RESUMEN
Regulatory T cells (Tregs) control adaptive immunity and restrain type 2 inflammation in allergic disease. Interleukin-33 promotes the expansion of tissue-resident Tregs and group 2 innate lymphoid cells (ILC2s); however, how Tregs locally coordinate their function within the inflammatory niche is not understood. Here, we show that ILC2s are critical orchestrators of Treg function. Using spatial, cellular, and molecular profiling of the type 2 inflamed niche, we found that ILC2s and Tregs engage in a direct (OX40L-OX40) and chemotaxis-dependent (CCL1-CCR8) cellular dialogue that enforces the local accumulation of Gata3high Tregs, which are transcriptionally and functionally adapted to the type 2 environment. Genetic interruption of ILC2-Treg communication resulted in uncontrolled type 2 lung inflammation after allergen exposure. Mechanistically, we found that Gata3high Tregs can modulate the local bioavailability of the costimulatory molecule OX40L, which subsequently controlled effector memory T helper 2 cell numbers. Hence, ILC2-Treg interactions represent a critical feedback mechanism to control adaptive type 2 immunity.
Asunto(s)
Inmunidad Adaptativa , Factor de Transcripción GATA3 , Ratones Endogámicos C57BL , Linfocitos T Reguladores , Animales , Linfocitos T Reguladores/inmunología , Factor de Transcripción GATA3/inmunología , Factor de Transcripción GATA3/metabolismo , Ratones , Inmunidad Adaptativa/inmunología , Linfocitos/inmunología , Inmunidad Innata/inmunología , Ratones Noqueados , Células Th2/inmunología , FemeninoRESUMEN
Transmembrane protein GARP binds latent TGF-ß1 to form GARP:(latent)TGF-ß1 complexes on the surface of several cell types including Tregs, B-cells, and platelets. Upon stimulation, these cells release active TGF-ß1. Blocking TGF-ß1 activation by Tregs with anti-GARP:TGF-ß1 mAbs overcomes resistance to PD1/PD-L1 blockade and induces immune-mediated regressions of murine tumors, indicating that Treg-derived TGF-ß1 inhibits anti-tumor immunity. TGF-ß1 exerts a vast array of effects on immune responses. For example, it favors differentiation of TH17 cells and B-cell switch to IgA production, two important processes for mucosal immunity. Here, we sought to determine whether treatment with anti-GARP:TGF-ß1 mAbs would perturb immune responses to intestinal bacterial infection. We observed no aggravation of intestinal disease, no systemic dissemination, and no alteration of innate or adaptative immune responses upon oral gavage of C. rodentium in highly susceptible Il22r-/- mice treated with anti-GARP:TGF-ß1 mAbs. To examine the effects of GARP:TGF-ß1 blockade on Ig production, we compared B cell- and TH cell- responses to OVA or CTB protein immunization in mice carrying deletions of Garp in Tregs, B cells, or platelets. No alteration of adaptive immune responses to protein immunization was observed in the absence of GARP on any of these cells. Altogether, we show that antibody-mediated blockade of GARP:TGF-ß1 or genetic deletion of Garp in Tregs, B cells or platelets, do not alter innate or adaptive immune responses to intestinal bacterial infection or protein immunization in mice. Anti-GARP:TGF-ß1 mAbs, currently tested for cancer immunotherapy, may thus restore anti-tumor immunity without severely impairing other immune defenses. PRéCIS: Immunotherapy with GARP:TGF-ß1 mAbs may restore anti-tumor immunity without impairing immune or inflammatory responses required to maintain homeostasis or host defense against infection, notably at mucosal barriers.
