RESUMEN
Tumor-associated eosinophilia is frequently observed in cancer. However, despite numerous studies of patients with cancer and mouse models of cancer, it has remained uncertain if eosinophils contribute to tumor immunity or are mere bystander cells. Here we report that activated eosinophils were essential for tumor rejection in the presence of tumor-specific CD8(+) T cells. Tumor-homing eosinophils secreted chemoattractants that guided T cells into the tumor, which resulted in tumor eradication and survival. Activated eosinophils initiated substantial changes in the tumor microenvironment, including macrophage polarization and normalization of the tumor vasculature, which are known to promote tumor rejection. Thus, our study presents a new concept for eosinophils in cancer that may lead to novel therapeutic strategies.
Asunto(s)
Vasos Sanguíneos/inmunología , Linfocitos T CD8-positivos/inmunología , Factores Quimiotácticos/inmunología , Eosinófilos/inmunología , Melanoma/inmunología , Neoplasias Cutáneas/inmunología , Animales , Diferenciación Celular , Movimiento Celular , Citotoxicidad Inmunológica , Melanoma/irrigación sanguínea , Melanoma Experimental , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neovascularización Patológica/inmunología , Neovascularización Fisiológica , Neoplasias Cutáneas/irrigación sanguínea , Carga Tumoral/inmunología , Microambiente TumoralRESUMEN
CD8(+) T cells recognizing tumor-specific antigens are detected in cancer patients but are dysfunctional. Here we developed a tamoxifen-inducible liver cancer mouse model with a defined oncogenic driver antigen (SV40 large T-antigen) to follow the activation and differentiation of naive tumor-specific CD8(+) T (TST) cells after tumor initiation. Early during the pre-malignant phase of tumorigenesis, TST cells became dysfunctional, exhibiting phenotypic, functional, and transcriptional features similar to dysfunctional T cells isolated from late-stage human tumors. Thus, T cell dysfunction seen in advanced human cancers may already be established early during tumorigenesis. Although the TST cell dysfunctional state was initially therapeutically reversible, it ultimately evolved into a fixed state. Persistent antigen exposure rather than factors associated with the tumor microenvironment drove dysfunction. Moreover, the TST cell differentiation and dysfunction program exhibited features distinct from T cell exhaustion in chronic infections. Strategies to overcome this antigen-driven, cell-intrinsic dysfunction may be required to improve cancer immunotherapy.
Asunto(s)
Antígenos Transformadores de Poliomavirus/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Inmunoterapia Adoptiva/métodos , Neoplasias Hepáticas/inmunología , Animales , Carcinogénesis , Diferenciación Celular , Células Cultivadas , Senescencia Celular , Modelos Animales de Enfermedad , Humanos , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/terapia , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Tamoxifeno , Microambiente TumoralRESUMEN
The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long-term repopulating HSCs (LT-HSC) are routinely enriched as Lin- Sca1+ c-Kit+ CD34- Flt3- CD150+ CD48- cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L-eGFP reporter mice, we show that endogenous Flt3L-eGFP-reporter RNA expression correlates with eGFP-protein expression. This Flt3L-eGFP-reporter expression distinguishes two LT-HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L-eGFP-reporterlow cells are identified as predominantly resting HSCs with long-term repopulating capacities. In contrast, Flt3L-eGFP-reporterhigh cells are in majority proliferating HSCs with only short-term repopulating capacities. Flt3L-eGFP-reporterlow cells express hypoxia, autophagy-inducing, and the LT-HSC-associated genes HoxB5 and Fgd5, while Flt3L-eGFP-reporterhigh HSCs upregulate genes involved in HSC differentiation. Flt3L-eGFP-reporterlow cells develop to Flt3L-eGFP-reporterhigh cells in vitro, although Flt3L-eGFP-reporterhigh cells remain Flt3L-eGFP-reporterhigh . CD150+ Flt3L-eGFP-reporterlow cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L-eGFP-reporterhigh cells do express EPCR but not CD41. Thus, FACS-enrichment of Flt3/ Flt3L-eGFP-reporter negative, Lin- CD150+ CD48- EPCR+ CD41+ HSCs allows a further 5-fold enrichment of functional LT-HSCs.
