RESUMO
Tn is a tumor-associated carbohydrate antigen that constitutes both a diagnostic tool and an immunotherapeutic target. It originates from interruption of the mucin O-glycosylation pathway through defects involving, at least in part, alterations in core-1 synthase activity, which is highly dependent on Cosmc, a folding chaperone. Tn antigen is recognized by the Macrophage Galactose-type Lectin (MGL), a C-type lectin receptor present on dendritic cells and macrophages. Specific interactions between Tn and MGL shape anti-tumoral immune responses by regulating several innate and adaptive immune cell programs. In this work, we generated and characterized a variant of the lung cancer murine cell line LL/2 that expresses Tn by mutation of the Cosmc chaperone gene (Tn+ LL/2). We confirmed Tn expression by lectin glycophenotyping and specific anti-Tn antibodies, verified abrogation of T-synthase activity in these cells, and confirmed its recognition by the murine MGL2 receptor. Interestingly, Tn+ LL/2 cells were more aggressive in vivo, resulting in larger and highly vascularized tumors than those generated from wild type Tn- LL/2 cells. In addition, Tn+ tumors exhibited an increase in CD11c+ F4/80+ cells with high expression of MGL2, together with an augmented expression of IL-10 in infiltrating CD4+ and CD8+ T cells. Importantly, this immunosuppressive microenvironment was dependent on the presence of MGL2+ cells, since depletion of these cells abrogated tumor growth, vascularization and recruitment of IL-10+ T cells. Altogether, our results suggest that expression of Tn in tumor cells and its interaction with MGL2-expressing CD11c+F4/80+ cells promote immunosuppression and angiogenesis, thus favoring tumor progression.
Assuntos
Antígenos Glicosídicos Associados a Tumores/imunologia , Galactose/imunologia , Lectinas Tipo C/imunologia , Neoplasias Pulmonares/imunologia , Macrófagos/imunologia , Neovascularização Patológica/imunologia , Animais , Antígeno CD11c/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Feminino , Terapia de Imunossupressão/métodos , Interleucina-10/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microambiente Tumoral/imunologiaRESUMO
IL-4 and IL-13 cytokines have been associated with a non-healing phenotype in murine leishmaniasis in L. mexicana -infected BALB/c mice as demonstrated in IL-4-/-, IL-13-/- and IL-4Rα-/- global knockout mouse studies. However, it is unclear from the studies which cell-type-specific IL-4/IL-13 signaling mediates protection to L. mexicana. Previous studies have ruled out a role for IL-4-mediated protection on CD4+ T cells during L. mexicana infections. A candidate for this role may be non-lymphocyte cells, particularly DCs, as was previously shown in L. major infections, where IL-4 production drives dendritic cell-IL-12 production thereby mediating a type 1 immune response. However, it is unclear if this IL-4-instruction of type 1 immunity also occurs in CL caused by L. mexicana, since the outcome of cutaneous leishmaniasis often depends on the infecting Leishmania species. Thus, BALB/c mice with cell-specific deletion of the IL-4Rα on CD11c+ DCs (CD11ccreIL-4Rα-/lox) were infected with L. mexicana promastigotes in the footpad and the clinical phenotype, humoral and cellular immune responses were investigated, compared to the littermate control. Our results show that CL disease progression in BALB/c mice is independent of IL-4Rα signaling on DCs as CD11ccreIL-4Rα-/lox mice had similar footpad lesion progression, parasite loads, humoral responses (IgE, IgG1, IgG 2a/b), and IFN-γ cytokine secretion in comparison to littermate controls. Despite this comparable phenotype, surprisingly, IL-4 production in CD11ccreIL-4Rα-/lox mice was significantly increased with an increasing trend of IL-13 when compared to littermate controls. Moreover, the absence of IL-4Rα signaling did not significantly alter the frequency of CD4 and CD8 lymphocytes nor their activation, or memory phenotype compared to littermate controls. However, these populations were significantly increased in CD11ccreIL-4Rα-/lox mice due to greater total cell infiltration into the lymph node. A similar trend was observed for B cells whereas the recruitment of myeloid populations (macrophages, DCs, neutrophils, and Mo-DCs) into LN was comparable to littermate IL-4Rα-/lox mice. Interestingly, IL-4Rα-deficient bone marrow-derived dendritic cells (BMDCs), stimulated with LPS or L. mexicana promastigotes in presence of IL-4, showed similar levels of IL-12p70 and IL-10 to littermate controls highlighting that IL-4-mediated DC instruction was not impaired in response to L. mexicana. Similarly, IL-4 stimulation did not affect the maturation or activation of IL-4Rα-deficient BMDCs during L. mexicana infection nor their effector functions in production of nitrite and arginine-derived metabolite (urea). Together, this study suggests that IL-4 Rα signaling on DCs is not key in the regulation of immune-mediated protection in mice against L. mexicana infection.
