RESUMEN
Tumor-associated macrophages (TAMs) can have protumor properties, including suppressing immune responses, promoting vascularization and, consequently, augmenting tumor progression. To stop TAM-mediated immunosuppression, we use a novel treatment by injecting antibodies specific for scavenger receptor MARCO, which is expressed on a specific subpopulation of TAMs in the tumor. We now report the location of this TAM as well as the pleiotropic mechanism of action of anti-MARCO antibody treatment on tumor progression and further show that this is potentially relevant to humans. Using specific targeting, we observed decreased tumor vascularization, a switch in the metabolic program of MARCO-expressing macrophages, and activation of natural killer (NK) cell killing through TNF-related apoptosis-inducing ligand (TRAIL). This latter activity reverses the effect of melanoma cell-conditioned macrophages in blocking NK activation and synergizes with T cell-directed immunotherapy, such as antibodies to PD-1 or PD-L1, to enhance tumor killing. Our study thus reveals an approach to targeting the immunosuppressive tumor microenvironment with monoclonal antibodies to enhance NK cell activation and NK cell-mediated killing. This can complement existing T cell-directed immunotherapy, providing a promising approach to combinatorial immunotherapy for cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Células Asesinas Naturales/inmunología , Melanoma/tratamiento farmacológico , Receptores Inmunológicos/antagonistas & inhibidores , Macrófagos Asociados a Tumores/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Células Asesinas Naturales/metabolismo , Masculino , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Noqueados , Cultivo Primario de Células , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismoRESUMEN
Lymphocyte-based immunotherapy has emerged as a breakthrough in cancer therapy for both hematologic and solid malignancies. In a subpopulation of cancer patients, this powerful therapeutic modality converts malignancy to clinically manageable disease. However, the T cell- and chimeric antigen receptor T (CAR-T) cell-mediated antimetastatic activity, especially their impacts on microscopic metastatic lesions, has not yet been investigated. Here we report a living zebrafish model that allows us to visualize the metastatic cancer cell killing effect by tumor- infiltrating lymphocytes (TILs) and CAR-T cells in vivo at the single-cell level. In a freshly isolated primary human melanoma, specific TILs effectively eliminated metastatic cancer cells in the living body. This potent metastasis-eradicating effect was validated using a human lymphoma model with CAR-T cells. Furthermore, cancer-associated fibroblasts protected metastatic cancer cells from T cell-mediated killing. Our data provide an in vivo platform to validate antimetastatic effects by human T cell-mediated immunotherapy. This unique technology may serve as a precision medicine platform for assessing anticancer effects of cellular immunotherapy in vivo before administration to human cancer patients.
Asunto(s)
Inmunoterapia/métodos , Linfocitos Infiltrantes de Tumor/metabolismo , Análisis de la Célula Individual/métodos , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Inmunoterapia Adoptiva/métodos , Activación de Linfocitos/fisiología , Modelos Animales , Metástasis de la Neoplasia/patología , Neoplasias/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Pez CebraRESUMEN
The efficacy of immunotherapies for malignant melanoma is severely hampered by local and systemic immunosuppression mediated by myeloid-derived suppressor cells (MDSC). Inhibitor of differentiation 1 (ID1) is a transcriptional regulator that was shown to be centrally involved in the induction of immunosuppressive properties in myeloid cells in mice, while it was overexpressed in CD11b+ cells in the blood of late-stage melanoma patients. Therefore, we comprehensively assessed ID1 expression in PBMC from stage III and IV melanoma patients, and studied ID1 regulation in models for human monocyte differentiation towards monocyte-derived dendritic cells. A highly significant elevation of ID1 was observed in CD33+CD11b+CD14+HLA-DRlow monocytic MDSC in the blood of melanoma patients compared to their HLA-DRhigh counterparts, while expression of ID1 correlated positively with established MDSC markers S100A8/9 and iNOS. Moreover, expression of ID1 in monocytes significantly decreased in PBMC samples taken after surgical removal of melanoma metastases, compared to those taken before surgery. Finally, maturation of monocyte-derived DC coincided with a significant downregulation of ID1. Together, these data indicate that increased ID1 expression is strongly associated with expression of phenotypic and immunosuppressive markers of monocytic MDSC, while downregulation is associated with a more immunogenic myeloid phenotype. As such, ID1 may be an additional phenotypic marker for monocytic MDSC. Investigation of ID1 as a pharmacodynamic biomarker or its use as a target for modulating MDSC is warranted.
