RESUMEN
Immune checkpoint blocking (ICB) is a promising new tool of cancer treatment. Yet, the underlying therapeutic mechanisms are not fully understood. Here we investigated the role of dendritic cells (DCs) for the therapeutic effect of ICB in a λ-MYC-transgenic mouse model of endogenously arising B-cell lymphoma. The growth of these tumors can be effectively delayed by antibodies against CTLA-4 and PD-1. Tumor-infiltrating DCs from mice having received therapy showed an upregulation of costimulatory molecules as well as an augmented IL-12/IL-10 ratio as compared to untreated controls. Both alterations seemed to be induced by interferon-γ (IFN-γ), which is upregulated in T cells and natural killer cells upon ICB. Furthermore, the enhanced IL-12/IL-10 ratio, which favors Th1-prone antitumor T-cell responses, was a consequence of direct interaction of ICB antibodies with DCs. Importantly, the capability of tumor-infiltrating DCs of stimulating peptide-specific or allogeneic T-cell responses in vitro was improved when DCs were derived from ICB-treated mice. The data indicate that ICB therapy is not only effective by directly activating T cells, but also by triggering a complex network, in which DCs play a pivotal role at the interface between innate and adaptive antitumor responses.
Asunto(s)
Células Dendríticas/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Linfoma de Células B/inmunología , Animales , Antígeno CTLA-4/inmunología , Células Cultivadas , Modelos Animales de Enfermedad , Quimioterapia Combinada , Genes myc/genética , Humanos , Linfoma de Células B/cirugía , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/inmunologíaRESUMEN
Little is known on the control of lymphomas by NK cells. Here, we study the role of the NK group 2D (NKG2D) receptor for the immunosurveillance of lymphoma. By using transplantable tumors as well as a λ-myc-transgenic model of endogenously arising lymphoma and NKG2D-deficient mice, we show that NK cells eliminate tumor cells in vivo after receiving two signals. One step involved the activation of NK cells giving rise to IFN-γ expression, which was effected by MHCI(low) tumor cells or DCs. However, this was necessary but not sufficient to mediate cytotoxicity. Triggering cytotoxicity additionally required a second step, which could be mediated by engagement of the NKG2D receptor. Thus, NKG2D-deficient NK cells could become activated in vivo, but they were not able to reject transplanted lymphomas or to degranulate in animals bearing autochthonous lymphomas. Tumor growth in NKG2D-deficient λ-myc-transgenic mice was significantly accelerated compared to NKG2D-competent animals. Whereas the latter developed tumors that lost expression of NKG2D ligands (NKG2D-L) in late disease stages, this did not occur in NKG2D-deficient mice. This indicates that NK cells and the NKG2D receptor play a role for control of lymphomas and that selection for NKG2D-L loss mutants provides a mechanism of tumor escape.
Asunto(s)
Proteínas Portadoras/inmunología , Regulación Neoplásica de la Expresión Génica , Antígenos de Histocompatibilidad Clase I/inmunología , Células Asesinas Naturales/inmunología , Linfoma de Células B/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Escape del Tumor/genética , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase I/genética , Interferón gamma/genética , Interferón gamma/inmunología , Células Asesinas Naturales/patología , Activación de Linfocitos , Linfoma de Células B/genética , Linfoma de Células B/patología , Proteínas de la Membrana , Ratones , Ratones Transgénicos , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Trasplante de Neoplasias , Transducción de SeñalRESUMEN
It is well established that an interplay between natural killer (NK) cells and dendritic cells (DCs) gives rise to their reciprocal activation and provides a Th1-biased cytokine milieu that fosters antitumor T-cell responses. Ex vivo-differentiated DCs transferred into mice strongly stimulate endogenous NK cells to produce interferon (IFN)-γ and initiate a cascade that eventually leads to cytotoxic T-lymphocyte responses. We show that the ability of exogenous DCs to trigger this pathway obviates CD40 signaling and CD4(+) T-cell help and depends on a preceding maturation step. Importantly, this mechanism was also effective in endogenously arising tumors where IFN-γ production is compromised in contrast to transplantable tumors. In c-myc-transgenic mice developing spontaneous lymphomas, injection of unpulsed DCs caused NK-cell activation and induced CD8(+) T cells capable of recognizing the lymphoma cells. Animals treated with unpulsed DCs showed a survival benefit compared to untreated myc mice. Hence, tumor immunity induced by DC-based vaccines not only depends on specific antigens loaded on the DCs. Rather, DC vaccines generate broader immune responses, because endogenous DCs presenting tumor antigens may also become stimulated by NK cells that were activated by exogenous DCs. Thus, the DC/NK-cell/cytotoxic T lymphocyte axis may commonly have relevance for DC-based vaccination protocols in clinical settings.
