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Objectives: Autologous chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies achieves long-term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR-T manufacturing, the compromised fitness of patient-derived T cells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR-NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. Methods: To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in-depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR-NK manufacturing pipeline. Results: We identified adult peripheral blood-derived NK cells to be the superior source for generating a CAR-NK cell product because of a higher maximum yield of CAR-expressing NK cells combined with potent natural, as well as CAR-mediated anti-tumor effector functions. Conclusions: Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre- and post-transduction and high on-target cytotoxicity make peripheral blood-derived NK cells a feasible and attractive CAR-NK cell product for clinical utility.
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NKTR-255 is a PEG conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans or cis presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naive and memory OVA-specific CD8+ T cells (OT-I) and NK cells in mice. NKTR-255 induced a 2.5- and 2.0-fold expansion of CD8+ T and NK cells, respectively, in WT mice. In adoptive transfer studies, proliferation of naive and memory WT OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα-/- mice, suggesting trans presentation was not utilized by NKTR-255. Interestingly, naive IL-15Rα-/- OT-I cells had deficient responses to NKTR-255, while memory IL-15Rα-/- OT-I cell responses were partially impaired, suggesting that naive CD8+ T cells are more dependent on cis presentation of NKTR-255 than memory CD8+ T cells. In bone marrow chimera studies, IL-15Rα-/- and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells, showing that conversion to IL-15Rß agonist biases the response toward NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis presentation and act as an IL-15Rαß agonist on CD8+ T cells.
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Linfocitos T CD8-positivos/efectos de los fármacos , Interleucina-15/farmacología , Células Asesinas Naturales/efectos de los fármacos , Animales , Linfocitos T CD8-positivos/inmunología , Femenino , Memoria Inmunológica , Interleucina-15/química , Subunidad alfa del Receptor de Interleucina-15/fisiología , Subunidad beta del Receptor de Interleucina-2/agonistas , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Polietilenglicoles/químicaRESUMEN
Expression of tissue-restricted antigens (TRAs) in thymic epithelial cells (TECs) ensures negative selection of highly self-reactive T cells to establish central tolerance. Whether some of these TRAs could exert their canonical biological functions to shape thymic environment to regulate T cell development is unclear. Analyses of publicly available databases have revealed expression of transcripts at various levels of many cytokines and cytokine receptors such as IL-15, IL-15Rα, IL-13, and IL-23a in both human and mouse TECs. Ablation of either IL-15 or IL-15Rα in TECs selectively impairs type 1 innate like T cell, such as iNKT1 and γδT1 cell, development in the thymus, indicating that TECs not only serve as an important source of IL-15 but also trans-present IL-15 to ensure type 1 innate like T cell development. Because type 1 innate like T cells are proinflammatory, our data suggest the possibility that TEC may intrinsically control thymic inflammatory innate like T cells to influence thymic environment.
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Células Epiteliales/metabolismo , Inmunidad Innata , Interleucina-15/metabolismo , Receptores de Interleucina-15/metabolismo , Subgrupos de Linfocitos T/metabolismo , Timocitos/metabolismo , Timo/metabolismo , Animales , Comunicación Celular , Microambiente Celular , Bases de Datos Genéticas , Células Epiteliales/inmunología , Humanos , Interleucina-15/genética , Linfocitos Intraepiteliales/inmunología , Linfocitos Intraepiteliales/metabolismo , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/metabolismo , Receptores de Interleucina-15/genética , Transducción de Señal , Subgrupos de Linfocitos T/inmunología , Timocitos/inmunología , Timo/citología , Timo/inmunologíaRESUMEN
Type I interferon (IFN-I) has potent anti-tumor effects against urothelial carcinoma (UC) and may be an alternative treatment option for patients who do not respond to Bacillus Calmette-Guerin. However, the mechanisms that mediate the IFN-I-stimulated immune responses against UC have yet to be elucidated. Herein, we evaluated the anti-tumor mechanisms of IFN-I in UC in human patients and in mice. Patient tumors from a Phase I clinical trial with adenoviral interferon-α (Ad-IFNα/Syn3) showed increased expression of T cell and checkpoint markers following treatment with Ad-IFNα/Syn3 by RNAseq and immunohistochemistry analysis in 25% of patients. In mice, peritumoral injections of poly(I:C) into MB49 UC tumors was used to incite an IFN-driven inflammatory response that significantly inhibited tumor growth. IFN-I engaged both innate and adaptive cells, seen in increased intratumoral CD8 T cells, NK cells, and CD11b+Ly6G+ cells, but tumor inhibition was not reliant on any one immune cell type. Nonetheless, poly(I:C)-mediated tumor regression and change in the myeloid cell landscape was dependent on IL-6. Mice were also treated with poly(I:C) in combination with anti-PD-1 monoclonal antibody (mAb) to assess for additional benefit to tumor growth and animal survival. When used in combination with anti-PD-1 mAb, IFN-I stimulation prolonged survival, coinciding with inhibition of angiogenesis and enriched gene signatures of metabolism, extracellular matrix organization, and MAPK/AKT signaling. Altogether, these findings suggest IFN-I's immune-driven antitumor response in UC is mediated by IL-6 and a collaboration of immune cells, and its use in combination with checkpoint blockade therapy can increase clinical benefit.