Asunto(s)
Inmunidad Adaptativa , Infecciones Bacterianas , Proteínas de la Membrana , Factor de Crecimiento Transformador beta1 , Animales , Anticuerpos Monoclonales/metabolismo , Infecciones Bacterianas/inmunología , Infecciones Bacterianas/metabolismo , Inmunidad , Inmunización , Proteínas de la Membrana/metabolismo , Ratones , Linfocitos T Reguladores , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
It is unclear how genetic aberrations impact the state of nascent tumour cells and their microenvironment. BRCA1 driven triple negative breast cancer (TNBC) has been shown to arise from luminal progenitors yet little is known about how BRCA1 loss-of-function (LOF) and concomitant mutations affect the luminal progenitor cell state. Here we demonstrate how time-resolved single-cell profiling of genetically engineered mouse models before tumour formation can address this challenge. We found that perturbing Brca1/p53 in luminal progenitors induces aberrant alveolar differentiation pre-malignancy accompanied by pro-tumourigenic changes in the immune compartment. Unlike alveolar differentiation during gestation, this process is cell autonomous and characterised by the dysregulation of transcription factors driving alveologenesis. Based on our data we propose a model where Brca1/p53 LOF inadvertently promotes a differentiation program hardwired in luminal progenitors, highlighting the deterministic role of the cell-of-origin and offering a potential explanation for the tissue specificity of BRCA1 tumours.
Asunto(s)
Proteína BRCA1/genética , Transformación Celular Neoplásica/genética , Neoplasias Mamarias Experimentales/genética , Fenobarbital/metabolismo , Análisis de la Célula Individual/métodos , Células Madre/patología , Animales , Proteína BRCA1/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Comunicación Celular/fisiología , Diferenciación Celular/fisiología , Transformación Celular Neoplásica/metabolismo , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/patología , Ratones , Mutación , Células Madre/fisiología , Microambiente Tumoral/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Metastasis constitutes the primary cause of cancer-related deaths, with the lung being a commonly affected organ. We found that activation of lung-resident group 2 innate lymphoid cells (ILC2s) orchestrated suppression of natural killer (NK) cell-mediated innate antitumor immunity, leading to increased lung metastases and mortality. Using multiple models of lung metastasis, we show that interleukin (IL)-33-dependent ILC2 activation in the lung is involved centrally in promoting tumor burden. ILC2-driven innate type 2 inflammation is accompanied by profound local suppression of interferon-γ production and cytotoxic function of lung NK cells. ILC2-dependent suppression of NK cells is elaborated via an innate regulatory mechanism, which is reliant on IL-5-induced lung eosinophilia, ultimately limiting the metabolic fitness of NK cells. Therapeutic targeting of IL-33 or IL-5 reversed NK cell suppression and alleviated cancer burden. Thus, we reveal an important function of IL-33 and ILC2s in promoting tumor metastasis via their capacity to suppress innate type 1 immunity.
Asunto(s)
Eosinófilos/inmunología , Células Asesinas Naturales/inmunología , Neoplasias Pulmonares/inmunología , Pulmón/inmunología , Linfocitos/inmunología , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Interleucina-33/metabolismo , Interleucina-5/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Metástasis de la Neoplasia , Células Th2/inmunologíaRESUMEN
The inflammatory response to transformed cells forms the cornerstone of natural or therapeutically induced protective immunity to cancer. Regulatory T (Treg) cells are known for their critical role in suppressing inflammation, and therefore can antagonize effective anti-cancer immune responses. As such, Treg cells can play detrimental roles in tumour progression and in the response to both conventional and immune-based cancer therapies. Recent advances in our understanding of Treg cells reveal complex niche-specific regulatory programmes and functions, which are likely to extrapolate to cancer. The regulation of Treg cells is reliant on upstream cues from haematopoietic and non-immune cells, which dictates their genetic, epigenetic and downstream functional programmes. In this review we will discuss how Treg cells are themselves regulated in normal and transformed tissues, and the implications of this cross talk on tumour growth.
Asunto(s)
Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Escape del Tumor , Animales , Humanos , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Linfocitos Infiltrantes de Tumor/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Fenotipo , Transducción de Señal , Linfocitos T Reguladores/metabolismo , Microambiente TumoralRESUMEN
Transforming growth factor-ß1 (TGF-ß1) is one of very few cytokines produced in a latent form, requiring activation to exert any of its vastly diverse effects on development, immunity, and cancer. Regulatory T cells (Tregs) suppress immune cells within close proximity by activating latent TGF-ß1 presented by GARP (glycoprotein A repetitions predominant) to integrin αVß8 on their surface. We solved the crystal structure of GARP:latent TGF-ß1 bound to an antibody that stabilizes the complex and blocks release of active TGF-ß1. This finding reveals how GARP exploits an unusual medley of interactions, including fold complementation by the amino terminus of TGF-ß1, to chaperone and orient the cytokine for binding and activation by αVß8. Thus, this work further elucidates the mechanism of antibody-mediated blockade of TGF-ß1 activation and immunosuppression by Tregs.