Asunto(s)
Biomarcadores/metabolismo , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Melanoma/metabolismo , Monocitos/metabolismo , Células Supresoras de Origen Mieloide/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular Tumoral , Células Cultivadas , Femenino , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Melanoma/sangre , Melanoma/cirugía , Ratones , Persona de Mediana Edad , FenotipoRESUMEN
Adoptive cell therapy (ACT) is becoming a prominent alternative therapeutic treatment for cancer patients relapsing on traditional therapies. In parallel, antibodies targeting immune checkpoint molecules, such as cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) and cell death protein 1 pathway (PD-1), are rapidly being approved for multiple cancer types, including as first line therapy for PD-L1-expressing non-small-cell lung cancer. The combination of ACT and checkpoint blockade could substantially boost the efficacy of ACT. In this study, we generated a novel self-delivering small interfering RNA (siRNA) (sdRNA) that knocked down PD-1 expression on healthy donor T cells as well as patient-derived tumor-infiltrating lymphocytes (TIL). We have developed an alternative chemical modification of RNA backbone for improved stability and increased efficacy. Our results show that T cells treated with sdRNA specific for PD-1 had increased interferon γ (IFN-γ) secreting capacity and that this modality of gene expression interference could be utilized in our rapid expansion protocol for production of TIL for therapy. TIL expanded in the presence of PD-1-specific sdRNA performed with increased functionality against autologous tumor as compared to control TIL. This method of introducing RNAi into T cells to modify the expression of proteins could easily be adopted into any ACT protocol and will lead to the exploration of new combination therapies.
Asunto(s)
Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/terapia , Melanoma/inmunología , Melanoma/terapia , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T/metabolismo , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Citometría de Flujo , Células HeLa , Humanos , Inmunoterapia Adoptiva/métodos , Interferón gamma/genética , Interferón gamma/metabolismo , Neoplasias Pulmonares/inmunología , Melanoma/metabolismo , Receptor de Muerte Celular Programada 1/genética , Interferencia de ARN/fisiologíaRESUMEN
Treatment of hematological malignancies by adoptive transfer of activated natural killer (NK) cells is limited by poor postinfusion persistence. We compared the ability of interleukin-2 (IL-2) and IL-15 to sustain human NK-cell functions following cytokine withdrawal to model postinfusion performance. In contrast to IL-2, IL-15 mediated stronger signaling through the IL-2/15 receptor complex and provided cell function advantages. Genome-wide analysis of cytosolic and polysome-associated messenger RNA (mRNA) revealed not only cytokine-dependent differential mRNA levels and translation during cytokine activation but also that most gene expression differences were primed by IL-15 and only manifested after cytokine withdrawal. IL-15 augmented mammalian target of rapamycin (mTOR) signaling, which correlated with increased expression of genes related to cell metabolism and respiration. Consistently, mTOR inhibition abrogated IL-15-induced cell function advantages. Moreover, mTOR-independent STAT-5 signaling contributed to improved NK-cell function during cytokine activation but not following cytokine withdrawal. The superior performance of IL-15-stimulated NK cells was also observed using a clinically applicable protocol for NK-cell expansion in vitro and in vivo. Finally, expression of IL-15 correlated with cytolytic immune functions in patients with B-cell lymphoma and favorable clinical outcome. These findings highlight the importance of mTOR-regulated metabolic processes for immune cell functions and argue for implementation of IL-15 in adoptive NK-cell cancer therapy.