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Previous studies have provided evidence that IL-15 expression within human tumors is crucial for optimal antitumor responses; however, the regulation of IL-15 within the tumor microenvironment (TME) is unclear. We report herein, in analyses of mice implanted with various tumor cell lines, soluble IL-15/IL-15Rα complexes (sIL-15 complexes) are abundant in the interstitial fluid of tumors with expression preceding the infiltration of tumor-infiltrating lymphocytes. Moreover, IL-15 as well as type I IFN, which regulates IL-15, was required for establishing normal numbers of CD8 T cells and natural killer cells in tumors. Depending on tumor type, both the tumor and the stroma are sources of sIL-15 complexes. In analyses of IL-15 reporter mice, most myeloid cells in the TME express IL-15 with CD11b+Ly6Chi cells being the most abundant, indicating there is a large source of IL-15 protein in tumors that lies sequestered within the tumor stroma. Despite the abundance of IL-15-expressing cells, the relative levels of sIL-15 complexes are low in advanced tumors but can be up-regulated by local stimulator of IFN genes (STING) activation. Furthermore, while treatment of tumors with STING agonists leads to tumor regression, optimal STING-mediated immunity and regression of distant secondary tumors required IL-15 expression. Overall, our study reveals the dynamic regulation of IL-15 in the TME and its importance in antitumor immunity. These findings provide insight into an unappreciated attribute of the tumor landscape that contributes to antitumor immunity, which can be manipulated therapeutically to enhance antitumor responses.
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Regulación Neoplásica de la Expresión Génica/inmunología , Interleucina-15/inmunología , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Microambiente Tumoral/inmunología , Animales , Línea Celular Tumoral , Inflamación/genética , Inflamación/inmunología , Inflamación/patología , Interleucina-15/genética , Melanoma/genética , Melanoma/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Receptores de Interleucina-15/genética , Receptores de Interleucina-15/inmunología , Microambiente Tumoral/genéticaRESUMEN
BACKGROUND AND PURPOSE: BCG unresponsive bladder cancer is an inherently resistant disease state for which the preferred treatment is radical cystectomy. To date, no effective intravesical therapies exist for patients who possess these resistant tumors. For this reason, many research groups are actively investigating/testing novel therapeutic agents to aid in bladder preservation for this patient population. This review article describes our 15-year experience developing and testing IFN-based gene therapy. METHODS: A comprehensive review was performed of all studies pertaining to IFN-based gene therapy for non-muscle invasive bladder cancer from 2003 to 2018. RESULTS AND CONCLUSIONS: Over the past two decades, gene therapy has evolved into a powerful tool in our fight against cancer. After overcoming the initial barriers associated with gene delivery to the bladder, we have made significant strides forward in developing this novel therapeutic strategy for the treatment of this inherently resistant disease state. Our results to date are very encouraging; however, much work lies ahead to better understand and optimize this novel approach for treating non-muscle invasive bladder.
