RESUMEN
The ability to treat severe viral infections is limited by our understanding of the mechanisms behind virus-induced immunopathology. While the role of type I interferons (IFNs) in early control of viral replication is clear, less is known about how IFNs can regulate the development of immunopathology and affect disease outcomes. Here, we report that absence of type I IFN receptor (IFNAR) is associated with extensive immunopathology following mucosal viral infection. This pathology occurred independent of viral load or type II immunity but required the presence of macrophages and IL-6. The depletion of macrophages and inhibition of IL-6 signaling significantly abrogated immunopathology. Tissue destruction was mediated by macrophage-derived matrix metalloproteinases (MMPs), as MMP inhibition by doxycycline and Ro 28-2653 reduced the severity of tissue pathology. Analysis of post-mortem COVID-19 patient lungs also displayed significant upregulation of the expression of MMPs and accumulation of macrophages. Overall, we demonstrate that IFNs inhibit macrophage-mediated MMP production to prevent virus-induced immunopathology and uncover MMPs as a therapeutic target towards viral infections.
Asunto(s)
COVID-19 , Interferón Tipo I , Infecciones por Orthomyxoviridae , Humanos , Interleucina-6/metabolismo , Macrófagos/metabolismo , ProteolisisRESUMEN
Despite the remarkable success of chimeric antigen receptor (CAR)-T cells against hematologic malignancies, severe off-tumor effects have constrained their use against solid tumors. Recently, CAR-engineered natural killer (NK) cells have emerged as an effective and safe alternative. Here, we demonstrate that HER2 CAR-expression in NK cells from healthy donors and patients with breast cancer potently enhances their anti-tumor functions against various HER2-expressing cancer cells, regardless of MHC class I expression. Moreover, HER2 CAR-NK cells exert higher cytotoxicity than donor-matched HER2 CAR-T cells against tumor targets. Importantly, unlike CAR-T cells, HER2 CAR-NK cells do not elicit enhanced cytotoxicity or inflammatory cytokine production against non-malignant human lung epithelial cells with basal HER2 expression. Further, HER2 CAR-NK cells maintain high cytotoxic function in the presence of immunosuppressive factors enriched in solid tumors. These results show that CAR-NK cells may be a highly potent and safe source of immunotherapy in the context of solid tumors.
RESUMEN
Lung cancer remains the leading cause of cancer death worldwide despite the significant progress made by immune checkpoint inhibitors, including programmed death receptor-1 (PD1)/PD ligand 1 (PDL1)-blockade therapy. PD1/PDL1-blockade has achieved unprecedented tumor regression in some patients with advanced lung cancer. However, the majority of patients fail to respond to PD1/PDL1 inhibitors. The high rate of therapy non-response results from insufficient PDL1 expression on most patients' tumors and the presence of further immunosuppressive mechanisms in the tumor microenvironment. Here, we sensitize non-responding tumors from patients with lung cancer to PD1-blockade therapy using highly cytotoxic expanded natural killer (NK) cells. We uncover that NK cells expanded from patients with lung cancer dismantle the immunosuppressive tumor microenvironment by maintaining strong antitumor activity against both PDL1+ and PDL1- patient tumors. In the process, through a contact-independent mechanism involving interferon γ, expanded NK cells rescued tumor killing by exhausted endogenous TILs and upregulated the tumor proportion score of PDL1 across patient tumors. In contrast, unexpanded NK cells, which are susceptible to tumor-induced immunosuppression, had no effect on tumor PDL1. As a result, combined treatment of expanded NK cells and PD1-blockade resulted in robust synergistic tumor destruction of initially non-responding patient tumors. Thus, expanded NK cells may overcome the critical roadblocks to extending the prodigious benefits of PD1-blockade therapy to more patients with lung cancer and other tumor types.
