RESUMO
New adjuvant strategies are needed to improve protein-based subunit vaccine immunogenicity. We examined the potential to use nanostructure of 6-O-ascorbyl palmitate to formulate ovalbumin (OVA) protein and an oligodeoxynucleotide (CpG-ODN) (OCC). In mice immunized with a single dose, OCC elicited an OVA-specific immune response superior to OVA/CpG-ODN solution (OC). Rheological studies demonstrated OCC's self-assembling viscoelastic properties. Biodistribution studies indicated that OCC prolonged OVA and CpG-ODN retention at injection site and lymph nodes, reducing systemic spread. Flow-cytometry assays demonstrated that OCC promoted OVA and CpG-ODN co-uptake by Ly6ChiCD11bhiCD11c+ monocytes. OCC and OC induced early IFN-γ in lymph nodes, but OCC led to higher concentration. Conversely, mice immunized with OC showed higher serum IFN-γ concentration compared to those immunized with OCC. In mice immunized with OCC, NK1.1+ cells were the IFN-γ major producers, and IFN-γ was essential for OVA-specific IgG2c switching. These findings illustrate how this nanostructure improves vaccine's response.
Assuntos
Nanoestruturas , Oligodesoxirribonucleotídeos , Ovalbumina , Vacinas de Subunidades Antigênicas , Animais , Nanoestruturas/química , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/química , Vacinas de Subunidades Antigênicas/farmacocinética , Camundongos , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/farmacocinética , Ovalbumina/imunologia , Ovalbumina/química , Feminino , Camundongos Endogâmicos C57BL , Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacocinética , Interferon gama/metabolismo , Distribuição Tecidual , Ácido Ascórbico/análogos & derivadosRESUMO
Introduction: Identification of tumor specific neoantigen (TSN) immunogenicity is crucial to develop peptide/mRNA based anti-tumoral vaccines and/or adoptive T-cell immunotherapies; thus, accurate in-silico classification/prioritization proves critical for cost-effective clinical applications. Several methods were proposed as TSNs immunogenicity predictors; however, comprehensive performance comparison is still lacking due to the absence of well documented and adequate TSN databases. Methods: Here, by developing a new curated database having 199 TSNs with experimentally-validated MHC-I presentation and positive/negative immune response (ITSNdb), sixteen metrics were evaluated as immunogenicity predictors. In addition, by using a dataset emulating patient derived TSNs and immunotherapy cohorts containing predicted TSNs for tumor neoantigen burden (TNB) with outcome association, the metrics were evaluated as TSNs prioritizers and as immunotherapy response biomarkers. Results: Our results show high performance variability among methods, highlighting the need for substantial improvement. Deep learning predictors were top ranked on ITSNdb but show discrepancy on validation databases. In overall, current predicted TNB did not outperform existing biomarkers. Conclusion: Recommendations for their clinical application and the ITSNdb are presented to promote development and comparison of computational TSNs immunogenicity predictors.
Assuntos
Neoplasias , Humanos , Antígenos de Neoplasias , PeptídeosRESUMO
The tumoral immune microenvironment (TIME) plays a key role in prognosis, therapeutic approach and pathophysiological understanding over oncological processes. Several computational immune cell-type deconvolution methods (DM), supported by diverse molecular signatures (MS), have been developed to uncover such TIME interplay from RNA-seq tumor biopsies. MS-DM pairs were benchmarked against each other by means of different metrics, such as Pearson's correlation, R2 and RMSE, but these only evaluate the linear association of the estimated proportion related to the expected one, missing the analysis of prediction-dependent bias trends and cell identification accuracy. We present a novel protocol composed of four tests allowing appropriate evaluation of the cell type identification performance and proportion prediction accuracy of molecular signature-deconvolution method pair by means of certainty and confidence cell-type identification scores (F1-score, distance to the optimal point and error rates) as well the Bland-Altman method for error-trend analysis. Our protocol was used to benchmark six state-of-the-art DMs (CIBERSORTx, DCQ, DeconRNASeq, EPIC, MIXTURE and quanTIseq) paired to five murine tissue-specific MSs, revealing a systematic overestimation of the number of different cell types across almost all methods.