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Terapia Genética/métodos , Interferones/uso terapéutico , Neoplasias de la Vejiga Urinaria/terapia , Adyuvantes Inmunológicos/uso terapéutico , Vacuna BCG/uso terapéutico , Investigación Biomédica , Ensayos Clínicos como Asunto , Humanos , Insuficiencia del Tratamiento , Neoplasias de la Vejiga Urinaria/tratamiento farmacológicoRESUMEN
Anticancer vaccination is a promising approach to increase the efficacy of cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and programmed death ligand 1 (PD-L1) checkpoint blockade therapies. However, the landmark FDA registration trial for anti-CTLA-4 therapy (ipilimumab) revealed a complete lack of benefit of adding vaccination with gp100 peptide formulated in incomplete Freund's adjuvant (IFA). Here, using a mouse model of melanoma, we found that gp100 vaccination induced gp100-specific effector T cells (Teffs), which dominantly forced trafficking of anti-CTLA-4-induced, non-gp100-specific Teffs away from the tumor, reducing tumor control. The inflamed vaccination site subsequently also sequestered and destroyed anti-CTLA-4-induced Teffs with specificities for tumor antigens other than gp100, reducing the antitumor efficacy of anti-CTLA-4 therapy. Mechanistically, Teffs at the vaccination site recruited inflammatory monocytes, which in turn attracted additional Teffs in a vicious cycle mediated by IFN-γ, CXCR3, ICAM-1, and CCL2, dependent on IFA formulation. In contrast, nonpersistent vaccine formulations based on dendritic cells, viral vectors, or water-soluble peptides potently synergized with checkpoint blockade of both CTLA-4 and PD-L1 and induced complete tumor regression, including in settings of primary resistance to dual checkpoint blockade. We conclude that cancer vaccine formulation can dominantly determine synergy, or lack thereof, with CTLA-4 and PD-L1 checkpoint blockade therapy for cancer.
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Antígeno B7-H1/inmunología , Antígeno CTLA-4/inmunología , Vacunas contra el Cáncer/farmacología , Puntos de Control del Ciclo Celular/inmunología , Melanoma/terapia , Neoplasias Experimentales/terapia , Péptidos/inmunología , Antígeno gp100 del Melanoma/farmacología , Animales , Antígeno B7-H1/genética , Antígeno CTLA-4/genética , Vacunas contra el Cáncer/inmunología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Línea Celular Tumoral , Quimiocina CCL2/genética , Quimiocina CCL2/inmunología , Células Dendríticas/inmunología , Células Dendríticas/patología , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/inmunología , Melanoma/genética , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Transgénicos , Monocitos/inmunología , Monocitos/patología , Neoplasias Experimentales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Péptidos/farmacología , Receptores CXCR3/genética , Receptores CXCR3/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Antígeno gp100 del Melanoma/inmunologíaRESUMEN
T-cell tolerance is a major obstacle to successful cancer immunotherapy; thus, developing strategies to break immune tolerance is a high priority. Here we show that expression of the E3 ubiquitin ligase Grail is upregulated in CD8+ T cells that have infiltrated into transplanted lymphoma tumours, and Grail deficiency confers long-term tumour control. Importantly, therapeutic transfer of Grail-deficient CD8+ T cells is sufficient to repress established tumours. Mechanistically, loss of Grail enhances anti-tumour reactivity and functionality of CD8+ T cells. In addition, Grail-deficient CD8+ T cells have increased IL-21 receptor (IL-21R) expression and hyperresponsiveness to IL-21 signalling as Grail promotes IL-21R ubiquitination and degradation. Moreover, CD8+ T cells isolated from lymphoma patients express higher levels of Grail and lower levels of IL-21R, compared with CD8+ T cells from normal donors. Our data demonstrate that Grail is a crucial factor controlling CD8+ T-cell function and is a potential target to improve cytotoxic T-cell activity.Grail is an E3 ubiquitin ligase that inhibits T-cell receptor signalling in CD4+ T cells. Here the authors show Grail also limits IL-21 receptor expression and function in CD8+ T cells, is overactive in these cells in patients with lymphoma, and promotes tumour development in a lymphoma transplant mouse model.