Asunto(s)
Citotoxicidad Inmunológica/inmunología , Inmunoterapia Adoptiva , Interleucina-15/farmacología , Células Asesinas Naturales/inmunología , Neoplasias Experimentales/terapia , Serina-Treonina Quinasas TOR/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Citocinas/metabolismo , Humanos , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Activación de Linfocitos , Ratones Endogámicos NOD , Ratones SCID , Proteínas Mitocondriales/genética , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patología , Transducción de SeñalRESUMEN
Treatment of cancer patients by adoptive T cell therapy has yielded promising results. In solid tumors, however, T cells encounter a hostile environment, in particular with increased inflammatory activity as a hallmark of the tumor milieu that goes along with abundant reactive oxygen species (ROS) that substantially impair antitumor activity. We present a strategy to render antitumor T cells more resilient toward ROS by coexpressing catalase along with a tumor specific chimeric Ag receptor (CAR) to increase their antioxidative capacity by metabolizing H2O2. In fact, T cells engineered with a bicistronic vector that concurrently expresses catalase, along with the CAR coexpressing catalase (CAR-CAT), performed superior over CAR T cells as they showed increased levels of intracellular catalase and had a reduced oxidative state with less ROS accumulation in both the basal state and upon activation while maintaining their antitumor activity despite high H2O2 levels. Moreover, CAR-CAT T cells exerted a substantial bystander protection of nontransfected immune effector cells as measured by CD3ζ chain expression in bystander T cells even in the presence of high H2O2 concentrations. Bystander NK cells, otherwise ROS sensitive, efficiently eliminate their K562 target cells under H2O2-induced oxidative stress when admixed with CAR-CAT T cells. This approach represents a novel means for protecting tumor-infiltrating cells from tumor-associated oxidative stress-mediated repression.
Asunto(s)
Catalasa/inmunología , Inmunoterapia Adoptiva/métodos , Células Asesinas Naturales/inmunología , Estrés Oxidativo/fisiología , Linfocitos T/inmunología , Western Blotting , Efecto Espectador/inmunología , Línea Celular , Separación Celular , Humanos , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas Recombinantes de Fusión/inmunología , TransfecciónRESUMEN
Dendritic cell (DC) vaccines have been demonstrated to elicit immunological responses in numerous cancer immunotherapy trials. However, long-lasting clinical effects are infrequent. We therefore sought to establish a protocol to generate DC with greater immunostimulatory capacity. Immature DC were generated from healthy donor monocytes by culturing in the presence of IL-4 and GM-CSF and were further differentiated into mature DC by the addition of cocktails containing different cytokines and toll-like receptor (TLR) agonists. Overall, addition of IFNγ and the TLR7/8 agonist R848 during maturation was essential for the production of high levels of IL-12p70 which was further augmented by adding the TLR3 agonist poly I:C. In addition, the DC matured with IFNγ, R848, and poly I:C also induced upregulation of several other pro-inflammatory and Th1-skewing cytokines/chemokines, co-stimulatory receptors, and the chemokine receptor CCR7. For most cytokines and chemokines the production was even further potentiated by addition of the TLR4 agonist LPS. Concurrently, upregulation of the anti-inflammatory cytokine IL-10 was modest. Most importantly, DC matured with IFNγ, R848, and poly I:C had the ability to activate IFNγ production in allogeneic T cells and this was further enhanced by adding LPS to the cocktail. Furthermore, epitope-specific stimulation of TCR-transduced T cells by peptide- or whole tumor lysate-loaded DC was efficiently stimulated only by DC matured in the full maturation cocktail containing IFNγ and the three TLR ligands R848, poly I:C, and LPS. We suggest that this cocktail is used for future clinical trials of anti-cancer DC vaccines.