Assuntos
Neoplasias , Humanos , Animais , Camundongos , Análise de Sequência de RNA/métodos , RNA-Seq , Neoplasias/diagnóstico , Neoplasias/genética , Benchmarking , Microambiente TumoralRESUMO
Background: COVID-19 severity has been linked to an increased production of inflammatory mediators called "cytokine storm". Available data is mainly restricted to the first international outbreak and reports highly variable results. This study compares demographic and clinical features of patients with COVID-19 from Córdoba, Argentina, during the first two waves of the pandemic and analyzes association between comorbidities and disease outcome with the "cytokine storm", offering added value to the field. Methods: We investigated serum concentration of thirteen soluble mediators, including cytokines and chemokines, in hospitalized patients with moderate and severe COVID-19, without previous rheumatic and autoimmune diseases, from the central region of Argentina during the first and second infection waves. Samples from healthy controls were also assayed. Clinical and biochemical parameters were collected. Results: Comparison between the two first COVID-19 waves in Argentina highlighted that patients recruited during the second wave were younger and showed less concurrent comorbidities than those from the first outbreak. We also recognized particularities in the signatures of systemic cytokines and chemokines in patients from both infection waves. We determined that concurrent pre-existing comorbidities did not have contribution to serum concentration of systemic cytokines and chemokines in COVID-19 patients. We also identified immunological and biochemical parameters associated to inflammation which can be used as prognostic markers. Thus, IL-6 concentration, C reactive protein level and platelet count allowed to discriminate between death and discharge in patients hospitalized with severe COVID-19 only during the first but not the second wave. Conclusions: Our data provide information that deepens our understanding of COVID-19 pathogenesis linking demographic features of a COVID-19 cohort with cytokines and chemokines systemic concentration, presence of comorbidities and different disease outcomes. Altogether, our findings provide information not only at local level by delineating inflammatory/anti-inflammatory response of patients but also at international level addressing the impact of comorbidities and the infection wave in the variability of cytokine and chemokine production upon SARS-CoV-2 infection.
Assuntos
COVID-19 , Humanos , Citocinas/metabolismo , SARS-CoV-2/metabolismo , Argentina , Quimiocinas , Síndrome da Liberação de Citocina , PandemiasRESUMO
Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.
Assuntos
Antineoplásicos , Baculoviridae , Baculoviridae/genética , Baculoviridae/metabolismo , Capsídeo , Receptor Toll-Like 9/genética , Receptor Toll-Like 9/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas do Capsídeo/genéticaRESUMO
T cell activation requires the processing and presentation of antigenic peptides in the context of a major histocompatibility complex (MHC complex). Cross-dressing is a non-conventional antigen presentation mechanism, involving the transfer of preformed peptide/MHC complexes from whole cells, such as apoptotic cells (ACs) to the cell membrane of professional antigen-presenting cells (APCs), such as dendritic cells (DCs). This is an essential mechanism for the induction of immune response against viral antigens, tumors, and graft rejection, which until now has not been clarified. Here we show for first time that the P2X7 receptor (P2X7R) is crucial to induce cross-dressing between ACs and Bone-Marrow DCs (BMDCs). In controlled ex vivo assays, we found that the P2X7R in both ACs and BMDCs is required to induce membrane and fully functional peptide/MHC complex transfer to BMDCs. These findings show that acquisition of ACs-derived preformed antigen/MHC-I complexes by BMDCs requires P2X7R expression.
RESUMO
Echinococcus granulosus is a cestode parasite which causes cystic echinococcosis disease. Previously we observed that vaccination with E. granulosus antigens from human hydatid cyst fluid (HCF) significantly inhibits colon cancer growth. In the present work, we evaluate the anti-tumor immune response induced by human HCF against LL/2 lung cancer in mice. HCF vaccination protected from tumor growth, both in prophylactic and therapeutic settings, and significantly increased mouse survival compared to control mice. Considering that tumor-associated carbohydrate antigens are expressed in E. granulosus, we oxidized terminal carbohydrates in HCF with sodium periodate. This treatment abrogates the anti-tumor activity induced by HCF vaccination. We found that HCF vaccination-induced IgG antibodies that recognize LL/2 tumor cells by flow cytometry. An antigen-specific immune response is induced with HCF vaccination in the tumor-draining lymph nodes and spleen characterized by the production of IL-5 and, in less extent, IFNÉ£. In the tumor microenvironment, we found that NK1.1 positive cells from HCF-treated mice showed higher expression of CD69 than control mice ones, indicating a higher level of activation. When we depleted these cells by administrating the NK-specific antibody NK1.1, a significantly decreased survival was observed in HCF-induced mice, suggesting that NK1.1+ cells mediate the anti-tumor protection induced by HCF. These results suggest that HCF can evoke an integrated anti-tumor immune response involving both, the innate and adaptive components, and provide novel insights into the understanding of the intricate relationship between HCF vaccination and tumor growth.