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Linfocitos T CD8-positivos/enzimología , Linfoma/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Linfocitos T CD8-positivos/inmunología , Humanos , Tolerancia Inmunológica , Interleucinas/genética , Interleucinas/inmunología , Linfoma/enzimología , Linfoma/genética , Ratones , Ratones Noqueados , Infiltración Neutrófila , Receptores de Interleucina-21/genética , Receptores de Interleucina-21/inmunología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
This review provides an in-depth description of the preclinical and clinical studies demonstrating the effectiveness and limitations of IL-15 and IL-15 analogs given as an exogenous immuno-oncology agent. IL-15 is a cytokine that primarily stimulates the proliferation and cytotoxic functions of CD8T cells and NK cells leading to enhanced anti-tumor responses. While initially showing promise as a cancer therapeutic, the efficacy of IL-15 was limited by its short in vivo half-life. More recently, various approaches have been developed to improve the in vivo half-life and efficacy of IL-15, largely by generating IL-15/IL-15Rα conjugates. These new IL-15 based agents renew the prospect of IL-15 as a cancer immunotherapeutic agent. While having some efficacy in inducing tumor regression as a monotherapy, IL-15 agents also show great potential in being used in combination with other immuno-oncological therapies. Indeed, IL-15 used in combination therapy yields even better anti-tumor responses and prolongs survival than IL-15 treatment alone in numerous murine cancer models. The promising results from these preclinical studies have led to the implementation of several clinical trials to test the safety and efficacy of IL-15-based agents as a stand-alone treatment or in conjunction with other therapies to treat both advanced solid tumors and hematological malignancies.
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Linfocitos T CD8-positivos/inmunología , Inmunoterapia/métodos , Interleucina-15/uso terapéutico , Células Asesinas Naturales/inmunología , Neoplasias/terapia , Animales , Proliferación Celular , Ensayos Clínicos como Asunto , Citotoxicidad Inmunológica , Humanos , Neoplasias/inmunología , Estabilidad Proteica , Receptores de Interleucina-15/metabolismoRESUMEN
Induction of lymphopenia has been exploited therapeutically to improve immune responses to cancer therapies and vaccinations. Whereas IL-15 has well-established roles in stimulating lymphocyte responses after lymphodepletion, the mechanisms regulating these IL-15 responses are unclear. We report that cell surface IL-15 expression is upregulated during lymphopenia induced by total body irradiation (TBI), cyclophosphamide, or Thy1 Ab-mediated T cell depletion, as well as in RAG(-/-) mice; interestingly, the cellular profile of surface IL-15 expression is distinct in each model. In contrast, soluble IL-15 (sIL-15) complexes are upregulated only after TBI or αThy1 Ab. Analysis of cell-specific IL-15Rα conditional knockout mice revealed that macrophages and dendritic cells are important sources of sIL-15 complexes after TBI but provide minimal contribution in response to Thy1 Ab treatment. Unlike with TBI, induction of sIL-15 complexes by αThy1 Ab is sustained and only partially dependent on type I IFNs. The stimulator of IFN genes pathway was discovered to be a potent inducer of sIL-15 complexes and was required for optimal production of sIL-15 complexes in response to Ab-mediated T cell depletion and TBI, suggesting products of cell death drive production of sIL-15 complexes after lymphodepletion. Lastly, we provide evidence that IL-15 induced by inflammatory signals in response to lymphodepletion drives lymphocyte responses, as memory CD8 T cells proliferated in an IL-15-dependent manner. Overall, these studies demonstrate that the form in which IL-15 is expressed, its kinetics and cellular sources, and the inflammatory signals involved are differentially dictated by the manner in which lymphopenia is induced.
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Interleucina-15/inmunología , Depleción Linfocítica , Linfopenia/inmunología , Animales , Modelos Animales de Enfermedad , Inflamación/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Immunization strategies generating large numbers of antigen-specific T cells in the female reproductive tract (FRT) can provide barrier protection against sexually-transmitted pathogens, such as the human immunodeficiency virus (HIV) and human papillomaviruses (HPV). The kinetics and mechanisms of regulation of vaccine-induced adaptive T cell-mediated immune responses in FRT are less well defined. We present here evidence for intranasal delivery of the model antigen ovalbumin (OVA) along with alpha-galactosylceramide adjuvant as a protein vaccine to induce significantly higher levels of antigen-specific effector and memory CD8⺠T cells in the FRT, relative to other systemic and mucosal tissues. Antibody blocking of the CXCR3 receptor significantly reduced antigen-specific CD8⺠T cells subsequent to intranasal delivery of the protein vaccine suggesting an important role for the CXCR3 chemokine-receptor signaling for T cell trafficking. Further, intranasal vaccination with an adenoviral vector expressing OVA or HIV-1 envelope was as effective as intramuscular vaccination for generating OVA- or ENV-specific immunity in the FRT. These results support the application of the needle-free intranasal route as a practical approach to delivering protein as well as DNA/virus vector-based vaccines for efficient induction of effector and memory T cell immunity in the FRT.