Assuntos
Células Dendríticas/imunologia , Interleucina-4/imunologia , Leishmania mexicana/imunologia , Leishmaniose Cutânea/imunologia , Animais , Linfócitos B/imunologia , Antígeno CD11c/imunologia , Feminino , Interleucina-10/imunologia , Leishmania major/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interleucina-4/imunologia , Transdução de Sinais/imunologiaRESUMO
Dendritic cells (DCs) are potent antigen-presenting cells that possess the ability to stimulate naïve T cells, initiating the adaptive immune response. Ex vivo DC cultures are useful to evaluate how helminths regulate DC maturation and stimulatory activity. Here, we describe how to isolate CD11c+ from F. hepatica-infected mice to evaluate their activation state, cytokine production and regulatory function in an allogeneic T cell assay.
Assuntos
Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Fasciola hepatica/imunologia , Fasciolíase/imunologia , Animais , Antígenos de Helmintos/imunologia , Antígeno CD11c/imunologia , Fasciolíase/parasitologia , Feminino , Fatores Imunológicos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T/imunologia , Linfócitos T/parasitologiaRESUMO
INTRODUCTION: In addition to conventional therapies, several new strategies have been proposed for modulating autoimmune diseases, including the adoptive transfer of immunological cells. In this context, dendritic cells (DCs) appear to be one of the most promising treatments for autoimmune disorders. The present study aimed to evaluate the effects of adoptive transfer of DCs obtained from both naïve and ovalbumin (OVA)-tolerant mice on the severity of TNBS induced colitis and analyze the eventual protective mechanisms. METHODS AND RESULTS: To induce oral tolerance, BALB/c mice were fed 4mg/mL OVA solution for seven consecutive days. Spleen DCs were isolated from tolerant (tDC) and naïve (nDC) mice, and then adoptively transferred to syngeneic mice. Three days later, colitis was induced in DC treated mice by intrarectal instillation of 100µg2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol. Control subjects received only intrarectal instillation of either TNBS solution or a vehicle. Five days later, mice from all groups were euthanized and examined for physiological and immunological parameters. Regarding the phenotype, we observed that the frequencies of CD11+ MHC II+ and CD11+ MHCII+ CD86+ cells were significantly lower in DCs isolated from tolerant mice than in those from naive mice. However, pretreatment with both types of DCs was able to significantly reduce clinical signs of colitis such as diarrhea, rectal prolapse, bleeding, and cachexia, although only treatment with tDCs was able to prevent weight loss from instillation of TNBS. In vitro proliferation of spleen cells from mice treated with either type of DCs was significantly lower than that observed in splenic cell cultures of naïve mice. Although no significant difference was observed in the frequencies of Treg cells in the experimental groups, the frequency of Th17+CD4+cellsand the secretion of IL-17 were more reduced in the cultures of spleen cells from mice treated with either type of DCs. The levels of IL-9 and IFN-γ were lower in supernatants of cells from mice treated with nDCs. CONCLUSION: The results allow us to conclude that the adoptive transfer of cells expressing CD11c is able to reduce the clinical and immunological signs of drug-induced colitis. Adoptive transfer of CD11c+DC isolated from both naive and tolerant mice altered the proliferative and T cell responses. To the best of our knowledge, there is no previously published data showing the protective effects of DCs from naïve or tolerant mice in the treatment of colitis.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Colite/terapia , Células Dendríticas/transplante , Tolerância Imunológica , Transferência Adotiva/métodos , Animais , Antígeno B7-2/imunologia , Antígeno CD11c/imunologia , Colite/induzido quimicamente , Colite/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Camundongos , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4(+) populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c(+) CD11b(-) CD103(+) cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c(+) CD11b(+) CD103(-) cells. CD11c(+) CD11b(-) CD103(+) cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4(+) cells. Moreover, CD4(+) cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4(+) cells is dependent on CD11c(+) CD11b(-) CD103(+) cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.