Asunto(s)
Células Dendríticas/inmunología , Interferón gamma/farmacología , Linfocitos T/inmunología , Receptores Toll-Like/agonistas , Diferenciación Celular , HumanosRESUMEN
Dendritic cell (DC) vaccines induce T-cell responses in cancer patients. However, there is a paucity of data regarding the role of DC vaccines in shaping natural killer (NK) cell responses. Here, we observe that NK cells are less activated following DC vaccination. In vitro, DC-mediated inhibition of NK cells did not require cell-to-cell contact, but required increased Signal transducer and activator of transcription 3 (STAT3) phosphorylation (pSTAT3) in DCs. When phosphorylation of STAT3 was inhibited in DCs, we found that DCs did not suppress NK cells, and observed an increase in the production of lymphotoxin-alpha (LTα) and interleukin-12 (IL-12) as well as reduced release of transforming growth factor beta (TGF-ß). The addition of recombinant LTα or IL-12 to the DC-NK-cell cocultures restored NK-cell activity, and neutralization of TGF-ß resulted in elevated production of LTα and IL-12 from DCs. Compared with LPS, DCs matured with a cocktail of R848, poly I:C, and IFN-γ showed reduced levels of pSTAT3 and higher levels of LTα and IL-12 and did not inhibit NK-cell activity. These results show that LTα, IL-12, and TGF-ß are involved in the cross-talk between NK cells and DCs. Our findings have important implications for the development of DC-based vaccination strategies to potentiate NK-cell responses in patients with cancer.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Interleucina-12/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Linfotoxina-alfa/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Comunicación Autocrina/efectos de los fármacos , Comunicación Autocrina/inmunología , Vacunas contra el Cáncer , Comunicación Celular/inmunología , Células Dendríticas/efectos de los fármacos , Humanos , Inmunomodulación/efectos de los fármacos , Inmunoterapia , Interferón gamma/biosíntesis , Interleucina-12/farmacología , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Linfotoxina-alfa/farmacología , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/terapia , Fenotipo , Fosforilación , Factor de Transcripción STAT3/metabolismo , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The fourth "Melanoma Bridge Meeting" took place in Naples, December 3-6th, 2014. The four topics discussed at this meeting were: Molecular and Immunological Advances, Combination Therapies, News in Immunotherapy, and Tumor Microenvironment and Biomarkers. Until recently systemic therapy for metastatic melanoma patients was ineffective, but recent advances in tumor biology and immunology have led to the development of new targeted and immunotherapeutic agents that prolong progression-free survival (PFS) and overall survival (OS). New therapies, such as mitogen-activated protein kinase (MAPK) pathway inhibitors as well as other signaling pathway inhibitors, are being tested in patients with metastatic melanoma either as monotherapy or in combination, and all have yielded promising results. These include inhibitors of receptor tyrosine kinases (BRAF, MEK, and VEGFR), the phosphatidylinositol 3 kinase (PI3K) pathway [PI3K, AKT, mammalian target of rapamycin (mTOR)], activators of apoptotic pathway, and the cell cycle inhibitors (CDK4/6). Various locoregional interventions including radiotherapy and surgery are still valid approaches in treatment of advanced melanoma that can be integrated with novel therapies. Intrinsic, adaptive and acquired resistance occur with targeted therapy such as BRAF inhibitors, where most responses are short-lived. Given that the reactivation of the MAPK pathway through several distinct mechanisms is responsible for the majority of acquired resistance, it is logical to combine BRAF inhibitors with inhibitors of targets downstream in the MAPK pathway. For example, combination of BRAF/MEK inhibitors (e.g., dabrafenib/trametinib) have been demonstrated to improve survival compared to monotherapy. Application of novel technologies such sequencing have proven useful as a tool for identification of MAPK pathway-alternative resistance mechanism and designing other combinatorial therapies such as those between BRAF and AKT inhibitors. Improved survival rates have also been observed with immune-targeted therapy for patients with metastatic melanoma. Immune-modulating antibodies came to the forefront with anti-CTLA-4, programmed cell death-1 (PD-1) and PD-1 ligand 1 (PD-L1) pathway blocking antibodies that result in durable responses in a subset of melanoma patients. Agents targeting other immune inhibitory (e.g., Tim-3) or immune stimulating (e.g., CD137) receptors and other approaches such as adoptive cell transfer demonstrate clinical benefit in patients with melanoma as well. These agents are being studied in combination with targeted therapies in attempt to produce longer-term responses than those more typically seen with targeted therapy. Other combinations with cytotoxic chemotherapy and inhibitors of angiogenesis are changing the evolving landscape of therapeutic options and are being evaluated to prevent or delay resistance and to further improve survival rates for this patient population. This meeting's specific focus was on advances in combination of targeted therapy and immunotherapy. Both combination targeted therapy approaches and different immunotherapies were discussed. Similarly to the previous meetings, the importance of biomarkers for clinical application as markers for diagnosis, prognosis and prediction of treatment response was an integral part of the meeting. The overall emphasis on biomarkers supports novel concepts toward integrating biomarkers into contemporary clinical management of patients with melanoma across the entire spectrum of disease stage. Translation of the knowledge gained from the biology of tumor microenvironment across different tumors represents a bridge to impact on prognosis and response to therapy in melanoma.