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Here, we examine the mechanism by which plasmacytoid dendritic cells (pDCs) and type I interferons promote humoral autoimmunity. In an amyloid-induced experimental autoimmune model, neutrophil depletion enhanced anti-nuclear antibody development, which correlated with heightened IFN-γ production by natural killer (NK) cells. IFN-α/ß produced by pDCs activated NK cells via IL-15 induction. Neutrophils released reactive oxygen species (ROS), which negatively modulated the levels of IL-15, thereby inhibiting IFN-γ production. Mice deficient in NADPH oxidase 2 produced increased amounts of IFN-γ and developed augmented titers of autoantibodies. Both the pDC-IFN-α/ß pathway and IFN-γ were indispensable in stimulating humoral autoimmunity. Male NZB/W F1 mice expressed higher levels of superoxide than their female lupus-prone siblings, and depletion of neutrophils resulted in spontaneous NK cell and autoimmune B cell activation. Our findings suggest a regulatory role for neutrophils in vivo and highlight the importance of an NK-IFN-γ axis downstream of the pDC-IFN-α/ß pathway in systemic autoimmunity.
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Autoinmunidad , Células Dendríticas/inmunología , Inmunidad Humoral , Interferón gamma/metabolismo , Lupus Eritematoso Sistémico/inmunología , Neutrófilos/inmunología , Especies Reactivas de Oxígeno/metabolismo , Animales , Linfocitos B/inmunología , Femenino , Interferón gamma/genética , Interleucina-15/genética , Interleucina-15/metabolismo , Células Asesinas Naturales/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BLRESUMEN
Interleukin (IL)-15 associates with IL-15Rα on the cell surface where it can be cleaved into soluble cytokine/receptor complexes that have the potential to stimulate CD8 T cells and NK cells. Unfortunately, little is known about the in vivo production of soluble IL-15Rα/IL-15 complexes (sIL-15 complexes), particularly regarding the circumstances that induce them and the mechanisms responsible. The main objective of this study was to elucidate the signals leading to the generation of sIL-15 complexes. In this study, we show that sIL-15 complexes are increased in the serum of mice in response to Interferon (IFN)-α. In bone marrow derived dendritic cells (BMDC), IFN-α increased the activity of ADAM17, a metalloproteinase implicated in cleaving IL-15 complexes from the cell surface. Moreover, knocking out ADAM17 in BMDCs prevented the ability of IFN-α to induce sIL-15 complexes demonstrating ADAM17 as a critical protease mediating cleavage of IL-15 complexes in response to type I IFNs. Type I IFN signaling was required for generating sIL-15 complexes as in vivo induction of sIL-15 complexes by Poly I:C stimulation or total body irradiation (TBI) was impaired in IFNAR-/- mice. Interestingly, serum sIL-15 complexes were also induced in mice infected with Vesicular stomatitis virus (VSV) or mice treated with agonistic CD40 antibodies; however, sIL-15 complexes were still induced in IFNAR-/- mice after VSV infection or CD40 stimulation indicating pathways other than type I IFNs induce sIL-15 complexes. Overall, this study has shown that type I IFNs, VSV infection, and CD40 stimulation induce sIL-15 complexes suggesting the generation of sIL-15 complexes is a common event associated with immune activation. These findings reveal an unrealized mechanism for enhanced immune responses occurring during infection, vaccination, inflammation, and autoimmunity.