Asunto(s)
Células de la Médula Ósea/fisiología , Células Madre Hematopoyéticas/fisiología , Proteínas de la Membrana/metabolismo , Animales , Autofagia/genética , Diferenciación Celular , Proliferación Celular , Autorrenovación de las Células , Células Cultivadas , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/genética , Factores de Intercambio de Guanina Nucleótido/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hipoxia/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miembro 1 de la Familia de Moléculas Señalizadoras de la Activación Linfocitaria/metabolismoRESUMEN
In adult mice, lymphopenia-induced proliferation (LIP) leads to T cell activation, memory differentiation, tissue destruction, and a loss of TCR diversity. Neonatal mice are lymphopenic within the first week of life. This enables some recent thymic emigrants to undergo LIP and convert into long-lived memory T cells. Surprisingly, however, most neonatal T cells do not undergo LIP. We therefore asked whether neonate-specific mechanisms prevent lymphopenia-driven T cell activation. In this study, we show that IL-7R-dependent innate lymphoid cells (ILCs) block LIP of CD8(+) T cells in neonatal but not adult mice. Importantly, CD8(+) T cell responses against a foreign Ag are not inhibited by neonatal ILCs. This ILC-based inhibition of LIP ensures the generation of a diverse naive T cell pool in lymphopenic neonates that is mandatory for the maintenance of T cell homeostasis and immunological self-tolerance later in life.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica , Memoria Inmunológica , Activación de Linfocitos , Linfopenia/inmunología , Traslado Adoptivo , Animales , Animales Recién Nacidos , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Proliferación Celular , Homeostasis , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Receptores de Interleucina-7/inmunología , Transducción de SeñalRESUMEN
BACKGROUND: Angiogenesis is crucial for many pathological processes and becomes a therapeutic strategy against diseases ranging from inflammation to cancer. The regulatory mechanism of angiogenesis remains unclear. Although tetraspanin CD82 is widely expressed in various endothelial cells (ECs), its vascular function is unknown. METHODS AND RESULTS: Angiogenesis was examined in Cd82-null mice with in vivo and ex vivo morphogenesis assays. Cellular functions, molecular interactions, and signaling were analyzed in Cd82-null ECs. Angiogenic responses to various stimuli became markedly increased upon Cd82 ablation. Major changes in Cd82-null ECs were enhanced migration and invasion, likely resulting from the upregulated expression of cell adhesion molecules such as CD44 and integrins at the cell surface and subsequently elevated outside-in signaling. Gangliosides, lipid raft clustering, and CD44-membrane microdomain interactions were increased in the plasma membrane of Cd82-null ECs, leading to less clathrin-independent endocytosis and then more surface presence of CD44. CONCLUSIONS: Our study reveals that CD82 restrains pathological angiogenesis by inhibiting EC movement, that lipid raft clustering and cell adhesion molecule trafficking modulate angiogenic potential, that transmembrane protein modulates lipid rafts, and that the perturbation of CD82-ganglioside-CD44 signaling attenuates pathological angiogenesis.
Asunto(s)
Células Endoteliales/metabolismo , Receptores de Hialuranos/metabolismo , Proteína Kangai-1/metabolismo , Microdominios de Membrana/metabolismo , Neovascularización Patológica/metabolismo , Animales , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Endocitosis/fisiología , Células Endoteliales/patología , Gangliósidos/metabolismo , Proteína Kangai-1/genética , Microdominios de Membrana/patología , Ratones Noqueados , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Transporte de Proteínas/fisiología , Transducción de Señal/fisiologíaRESUMEN
Transgenic mice expressing the diphtheria toxin receptor (DTR) in specific cell types are key tools for functional studies in several biological systems. B6.FVB-Tg(Itgax-DTR/EGFP)57Lan/J (CD11c.DTR) and B6.Cg-Tg(Itgax-DTR/OVA/EGFP)1Gjh/Crl (CD11c.DOG) mice express the DTR in CD11c(+) cells, allowing conditional depletion of dendritic cells. We report that dendritic-cell depletion in these models caused polymorphonuclear neutrophil (PMN) release from the bone marrow, which caused chemokine-dependent neutrophilia after 6-24 h and increased bacterial clearance in a mouse pyelonephritis model. We present a transgenic mouse line, B6.Cg-Tg(Itgax-EGFP-CRE-DTR-LUC)2Gjh/Crl (CD11c.LuciDTR), which is unaffected by early neutrophilia. However, CD11c.LuciDTR and CD11c.DTR mice showed late neutrophilia 72 h after dendritic cell depletion, which was independent of PMN release and possibly resulted from increased granulopoiesis. Thus, the time point of dendritic cell depletion and the choice of DTR transgenic mouse line must be considered in experimental settings where neutrophils may be involved.
