Animales , Especificidad de Anticuerpos , Sitios de Unión de Anticuerpos , Neoplasias de la Mama/metabolismo , Movimiento Celular/fisiología , Femenino , Humanos , Inmunohistoquímica , Ratones , Persona de Mediana Edad , Análisis de Matrices TisularesRESUMEN
We investigated the influence of the site of administration of DNA vaccine on the induced immune response. DNA vaccines were administered by electroporation at three different sites: tibial cranial muscle, abdominal skin and ear pinna. Aiming to draw general conclusions about DNA vaccine delivery, we successively used several plasmids encoding either luciferase and ovalbumin as models or gp160 and P1A as vaccines against HIV and P815 mastocytoma, respectively. Low levels and duration of luciferase transgene expression were observed after electroporation of the abdominal skin, partly explaining its lower immunogenic performance as compared to the other sites of administration. Analyses of OT-I CD8+ and OT-II CD4+ T cell responses highlighted the differential impact of the delivery site on the elicited immune response. Muscle electroporation induced the strongest humoral immune response and both muscle and ear pinna sites induced cellular immunity against gp160. Ear pinna delivery generated the highest level of CTL responses against P1A but electroporation of muscle and ear pinna were equally efficient in delaying P815 growth and improving mice survival. The present study demonstrated that the site of administration is a key factor to be tested in the development of DNA vaccine.
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Electroporación , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas contra el SIDA/administración & dosificación , Vacunas contra el SIDA/inmunología , Abdomen , Administración Cutánea , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Vacunas contra el Cáncer/inmunología , Pabellón Auricular , Femenino , Anticuerpos Anti-VIH/sangre , Anticuerpos Anti-VIH/inmunología , Proteínas gp160 de Envoltorio del VIH/genética , VIH-1/inmunología , Miembro Posterior , Inmunidad Celular , Inmunidad Humoral , Luciferasas/genética , Ratones Endogámicos C57BL , Músculo Esquelético , Plásmidos , Piel/inmunologíaRESUMEN
Inhalation aerosols offer a targeted therapy for respiratory diseases. However, the therapeutic efficacy of inhaled biopharmaceuticals is limited by the rapid clearance of macromolecules in the lungs. The aim of this research was to study the effects of the PEGylation of antibody fragments on their local residence time after administration to the respiratory tract. We demonstrate that the conjugation of a two-armed 40-kDa polyethylene glycol (PEG) chain to anti-interleukin-17A (IL-17A) F(ab')2 and anti-IL-13 Fab' greatly prolonged the presence of these fragments within the lungs of mice. The content of PEGylated antibody fragments within the lungs plateaued up to 4h post-delivery, whereas the clearance of unconjugated proteins started immediately after administration. Forty-eight hours post-delivery, F(ab')2 and Fab' contents in the lungs had decreased to 10 and 14% of the dose initially deposited, respectively. However, this value was 40% for both PEG40-F(ab')2 and PEG40-Fab'. The prolonged pulmonary residency of the anti-IL-17A PEG40-F(ab')2 translated into an improved efficacy in reducing lung inflammation in a murine model of house dust mite-induced lung inflammation. We demonstrate that PEGylated proteins were principally retained within the lung lumen rather than the nasal cavities or lung parenchyma. In addition, we report that PEG increased pulmonary retention of antibody fragments through mucoadhesion and escape from alveolar macrophages rather than increased hydrodynamic size or improved enzymatic stability. The PEGylation of proteins might find broad application in the local delivery of therapeutic proteins to diseased airways.