Asunto(s)
Antígeno B7-H1/metabolismo , Técnicas de Cocultivo/métodos , Inhibidores de Puntos de Control Inmunológico/farmacología , Células Asesinas Naturales/citología , Neoplasias Pulmonares/inmunología , Células A549 , Anciano , Anciano de 80 o más Años , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Interferón gamma/metabolismo , Células K562 , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/trasplante , Neoplasias Pulmonares/terapia , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Persona de Mediana Edad , Microambiente TumoralAsunto(s)
Citocinas/biosíntesis , Interferón beta/fisiología , Activación de Macrófagos/fisiología , Fagocitosis/fisiología , Animales , Escherichia coli/inmunología , Genes MHC Clase II/genética , Interleucina-10/fisiología , Interleucinas/biosíntesis , Ratones , Células RAW 264.7 , Transducción de Señal , Staphylococcus aureus/inmunología , Receptores Toll-Like/fisiologíaRESUMEN
NK cells are a key antiviral component of the innate immune response to HSV-2, particularly through their production of IFN-γ. It is still commonly thought that type I IFN activates NK cell function; however, rather than requiring the type I IFN receptor themselves, we have previously found that type I IFN activates NK cells through an indirect mechanism involving inflammatory monocytes and IL-18. Here, we further show that direct action of type I IFN on NK cells, rather than inducing IFN-γ, negatively regulates its production during HSV-2 infection and cytokine stimulation. During infection, IFN-γ is rapidly induced from NK cells at day 2 post-infection and then immediately downregulated at day 3 post-infection. We found that this downregulation of IFN-γ release was not due to a loss of NK cells at day 3 post-infection, but negatively regulated through IFN signaling on NK cells. Absence of IFNAR on NK cells led to a significantly increased level of IFN-γ compared to WT NK cells after HSV-2 infection in vitro. Further, priming of NK cells with type I IFN was able to suppress cytokine-induced IFN-γ production from both human and mouse NK cells. We found that this immunosuppression was not mediated by IL-10. Rather, we found that type I IFN induced a significant increase in Axl expression on human NK cells. Overall, our data suggests that type I IFN negatively regulates NK cell IFN-γ production through a direct mechanism in vitro and during HSV-2 infection.
Asunto(s)
Interferón gamma/biosíntesis , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Receptor de Interferón alfa y beta/metabolismo , Animales , Biomarcadores , Citocinas/metabolismo , Regulación de la Expresión Génica , Herpes Simple/inmunología , Herpes Simple/metabolismo , Herpes Simple/virología , Herpesvirus Humano 2/fisiología , Humanos , Inmunofenotipificación , Interleucina-10/biosíntesis , Recuento de Linfocitos , Ratones , Ratones Transgénicos , Receptor de Interferón alfa y beta/genéticaRESUMEN
Despite effective new treatments for Hepatitis C virus (HCV) infection, development of drug resistance, safety concerns and cost are remaining challenges. More importantly, there is no vaccine available against hepatitis C infection. Recent data suggest that there is a strong correlation between spontaneous HCV clearance and human NK cell function, particularly IFN-γ production. Further, IL-15 has innate antiviral activity and is also one of the main factors that activates NK cells to produce IFN-γ. To examine whether IL-15 and IFN-γ have direct antiviral activity against HCV, Huh7.5 cells were treated with either IFN-γ or IL-15 prior to HCV infection. Our data demonstrate that IFN-γ and IL-15 block HCV replication in vitro. Additionally, we show that IL-15 and IFN-γ do not induce anti-HCV effects through the type I interferon signaling pathway or nitric oxide (NO) production. Instead, IL-15 and IFN-γ provide protection against HCV via the ERK pathway. Treatment of Huh7.5 cells with a MEK/ERK inhibitor abrogated the anti-HCV effects of IL-15 and IFN-γ and overexpression of ERK1 prevented HCV replication compared to control transfection. Our in vitro data support the hypothesis that early production of IL-15 and activation of NK cells in the liver lead to control of HCV replication.