Asunto(s)
Vacunas contra el Cáncer/inmunología , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Linfoma de Células B/inmunología , Linfoma de Células B/terapia , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos CD40/metabolismo , Línea Celular Tumoral , Citocinas/metabolismo , Femenino , Genes myc , Interferón gamma/metabolismo , Células Asesinas Naturales/citología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Trasplante de NeoplasiasRESUMEN
Costimulatory surface molecules and instructive cytokines expressed by dendritic cells (DCs) determine the outcome of an immune response. In malignant disease, DCs are often functionally compromised. In most tumors studied so far, the deficient induction of effective T cell responses has been associated with a blockade of DC maturation, but little has been known on DCs infiltrating malignant B cell lymphoma. Here, we investigated for the first time the phenotypic and functional status of DCs in B cell lymphoma, and we analyzed the network of DCs, tumor cells, natural killer (NK) cells and cytokines present in the tumor micromilieu. Therefor, we used an endogenous myc-transgenic mouse lymphoma model, because transplanted tumor cells foster an IFN-γ-driven Th1 antitumor response rather than an immunosuppressive environment, which is observed in autochthonous neoplasias. Lymphoma-infiltrating DCs showed a mature phenotype and a Th2-inducing cytokine pattern. This situation is in contrast to most human malignancies and mouse models described. Cellular contacts between DCs and tumor cells, which involved CD62L on the lymphoma, caused upregulation of costimulatory molecules, whereas IL-10 primarily derived from lymphoma cells induced an IL-12/IL-10 shift in DCs. Thus, alteration of costimulatory molecules and instructive cytokines was mediated by distinct mechanisms. Normal NK cells were able to additionally modulate DC maturation but this effect was absent in the lymphoma environment where IFN-γ production by NK cells was severely impaired. These data are relevant for establishing novel immunotherapeutic approaches against B cell lymphoma.
Asunto(s)
Citocinas/inmunología , Células Dendríticas/inmunología , Células Asesinas Naturales/inmunología , Linfoma de Células B/inmunología , Microambiente Tumoral/inmunología , Animales , Células 3T3 BALB , Citocinas/biosíntesis , Citometría de Flujo , Genes myc , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , OncogenesRESUMEN
To establish strategies for immunotherapy of B-cell lymphoma, it is mandatory to gain deeper insights into the mechanisms of tumor immune escape. In a mouse model of endogenously arising lymphoma, we investigated the impact of IL-10 on the regulation of antitumor responses. Despite progressive functional impairment of NK cells and lack of IFN-γ in the tumor milieu, we found an augmented fraction of T helper type 1 (Th1) cells, which continued to express IFN-γ but also upregulated IL-10 during disease development. Using a lymphoma microenvironment in vitro, we showed that Th1 cells were converted to Foxp3-negative T regulatory type 1 (Tr1) cells, which coexpressed IFN-γ and IL-10 and upregulated PD-1. This differentiation required pre-existing IL-10, which was primarily provided by malignant B cells and dendritic cells. IFN-γ only declined in cells with the uppermost PD-1 levels. Importantly, antibody-mediated IL-10 ablation in vivo improved effector cell functions and significantly suppressed tumor development. While the contribution of IL-10 to cancer immune escape has been controversially discussed in the past, we show that IL-10 suppresses ongoing, potentially protective immune responses in lymphoma and might be a target for immunotherapy.