Asunto(s)
Melanoma/patología , Biomarcadores de Tumor , Humanos , Inmunoterapia , Italia , Melanoma/inmunología , Melanoma/terapia , Microambiente TumoralRESUMEN
Downregulation of HLA class I expression may contribute to a poor prognosis in cancer patients. There is limited information about epigenetic and oncogenic regulation of HLA class I, and multiple mechanisms may be involved. In the current study, we examined the relationship between the HER2-signaling pathway (MAPK and PI3K-Akt) and the expression of HLA class I and Ag-processing machinery (APM) components. A panel of gastric and esophageal cancer cell lines was treated with wortmannin as an Akt-signal inhibitor; the MAPK signal inhibitor PD98059; lapatinib, which inhibits both the epidermal growth factor receptor and HER2 tyrosine kinase; or siRNA for MAPK. The levels of HER2-signaling molecules, APM components, and HLA class I were evaluated by Western blot, quantitative PCR, and flow cytometry. Resected gastric tumor tissues (n = 102) were analyzed for p-Erk and HLA class I expression by immunohistochemistry. As a result, inhibition of the MAPK pathway induced upregulation of HLA-A02 and HLA-A24 expression in parallel with an increase in APM components and enhanced target sensitivity to tumor Ag-specific CTL lysis. HLA-A expression was predominantly regulated by the MAPK pathway, but it was also influenced, in part, by the Akt pathway. There was a strong inverse correlation between p-Erk expression and HLA class I expression in clinical tumor samples. In conclusion, HLA-A expression is predominantly regulated by the MAPK pathway in gastric and esophageal cancer.
Asunto(s)
Antígenos de Neoplasias/biosíntesis , Neoplasias Esofágicas/inmunología , Regulación Neoplásica de la Expresión Génica/fisiología , Antígenos HLA-A/biosíntesis , Sistema de Señalización de MAP Quinasas/fisiología , Neoplasias Gástricas/inmunología , Androstadienos/farmacología , Presentación de Antígeno/genética , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/antagonistas & inhibidores , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Flavonoides/farmacología , Genes MHC Clase I , Antígenos HLA-A/genética , Humanos , Lapatinib , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/fisiología , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/fisiología , Quinazolinas/farmacología , ARN Interferente Pequeño/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , WortmaninaRESUMEN
Immune checkpoints are a series of inhibitory pathways that are crucial for modulating the intensity and duration of immune response. Among these checkpoints, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) has been shown to be a key regulator of the early activation of naïve and memory T cells. Immune checkpoint blockade is emerging as one of the most promising therapeutic approaches directed toward the activation of the immune response against tumors. The first of these therapies that has been FDA approved is ipilimumab, a fully human monoclonal antibody that blocks CTLA-4. The in cis effects that CTLA-4 blockade has on T cells have been properly described, but there are still questions to be answered regarding the indirect or in trans effects. One of the alternative cellular populations that may play a role in the outcome of CTLA-4 blockade therapy is myeloid-derived suppressor cells (MDSCs), which have recently been associated with clinical outcome in advanced melanoma. In addition to this, MDSCs have been shown to be decreased in number and functional potential after treatment with ipilimumab. A better clarification of what effects CTLA-4 blockade may have on these cellular populations is likely to provide insights on possible predictive biomarkers for CTLA-4 blockade therapy.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígeno CTLA-4/antagonistas & inhibidores , Células Mieloides/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Animales , Anticuerpos Monoclonales/uso terapéutico , Antígeno CTLA-4/inmunología , Humanos , Ipilimumab , Células Mieloides/efectos de los fármacos , Escape del Tumor/efectos de los fármacos , Escape del Tumor/inmunologíaRESUMEN
Adoptive transfer of in vitro-expanded tumor-infiltrating lymphocytes (TIL) has shown great clinical benefit in patients with malignant melanoma. TIL therapy itself has little side effects, but conditioning chemo- or radiotherapy and postinfusion interleukin 2 (IL-2) injections are associated with severe adverse advents. We reasoned that combining TIL infusion with dendritic cell (DC) vaccination could circumvent the need for conditioning and IL-2 support and thus represent a milder treatment approach. Eight patients with stage IV melanoma were enrolled in the MAT01 study, consisting of vaccination with autologous tumor-lysate-loaded DC, followed by TIL infusion. Six of eight patients were treated according to protocol, while