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Hepacivirus/fisiología , Interferón gamma/farmacología , Interleucina-15/farmacología , Células Asesinas Naturales/inmunología , Hígado/inmunología , Hígado/virología , Sistema de Señalización de MAP Quinasas/inmunología , Replicación Viral , Antivirales/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Hepacivirus/efectos de los fármacos , Hepacivirus/inmunología , Humanos , Inmunidad Innata/efectos de los fármacos , Interferón Tipo I/metabolismo , Interferón Tipo I/farmacología , Interferón-alfa/farmacología , Hígado/efectos de los fármacos , Hígado/fisiopatología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/genética , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Óxido Nítrico/farmacología , Regulación hacia Arriba , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
Natural killer (NK) cells are useful for cancer immunotherapy and have proven clinically effective against hematologic malignancies. However, immunotherapies for poor prognosis solid malignancies, including ovarian cancer, have not been as successful due to immunosuppression by solid tumors. Although rearming patients' own NK cells to treat cancer is an attractive option, success of that strategy is limited by the impaired function of NK cells from cancer patients and by inhibition by self-MHC. In this study, we show that expansion converts healthy donor and immunosuppressed ovarian cancer patient NK cells to a cytotoxic CD56superbrightCD16+ subset with activation state and antitumor functions that increase with CD56 brightness. We investigated whether these expanded NK cells may overcome the limitations of autologous NK cell therapy against solid tumors. Peripheral blood- and ascites-derived NK cells from ovarian cancer patients were expanded and then adoptively transferred into cell-line and autologous patient-derived xenograft models of human ovarian cancer. Expanded ovarian cancer patient NK cells reduced the burden of established tumors and prolonged survival. These results suggest that CD56bright NK cells harbor superior antitumor function compared with CD56dim cells. Thus, NK cell expansion may overcome limitations on autologous NK cell therapy by converting the patient's NK cells to a cytotoxic subset that exerts a therapeutic effect against autologous tumor. These findings suggest that the value of expanded autologous NK cell therapy for ovarian cancer and other solid malignancies should be clinically assessed. Cancer Immunol Res; 6(10); 1174-85. ©2018 AACR.
Asunto(s)
Antígeno CD56/inmunología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Receptores de IgG/inmunología , Animales , Ascitis/inmunología , Línea Celular Tumoral , Células Cultivadas , Femenino , Humanos , Inmunoterapia Adoptiva , Ratones Transgénicos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Ovarian cancer (OC) is the leading cause of gynecological cancer-related death in North America. Most ovarian cancer patients (OCPs) experience disease recurrence after first-line surgery and chemotherapy; thus, there is a need for novel second-line treatments to improve the prognosis of OC. Although peripheral blood-derived NK cells are known for their ability to spontaneously lyse tumour cells without prior sensitization, ascites-derived NK cells (ascites-NK cells) isolated from OCPs exhibit inhibitory phenotypes, impaired cytotoxicity and may play a pro-tumourigenic role in cancer progression. Therefore, it is of interest to improve the cytotoxic effector function of impaired OCP ascites-NK cells at the tumour environment. We investigated the efficacy of using an artificial APC-based ex vivo expansion technique to generate cytotoxic, expanded NK cells from previously impaired OCP ascites-NK cells, for use in an autologous model of NK cell immunotherapy. We are the first to obtain a log-scale expansion of OCP ascites-NK cells that upregulate the surface expression of activating receptors NKG2D, NKp30, NKp44, produce robust amounts of anti-tumour cytokines in the presence of OC cells and mediate direct tumour cytotoxicity against ascites-derived, primary OC cells obtained from autologous patients. Our findings demonstrate that it is possible to generate cytotoxic OCP ascites-NK cells from previously impaired OCP ascites-NK cells, which presents a promising immunotherapeutic target for the second-line treatment of OC. Future work should focus on evaluating the in vivo efficacy of autologous NK cell immunotherapy through the intraperitoneal delivery of NK cell expansion factors to a preclinical xenograft mouse model of human OC.