Assuntos
Antígenos CD/imunologia , Antígeno CD11c/imunologia , Cadeias alfa de Integrinas/imunologia , Interferon gama/imunologia , Interleucina-17/imunologia , Pulmão/imunologia , Mycobacterium tuberculosis/imunologia , Células Th17/imunologia , Tuberculose Pulmonar/imunologia , Animais , Antígenos CD/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Antígeno CD11c/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/microbiologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Dendríticas/microbiologia , Modelos Animais de Doenças , Feminino , Genótipo , Imunidade Celular , Cadeias alfa de Integrinas/metabolismo , Interferon gama/metabolismo , Interleucina-17/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/patogenicidade , Fenótipo , Transdução de Sinais , Especificidade da Espécie , Células Th17/metabolismo , Células Th17/microbiologia , Fatores de Tempo , Tuberculose Pulmonar/genética , Tuberculose Pulmonar/metabolismo , Tuberculose Pulmonar/microbiologiaRESUMO
We have initially shown that DC/ApoNec vaccine can induce protection against the poorly immunogenic B16F1 melanoma in mice. The population of DC obtained for vaccination after 7days culture with murine GM-CSF is heterogeneous and presents about 60% of CD11c+ DC. Therefore, our purpose was to identify the phenotype of the cells obtained after differentiation and its immunogenicity once injected. DC were separated with anti-CD11c microbeads and the two populations identified in terms of CD11c positivity (DC+ and DC-) were also studied. Approximately 26.6% of the cells in DC+ fraction co-expressed CD11c+ and F4/80 markers and 75.4% were double positive for CD11c and CD11b markers. DC+ fraction also expressed Ly6G. DC- fraction was richer in CD11c-/F4/80+ macrophages (44.7%), some of which co-expressed Ly6G (41.8%), and F4/80-/Ly6-G+ neutrophils (34.6%). Both DC+ and DC- fractions displayed similar capacity to phagocyte and endocyte antigens and even expressed levels of MHC Class II and CD80, CD83 and CD86 costimulatory molecules similar to those in the DC fraction. However, only DC/ApoNec vaccine was capable to induce protection in mice (p<0.01). After 24h co-culture, no detectable level of IL-12 was recorded in DC/ApoNec vaccine, either in supernatant or intracellularly. Therefore, the protection obtained with DC/ApoNec vaccine seemed to be independent of the vaccine's ability to secrete this inflammatory cytokine at the time of injection. In conclusion, we demonstrated that all cell types derived from the culture of mouse bone marrow with GM-CSF are necessary to induce antitumor protection in vivo.
Assuntos
Apresentação de Antígeno/imunologia , Células da Medula Óssea/imunologia , Antígeno CD11c/imunologia , Vacinas Anticâncer/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Melanoma Experimental/imunologia , Melanoma Experimental/prevenção & controle , Animais , Apresentação de Antígeno/genética , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Células da Medula Óssea/metabolismo , Antígeno CD11c/genética , Vacinas Anticâncer/genética , Vacinas Anticâncer/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-12/genética , Interleucina-12/imunologia , Interleucina-12/metabolismo , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neutrófilos/imunologia , Neutrófilos/metabolismoRESUMO
Sporotrichosis is a disease caused by the dimorphic fungus Sporothrix schenckii. The main clinical manifestations occur in the skin, however the number of systemic and visceral cases has increased, especially in immunocompromised patients. Dendritic cells (DCs) are highly capable to recognize the fungus associated data and translate it into differential T cells responses both in vivo and in vitro. Although, the mechanisms involved in the interaction between DCs and S. schenckii are not fully elucidated. The present study investigated the phenotypic and functional changes in bone marrow dendritic cells (BMDCs) stimulated in vitro with the yeast form of S. schenckii or exoantigen (ExoAg) and its ability to trigger a cellular immune response in vitro. Our results demonstrated that the live yeast of S. schenckii and its exoantigen, at a higher dose, were able to activate BMDCs and made them capable of triggering T cell responses in vitro. Whereas the yeast group promoted more pronounced IFN-γ production rather than IL-17, the Exo100 group generated similar production of both cytokines. The exoantigen stimulus suggests a capability to deviate the immune response from an effector Th1 to an inflammatory Th17 response. Interestingly, only the Exo100 group promoted the production of IL-6 and a significant increase of TGF-ß, in addition to IL-23 production. Interestingly, only Exo100 group was capable to promote the production of IL-6 and a significant increase on TGF-ß, in addition with IL-23 detection. Our results demonstrated the plasticity of DCs in translating the data associated with the fungus S. schenckii and ExoAg into differential T cell responses in vitro. The possibility of using ex vivo-generated DCs as vaccinal and therapeutic tools for sporotrichosis is a challenge for the future.