Asunto(s)
Antígeno CD11c/inmunología , Neutrófilos/inmunología , Animales , Antígeno CD11c/genética , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Células Dendríticas/patología , Toxina Diftérica/farmacología , Modelos Animales de Enfermedad , Femenino , Macrófagos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Inmunológicos , Neutrófilos/citología , Pielonefritis/inmunología , Pielonefritis/microbiología , Pielonefritis/patología , Escherichia coli Uropatógena/inmunología , Escherichia coli Uropatógena/fisiologíaRESUMEN
In healthy individuals, T cells react against incoming pathogens, but remain tolerant to self-antigens, thereby preventing autoimmune reactions. CD4 regulatory T cells are major contributors in induction and maintenance of peripheral tolerance, but a regulatory role has been also reported for several subsets of CD8 T cells. To determine the molecular basis of peripheral CD8 T-cell tolerance, we exploited a double transgenic mouse model in which CD8 T cells are neonatally tolerized following interaction with a parenchymal self-antigen. These tolerant CD8 T cells have regulatory capacity and can suppress T cells in an antigen-specific manner during adulthood. Dickkopf-3 (DKK3) was found to be expressed in the tolerant CD8 T cells and to be essential for the observed CD8 T-cell tolerance. In vitro, genetic deletion of DKK3 or blocking with antibodies restored CD8 T-cell proliferation and IL-2 production in response to the tolerizing self-antigen. Moreover, exogenous DKK3 reduced CD8 T-cell reactivity. In vivo, abrogation of DKK3 function reversed tolerance, leading to eradication of tumors expressing the target antigen and to rejection of autologous skin grafts. Thus, our findings define DKK3 as a immune modulator with a crucial role for CD8 T-cell tolerance.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Tolerancia Inmunológica/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Western Blotting , Linfocitos T CD8-positivos/citología , Proliferación Celular , Citotoxicidad Inmunológica , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Inflammatory mechanisms contribute substantially to secondary tissue injury after brain ischemia. Regulatory T cells (Tregs) are key endogenous modulators of postischemic neuroinflammation. We investigated the potential of histone deacetylase inhibition (HDACi) to enhance Treg potency for experimental stroke in mice. HDACi using trichostatin A increased the number of Tregs and boosted their immunosuppressive capacity and interleukin (IL)-10 expression. In vivo treatment reduced infarct volumes and behavioral deficits after cortical brain ischemia, attenuated cerebral proinflammatory cytokine expression, and increased numbers of brain-invading Tregs. A similar effect was obtained using tubastatin, a specific inhibitor of HDAC6 and a key HDAC in Foxp3 regulation. The neuroprotective effect of HDACi depended on the presence of Foxp3(+) Tregs, and in vivo and in vitro studies showed that the anti-inflammatory cytokine IL-10 was their main mediator. In summary, modulation of Treg function by HDACi is a novel and potent target to intervene at the center of neuroinflammation. Furthermore, this novel concept of modulating endogenous immune mechanisms might be translated to a broad spectrum of diseases, including primary neuroinflammatory and neurodegenerative disorders.