Asunto(s)
Ascitis/inmunología , Citotoxicidad Inmunológica/inmunología , Inmunoterapia , Células Asesinas Naturales/inmunología , Neoplasias Ováricas/inmunología , Ascitis/metabolismo , Proliferación Celular , Citocinas/metabolismo , Femenino , Humanos , Células Asesinas Naturales/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/terapia , Células Tumorales CultivadasRESUMEN
With over 600,000 units of umbilical cord blood (CB) stored on a global scale, it is important to elucidate the therapeutic abilities of this cryopreserved reservoir. In the advancing field of natural killer (NK) cell cancer immunotherapy, CB has proven to be a promising and noninvasive source of therapeutic NK cells. Although studies have proven the clinical efficacy of using long-term cryopreserved CB in the context of hematopoietic stem cell transplantations, little is known about its use for the ex vivo expansion of effector immune cells. Therefore, our group sought to derive ex vivo-expanded NK cells from long-term cryopreserved CB, using an artificial antigen presenting cell-mediated expansion technique. We compared the expansion potential and antitumor effector function of CB-derived NK (CB-NK) cells expanded from fresh (n=4), short-term cryopreserved (<1-year old, n=5), and long-term cryopreserved (1-10-year old, n=5) CB. Here, we demonstrated it is possible to obtain an exponential amount of expanded CB-NK cells from long-term cryopreserved CB. Ex vivo-expanded CB-NK cells had an increased surface expression of activating markers and showed potent antitumor function by producing robust levels of proinflammatory cytokines, interferon-γ, and tumor necrosis factor-α. Moreover, expanded CB-NK cells (n=3-5) demonstrated cytotoxicity towards primary breast cancer cells (n=2) derived from a triple-negative breast cancer and an estrogen receptor-positive/progesterone receptor-positive breast cancer patient. Long-term cryopreservation had no effect on the expansion potential or effector function of expanded CB-NK cells. Therefore, we propose that long-term cryopreserved CB remains clinically useful for the ex vivo expansion of therapeutic NK cells.
Asunto(s)
Neoplasias de la Mama/inmunología , Citotoxicidad Inmunológica , Sangre Fetal/citología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Biomarcadores de Tumor , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Criopreservación , Citocinas/metabolismo , Humanos , Activación de LinfocitosRESUMEN
Adoptive immune cell therapy is emerging as a promising immunotherapy for cancer. Particularly, the adoptive transfer of NK cells has garnered attention due to their natural cytotoxicity against tumor cells and safety upon adoptive transfer to patients. Although strategies exist to efficiently generate large quantities of expanded NK cells ex vivo, it remains unknown whether these expanded NK cells can persist and/or proliferate in vivo in the absence of exogenous human cytokines. Here, we have examined the adoptive transfer of ex vivo expanded human cord blood-derived NK cells into humanized mice reconstituted with autologous human cord blood immune cells. We report that ex vivo expanded NK cells are able to survive and possibly proliferate in vivo in humanized mice without exogenous cytokine administration, but not in control mice that lack human immune cells. These findings demonstrate that the presence of autologous human immune cells supports the in vivo survival of ex vivo expanded human NK cells. These results support the application of ex vivo expanded NK cells in cancer immunotherapy and provide a translational humanized mouse model to test the lifespan, safety, and functionality of adoptively transferred cells in the presence of autologous human immune cells prior to clinical use.
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Traslado Adoptivo/métodos , Proliferación Celular , Citotoxicidad Inmunológica/inmunología , Células Asesinas Naturales/trasplante , Animales , Línea Celular Tumoral , Supervivencia Celular/inmunología , Células Cultivadas , Sangre Fetal/citología , Humanos , Inmunoterapia Adoptiva/métodos , Células K562 , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Ratones Endogámicos NOD , Ratones Noqueados , Neoplasias/inmunología , Neoplasias/terapiaRESUMEN
BACKGROUND: Natural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours. METHODS: We used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro. RESULTS: After 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro. CONCLUSION: NK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.