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Interferón-alfa/fisiología , Interleucina-15/sangre , Animales , Antígenos CD40/metabolismo , Células Cultivadas , Células Dendríticas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/metabolismo , Estomatitis Vesicular/sangre , Irradiación Corporal TotalRESUMEN
Sampling the mucosal tissues and analyses of immune responses are integral to vaccine-development strategies against human immunodeficiency virus (HIV), which is transmitted predominantly across the oro-genital mucosa. While immune assay development and standardization attempts employ mouse models, immunogenicity and protective efficacy that can be extrapolated to humans are realized only from experiments in nonhuman primates. Here, we describe commonly used practices for immunizations in rhesus macaques (Macaca mulatta) along with procedures for obtaining important mucosal tissues samples from macaques and mice. We also describe detailed protocols for two important assays applicable in mouse as well as primate experiments for determining antigen-specific T cells responses induced after vaccination.
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Vacunas contra el SIDA/administración & dosificación , Inmunidad Celular , Inmunización/métodos , Pruebas Inmunológicas/métodos , Linfocitos T/inmunología , Vacunas contra el SIDA/inmunología , Administración Intravenosa , Administración Tópica , Animales , Biopsia/métodos , Separación Celular/métodos , Femenino , VIH/inmunología , Infecciones por VIH/prevención & control , Humanos , Inyecciones Intramusculares , Macaca mulatta , Ratones , Membrana Mucosa/metabolismoRESUMEN
Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8+ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.
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Vacunas contra el Cáncer/administración & dosificación , Neoplasias Pulmonares/prevención & control , Melanoma Experimental/prevención & control , Inmunidad Adaptativa , Adyuvantes Inmunológicos/administración & dosificación , Administración Sublingual , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Femenino , Galactosilceramidas/administración & dosificación , Inmunidad Humoral , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Melanoma Experimental/inmunología , Melanoma Experimental/secundario , Ratones Endogámicos C57BL , Mucosa Bucal/inmunología , Células T Asesinas Naturales/inmunología , Trasplante de Neoplasias , Ovalbúmina/inmunología , VacunaciónRESUMEN
The differentiation of CD4(+) T cells into different Th lineages is driven by cytokine milieu in the priming site and the underlying transcriptional circuitry. Even though many positive regulators have been identified, it is not clear how this process is inhibited at transcriptional level. In this study, we report that the E-twenty six (ETS) transcription factor E74-like factor 4 (ELF4) suppresses the differentiation of Th17 cells both in vitro and in vivo. Culture of naive Elf4(-/-) CD4(+) T cells in the presence of IL-6 and TGF-ß (or IL-6, IL-23, and IL-1ß) resulted in increased numbers of IL-17A-positive cells compared with wild-type controls. In contrast, the differentiation to Th1, Th2, or regulatory T cells was largely unaffected by loss of ELF4. The increased expression of genes involved in Th17 differentiation observed in Elf4(-/-) CD4(+) T cells suggested that ELF4 controls their programming into the Th17 lineage rather than only IL-17A gene expression. Despite normal proliferation of naive CD4(+) T cells, loss of ELF4 lowered the requirement of IL-6 and TGF-ß signaling for IL-17A induction in each cell division. ELF4 did not inhibit Th17 differentiation by promoting IL-2 production as proposed for another ETS transcription factor, ETS1. Elf4(-/-) mice showed increased numbers of Th17 cells in the lamina propria at steady state, in lymph nodes after immunization, and, most importantly, in the CNS following experimental autoimmune encephalomyelitis induction, contributing to the increased disease severity. Collectively, our findings suggest that ELF4 restrains Th17 differentiation in dividing CD4(+) T cells by regulating commitment to the Th17 differentiation program.