Asunto(s)
Interferón gamma/metabolismo , Interleucina-10/metabolismo , Linfoma de Células B/genética , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Animales , Diferenciación Celular , Células Dendríticas/inmunología , Regulación Neoplásica de la Expresión Génica , Humanos , Interleucina-10/genética , Linfoma de Células B/inmunología , Ratones , Escape del Tumor , Microambiente Tumoral , Regulación hacia ArribaRESUMEN
BACKGROUND: Although antibodies blocking immune checkpoints have already been approved for clinical cancer treatment, the mechanisms involved are not yet completely elucidated. Here we used a λ-MYC transgenic model of endogenously growing B-cell lymphoma to analyze the requirements for effective therapy with immune checkpoint inhibitors. METHODS: Growth of spontaneous lymphoma was monitored in mice that received antibodies targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein-4, and the role of different immune cell compartments and cytokines was studied by in vivo depletion experiments. Activation of T and natural killer cells and the induction of tumor senescence were analyzed by flow cytometry. RESULTS: On immune checkpoint blockade, visible lymphomas developed at later time points than in untreated controls, indicating an enhanced tumor control. Importantly, 20% to 30% of mice were even long-term protected and did never develop clinical signs of tumor growth. The therapeutic effect was dependent on cytokine-induced senescence in malignant B cells. The proinflammatory cytokines interferon-γ (IFN-γ) and tumor necrosis factor (TNF) were necessary for the survival benefit as well as for senescence induction in the λ-MYC model. Antibody therapy improved T-cell functions such as cytokine production, and long-time survivors were only observed in the presence of T cells. Yet, NK cells also had a pronounced effect on therapy-induced delay of tumor growth. Antibody treatment enhanced numbers, proliferation and IFN-γ expression of NK cells in developing tumors. The therapeutic effect was fully abrogated only after depletion of both, T cells and NK cells, or after ablation of either IFN-γ or TNF. CONCLUSIONS: Tumor cell senescence may explain why patients responding to immune checkpoint blockade frequently show stable growth arrest of tumors rather than complete tumor regression. In the lymphoma model studied, successful therapy required both, tumor-directed T-cell responses and NK cells, which control, at least partly, tumor development through cytokine-induced tumor senescence.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Citocinas/metabolismo , Inhibidores de Puntos de Control Inmunológico/administración & dosificación , Células Asesinas Naturales/efectos de los fármacos , Linfoma/tratamiento farmacológico , Nivolumab/administración & dosificación , Linfocitos T/efectos de los fármacos , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Proliferación Celular , Senescencia Celular , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Interferón gamma/metabolismo , Células Asesinas Naturales/inmunología , Linfoma/inmunología , Ratones , Nivolumab/farmacología , Linfocitos T/inmunología , Resultado del Tratamiento , Factor de Necrosis Tumoral alfa/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Immune checkpoint blockade (ICB)-based or natural cancer immune responses largely eliminate tumours. Yet, they require additional mechanisms to arrest those cancer cells that are not rejected. Cytokine-induced senescence (CIS) can stably arrest cancer cells, suggesting that interferon-dependent induction of senescence-inducing cell cycle regulators is needed to control those cancer cells that escape from killing. Here we report in two different cancers sensitive to T cell-mediated rejection, that deletion of the senescence-inducing cell cycle regulators p16Ink4a/p19Arf (Cdkn2a) or p21Cip1 (Cdkn1a) in the tumour cells abrogates both the natural and the ICB-induced cancer immune control. Also in humans, melanoma metastases that progressed rapidly during ICB have losses of senescence-inducing genes and amplifications of senescence inhibitors. Metastatic cells also resist CIS. Such genetic and functional alterations are infrequent in metastatic melanomas regressing during ICB. Thus, activation of tumour-intrinsic, senescence-inducing cell cycle regulators is required to stably arrest cancer cells that escape from eradication.
Asunto(s)
Ciclo Celular , Senescencia Celular , Interferones/metabolismo , Melanoma/inmunología , Melanoma/patología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Humanos , Inmunoterapia , Antígeno Ki-67/metabolismo , Ganglios Linfáticos/patología , Melanoma/terapia , Melanoma/ultraestructura , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Transcripción STAT1/metabolismo , Análisis de Supervivencia , Carga TumoralRESUMEN
Foxp3+ regulatory T cells (Tregs) sustain immune homeostasis and may contribute to immune escape in malignant disease. As a prerequisite for developing immunologic approaches in cancer therapy, it is necessary to understand the ontogeny and the antigenic specificities of tumor-infiltrating Tregs. We addressed this question by using a λ-MYC transgenic mouse model of endogenously arising B-cell lymphoma, which mirrors key features of human Burkitt lymphoma. We show that Foxp3+ Tregs suppress antitumor responses in endogenous lymphoma. Ablation of Foxp3+ Tregs significantly delayed tumor development. The ratio of Treg to effector T cells was elevated in growing tumors, which could be ascribed to differential proliferation. The Tregs detected were mainly natural Tregs that apparently recognized self-antigens. We identified MHC class II-restricted nonmutated self-epitopes, which were more prevalent in lymphoma than in normal B cells and could be recognized by Tregs. These epitopes were derived from proteins that are associated with cellular processes related to malignancy and may be overexpressed in the tumor.