Asunto(s)
Tolerancia Inmunológica , Proteínas de la Membrana/química , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/química , Humanos , Activación de Linfocitos , Proteínas de la Membrana/inmunología , Conformación Proteica en Lámina beta , Pliegue de Proteína , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
Human regulatory T cells (Tregs) suppress other T cells by converting the latent, inactive form of TGF-ß1 into active TGF-ß1. In Tregs, TGF-ß1 activation requires GARP, a transmembrane protein that binds and presents latent TGF-ß1 on the surface of Tregs stimulated through their T cell receptor. However, GARP is not sufficient because transduction of GARP in non-Treg T cells does not induce active TGF-ß1 production. RGD-binding integrins were shown to activate TGF-ß1 in several non-T cell types. Here we show that αVß8 dimers are present on stimulated human Tregs but not in other T cells, and that antibodies against αV or ß8 subunits block TGF-ß1 activation in vitro. We also show that αV and ß8 interact with GARP/latent TGF-ß1 complexes in human Tregs. Finally, a blocking antibody against ß8 inhibited immunosuppression by human Tregs in a model of xenogeneic graft-vs.-host disease induced by the transfer of human T cells in immunodeficient mice. These results show that TGF-ß1 activation on the surface of human Tregs implies an interaction between the integrin αVß8 and GARP/latent TGF-ß1 complexes. Immunosuppression by human Tregs can be inhibited by antibodies against GARP or against the integrin ß8 subunit. Such antibodies may prove beneficial against cancer or chronic infections.
Asunto(s)
Enfermedad Injerto contra Huésped/inmunología , Tolerancia Inmunológica/efectos de los fármacos , Integrinas/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/uso terapéutico , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Integrinas/antagonistas & inhibidores , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Ratones , Ratones SCID , Neoplasias/inmunología , Neoplasias/terapia , Linfocitos T Reguladores/trasplante , Factor de Crecimiento Transformador beta1/metabolismo , Trasplante HeterólogoRESUMEN
TGF-ß1, 2 and 3 cytokines are involved in many cellular processes including cell proliferation, differentiation, migration and survival. Whereas TGF-ß2 and 3 play important roles in embryonic development, TGF-ß1 is mostly implicated in controlling immune responses after birth. The production of TGF-ß1 is a tightly regulated process, occurring mostly at a post-translational level. Virtually all cells produce the latent, inactive form of TGF-ß1. In latent TGF-ß1, the mature TGF-ß1 dimer is non-covalently associated to the Latency Associated Peptide, or LAP, which prevents binding to the TGF-ß1 receptor. Activation of the cytokine implies release of mature TGF-ß1 from LAP. Only a few cell types activate latent TGF-ß1, via mechanisms that are cell type specific. Proteins such as integrins, proteases and thrombospondin-1 activate TGF-ß1 in epithelial cells, fibroblasts and dendritic cells. More recently, the protein GARP was shown to be involved in TGF-ß1 activation by regulatory T cells (Treg), a subset of CD4+ T lymphocytes specialized in suppression of immune responses. GARP is a transmembrane protein that binds latent-TGF-ß1 and tethers it on the Treg surface. The role of GARP was studied mostly in Tregs, and this was recently reviewed in L. Sun, H. Jin and H. Li, Oncotarget, 2016, 7, 42826-42836. However, GARP is also expressed in non-immune cells. This review focuses on the roles of GARP in latent TGF-ß1 activation by immune and non-immune cells.
Asunto(s)
Proteínas de la Membrana/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Humanos , Proteínas de la Membrana/genética , Factor de Crecimiento Transformador beta2/genética , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta3/genética , Factor de Crecimiento Transformador beta3/metabolismoRESUMEN
Production of active TGF-ß is regulated at a posttranslational level and implies release of the mature cytokine dimer from the inactive, latent TGF-ß precursor. There are several cell-type specific mechanisms of TGF-ß activation. We identified a new mechanism operating on the surface of human regulatory T cells and involving membrane protein GARP, which binds latent TGF-ß1. The paracrine activity of regulatory T cell-derived TGF-ß1 contributes to immunosuppression and can be inhibited with anti-GARP Abs. Whether other immune cell types use surface GARP to activate latent TGF-ß1 was not known. We show in this study that stimulated, human B lymphocytes produce active TGF-ß1 from surface GARP/latent TGF-ß1 complexes with isotype switching to IgA production.