Assuntos
Antígenos Ly/imunologia , Equinococose/imunologia , Echinococcus granulosus/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Humanos , Imunidade/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Microambiente Tumoral/imunologiaRESUMO
It is well known that neutrophils are rapidly recruited to a site of injury or infection and perform a critical role in pathogen clearance and inflammation. However, they are also able to interact with and regulate innate and adaptive immune cells and some stimuli induce the migration of neutrophils to lymph nodes (LNs). Previously, we demonstrated that the immune complex (IC) generated by injecting OVA into the footpad of OVA/CFA immunized mice induced the migration of OVA+ neutrophils to draining LNs (dLNs). Here we investigate the effects of these neutrophils which reach dLNs on CD4+ T cell response. Our findings here strongly support a dual role for neutrophils in dLNs regarding CD4+ T cell response modulation. On the one hand, the CD4+ T cell population expands after the influx of OVA+ neutrophils to dLNs. These CD4+ T cells enlarge their proliferative response, activation markers and IL-17 and IFN-γ cytokine production. On the other hand, these neutrophils also restrict CD4+ T cell expansion. The neutrophils in the dLNs upregulate PD-L1 molecules and are capable of suppressing CD4+ T cell proliferation. These results indicate that neutrophils migration to dLNs have an important role in the homeostasis of adaptive immunity. This report describes for the first time that the influx of neutrophils to dLNs dependent on IC presence improves CD4+ T cell response, at the same time controlling CD4+ T cell proliferation through a PD-L1 dependent mechanism.
Assuntos
Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/imunologia , Movimento Celular/imunologia , Linfonodos/citologia , Neutrófilos/imunologia , Imunidade Adaptativa/imunologia , Transferência Adotiva , Animais , Complexo Antígeno-Anticorpo/efeitos dos fármacos , Complexo Antígeno-Anticorpo/imunologia , Antígeno B7-H1/genética , Proliferação de Células/efeitos dos fármacos , Técnicas de Inativação de Genes , Interferon gama/análise , Interleucina-17/análise , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/farmacologiaRESUMO
There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines.
Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Interferon Tipo I/metabolismo , Oligodesoxirribonucleotídeos/imunologia , Adjuvantes Imunológicos , Animais , Antígenos/química , Citocinas/metabolismo , Humanos , Imunidade Humoral , Camundongos , Camundongos Knockout , Nanoestruturas , Oligodesoxirribonucleotídeos/química , Ovalbumina/imunologia , Transdução de Sinais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, although it also has been used as display vector for vaccine development. In this work, we evaluated the effectiveness of repetitive doses of AcMNPV-based vectors on the cytotoxic immune response specific to the capsid-displayed heterologous antigen ovalbumin (OVA). Our results demonstrate that baculovirus vectors induce a boosting effect in the cytotoxic immune response to OVA, making possible to recover the levels obtained in the primary response. Moreover, mice preimmunized with wild-type baculovirus showed a complete lack of antigen-specific CD8 cytotoxic T lymphocytes (CTLs) that may be related to the presence of antibodies directed to baculoviral surface proteins, particularly to GP64. However, baculovirus was able to induce the innate immune response in spite of a previous response against this vector, although some quantitative differences reflect a distinct activation of the immune cells in prime and boost. This is the first report in which the novel capsid display strategy is evaluated in prime-boost schemes to improve efficient CTL responses.
Assuntos
Proteínas do Capsídeo/imunologia , Capsídeo/imunologia , Nucleopoliedrovírus/imunologia , Vacinação , Animais , Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular , Citocinas/sangue , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologia , Spodoptera/imunologia , Spodoptera/virologia , Linfócitos T Citotóxicos/imunologiaRESUMO
The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8+ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8+ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α+ cDCs from old mice have an impaired ability to activate naïve CD8+ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8+ T cells and the generation of effector cytotoxic T cells.