one patient received only TIL and one only DC. Treatments were well tolerated with a single grade 3 adverse event. The small study size precludes analysis of clinical responses, though interestingly one patient showed a complete remission and two had stable disease. Analysis of the infusion products revealed that mature DC were generated in all cases. TIL after expansion were CD3+ T cells, dominated by effector memory CD8+ cytotoxic T cells. Analysis of the T cell receptor repertoire revealed presence of highly dominant clones in most infusion products, and many of these could be detected in the circulation for weeks after T cell transfer. Here, we report the first combination of DC vaccination and TIL infusion in malignant melanoma. This combined treatment was safe and feasible, though after evaluating both clinical and immunological parameters, we expect that administration of lymphodepleting chemotherapy and IL-2 will likely increase treatment efficacy.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/trasplante , Inmunoterapia Adoptiva/métodos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/trasplante , Melanoma/inmunología , Melanoma/terapia , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Melanoma/patología , Persona de Mediana Edad , Estadificación de Neoplasias , Vacunación/métodos , Adulto JovenRESUMEN
Adoptive transfer of third-party mesenchymal stromal cells (MSCs) has emerged as a promising tool for the treatment of steroid-refractory graft-versus-host disease (GVHD). Despite numerous in vitro studies and preclinical models, little is known about their effects on the patients' immune system. We assessed immune alterations in the T-cell, B-cell, natural killer cell, dendritic cell, and monocytic compartments of steroid-refractory GVHD patients 30, 90, and 180 days after MSC (n = 6) or placebo (n = 5) infusion, respectively. Infused MSCs were bioactive as suggested by the significant reduction in epithelial cell death, which represents a biomarker for acute GVHD. There were several indications that MSCs shift the patients' immune system toward a more tolerogenic profile. Most importantly, infusion of MSCs was associated with increased levels of regulatory (forkhead box P3 (FOXP3)(+) and interleukin (IL)-10(+) ) T-cells, reduced pro-inflammatory IL-17(+) T(Th17)-cells, and skewing toward type-2 T-helper cell responses. Furthermore, IL-2, which has been recently shown to exert a positive immune modulating effect in GVHD patients, was higher in the MSC patients at all evaluated time points during 6 months after MSC-infusion. Overall, our findings will contribute to the refinement of monitoring tools, for assessing MSC treatment-efficacy and increase our understanding regarding the MSCs' in vivo effects.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/inmunología , Adulto , Anciano , Femenino , Neoplasias Hematológicas/inmunología , Neoplasias Hematológicas/patología , Neoplasias Hematológicas/cirugía , Humanos , Inmunidad Celular/inmunología , Inmunoterapia Adoptiva/métodos , Masculino , Persona de Mediana Edad , Trasplante HomólogoRESUMEN
Overexpression of the receptor tyrosine kinases HER2 and HER3 is associated with a poor prognosis in several types of cancer. Presently, HER2- as well as HER3-targeted therapies are in clinical practice or evaluated within clinical trials, including treatment with mAbs mediating growth inhibition and/or activation of Ab-induced innate or adaptive cellular immunity. A better understanding of how HER2/HER3 signaling in tumors influences cellular immune mechanisms is therefore warranted. In this study, we demonstrate that HER2/HER3 signaling regulates the expression of MHC class I-related chain A and B (MICA and MICB) in breast cancer cell lines. The MICA and MICB (MICA/B) molecules act as key ligands for the activating receptor NK group 2, member D (NKG2D) and promote NK cell-mediated recognition and cytolysis. Genetic silencing of HER3 but not HER2 downregulated the expression of MICA/B, and HER3 overexpression significantly enhanced MICA expression. Among the major pathways activated by HER2/HER3 signaling, the PI3K/AKT pathway was shown to predominantly regulate MICA/B expression. Treatment with the HER3-specific ligand neuregulin 1ß promoted the expression in a process that was antagonized by pharmacological and genetic interference with HER3 but not by the ataxia-telangiectasia-mutated (ATM) and ATM and Rad3-related protein kinases inhibitor caffeine. These observations further emphasize that HER2/HER3 signaling directly, and not via genotoxic stress, regulates MICA/B expression. As anticipated, stimulating HER2/HER3 enhanced the NKG2D-MICA/B-dependent NK cell-mediated cytotoxicity. Taken together, we conclude that signaling via the HER2/HER3 pathway in breast carcinoma cell lines may lead to enhanced NKG2D-MICA/B recognition by NK cells and T cells.