Asunto(s)
Linfocitos B/inmunología , Inmunoglobulina A/biosíntesis , Cambio de Clase de Inmunoglobulina , Proteínas de la Membrana/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Linfocitos B/efectos de los fármacos , Citocinas/inmunología , Humanos , Inmunoglobulina A/inmunología , Activación de Linfocitos , Oligodesoxirribonucleótidos/inmunología , Unión Proteica , Recombinación Genética , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/inmunologíaRESUMEN
Reducing Treg function in cancer patients should augment antitumor immune responses. We recently uncovered a mechanism of immunosuppression by human Tregs that implies transmembrane protein GARP and production of active TGF-ß1. We obtained monoclonal antibodies that block this process and could thus serve as a novel approach for cancer immunotherapy.
RESUMEN
Production of active TGF-ß1 is one mechanism by which human regulatory T cells (Tregs) suppress immune responses. This production is regulated by glycoprotein A repetitions predominant (GARP), a transmembrane protein present on stimulated Tregs but not on other T lymphocytes (Th and CTLs). GARP forms disulfide bonds with proTGF-ß1, favors its cleavage into latent inactive TGF-ß1, induces the secretion and surface presentation of GARP·latent TGF-ß1 complexes, and is required for activation of the cytokine in Tregs. We explored whether additional Treg-specific protein(s) associated with GARP·TGF-ß1 complexes regulate TGF-ß1 production in Tregs. We searched for such proteins by yeast two-hybrid assay, using GARP as a bait to screen a human Treg cDNA library. We identified lysosomal-associated transmembrane protein 4B (LAPTM4B), which interacts with GARP in mammalian cells and is expressed at higher levels in Tregs than in Th cells. LAPTM4B decreases cleavage of proTGF-ß1, secretion of soluble latent TGF-ß1, and surface presentation of GARP·TGF-ß1 complexes by Tregs but does not contribute to TGF-ß1 activation. Therefore, LAPTM4B binds to GARP and is a negative regulator of TGF-ß1 production in human Tregs. It may play a role in the control of immune responses by decreasing Treg immunosuppression.
Asunto(s)
Proteínas de la Membrana/fisiología , Proteínas Oncogénicas/fisiología , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Regulatory T cells (Tregs) are essential to prevent autoimmunity, but excessive Treg function contributes to cancer progression by inhibiting antitumor immune responses. Tregs exert contact-dependent inhibition of immune cells through the production of active transforming growth factor-ß1 (TGF-ß1). On the Treg cell surface, TGF-ß1 is in an inactive form bound to membrane protein GARP and then activated by an unknown mechanism. We demonstrate that GARP is involved in this activation mechanism. Two anti-GARP monoclonal antibodies were generated that block the production of active TGF-ß1 by human Tregs. These antibodies recognize a conformational epitope that requires amino acids GARP137-139 within GARP/TGF-ß1 complexes. A variety of antibodies recognizing other GARP epitopes did not block active TGF-ß1 production by Tregs. In a model of xenogeneic graft-versus-host disease in NSG mice, the blocking antibodies inhibited the immunosuppressive activity of human Tregs. These antibodies may serve as therapeutic tools to boost immune responses to infection or cancer via a mechanism of action distinct from that of currently available immunomodulatory antibodies. Used alone or in combination with tumor vaccines or antibodies targeting the CTLA4 or PD1/PD-L1 pathways, blocking anti-GARP antibodies may improve the efficiency of cancer immunotherapy.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunosupresores/química , Proteínas de la Membrana/química , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/química , Animales , Autoinmunidad , Epítopos/química , Enfermedad Injerto contra Huésped , Humanos , Proteínas de la Membrana/metabolismo , Metilación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Unión Proteica , Conformación Proteica , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