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Neoplasias de la Mama/inmunología , Citotoxicidad Inmunológica , Células Asesinas Naturales/inmunología , Animales , Biomarcadores , Neoplasias de la Mama/terapia , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Femenino , Xenoinjertos , Humanos , Inmunofenotipificación , Inmunoterapia Adoptiva , Células Asesinas Naturales/metabolismo , Ratones , Ratones Noqueados , Receptor ErbB-2/metabolismoRESUMEN
The requirement of type I interferon (IFN) for natural killer (NK) cell activation in response to viral infection is known, but the underlying mechanism remains unclear. Here, we demonstrate that type I IFN signaling in inflammatory monocytes, but not in dendritic cells (DCs) or NK cells, is essential for NK cell function in response to a mucosal herpes simplex virus type 2 (HSV-2) infection. Mice deficient in type I IFN signaling, Ifnar-/- and Irf9-/- mice, had significantly lower levels of inflammatory monocytes, were deficient in IL-18 production, and lacked NK cell-derived IFN-γ. Depletion of inflammatory monocytes, but not DCs or other myeloid cells, resulted in lower levels of IL-18 and a complete abrogation of NK cell function in HSV-2 infection. Moreover, this resulted in higher susceptibility to HSV-2 infection. Although Il18-/- mice had normal levels of inflammatory monocytes, their NK cells were unresponsive to HSV-2 challenge. This study highlights the importance of type I IFN signaling in inflammatory monocytes and the induction of the early innate antiviral response.
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Herpes Simple/inmunología , Interferón Tipo I/fisiología , Interleucina-18/fisiología , Células Asesinas Naturales/inmunología , Monocitos/fisiología , Transducción de Señal/fisiología , Animales , Herpesvirus Humano 2/inmunología , Inmunidad Innata , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/fisiología , Interferón gamma/biosíntesis , Interleucina-15/fisiología , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Receptor de Interferón alfa y beta/fisiologíaRESUMEN
Endotoxin, or LPS tolerance, is an immunomodulatory mechanism that results in a significantly diminished response to secondary LPS exposure, which may serve to protect the host against endotoxic shock. Type I interferons (IFNs) are cytokines released upon LPS binding to TLR4 and have been shown to have immunomodulatory properties. Due to this regulatory function of type I IFN, we aimed to investigate the role of type I IFN signalling in LPS tolerance. Our data suggests that type I IFN does not play a role in LPS tolerance in vitro, as both wild type and IFNAR1-/- peritoneal macrophages showed reduced cytokine production after secondary LPS exposure. Furthermore, both wild type and IFNAR1-/- mice were protected from a lethal dose of LPS after receiving three small doses to induce tolerance. However, IFNAR-/- mice seemed to recover faster than wild type mice, suggesting type I IFN signalling plays a detrimental role in LPS-induced sepsis. Although type I IFN may have a regulatory function in microbial infections, it does not seem to play a role in endotoxin tolerance. Type I IFN involvement in bacterial infection remains complex and further studies are needed to define the regulatory function of type I IFN signalling.
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Interferón Tipo I/fisiología , Lipopolisacáridos/toxicidad , Choque Séptico/inmunología , Transducción de Señal , Animales , Células Cultivadas , Tolerancia a Medicamentos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/genéticaRESUMEN
Natural killer (NK) cells have an important role in mounting protective innate responses against genital herpes simplex virus type 2 (HSV-2) infections. However their role as effectors in adaptive immune responses against HSV-2 is unclear. Here, we demonstrate that NK cells from C57BL/6 mice in an ex vivo splenocyte culture produce significantly more interferon γ (IFN-γ) upon re-exposure to HSV-2 antigens in a mouse model of genital HSV-2 immunization. We find that naïve NK cells do not require any prior stimulation or priming to be activated to produce IFN-γ. Our results demonstrate that HSV-2-experienced CD4+ T cells have a crucial role in coordinating NK cell activation and that their presence during HSV-2 antigen presentation is required to activate NK cells in this model of secondary immune response. We also examined the requirement of cell-to-cell contacts for both CD4+ T cells and NK cells. NK cells are dependent on direct interactions with other HSV-2-experienced splenocytes, and CD4+ T cells need to be in close proximity to NK cells to activate them. This study revealed that NK cells do not exhibit any memory toward HSV-2 antigens and, in fact, require specific interactions with HSV-2-experienced CD4+ T cells to produce IFN-γ.