Asunto(s)
Neoplasias de la Mama/inmunología , Pruebas Inmunológicas de Citotoxicidad , Antígenos de Histocompatibilidad Clase I/biosíntesis , Células Asesinas Naturales/inmunología , Receptor ErbB-2/fisiología , Receptor ErbB-3/fisiología , Transducción de Señal/inmunología , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/genética , Línea Celular Tumoral , Pruebas Inmunológicas de Citotoxicidad/métodos , Femenino , Regulación Neoplásica de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/patología , Sistema de Señalización de MAP Quinasas/inmunología , Receptor ErbB-2/biosíntesis , Receptor ErbB-3/biosíntesis , Transducción de Señal/genética , Subgrupos de Linfocitos T/enzimología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/patología , Escape del Tumor/genéticaRESUMEN
Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell-to-myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell-derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m-knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell-mediated mechanism that drives the development of MDSCs during tumor immune escape.
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Tolerancia Inmunológica , Interleucina-6 , Células Asesinas Naturales , Células Supresoras de Origen Mieloide , Factor de Transcripción STAT3 , Factor de Transcripción STAT3/metabolismo , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Interleucina-6/metabolismo , Células Supresoras de Origen Mieloide/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Animales , Humanos , Transducción de Señal , Microambiente Tumoral/inmunología , Ratones Noqueados , Línea Celular Tumoral , Femenino , Ratones , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/patologíaRESUMEN
The extracellular matrix protein biglycan (Bgn) is a leucine-rich proteoglycan that is involved in the matrix assembly, cellular migration and adhesion, cell growth, and apoptosis. Although a distinct expression of Bgn was found in a number of human tumors, the role of this protein in the initiation and/or maintenance of neoplastic transformation has not been studied in detail. Using an in vitro model of oncogenic transformation, a down-regulation of Bgn expression as well as an altered secretion of different Bgn isoforms was found both in murine and human HER-2/neu oncogene-transformed cells when compared with HER-2/neu(-) cells. This was associated with a reduced growth, wound closure, and migration capacity. Vice versa, silencing of Bgn in HER-2/neu(-) fibroblasts increased the growth rate and migration capacity of these cells. Bgn expression was neither modulated in HER-2/neu(+) cells by transforming growth factor-ß(1) nor by inhibition of the phosphoinositol 3-kinase and MAP kinase pathways. In contrast, inhibition of the protein kinase C (PKC) pathway led to the reconstitution of Bgn expression. In particular, the PKC target protein cAMP response element binding protein (CREB) is a major regulator of Bgn expression as the silencing of CREB by RNA interference was accompanied by â¼5000-fold increase in Bgn-mRNA expression in HER-2/neu(+) cells. Thus, Bgn inhibits the major properties of HER-2/neu-transformed cells, which is inversely modulated by the PKC signaling cascade.