Transforming growth factor-ß (TGFß) is a key mediator of fibrogenesis. TGFß is overexpressed and activated in fibrotic diseases, regulates fibroblast differentiation into myofibroblasts and induces extracellular matrix deposition. Platelet-derived growth factor (PDGF) is also a regulator of fibrogenesis. Some studies showed a link between TGFß and PDGF in certain fibrotic diseases. TGFß induces PDGF receptor alpha expression in scleroderma fibroblasts. PDGF-C and -D are the most recently discovered ligands and also play a role in fibrosis. In this study, we report the first link between TGFß and PDGF-D and -C ligands. In normal fibroblasts, TGFß down-regulated PDGF-D expression and up-regulated PDGF-C expression at the mRNA and protein levels. This phenomenon is not limited to TGFß since other growth factors implicated in fibrosis, such as FGF, EGF and PDGF-B, also regulated PDGF-D and PDGF-C expression. Among different kinase inhibitors, only TGFß receptor inhibitors and the IκB kinase (IKK) inhibitor BMS-345541 blocked the effect of TGFß. However, activation of the classical NF-κB pathway was not involved. Interestingly, in a model of lung fibrosis induced by either bleomycin or silica, PDGF-D was down-regulated, which correlates with the production of TGFß and other fibrotic growth factors. In conclusion, the down-regulation of PDGF-D by TGFß and other growth factors may serve as a negative feedback in the network of cytokines that control fibrosis.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Linfocinas/metabolismo , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Factor de Crecimiento Transformador beta/farmacología , Línea Celular , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Prepucio/citología , Prepucio/efectos de los fármacos , Prepucio/metabolismo , Humanos , Linfocinas/genética , Masculino , Factor de Crecimiento Derivado de Plaquetas/genética , Proteína Smad4/genética , Proteína Smad4/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
GARP is a transmembrane protein present on stimulated human regulatory T lymphocytes (Tregs), but not on other T lymphocytes (Th cells). It presents the latent form of TGF-ß1 on the Treg surface. We report here that GARP favors the cleavage of the pro-TGF-ß1 precursor and increases the amount of secreted latent TGF-ß1. Stimulated Tregs, which naturally express GARP, and Th cells transfected with GARP secrete a previously unknown form of latent TGF-ß1 that is disulfide-linked to GARP. These GARP/TGF-ß1 complexes are possibly shed from the T cell surface. Secretion of GARP/TGF-ß1 complexes was not observed with transfected 293 cells and may thus be restricted to the T cell lineage. We conclude that in stimulated human Tregs, GARP not only displays latent TGF-ß1 at the cell surface, but also increases its secretion by forming soluble disulfide-linked complexes. Moreover, we identified six microRNAs (miRNAs) that are expressed at lower levels in Treg than in Th clones and that target a short region of the GARP 3' UTR. In transfected Th cells, the presence of this region decreased GARP levels, cleavage of pro-TGF-ß1, and secretion of latent TGF-ß1.
Asunto(s)
Proteínas de la Membrana/inmunología , MicroARNs/metabolismo , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Células HEK293 , Humanos , Inmunoprecipitación , Luciferasas , Proteínas de la Membrana/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T Reguladores/metabolismoRESUMEN
Arsenic trioxide (As(2)O(3)) is an effective therapeutic against acute promyelocytic leukemia and certain solid tumors. Because As(2)O(3) inhibits mitochondrial respiration in leukemia cells, we hypothesized that As(2)O(3) might enhance the radiosensitivity of solid tumors by increasing tumor oxygenation [partial pressure of oxygen (pO(2))] via a decrease in oxygen consumption. Two murine models of radioresistant hypoxic cancer were used to study the effects of As(2)O(3). We measured pO(2) and the oxygen consumption rate in vivo by electron paramagnetic resonance oximetry and (19)fluorine-MRI relaxometry. Tumor perfusion was assessed by Patent blue staining. In both models, As(2)O(3) inhibited mitochondrial respiration, leading to a rapid increase in pO(2). The decrease in oxygen consumption could be explained by an observed decrease in glutathione in As(2)O(3)-treated cells, as this could increase intracellular reactive oxygen species that can disrupt mitochondrial membrane potential. When tumors were irradiated during periods of As(2)O(3)-induced augmented oxygenation, radiosensitivity increased by 2.2-fold compared with control mice. Notably, this effect was abolished when temporarily clamped tumors were irradiated. Together, our findings show that As(2)O(3) acutely increases oxygen consumption and radiosensitizes tumors, providing a new rationale for clinical investigations of As(2)O(3) in irradiation protocols to treat solid tumors.