Asunto(s)
Inflamación/inmunología , Inflamación/prevención & control , Accidente Cerebrovascular/inmunología , Accidente Cerebrovascular/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Animales , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/farmacología , Ácidos Hidroxámicos/uso terapéutico , Inflamación/patología , Interleucina-10/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Accidente Cerebrovascular/genéticaRESUMEN
BACKGROUND: Tumor cell migration and metastasis require dynamic rearrangements of the actin cytoskeleton. Interestingly, the F-actin cross-linking and stabilizing protein L-plastin, originally described as a leukocyte specific protein, is aberrantly expressed in several non-hematopoietic malignant tumors. Therefore, it has been discussed as a tumor marker. However, systematic in vivo analyses of the functional relevance of L-plastin for tumor cell metastasis were so far lacking. METHODS: We investigated the relevance of L-plastin expression and phosphorylation by ectopical expression of L-plastin in human melanoma cells (MV3) and knock-down of endogenous L-plastin in prostate cancer (PC3M). The growth and metastatic potential of tumor cells expressing no L-plastin, phosphorylatable or non-phosphorylatable L-plastin was analyzed in a preclinical mouse model after subcutaneous and intracardial injection of the tumor cells. RESULTS: Knock-down of endogenous L-plastin in human prostate carcinoma cells led to reduced tumor cell growth and metastasis. Vice versa, and in line with these findings, ectopic expression of L-plastin in L-plastin negative melanoma cells significantly increased the number of metastases. Strikingly, the metastasis promoting effect of L-plastin was not observed if a non-phosphorylatable L-plastin mutant was expressed. CONCLUSIONS: Our data provide the first in vivo evidence that expression of L-plastin promotes tumor metastasis and, importantly, that this effect depends on an additionally required phosphorylation of L-plastin. In conclusion, these findings imply that for determining the importance of tumor-associated proteins like L-plastin a characterization of posttranslational modifications is indispensable.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Neoplasias de la Próstata/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Citoesqueleto/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Masculino , Melanoma/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Invasividad Neoplásica , Fosforilación , Neoplasias de la Próstata/patología , Transfección , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.
Asunto(s)
Células Dendríticas/patología , Inmunidad Innata/fisiología , Fagocitos/fisiología , Yersiniosis/inmunología , Yersinia enterocolitica/inmunología , Traslado Adoptivo , Animales , Bacterias/inmunología , Separación Celular , Células Cultivadas , Femenino , Homeostasis/inmunología , Ratones , Ratones Transgénicos , Neutrófilos/trasplante , Fagocitos/inmunología , Regulación hacia Arriba/inmunología , Yersiniosis/patología , Yersiniosis/terapiaRESUMEN
FoxP3(+) confers suppressive properties and is confined to regulatory T cells (T(reg)) that potently inhibit autoreactive immune responses. In the transplant setting, natural CD4(+) T(reg) are critical in controlling alloreactivity and the establishment of tolerance. We now identify an important CD8(+) population of FoxP3(+) T(reg) that convert from CD8(+) conventional donor T cells after allogeneic but not syngeneic bone marrow transplantation. These CD8(+) T(reg) undergo conversion in the mesenteric lymph nodes under the influence of recipient dendritic cells and TGF-ß. Importantly, this population is as important for protection from GVHD as the well-studied natural CD4(+)FoxP3(+) population and is more potent in exerting class I-restricted and antigen-specific suppression in vitro and in vivo. Critically, CD8(+)FoxP3(+) T(reg) are exquisitely sensitive to inhibition by cyclosporine but can be massively and specifically expanded in vivo to prevent GVHD by coadministering rapamycin and IL-2 antibody complexes. CD8(+)FoxP3(+) T(reg) thus represent a new regulatory population with considerable potential to preferentially subvert MHC class I-restricted T-cell responses after bone marrow transplantation.