Assuntos
Envelhecimento/imunologia , Apresentação de Antígeno , Linfócitos T CD8-Positivos/enzimologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Imunidade Celular , Glicoproteínas de Membrana/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Antígenos/imunologia , Antígenos/farmacologia , Apresentação Cruzada/efeitos dos fármacos , Feminino , Camundongos , Poli U/imunologia , Poli U/farmacologiaRESUMO
Modern subunit vaccines require the development of new adjuvant strategies. Recently, we showed that CpG-ODN formulated with a liquid crystal nanostructure formed by self-assembly of 6-O-ascorbyl palmitate (Coa-ASC16) is an attractive system for promoting an antigen-specific immune response to weak antigens. Here, we showed that after subcutaneous injection of mice with near-infrared fluorescent dye-labeled OVA antigen formulated with Coa-ASC16, the dye-OVA was retained at the injection site for a longer period than when soluble dye-OVA was administered. Coa-ASC16 alone elicited a local inflammation, but how this material triggers this response has not been described yet. Although it is known that some materials used as a platform are not immunologically inert, very few studies have directly focused on this topic. In this study, we explored the underlying mechanisms concerning the interaction between Coa-ASC16 and the immune system and we found that the whole inflammatory response elicited by Coa-ASC16 (leukocyte recruitment and IL-1ß, IL-6 and IL-12 production) was dependent on the MyD88 protein. TLR2, TLR4, TLR7 and NLRP3-inflammasome signaling were not required for induction of this inflammatory response. Coa-ASC16 induced local release of self-DNA, and in TLR9-deficient mice IL-6 production was absent. In addition, Coa-ASC16 revealed an intrinsic adjuvant activity which was affected by MyD88 and IL-6 absence. Taken together these results indicate that Coa-ASC16 used as a vaccine platform is effective due to the combination of the controlled release of antigen and its intrinsic pro-inflammatory activity. Understanding how Coa-ASC16 works might have significant implications for rational vaccine design.
Assuntos
Adjuvantes Imunológicos/química , Antígenos/administração & dosagem , Ácido Ascórbico/análogos & derivados , Fator 88 de Diferenciação Mieloide/metabolismo , Vacinas/administração & dosagem , Animais , Ácido Ascórbico/química , Preparações de Ação Retardada , Humanos , Inflamassomos/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/patologia , Interleucinas/biossíntese , Leucócitos/efeitos dos fármacos , Cristais Líquidos , Camundongos , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Ovalbumina/imunologia , Receptor Toll-Like 9/biossíntese , Receptor Toll-Like 9/genética , Receptores Toll-Like/biossínteseRESUMO
As we age, the homeostatic function of many systems in the body, such as the immune function declines, which in turn contributes to augment susceptibility to disease. Here we describe that challenging aged mice with synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) emulsified in incomplete Freund's adjuvant (IFA), (CpG-ODN+IFA) an inflammatory stimulus, led to the expansion of CD11b+Gr1+ myeloid cells with augmented expression of CD124 and CD31. These myeloid cells lasted longer in the spleen of aged mice than in their younger counterparts after CpG-ODN+IFA treatment and were capable of suppressing T cell proliferative response by arginase induction. Myeloid cells from aged CpG-ODN+IFA-treated mice presented increased arginase-1 expression and enzyme activity. In addition, we found a different requirement of cytokines for arginase induction according to mice age. In myeloid cells from young treated mice, arginase-1 expression and activity is induced by the presence of each IL-4 or IL-6 in their extracellular medium, unlike myeloid cells from aged treated mice which need the presence of both IL-4 and IL-6 together for arginase induction and suppressor function.
Assuntos
Arginase/metabolismo , Adjuvante de Freund/farmacologia , Lipídeos/farmacologia , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Oligodesoxirribonucleotídeos/farmacologia , Adjuvantes Imunológicos/farmacologia , Fatores Etários , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Células Mieloides/enzimologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/enzimologiaRESUMO
BACKGROUND: Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. METHODS: Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification. RESULTS: The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. CONCLUSIONS: This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. GENERAL SIGNIFICANCE: This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.