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Carcinoma Pulmonar de Lewis/tratamiento farmacológico , Neoplasias Hepáticas Experimentales/tratamiento farmacológico , Óxidos/farmacología , Consumo de Oxígeno/efectos de los fármacos , Fármacos Sensibilizantes a Radiaciones/farmacología , Animales , Trióxido de Arsénico , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/radioterapia , Terapia Combinada , Modelos Animales de Enfermedad , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/radioterapia , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
PURPOSE: Regulatory T cells (Tregs) are thought to inhibit antitumor immune responses, and their depletion could therefore have a synergistic effect with therapeutic cancer vaccines. We investigated the impact of three medications on blood Treg frequency in vaccinated cancer patients. EXPERIMENTAL DESIGN: To date, the most specific marker for human Tregs is demethylation in the DNA that encodes the transcription factor FOXP3. Thus, we used a FOXP3 methylation-specific quantitative PCR assay (MS-qPCR) to measure Treg frequencies in the peripheral blood mononuclear cells (PBMCs) of melanoma patients. The patients participated in three clinical trials that combined tumor vaccines with potential Treg-depleting agents: low-dose cyclophosphamide, anti-CD25 monoclonal antibody daclizumab, and the IL-2/diphtheria toxin fusion protein denileukin diftitox. RESULTS: In the nine control patients, blood Treg frequencies varied over time; there was a 46% reduction in one patient. In treated patients, a more than 2-fold decrease in Tregs was observed in one out of 11 patients receiving cyclophosphamide and in four out of 13 receiving daclizumab, but there was no such Treg decrease in any of the six patients who received denileukin diftitox. As a positive control, a more than 2-fold increase in blood Tregs was detected in four out of nine patients who were treated with interleukin-2. CONCLUSIONS: We used a MS-qPCR method that detects Tregs but not other activated T lymphocytes; however, none of the Treg-depleting strategies that we tested led, in the majority of patients, to a conservative 50% reduction in blood Tregs.
Asunto(s)
Vacunas contra el Cáncer/uso terapéutico , Metilación de ADN , Factores de Transcripción Forkhead/genética , Melanoma/patología , Linfocitos T Reguladores/metabolismo , Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Monoclonales Humanizados , Antineoplásicos/uso terapéutico , Recuento de Células , Ciclofosfamida/uso terapéutico , Daclizumab , Toxina Diftérica/uso terapéutico , Femenino , Marcadores Genéticos/genética , Humanos , Inmunoglobulina G/uso terapéutico , Interleucina-2/uso terapéutico , Intrones , Depleción Linfocítica , Masculino , Melanoma/inmunología , Melanoma/terapia , Proteínas Recombinantes de Fusión/uso terapéutico , Estudios Retrospectivos , Linfocitos T Reguladores/patologíaRESUMEN
Human Treg and Th clones secrete the latent form of TGF-beta, in which the mature TGF-beta protein is bound to the latency-associated peptide (LAP), and is thereby prevented from binding to the TGF-beta receptor. We previously showed that upon TCR stimulation, human Treg clones but not Th clones produce active TGF-beta and bear LAP on their surface. Here, we show that latent TGF-beta, i.e. both LAP and mature TGF-beta, binds to glycoprotein A repetitions predominant (GARP), a transmembrane protein containing leucine rich repeats, which is present on the surface of stimulated Treg clones but not on Th clones. Membrane localization of latent TGF-beta mediated by binding to GARP may be necessary for the ability of Treg to activate TGF-beta upon TCR stimulation. However, it is not sufficient as lentiviral-mediated expression of GARP in human Th cells induces binding of latent TGF-beta to the cell surface, but does not result in the production of active TGF-beta upon stimulation of these Th cells.