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Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos Fab de Inmunoglobulinas/química , Polietilenglicoles/química , Sistema Respiratorio/metabolismo , Alérgenos/inmunología , Animales , Líquido del Lavado Bronquioalveolar , Femenino , Interleucina-13/inmunología , Interleucina-17/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Neumonía/tratamiento farmacológico , Neumonía/metabolismo , Pyroglyphidae/inmunologíaRESUMEN
Acute graft versus host disease (aGVHD) remains a life-threatening complication of bone marrow transplantation. Here we show that IL-27, a member of the IL-12 cytokine family, plays an essential role in a parent-to-F1 murine aGVHD model, using B6 mice as parents and B6D2 mice as F1 recipients. IL-27 is transiently detectable in the serum of B6D2 recipients of B6 spleen cells, with a peak at day 10. Treatment with anti-IL-27p28 mAb MM27.7B1 (αp28Ab), at the time of and six days after B6 cell transfer, blocked GVHD. Protection was associated with host cell survival and undiminished engraftment of donor cells, lack of host B-cell depletion, increased Th2-type immunoglobulin production, a decrease in serum IFN-γ, a drop in anti-H-2D(d) cytotoxic T lymphocyte activity and an increase in Foxp3(+) T cells. We therefore conclude that IL-27 plays a critical role in the parent-to-F1 model of aGVHD and that blocking IL-27 could have therapeutic relevance.
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Enfermedad Injerto contra Huésped/etiología , Interleucinas/fisiología , Enfermedad Aguda , Animales , Proliferación Celular , Femenino , Interferón gamma/fisiología , Interleucinas/antagonistas & inhibidores , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Linfocitos T Citotóxicos/inmunología , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Pérdida de PesoRESUMEN
RATIONALE: Pseudomonas aeruginosa, a major problem pathogen responsible for severe infections in critically ill patients, triggers, through a functional type-3 secretion system (T3SS), the activation of an intracellular cytosolic sensor of innate immunity, NLRC4. Although the NLRC4-inflammasome-dependent response contributes to increased clearance of intracellular pathogens, it seems that NLRC4 inflammasome activation decreases the clearance of P. aeruginosa, a mainly extracellular pathogen. OBJECTIVES: We sought to determine the underlying mechanisms of this effect of the activation of NLRC4 by P. aeruginosa. METHODS: We established acute lung injury in wild-type and Nlrc4(-/-) mice using sublethal intranasal inocula of P. aeruginosa strain CHA expressing or not a functional T3SS. We studied 96-hour survival, lung injury, bacterial clearance from the lungs, cytokine secretion in bronchoalveolar lavage, lung antimicrobial peptide expression by quantitative polymerase chain reaction, and flow cytometry analysis of lung cells. MEASUREMENTS AND MAIN RESULTS: Nlrc4(-/-) mice showed enhanced bacterial clearance and decreased lung injury contributing to increased survival against extracellular P. aeruginosa strain expressing a functional T3SS. The mechanism involved decreased NLRC4-inflammasome-driven IL-18 secretion attenuating lung injury caused by excessive neutrophil recruitment. Additionally, in the lungs of Nlrc4(-/-) mice secretion of IL-17 by innate immune cells was increased and responsible for increased expression of lung epithelial antimicrobial peptides. Furthermore, IL-18 secretion was found to repress IL-17 and IL-17-driven lung antimicrobial peptide expression. CONCLUSIONS: We report a new role of the T3SS apparatus itself, independently of exotoxin translocation. Through NLRC4 inflammasome activation, the T3SS promotes IL-18 secretion, which dampens a beneficial IL-17-mediated antimicrobial host response.
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Lesión Pulmonar Aguda/microbiología , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas de Unión al Calcio/metabolismo , Inflamasomas/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Lesión Pulmonar Aguda/inmunología , Lesión Pulmonar Aguda/metabolismo , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/química , Caspasa 1/metabolismo , Células Cultivadas , Femenino , Citometría de Flujo , Inmunidad Innata , Interleucina-17/metabolismo , Interleucina-18/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Pseudomonas/inmunología , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Female mice of inbred strain CBA do not reject syngeneic male skin grafts even though they mount a T-cell response against the male-specific HY antigen. We show that local immunostimulation performed by injecting cytokines and Toll-like receptor ligands in close vicinity to the graft causes rejection. We feel that this approach should be tested in tumor-bearing human patients in combination with antitumor vaccination. Relief of intratumor immunosuppression may increase considerably the fraction of patients who respond to vaccination directed against tumor antigens recognized by T cells.