Asunto(s)
Antígenos Virales/inmunología , Linfocitos T CD4-Positivos/inmunología , Herpesvirus Humano 2/inmunología , Memoria Inmunológica , Células Asesinas Naturales/inmunología , Animales , Femenino , Inmunización , Interferón gamma/biosíntesis , Interleucina-2/metabolismo , Células Asesinas Naturales/citología , Activación de Linfocitos , Ratones Endogámicos C57BL , Bazo/citologíaRESUMEN
BACKGROUND: Humanized mouse models are an increasingly popular preclinical model to study the human immune response in a biological system. There are a variety of protocols to generate these mice, each differing in the strain of the recipient, source of hematopoietic stem cells, and mode of transplantation. Though there is well-documented reconstitution information regarding the spleen, blood, and bone marrow, there is little information regarding reconstitution of the lymph node and liver. In this report, we sought to compare reconstitution levels in a variety of immunological tissues, including the lymph node and liver, between mice engrafted intravenously as adults and intrahepatically in newborns. RESULTS: CD34+ cells were enriched from cord blood and transplanted intravenously into irradiated adult NOD-Rag1(-/-)IL2rγ(-/-) (NRG) mice or intra-hepatically into irradiated newborn NRG mice. At 9-28 weeks post-engraftment, immunological tissues were processed and analyzed for human lymphoid and myeloid subsets. Adult and newborn engrafted humanized mice were comparable in long-term reconstitution of human CD45 cells and subsequent lymphoid and myeloid subsets in the spleen, bone marrow, thymus, lymph node, and liver. Mice engrafted as newborns had a higher level of T-cells and a lower level of B-cells compared to mice engrafted as adults. We observed significant levels of human immune cell engraftment in both the lymph node and the liver, with a predominant adaptive immune population in both compartments. CONCLUSIONS: Human immune cells repopulate liver and mesenteric lymph nodes of NRG mice and can be used to study the human immune system in the gastrointestinal tract.
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Envejecimiento/inmunología , Linfocitos B/fisiología , Células Madre Hematopoyéticas/inmunología , Hígado/fisiología , Ganglios Linfáticos/fisiología , Linfocitos T/fisiología , Animales , Animales Recién Nacidos , Antígenos CD34/metabolismo , Autorrenovación de las Células , Supervivencia Celular , Células Cultivadas , Trasplante de Células Madre Hematopoyéticas , Proteínas de Homeodominio/genética , Humanos , Ratones , Ratones Noqueados , Quimera por TrasplanteRESUMEN
BACKGROUND: Natural Killer (NK) cells play an important role in tumor prevention, but once tumors form, the numbers as well as the cytotoxic functions of NK cells are reduced. IL-15 is a cytokine that increases and activates NK cells. Here we will examine the anti-tumor role of IL-15 in a spontaneous breast cancer model. METHODS: To achieve this, Polyoma Middle T (MT) mice that form spontaneous breast cancer were crossed with mice that either overexpress IL-15 (IL-15 transgenic (TG)) or mice that lack IL-15 (IL-15 knockout (KO)). We compared survival curves and tumor formation in IL-15 KO/MT, MT and IL-15 TG/MT groups. In addition, the phenotype, activation and contribution of NK cells and CD8 T cells to tumor formation were examined in each of these mouse strains via flow cytometry, ELISA, adoptive transfer and antibody depletion experiments. RESULTS: IL-15KO/MT tumors formed and progressed to endpoint more quickly than MT tumors. These tumors displayed little apoptosis and poor CD8 T cell infiltration. In contrast, IL-15 TG/MT mice had increased survival and the tumors displayed extensive cell death, high proportions of activated NK cells and a higher infiltration of CD8 T cells than MT tumors. CD8 T cells in IL-15 TG/MT tumors were capable of secreting IFNγ, possessed markers of memory, did not display an exhausted phenotype and were frequently NK1.1+. Long-term antibody depletion studies in IL-15 TG/MT mice revealed that NK1.1+, but not CD8 T cells, were critical for tumor destruction. Lastly, human NK cells, when exposed to a similar cytokine environment as that found in IL-15TG/MT tumors, were capable of killing human breast cancer cells. CONCLUSIONS: This study reveals that high levels of IL-15 can promote tumor destruction and reduce metastasis in breast cancer via effects on NK1.1+ cells. Our results suggest that strategies aimed at increasing NK cell activation may be effective against solid epithelial cancers.