Asunto(s)
Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Western Blotting , Antígenos CD4/metabolismo , Células Cultivadas , Citometría de Flujo , Humanos , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Subunidad alfa del Receptor de Interleucina-7/metabolismo , Células Jurkat , Proteínas de Unión a TGF-beta Latente/genética , Proteínas de Unión a TGF-beta Latente/metabolismo , Activación de Linfocitos/inmunología , Proteínas de la Membrana/genética , Modelos Biológicos , Muromonab-CD3/inmunología , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti-MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 x 10(-6) to 2 x 10(-3) among the CD4(+) blood T lymphocytes of the patients. The CD4(+) blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4(+) T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25(+) phenotype in the resting state. These CD25(+) clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-beta but none of cytokines IFN-gamma, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25(-) clones had a significant but lower suppressive activity. Most of the CD25(-) clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4(+) T cells that may exert regulatory T-cell function in vivo.
Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD4-Positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Melanoma/inmunología , Proteínas de Neoplasias/inmunología , Linfocitos T Reguladores/inmunología , Vacunas de Subunidad/uso terapéutico , Antígenos de Neoplasias/genética , Línea Celular Tumoral , Células Clonales/inmunología , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Humanos , Interleucina-2/farmacología , Interleucina-4/farmacología , Interleucina-7/farmacología , Activación de Linfocitos/efectos de los fármacos , Proteínas de Neoplasias/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Linfocitos T/inmunologíaRESUMEN
From cancerous and non-cancerous patients, we derived stable clones of CD4(+) Treg, defined as clones that expressed high CD25 at rest, were anergic in vitro, and suppressed the proliferation of co-cultured CD4(+) cells. A conserved region of FOXP3 intron 1 was demethylated in all Treg clones, whereas it was methylated in non-regulatory Th and CTL clones. In our panel of human clones, this stable epigenetic mark correlated better with suppressive activity than did FOXP3 mRNA or protein expression. We used expression microarrays to compare Treg and Th clones after activation, which is required for suppressive function. The transcriptional profile that is specific of activated Treg clones includes a TGF-beta signature. Both activated Treg and Th clones produced the latent form of TGF-beta. However, SMAD2 phosphorylation was observed after activation in the Treg but not in the Th clones, indicating that only activated Treg clones produced the bioactive form of TGF-beta. A TGF-beta signature was also displayed by a Th clone "suppressed" by a Treg clone. In conclusion, the hallmark of our panel of activated human Treg clones is to produce bioactive TGF-beta which has autocrine actions on Tregs and can have paracrine actions on other T cells.
Asunto(s)
Factores de Transcripción Forkhead/inmunología , Proteína Smad2/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/inmunología , Células Clonales/inmunología , Células Clonales/metabolismo , Metilación de ADN/genética , Metilación de ADN/inmunología , Femenino , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Perfilación de la Expresión Génica , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Masculino , Melanoma/inmunología , Melanoma/metabolismo , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/metabolismo , Proteína Smad2/metabolismo , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Among different features of cancer cells, two of them have retained our interest: their nearly universal glycolytic phenotype and their sensitivity towards an oxidative stress. Therefore, we took advantage of these features to develop an experimental approach by selectively exposing cancer cells to an oxidant insult induced by the combination of menadione (vitamin K(3)) and ascorbate (vitamin C). Ascorbate enhances the menadione redox cycling, increases the formation of reactive oxygen species and kills K562 cells as shown by more than 65% of LDH leakage after 24 hr of incubation. Since both lactate formation and ATP content are depressed by about 80% following ascorbate/menadione exposure, we suggest that the major intracellular event involved in such a cytotoxicity is related to the impairment of glycolysis. Indeed, NAD(+) is rapidly and severely depleted, a fact most probably related to a strong Poly(ADP-ribose) polymerase (PARP) activation, as shown by the high amount of poly-ADP-ribosylated proteins. The addition of N-acetylcysteine (NAC) restores most of the ATP content and the production of lactate as well. The PARP inhibitor dihydroxyisoquinoline (DiQ) was able to partially restore both parameters as well as cell death induced by ascorbate/menadione. These results suggest that the PARP activation induced by the oxidative stress is a major but not the only intracellular event involved in cell death by ascorbate/menadione. Due to the high energetic dependence of cancer cells on glycolysis, the impairment of such an essential pathway may explain the effectiveness of this combination to kill cancer cells.