Assuntos
Células Dendríticas/imunologia , Receptores de Superfície Celular/imunologia , Anticorpos de Cadeia Única/imunologia , Anticorpos de Domínio Único/imunologia , Animais , Biotina/imunologia , Antígeno CD11b/imunologia , Antígenos CD18/imunologia , Camelídeos Americanos , Linhagem Celular , Técnicas de Visualização da Superfície Celular/métodos , Células Cultivadas , Células Dendríticas/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunização/métodos , Antígenos Comuns de Leucócito/imunologia , Camundongos Endogâmicos BALB C , Peptídeos/imunologia , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.
Assuntos
Complexo Antígeno-Anticorpo/imunologia , Doenças do Sistema Imunitário/imunologia , Transtornos Leucocíticos/imunologia , Linfonodos/imunologia , Neutrófilos/imunologia , Transferência Adotiva , Animais , Movimento Celular/imunologia , Feminino , Cloridrato de Fingolimode , Imunossupressores/farmacologia , Inflamação/imunologia , Selectina L/imunologia , Linfonodos/citologia , Vasos Linfáticos/imunologia , Antígeno-1 Associado à Função Linfocitária/imunologia , Lisofosfolipídeos/agonistas , Antígeno de Macrófago 1/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos/transplante , Selectina-P/imunologia , Propilenoglicóis/farmacologia , Receptores CXCR4/imunologia , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/agonistas , Esfingosina/análogos & derivados , Esfingosina/farmacologiaRESUMO
The adjuvants approved in human vaccine with recombinant/purified antigens induce weak cellular immune response and so the development of new adjuvant strategies is critical. CpG-ODN has successfully been used as an adjuvant (phase I-III clinical trials) but its bioavailability needs to be improved. We investigated the adjuvant ability of CpG-ODN formulated with a liquid crystal nanostructure of 6-O-ascorbyl palmitate (Coa-ASC16). Mice immunized with OVA/CpG-ODN/Coa-ASC16 elicited a potent specific IgG1, IgG2a, Th1 and Th17 cellular response without systemic adverse effects. These responses were superior to those induced by OVA/CpG-ODN (solution of OVA with CpG-ODN) and to those induced by the formulation OVA/CpG-ODN/Al(OH)3. Immunization with OVA/CpG-ODN/Coa-ASC16 resulted in a long-lasting cell-mediated immune response (at least 6.5 months). Furthermore, Coa-ASC16 alone allows a controlled release of CpG-ODN in vitro and induces local inflammatory response, independent of TLR4 signaling, characterized by an influx of neutrophils and Ly6C(high) monocytes and pro-inflammatory cytokines. Remarkably, the adjuvant capacity of CpG-ODN co-injected with Coa-ASC16 (OVA/CpG-ODN plus Coa-ASC16) was similar to the adjuvant activity of OVA/CpG-ODN, supporting the requirement for whole formulation to help CpG-ODN adjuvanticity. These results show the potential of this formulation, opening a new avenue for the development of better vaccines.
Assuntos
Adjuvantes Imunológicos/farmacocinética , Imunidade Celular , Cristais Líquidos/química , Oligodesoxirribonucleotídeos/farmacocinética , Adjuvantes Imunológicos/química , Alanina Transaminase/sangue , Animais , Antígenos/imunologia , Ácido Ascórbico/análogos & derivados , Ácido Ascórbico/química , Ácido Ascórbico/farmacocinética , Aspartato Aminotransferases/sangue , Disponibilidade Biológica , Células Cultivadas , Feminino , Imunização , Imunoglobulina G/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Nanoestruturas/química , Oligodesoxirribonucleotídeos/química , Ovalbumina/imunologia , Transdução de Sinais , Baço/citologia , Baço/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Vacinas/química , Vacinas/imunologiaRESUMO
ssRNA can interact with dendritic cells (DCs) through binding to TLR7, inducing secretion of proinflammatory cytokines and type I IFN. Triggering TLR7 enhances cross-priming of CD8(+) T cells, which requires cross-presentation of exogenous Ag to DCs. However, how TLR triggering can affect Ag cross-presentation is still not clear. Using OVA as an Ag model, we observed that stimulation of TLR7 in DCs by polyuridylic acid (polyU), a synthetic ssRNA analog, generates a strong specific cytotoxic response in C57BL/6 mice. PolyU stimulate CD8α(+) DCs to cross-prime naive CD8(+) T cells in a type I IFN-dependent fashion. This enhanced cross-priming is accompanied by a higher density of OVA(256-264)/H-2K(b) complexes on CD8α(+) DCs treated with polyU, as well as by upregulation of costimulatory molecules and increased secretion of proinflammatory cytokines by DCs. Cross-priming of CD8(+) T cells by DCs treated with polyU requires proteasome and Ag translocation to cytosol through the Sec61 channel in DCs. The observed enhancement in OVA cross-presentation with polyU in DCs could be mediated by a limited Ag degradation in endophagosomal compartments and a higher permanence of OVA peptide/MHC class I complexes on DCs. These observations clearly reveal that key steps of Ag processing for cross-presentation can be modulated by TLR ligands, opening new avenues for understanding their mechanisms as adjuvants of the immune response.