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Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/metabolismo , Neoplasias Mamarias Animales/metabolismo , Animales , Antígenos Ly/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Interleucina-15/genética , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Activación de Linfocitos , Neoplasias Mamarias Animales/inmunología , Neoplasias Mamarias Animales/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Subfamilia B de Receptores Similares a Lectina de Células NK/metabolismoRESUMEN
NK cells play a vital role in innate anti-tumor immunity. Crosstalk between NK cells and dendritic cells (DCs) has come to the forefront in protection against tumors in the context of DC vaccines. We previously discovered that NK cell activation mediates the anti-tumor activity elicited by DC vaccines in response to melanoma tumor challenge in a murine lung metastasis model. In this study, we sought to explore the mechanism behind this NK-DC communication, specifically looking at the involvement of IL-15 and type I IFN signaling. Using DCs from IL-15(-/-) and IL-15Rα(-/-) mice, we found that the anti-tumor effect of the vaccine remained comparable with DCs from wild type mice. Moreover, DCs derived from IFN-α/ßR(-/-) mice also maintained their anti-tumor effect. Interestingly, endogenous DCs were found to accumulate in the draining lymph nodes post-immunization and their depletion abolished the anti-tumor effect of the vaccine. Our findings suggest the important role that type I IFN signaling and endogenous DCs play in DC vaccine-mediated anti-tumor protection. Our data suggest that type I IFNs from vaccine DCs activate host DCs to provide NK cell-mediated anti-tumor immunity.
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Células Dendríticas/fisiología , Interleucina-15/metabolismo , Células Asesinas Naturales/fisiología , Melanoma/terapia , Receptores de Interleucina-15/metabolismo , Neoplasias Cutáneas/terapia , Animales , Vacunas contra el Cáncer/inmunología , Comunicación Celular/genética , Células Dendríticas/trasplante , Femenino , Humanos , Inmunidad Innata , Interferón Tipo I/metabolismo , Interferón-alfa/genética , Interleucina-15/genética , Melanoma/inmunología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Metástasis de la Neoplasia , Receptores de Interleucina-15/genética , Transducción de Señal/genética , Neoplasias Cutáneas/inmunologíaRESUMEN
Interleukin-15 (IL-15) is an immunomodulatory cytokine that affects body mass regulation independent of lymphocytes; however, the underlying mechanism(s) involved remains unknown. In an effort to investigate these mechanisms, we performed metabolic cage studies, assessed intestinal bacterial diversity and macronutrient absorption, and examined adipose mitochondrial activity in cultured adipocytes and in lean IL-15 transgenic (IL-15tg), overweight IL-15 deficient (IL-15-/-), and control C57Bl/6 (B6) mice. Here we show that differences in body weight are not the result of differential activity level, food intake, or respiratory exchange ratio. Although intestinal microbiota differences between obese and lean individuals are known to impact macronutrient absorption, differing gut bacteria profiles in these murine strains does not translate to differences in body weight in colonized germ free animals and macronutrient absorption. Due to its contribution to body weight variation, we examined mitochondrial factors and found that IL-15 treatment in cultured adipocytes resulted in increased mitochondrial membrane potential and decreased lipid deposition. Lastly, IL-15tg mice have significantly elevated mitochondrial activity and mass in adipose tissue compared to B6 and IL-15-/- mice. Altogether, these results suggest that IL-15 is involved in adipose tissue regulation and linked to altered mitochondrial function.