Asunto(s)
Trasplante de Médula Ósea , Linfocitos T CD8-positivos/citología , Factores de Transcripción Forkhead/metabolismo , Tolerancia Inmunológica/inmunología , Linfocitos T Reguladores/citología , Animales , Anticuerpos/administración & dosificación , Anticuerpos/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/inmunología , Proliferación Celular/efectos de los fármacos , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Epítopos/inmunología , Femenino , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/patología , Tolerancia Inmunológica/efectos de los fármacos , Interleucina-2/inmunología , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Fenotipo , Sirolimus/administración & dosificación , Sirolimus/farmacología , Análisis de Supervivencia , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Factor de Crecimiento Transformador beta/farmacología , Trasplante HomólogoRESUMEN
Bone marrow-derived dendritic cell (DC) precursors seed peripheral organs, where they encounter diverse cellular environments during their final differentiation into DCs. Flt3 ligand (Flt3-L) is critical for instructing DC generation throughout different organs. However, it remains unknown which cells produce Flt3-L and, importantly, which cellular source drives DC development in such a variety of organs. Using a novel BAC transgenic Flt3-L reporter mouse strain coexpressing enhanced GFP and luciferase, we show ubiquitous Flt3-L expression in organs and cell types. These results were further confirmed at the protein level. Although Flt3-L was produced by immune and nonimmune cells, the source required for development of the DC compartment clearly differed among organs. In lymphoid organs such as the spleen and bone marrow, Flt3-L production by hemopoietic cells was critical for generation of normal DC numbers. This was unexpected for the spleen because both immune and nonimmune cells equally contributed to the Flt3-L content in that organ. Thus, localized production rather than the total tissue content of Flt3-L in spleen dictated normal splenic DC development. No differences were observed in the number of DC precursors, suggesting that the immune source of Flt3-L promoted pre-cDC differentiation in spleen. In contrast, DC generation in the lung, kidney, and pancreas was mostly driven by nonhematopoietic cells producing Flt3-L, with little contribution by immune cells. These findings demonstrate a high degree of flexibility in Flt3-L-dependent DC generation to adapt this process to organ-specific cellular environments encountered by DC precursors during their final differentiation.
Asunto(s)
Diferenciación Celular/inmunología , Células Dendríticas/citología , Animales , Médula Ósea , Células Madre Hematopoyéticas , Proteínas de la Membrana , Ratones , Ratones Endogámicos C57BL , Especificidad de ÓrganosRESUMEN
The vasculature of solid tumours is morphologically aberrant and characterized by dilated and fragile vessels, intensive vessel sprouting and loss of hierarchical architecture. Constant vessel remodelling leads to spontaneous haemorrhages and increased interstitial fluid pressure in the tumour environment. Tumour-related angiogenesis supports tumour growth and is also a major obstacle for successful immune therapy as it prevents migration of immune effector cells into established tumour parenchyma. The molecular mechanisms for these angiogenic alterations are largely unknown. Here we identify regulator of G-protein signalling 5 (Rgs5) as a master gene responsible for the abnormal tumour vascular morphology in mice. Loss of Rgs5 results in pericyte maturation, vascular normalization and consequent marked reductions in tumour hypoxia and vessel leakiness. These vascular and intratumoral changes enhance influx of immune effector cells into tumour parenchyma and markedly prolong survival of tumour-bearing mice. This is the first demonstration, to our knowledge, of reduced tumour angiogenesis and improved immune therapeutic outcome on loss of a vascular gene function and establishes a previously unrecognized role of G-protein signalling in tumour angiogenesis.
Asunto(s)
Neovascularización Patológica/prevención & control , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/inmunología , Proteínas RGS/deficiencia , Proteínas RGS/metabolismo , Animales , Permeabilidad Capilar , Hipoxia de la Célula/fisiología , Femenino , Masculino , Ratones , Oxígeno/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas RGS/genéticaRESUMEN
Natural killer (NK) cells are an important element in the immune defense against the orthopox family members vaccinia virus (VV) and ectromelia virus (ECTV). NK cells are regulated through inhibitory and activating signaling receptors, the latter involving NKG2D and the natural cytotoxicity receptors (NCR), NKp46, NKp44 and NKp30. Here we report that VV infection results in an upregulation of ligand structures for NKp30 and NKp46 on infected cells, whereas the binding of NKp44 and NKG2D was not significantly affected. Likewise, infection with ectromelia virus (ECTV), the mousepox agent, enhanced binding of NKp30 and, to a lesser extent, NKp46. The hemagglutinin (HA) molecules from VV and ECTV, which are known virulence factors, were identified as novel ligands for NKp30 and NKp46. Using NK cells with selectively silenced NCR expression and NCR-CD3ζ reporter cells, we observed that HA present on the surface of VV-infected cells, or in the form of recombinant soluble protein, was able to block NKp30-triggered activation, whereas it stimulated the activation through NKp46. The net effect of this complex influence on NK cell activity resulted in a decreased NK lysis susceptibility of infected cells at late time points of VV infection when HA was expression was pronounced. We conclude that poxviral HA represents a conserved ligand of NCR, exerting a novel immune escape mechanism through its blocking effect on NKp30-mediated activation at a late stage of infection.