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Biglicano/metabolismo , Transformación Celular Neoplásica/metabolismo , Receptor ErbB-2/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transformación Celular Neoplásica/inducido químicamente , Citometría de Flujo , Humanos , Ratones , Células 3T3 NIH , Receptor ErbB-2/genética , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
BACKGROUND: Malignancies induce changes in the levels of serum amino acids (AA), which may offer diagnostic potential. Furthermore, changes in AA levels are associated with immune cell function. In this study, serum AA levels were studied in breast cancer patients versus patients with benign breast lesions. METHODS: In a prospective study, serum levels of 15 AA were measured by high performance liquid chromatography before and after surgery in 41 breast cancer patients (BrCA) and nine patients with benign breast lesions (healthy donors, HD). Results were analyzed in relation to clinical tumor data and tested against immunological flow cytometry data. Principal component analysis was performed and the accuracy of AA levels as a potential diagnostic tool was tested. RESULTS: Pre- but not postoperative serum AA levels were increased in BrCA in eight out of 15 AA compared with HD. Serum AA levels were highest in the most aggressive (basal-like) as compared with the least aggressive tumor subtype (luminal A). A principal component (PC1) of all measured AA correlated with a mainly pro-inflammatory immune profile, while a second one (PC2, selectively considering AA preoperatively differing between HD and BrCA) could predict health state with an area under the curve of 0.870. CONCLUSIONS: Breast cancer shows a tumor-dependent impact on serum AA levels, which varies with intrinsic tumor subtypes and is associated with a pro-inflammatory state. Serum AA levels need further evaluation as a potential diagnostic tool.
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Aminoácidos/sangre , Neoplasias de la Mama/sangre , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/genética , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Persona de Mediana Edad , Análisis de Componente Principal , Estudios Prospectivos , Espectrometría de FluorescenciaRESUMEN
HLA abnormalities on tumour cells for immune escape have been widely described. In addition, cellular components of the tumour microenvironment, in particular myeloid derived suppressor cells (MDSC) and alternatively activated M2 tumour-associated macrophages (TAMs), are involved in tumour promotion, progression, angiogenesis and suppression of anti-tumour immunity. However, the role of HLA in these activities is poorly understood. This review details MHC class I characteristics and describes MHC class I receptors functions. This analysis established the basis for a reflection about the crosstalk among the tumour cells, the TAMs and the cells mediating an immune response.The tumour cells and TAMs exploit MHC class I molecules to modulate the surrounding immune cells. HLA A, B, C and G molecules down-regulate the macrophage myeloid activation through the interaction with the inhibitory LILRB receptors. HLA A, B, C are able to engage inhibitory KIR receptors negatively regulating the Natural Killer and cytotoxic T lymphocytes function while HLA-G induces the secretion of pro-angiogenic cytokines and chemokine thanks to an activator KIR receptor expressed by a minority of peripheral NK cells. The open conformer of classical MHC-I is able to interact with LILRA receptors described as being associated to the Th2-type cytokine response, triggering a condition for the M2 like TAM polarization. In addition, HLA-E antigens on the surface of the TAMs bind the inhibitory receptor CD94/NKG2A expressed by a subset of NK cells and activated cytotoxic T lymphocytes protecting from the cytolysis.Furthermore MHC class II expression by antigen presenting cells is finely regulated by factors provided with immunological capacities. Tumour-associated macrophages show an epigenetically controlled down-regulation of the MHC class II expression induced by the decoy receptor DcR3, a member of the TNFR, which further enhances the M2-like polarization. BAT3, a positive regulator of MHC class II expression in normal macrophages, seems to be secreted by TAMs, consequently lacking its intracellular function, it looks like acting as an immunosuppressive factor.In conclusion HLA could cover a considerable role in tumour-development orchestrated by tumour-associated macrophages.
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Antígenos HLA/inmunología , Macrófagos/inmunología , Macrófagos/patología , Neoplasias/inmunología , Neoplasias/patología , Humanos , Receptores Inmunológicos/metabolismo , Transducción de SeñalRESUMEN
Levels of regulatory T cells (Tregs) are increased in different cancer types as well as in inflammatory diseases, such as rheumatoid arthritis. Treg accumulation may result from aberrant proliferation and trafficking as well as greater resilience to oxidative stress compared with conventional T cells. This enhanced antioxidative capacity of Tregs possibly serves as feedback inhibition during inflammation and prevents uncontrolled immune reactions by favoring survival of suppressor rather than effector cells. In this study, we demonstrate that human Tregs express and secrete higher levels of thioredoxin-1, a major antioxidative molecule. Thioredoxin-1 has an essential role in maintaining their surface thiol density as the first line of antioxidative defense mechanisms and is sensitive to proinflammatory stimuli, mainly tumor necrosis factor-α, in a nuclear factor-κB-dependent fashion. The antiapoptotic and oncogenic potential of (secreted) Trx-1 suggests that it may exert effects in Tregs beyond redox regulation.