Asunto(s)
Tejido Adiposo/citología , Interleucina-15/metabolismo , Mitocondrias/metabolismo , Tamaño Mitocondrial , Células 3T3-L1 , Animales , Peso Corporal , Quimiocinas/biosíntesis , Femenino , Regulación de la Expresión Génica , Humanos , Interleucina-15/deficiencia , Interleucina-6/biosíntesis , Intestinos/microbiología , Masculino , Potencial de la Membrana Mitocondrial , Ratones , Ratones Transgénicos , Microbiota , Sobrepeso/metabolismo , Sobrepeso/patologíaRESUMEN
The immediate-early protein ICP0 from herpes simplex virus 1 (HSV-1) plays pleiotropic roles in promoting viral lytic replication and reactivation from latency. Most of the known actions of ICP0 occur in the nucleus and are thought to involve the E3 ubiquitin ligase activity of its RING finger domain, which targets proteins for degradation via the proteasome. Although ICP0 translocates to the cytoplasm as the infection progresses, little is known about its activities in this location. Here, we show that cytoplasmic ICP0 has two distinct functions. In primary cell cultures and in an intravaginal mouse model, cytoplasmic ICP0 promotes viral replication in the absence of an intact RING finger domain. Additionally, ICP0 blocks the activation of interferon regulatory factor 3 (IRF3), a key transcription factor of the innate antiviral response, in a mechanism that requires the RING finger domain but not the proteasome. To our knowledge, this is the first observation of a proteasome-independent function of the RING finger domain of ICP0. Collectively, these results underscore the importance of cytoplasm-localized ICP0 and the diverse nature of its activities. Importance: Despite ICP0 being a well-studied viral protein, the significance of its cytoplasmic localization has been largely overlooked. This is, in part, because common experimental manipulations result in the restriction of ICP0 to the nucleus. By overcoming this constraint, we both further characterize the ability of cytoplasmic ICP0 to inhibit antiviral signaling and show that ICP0 at this site has unexpected activities in promoting viral replication. This demonstrates the importance of considering location when analyzing protein function and adds a new perspective to our understanding of this multifaceted protein.
Asunto(s)
Citoplasma/virología , Herpesvirus Humano 1/inmunología , Herpesvirus Humano 1/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Factor 3 Regulador del Interferón/antagonistas & inhibidores , Dominios RING Finger , Ubiquitina-Proteína Ligasas/metabolismo , Replicación Viral , Animales , Línea Celular , Modelos Animales de Enfermedad , Femenino , Herpes Simple/virología , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
Obesity is a chronic inflammatory condition characterized by activation and infiltration of proinflammatory immune cells and a dysregulated production of proinflammatory cytokines. While known as a key regulator of immune natural killer (NK) cell function and development, we have recently demonstrated that reduced expression of the cytokine Interleukin-15 (IL-15) is closely linked with increased body weight and adiposity in mice and humans. Previously, we and others have shown that obese individuals have lower circulating levels of IL-15 and NK cells. Lean IL-15 overexpressing (IL-15 tg) mice had an accumulation in adipose NK cells compared to wildtype and NK cell deficient obese IL-15(-/-) mice. Since IL-15 induces weight loss in IL-15(-/-) and diet induced obese mice and has effects on various lymphocytes, the aim of this paper was to determine if lymphocytes, particularly NK cells, play a role in IL-15 mediated weight loss. Acute IL-15 treatment resulted in an increased accumulation of NK, NKT, and CD3(+) T cells in adipose tissue of B6 mice. Mice depleted of NK and NKT cells had similar weight loss comparable to controls treated with IL-15. Finally, IL-15 treatment induces significant weight loss in lymphocyte deficient RAG2(-/-)γc(-/-) mice independent of food intake. Fat pad cross-sections show decreased pad size with cytokine treatment is due to adipocyte shrinkage. These results clearly suggest that IL-15 mediates weight loss independent of lymphocytes.