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Modelos Animales de Enfermedad , Rechazo de Injerto/inducido químicamente , Tolerancia Inmunológica/inmunología , Inmunización/métodos , Neoplasias/terapia , Traslado Adoptivo , Animales , Citocinas/efectos adversos , Citocinas/inmunología , Cartilla de ADN/genética , Femenino , Inmunización/efectos adversos , Masculino , Ratones , Ratones Endogámicos CBA , Neoplasias/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores Sexuales , Trasplante de Piel/métodosRESUMEN
OBJECTIVE: Systemic juvenile idiopathic arthritis (JIA) is unique among the rheumatic diseases of childhood, given its distinctive systemic inflammatory character. Inappropriate control of innate immune responses following an initially harmless trigger is thought to account for the excessive inflammatory reaction. The aim of this study was to generate a similar systemic inflammatory syndrome in mice by injecting a relatively innocuous, yet persistent, immune system trigger: Freund's complete adjuvant (CFA), containing heat-killed mycobacteria. METHODS: Given the central role of interferon-γ (IFNγ) in immune regulation, we challenged wild-type (WT) and IFNγ-knockout (KO) BALB/c mice with CFA, and analyzed their clinical symptoms and biologic characteristics. The production of cytokines and the effects of anticytokine antibodies were investigated. RESULTS: In WT mice, CFA injection resulted in splenomegaly, lymphadenopathy, neutrophilia, thrombocytosis, and increased cytokine expression. In the absence of IFNγ, these symptoms were more pronounced and were accompanied by weight loss, arthritis, anemia, hemophagocytosis, abundance of immature blood cells, and increased levels of interleukin-6 (IL-6), all of which are reminiscent of the symptoms of systemic JIA. CFA-challenged IFNγ-KO mice showed increased expression of IL-17 by CD4+ T cells and by innate γ/δ T cells. Inflammatory and hematologic changes were prevented by treatment with anti-IL-12/IL-23p40 and anti-IL-17 antibodies. CONCLUSION: Immune stimulation of IFNγ-KO mice with CFA produces a systemic inflammatory syndrome reflecting the clinical, biologic, and histopathologic picture of systemic JIA. The protective function of IFNγ in preventing anemia and overall systemic inflammation is a striking observation. The finding that both adaptive and innate T cells are important sources of IL-17 may be of relevance in the pathogenesis of systemic JIA.
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Artritis Juvenil/inducido químicamente , Artritis Juvenil/fisiopatología , Modelos Animales de Enfermedad , Adyuvante de Freund/efectos adversos , Sistema Inmunológico/fisiopatología , Interferón gamma/deficiencia , Interferón gamma/fisiología , Inmunidad Adaptativa/fisiología , Anemia/metabolismo , Anemia/fisiopatología , Animales , Artritis Juvenil/metabolismo , Citocinas/metabolismo , Femenino , Adyuvante de Freund/farmacología , Sistema Inmunológico/efectos de los fármacos , Inmunidad Innata/fisiología , Inflamación/metabolismo , Inflamación/fisiopatología , Interferón gamma/genética , Interleucina-17/metabolismo , Interleucina-6/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , SíndromeRESUMEN
This study aimed to construct DNA vaccines encoding the mouse P1A tumor antigen and to generate a protective immune response against the P815 mastocytoma, as a model for vaccines against human MAGE-type tumor antigens. DNA vaccines were constructed and delivered to mice by intramuscular electroporation before tumor challenge. Immunization with a plasmid coding for the full-length P1A significantly delayed tumor growth and mice survived at least 10 days longer than untreated controls. 10% of the mice completely rejected the P815 tumors while 50% of them showed a regression phase followed by tumor regrowth. Mice immunized by electroporation of a P1A(35-43) minigene-encoding plasmid failed to reject tumor and even delay tumor growth. The P1A(35-43)-encoding plasmid was modified and helper epitope sequences were inserted. However, these modified plasmids were not able to improve the response against P815 mastocytoma. Consistent with these results, a 12-fold higher CTL activity was observed when the plasmid coding for full-length P1A was delivered as compared to the plasmid encoding the P1A(35-43) epitope. Our results demonstrated that electroporation is an efficient method to deliver DNA vaccines against P815 and suggested the superiority of full-length as compared to minigene constructs for DNA vaccines.