Asunto(s)
Virus de la Ectromelia/inmunología , Hemaglutininas/metabolismo , Células Asesinas Naturales/inmunología , Receptor 1 Gatillante de la Citotoxidad Natural/metabolismo , Receptor 3 Gatillante de la Citotoxidad Natural/metabolismo , Virus Vaccinia/inmunología , Animales , Línea Celular , Regulación Viral de la Expresión Génica , Humanos , Células Asesinas Naturales/metabolismo , Ratones , Receptor 1 Gatillante de la Citotoxidad Natural/genética , Receptor 3 Gatillante de la Citotoxidad Natural/genética , Plásmidos , ARN Interferente Pequeño , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Regulación hacia ArribaRESUMEN
Gastrointestinal helminth infections are extremely prevalent in many human populations and are associated with downmodulated immune responsiveness. In the experimental model system of Heligmosomoides polygyrus, a chronic infection establishes in mice, accompanied by a modulated Th2 response and increased regulatory T cell (Treg) activity. To determine if dendritic cell (DC) populations in the lymph nodes draining the intestine are responsible for the regulatory effects of chronic infection, we first identified a population of CD11c(lo) nonplasmacytoid DCs that expand after chronic H. polygyrus infection. The CD11c(lo) DCs are underrepresented in magnetic bead-sorted preparations and spared from deletion in CD11c-diptheria toxin receptor mice. After infection, CD11c(lo) DCs did not express CD8, CD103, PDCA, or Siglec-H and were poorly responsive to TLR stimuli. In DC/T cell cocultures, CD11c(lo) DCs from naive and H. polygyrus-infected mice could process and present protein Ag, but induced lower levels of Ag-specific CD4(+) T cell proliferation and effector cytokine production, and generated higher percentages of Foxp3(+) T cells in the presence of TGF-ß. Treg generation was also dependent on retinoic acid receptor signaling. In vivo, depletion of CD11c(hi) DCs further favored the dominance of the CD11c(lo) DC phenotype. After CD11c(hi) DC depletion, effector responses were inhibited dramatically, but the expansion in Treg numbers after H. polygyrus infection was barely compromised, showing a significantly higher regulatory/effector CD4(+) T cell ratio compared with that of CD11c(hi) DC-intact animals. Thus, the proregulatory environment of chronic intestinal helminth infection is associated with the in vivo predominance of a newly defined phenotype of CD11c(lo) tolerogenic DCs.
Asunto(s)
Células Dendríticas/inmunología , Helmintiasis/inmunología , Parasitosis Intestinales/inmunología , Animales , Antígenos CD/inmunología , Antígenos CD11/inmunología , Recuento de Células , Enfermedad Crónica , Inmunidad , Inmunofenotipificación , Cadenas alfa de Integrinas/inmunología , Ratones , Nematospiroides dubius/inmunología , Linfocitos T Reguladores/inmunologíaRESUMEN
Ag recognition is achieved through the communication across intercellular contacts between T cells and APCs such as dendritic cells (DC). Despite remarkable progress in delineating detailed molecular components at the intercellular contacts, little is known about the functional roles of physical cross-junctional adhesion between T and DC in shaping T cell responses. In addition, the mechanisms underlying sensitivity and specificity of Ag discrimination by T cells at intercellular contacts remain to be elucidated. In this study, we use single-cell force spectroscopy to probe the mechanical interactions between DC and T cells in response to stimulation with a panel of altered peptide ligands. The results show that intercellular interactions of DC-T cell conjugates exhibited different ranges of interaction forces in peptide-dependent manners that match the ability of the peptides to activate T cells. Elevated calcium mobilization and IL-2 secretion by T cells were only promoted in response to antigenic peptides that induce strong interaction forces, suggesting that mechanically stable DC-T cell contacts are crucial for driving T cell activation. Strong interactions were not solely dependent on cell-surface molecules such as TCRs and the adhesion molecule LFA-1, but were also controlled by cytoskeletal dynamics and the integrity of membrane lipid rafts. These data provide novel mechanical insights into the effect of Ag affinity on intercellular contacts that align with T cell responsiveness.