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Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Células Th17/citología , Células Th17/metabolismo , Factores de Transcripción/genética , Animales , Linfocitos T CD4-Positivos/inmunología , Línea Celular , Supervivencia Celular/genética , Citocinas/metabolismo , Humanos , Activación de Linfocitos/inmunología , Ratones , Ratones Noqueados , Células Th17/inmunologíaRESUMEN
Development of the natural killer (NK) cell lineage is dependent on the transcription factor Nfil3 (or E4BP4), which is thought to act downstream of IL-15 signaling. Nfil3-deficient mice lack NK cells, whereas other lymphocyte lineages (B, T, and NKT cells) remain largely intact. We report the appearance of Ly49H-expressing NK cells in Nfil3(-/-) mice infected with mouse cytomegalovirus (MCMV) or recombinant viruses expressing the viral m157 glycoprotein. Nfil3(-/-) NK cells at the peak of antigen-driven expansion were functionally similar to NK cells from infected wild-type mice with respect to IFN-γ production and cytotoxicity, and could comparably produce long-lived memory NK cells that persisted in lymphoid and nonlymphoid tissues for >60 d. We demonstrate that generation and maintenance of NK cell memory is an Nfil3-independent but IL-15-dependent process. Furthermore, specific ablation of Nfil3 in either immature NK cells in the bone marrow or mature peripheral NK cells had no observable effect on NK cell lineage maintenance or homeostasis. Thus, expression of Nfil3 is crucial only early in the development of NK cells, and signals through activating receptors and proinflammatory cytokines during viral infection can bypass the requirement for Nfil3, promoting the proliferation and long-term survival of virus-specific NK cells.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Células Asesinas Naturales/citología , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Linaje de la Célula , Separación Celular , Supervivencia Celular , Citocinas/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica , Homeostasis , Inflamación , Interferón gamma/metabolismo , Interleucina-15/metabolismo , Células Asesinas Naturales/virología , Ligandos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Muromegalovirus/inmunología , Subfamilia A de Receptores Similares a Lectina de Células NK/metabolismo , Bazo/citología , Tamoxifeno/químicaRESUMEN
Dendritic cells (DCs) are the most commonly studied source of the cytokine IL-15. Using an IL-15 reporter transgenic mouse, we have recently shown previously unappreciated differences in the levels of IL-15 expressed by subsets of conventional DCs (CD8⺠and CD8â»). In this study, we show that IL-15 promoter activity was differentially regulated in subsets of hematopoietically derived cells with IL-15 expression largely limited to myeloid lineages. In contrast, mature cells of the lymphoid lineages expressed little to no IL-15 activity. Surprisingly, we discovered that hematopoietic stem cells (lineageâ»Sca-1âºc-Kitâº) expressed high levels of IL-15, suggesting that IL-15 expression was extinguished during lymphoid development. In the case of T cells, this downregulation was Notch-dependent and occurred in a stepwise pattern coincident with increasing maturation and commitment to a T cell fate. Finally, we further demonstrate that IL-15 expression was also controlled throughout DC development, with key regulatory activity of IL-15 production occurring at the pre-DC branch point, leading to the generation of both IL-15âºCD8⺠and IL-15(â»/low)CD8â» DC subsets. Thus, IL-15 expression is coordinated with cellular fate in myeloid versus lymphoid immune cells.
Asunto(s)
Regulación de la Expresión Génica/inmunología , Hematopoyesis/inmunología , Células Madre Hematopoyéticas/inmunología , Interleucina-15/biosíntesis , Traslado Adoptivo , Animales , Diferenciación Celular/inmunología , Linaje de la Célula , Separación Celular , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Interleucina-15/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Células Mieloides/inmunología , Transcripción GenéticaRESUMEN
To understand why cancer vaccine-induced T cells often do not eradicate tumors, we studied immune responses in mice vaccinated with gp100 melanoma peptide in incomplete Freund's adjuvant (peptide/IFA), which is commonly used in clinical cancer vaccine trials. Peptide/IFA vaccination primed tumor-specific CD8(+) T cells, which accumulated not in tumors but rather at the persisting, antigen-rich vaccination site. Once there, primed T cells became dysfunctional and underwent antigen-driven, interferon-γ (IFN-γ)- and Fas ligand (FasL)-mediated apoptosis, resulting in hyporesponsiveness to subsequent vaccination. Provision of CD40-specific antibody, Toll-like receptor 7 (TLR7) agonist and interleukin-2 (IL-2) reduced T cell apoptosis but did not prevent vaccination-site sequestration. A nonpersisting vaccine formulation shifted T cell localization toward tumors, inducing superior antitumor activity while reducing systemic T cell dysfunction and promoting memory formation. These data show that persisting vaccine depots can induce specific T cell sequestration, dysfunction and deletion at vaccination sites; short-lived formulations may overcome these limitations and result in greater therapeutic efficacy of peptide-based cancer vaccines.