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Electroporación , Mastocitoma/inmunología , Mastocitoma/patología , Músculos/metabolismo , Plásmidos/genética , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/química , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Proliferación Celular , Epítopos de Linfocito T/inmunología , Femenino , Inmunización , Ratones , Músculos/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Factores de Tiempo , Vacunas de ADN/metabolismoRESUMEN
Th17 cells are a proinflammatory subset of effector T cells that have been implicated in the pathogenesis of asthma. Their production of the cytokine IL-17 is known to induce local recruitment of neutrophils, but the direct impact of IL-17 on the lung epithelium is poorly understood. In this study, we describe a novel mouse model of spontaneous IL-17-driven lung inflammation that exhibits many similarities to asthma in humans. We have found that STAT3 hyperactivity in T lymphocytes causes an expansion of Th17 cells, which home preferentially to the lungs. IL-17 secretion then leads to neutrophil infiltration and lung epithelial changes, in turn leading to a chronic inflammatory state with increased mucus production and decreased lung function. We used this model to investigate the effects of IL-17 activity on airway epithelium and identified CXCL5 and MIP-2 as important factors in neutrophil recruitment. The neutralization of IL-17 greatly reduces pulmonary neutrophilia, underscoring a key role for IL-17 in promoting chronic airway inflammation. These findings emphasize the role of IL-17 in mediating neutrophil-driven pulmonary inflammation and highlight a new mouse model that may be used for the development of novel therapies targeting Th17 cells in asthma and other chronic pulmonary diseases.
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Asma/inmunología , Enfermedades del Sistema Inmune/inmunología , Interleucina-17/inmunología , Trastornos Leucocíticos/inmunología , Neutrófilos/inmunología , Mucosa Respiratoria/inmunología , Animales , Asma/metabolismo , Separación Celular , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Interleucina-17/metabolismo , Ratones , Ratones Endogámicos C57BL , Neumonía/inmunología , Neumonía/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Mucosa Respiratoria/metabolismo , Células Th17/inmunología , Células Th17/metabolismo , TransfecciónRESUMEN
IPF is a chronic, progressive pulmonary disease, leading to respiratory failure. In search of mechanisms of IPF, we used the bleomycin-induced lung-injury model in mice, which causes acute inflammation that may progress to chronic lung inflammation and fibrosis. Here, we asked whether CXCL6/GCP-2, a member of the CXC chemokine superfamily, may be involved in IPF development. First, we reported an increase of CXCL6 levels in BALF from patients with IPF, as well as in the lung of mice, 24 h after bleomycin administration. To investigate whether CXCL6 played a role in experimental bleomycin-induced pulmonary fibrosis, we treated mice with an anti-mCXCL6 mAb that has been shown to inhibit neutrophil chemotaxis in vitro. CXCL6 antibody blockade attenuated acute inflammation with a reduced pulmonary neutrophil influx, IL-1ß, CXCL1, and TIMP-1 production. In the later phase (14 days after bleomycin exposure), lymphocyte recruitment and fibrosis markers, such as collagen and TIMP-1, were diminished, as well as collagen deposition and fibrotic lesion the lung. Therefore, the data suggest that CXCL6 contributes to experimental pulmonary fibrosis, and CXCL6 inhibition might be used to reduce lung toxicity associated with bleomycin treatment.