Asunto(s)
Comunicación Celular/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Comunicación Celular/genética , Membrana Celular/inmunología , Membrana Celular/metabolismo , Células Cultivadas , Colesterol/deficiencia , Colesterol/metabolismo , Citoesqueleto/inmunología , Citoesqueleto/patología , Antígenos de Histocompatibilidad Clase II/inmunología , Interleucina-2/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Microscopía de Fuerza Atómica , Datos de Secuencia Molecular , Ovalbúmina/inmunología , Fragmentos de Péptidos/inmunología , Péptidos/metabolismo , Péptidos/fisiologíaRESUMEN
Dendritic cells (DCs) are key components of the adaptive immune system contributing to initiation and regulation of T cell responses. T cells continuously scan DCs in lymphoid organs for the presence of foreign antigen. However, little is known about the functional consequences of these frequent T cell-DC interactions without cognate antigen. Here we demonstrate that these contacts in the absence of foreign antigen serve an important function, namely, induction of a basal activation level in T cells required for responsiveness to subsequent encounters with foreign antigens. This basal activation is provided by self-recognition of MHC molecules on DCs. Following DC depletion in mice, T cells became impaired in TCR signaling and immune synapse formation, and consequently were hyporesponsive to antigen. This process was reversible, as T cells quickly recovered when the number of DCs returned to a normal level. The extent of T cell reactivity correlated with the degree of DC depletion in lymphoid organs, suggesting that a full DC compartment guarantees optimal T cell responsiveness. These findings indicate that DCs are specialized cells that not only present foreign antigen, but also promote a "tonic" state in T cells for antigen responsiveness.
Asunto(s)
Células Dendríticas/inmunología , Linfocitos T/inmunología , Animales , Presentación de Antígeno , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Recuento de Células , Supervivencia Celular/inmunología , Células Dendríticas/citología , Factor de Crecimiento Similar a EGF de Unión a Heparina , Humanos , Sinapsis Inmunológicas/inmunología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/inmunología , Activación de Linfocitos , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Transducción de Señal/inmunología , Linfocitos T/citología , Quimera por Trasplante/inmunologíaRESUMEN
Infection with gastrointestinal helminths generates a dominant type 2 response among both adaptive (Th2) and innate (macrophage, eosinophil, and innate lymphoid) immune cell types. Two additional innate cell types, CD11c(high) dendritic cells (DCs) and basophils, have been implicated in the genesis of type 2 immunity. Investigating the type 2 response to intestinal nematode parasites, including Heligmosomoides polygyrus and Nippostrongylus brasiliensis, we first confirmed the requirement for DCs in stimulating Th2 adaptive immunity against these helminths through depletion of CD11c(high) cells by administration of diphtheria toxin to CD11c.DOG mice. In contrast, responsiveness was intact in mice depleted of basophils by antibody treatment. Th2 responses can be induced by adoptive transfer of DCs, but not basophils, exposed to soluble excretory-secretory products from these helminths. However, innate type 2 responses arose equally strongly in the presence or absence of CD11c(high) cells or basophils; thus, in CD11c.DOG mice, the alternative activation of macrophages, as measured by expression of arginase-1, RELM-α, and Ym-1 (Chi3L3) in the intestine following H. polygyrus infection or in the lung following N. brasiliensis infection, was unaltered by depletion of CD11c-expressing DCs and alveolar macrophages or by antibody-mediated basophil depletion. Similarly, goblet cell-associated RELM-ß in lung and intestinal tissues, lung eosinophilia, and expansion of innate lymphoid ("nuocyte") populations all proceeded irrespective of depletion of CD11c(high) cells or basophils. Thus, while CD11c(high) DCs initiate helminth-specific adaptive immunity, innate type 2 cells are able to mount an autonomous response to the challenge of parasite infection.