Assuntos
Antígenos de Fungos/imunologia , Células Dendríticas/imunologia , Sporothrix/imunologia , Esporotricose/imunologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Citocinas/imunologia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-23/imunologia , Interleucina-23/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Sporothrix/fisiologia , Esporotricose/metabolismo , Esporotricose/microbiologia , Células Th1/metabolismo , Células Th17/metabolismo , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
Vanadium (V) is a transition metal found in air adsorbed onto suspended particles. As a result, urban populations are often exposed to this element as a constituent of particulate matter (PM). One aspect of the myriad toxicities that might arise from these exposures is altered immune responses. Previous reports from the laboratory reported modifications in splenic architecture - with germinal center hyperplasia and a suppressed humoral immune response - in mice that had been exposed to vanadium agents via inhalation. This paper reports a decrease in the presence of the CD11c surface marker on mouse thymic dendritic cells (DC) as a result of host exposure to vanadium (here, in the form of vanadium pentoxide; V(2)O(5)) over a period of 4 weeks. All results were obtained using immunohistochemistry and flow cytometry. It is surmised that this decrease might induce a dysfunction, including possible negative selection of T-cells, which could increase the presence of autoreactive clones in the exposed host. Such an outcome could, in turn, increase the risk for development of autoimmune reactions in different organs specifically, and of autoimmune diseases in general in these V-exposed hosts.
Assuntos
Poluentes Atmosféricos/efeitos adversos , Doenças Autoimunes/etiologia , Antígeno CD11c/imunologia , Células Dendríticas/efeitos dos fármacos , Exposição Ambiental/efeitos adversos , Timo/efeitos dos fármacos , Vanádio/efeitos adversos , Animais , Doenças Autoimunes/imunologia , Separação Celular , Células Cultivadas , Células Dendríticas/imunologia , Regulação para Baixo , Citometria de Fluxo , Inalação , Masculino , Camundongos , Camundongos Endogâmicos , Timo/imunologiaRESUMO
Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 (lps-del)). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNß was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow-derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNß-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response.
Assuntos
Interferon beta/imunologia , Neoplasias Experimentais/imunologia , Transdução de Sinais/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Antígeno CD11c/imunologia , Antígeno CD11c/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Interferon beta/genética , Interferon beta/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Experimentais/patologia , Neoplasias Experimentais/prevenção & controle , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Cellular immune responses are a significant defence mechanism in human paracoccidioidomycosis (PCM), an endemic mycosis in Latin America; however, little is known about the role of dendritic cells (DCs) in human PCM. We investigated monocyte-derived DCs from patients with treated (TP) and active PCM (AP) compared with healthy non-PCM donors (CO). DCs from the TP group showed higher expression of HLA-DR, CD86 and DC-SIGN compared with CO, whereas AP showed similar expression to CO. Production of IL-10 was downregulated by TNF-α in all groups and lower levels were observed in untreated DCs from AP compared with CO. Conversely, IL-12p40 was significantly upregulated in the DCs of the TP group. TNF-α-activated DCs from the CO group produced significantly lower levels of IL-12p40 when differentiated from magnetic-sorted monocytes (MACS) compared with adhered monocyte-derived DCs. This comparison in the TP group revealed similar levels of IL-12p40, suggesting a T cell-independent increase in the production of IL-12p40. Higher expression of surface molecules with increased IL-12p40 may indicate a better activation of DCs after the treatment of PCM. Our findings suggest that DCs may be crucial in the protective response to Paracoccidioides brasiliensis and that in vitro-generated DCs might be useful in enhancing antifungal immunity, especially during active PCM.