Assuntos
Adjuvantes Imunológicos/farmacologia , Apresentação de Antígeno/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Antígenos H-2/imunologia , Glicoproteínas de Membrana/efeitos dos fármacos , Ovalbumina/imunologia , Fragmentos de Peptídeos/imunologia , Poli U/farmacologia , Receptor 7 Toll-Like/efeitos dos fármacos , Animais , Apresentação de Antígeno/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Compartimento Celular , Células Cultivadas/imunologia , Citotoxicidade Imunológica , Células Dendríticas/imunologia , Endossomos/imunologia , Feminino , Glicoproteínas de Membrana/imunologia , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagossomos/imunologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Proteico , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Canais de Translocação SEC , Baço/imunologia , Receptor 7 Toll-Like/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
The ability of synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) to induce both stimulatory and counter-regulatory responses offers novel opportunities for using these molecules as immunomodulatory agents in different therapeutic strategies. Here, we investigated the potential of CpG-ODN to activate the arginase (ARG) enzyme in vivo and focused on the consequences of this activation in T-cell proliferation. Challenging mice subcutaneously with CpG-ODN emulsified in incomplete Freund's adjuvant (IFA) induced ARG and reduced T-cell proliferation associated with CD3ζ chain downregulation. Interestingly, impaired T-cell expansion correlated with elevated levels of CD11b(+)Gr1(+) myeloid cells localized near T-cell areas in the spleen. In addition, purified CD11b(+) cells obtained from the spleen of CpG-ODN+IFA-treated mice exhibited increased ARG activity and ARG I expression along with an augmented [(3)H]-L-arginine uptake. CD11b(+) myeloid cells significantly suppressed T-cell proliferation and CD3ζ chain expression induced by a polyclonal stimulus. Furthermore, these effects could be recovered by the addition of excess L-arginine or by treatment of CD11b(+) cells with a specific ARG inhibitor. This study provides a novel evidence that CpG-ODN+IFA are able to induce splenic CD11b(+) cells with ARG activity, with this population being responsible for the impaired T-cell proliferation observed after the treatment with CpG-ODN+IFA. These results underscore a key role of CpG-ODN on ARG activity in vivo and add support to the growing body of evidence in favor of a counter-regulatory role for CpG-ODN in an immune response.
Assuntos
Arginase/imunologia , Adjuvante de Freund/imunologia , Lipídeos/imunologia , Células Mieloides/imunologia , Oligodesoxirribonucleotídeos/imunologia , Animais , Arginase/metabolismo , Arginina/metabolismo , Antígeno CD11b/imunologia , Antígeno CD11b/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Feminino , Citometria de Fluxo , Adjuvante de Freund/farmacologia , Lipídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Baço/citologia , Baço/imunologia , Baço/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/metabolismo , TrítioRESUMO
Baculoviruses (BV) are DNA viruses that are pathogenic for insects. Although BV infect a range of mammalian cell types, they do not replicate in these cells. Indeed, the potential effects of these insect viruses on the immune responses of mammals are only just beginning to be studied. We show in this paper that a recombinant Autographa californica multiple nuclear polyhedrosis virus carrying a fragment of ovalbumin (OVA) on the VP39 capsid protein (BV-OVA) has the capacity to act as an adjuvant and vector of antigens in mice, thereby promoting specific CD4 and cytotoxic T cell responses against OVA. BV also induced in vivo maturation of dendritic cells and the production of inflammatory cytokines, thus promoting innate and adaptive immune responses. The OVA-specific response induced by BV-OVA was strong enough to reject a challenge with OVA-expressing melanoma cells (MO5 cells) and effectively prolonged survival of MO5 bearing mice. All these findings, together with the absence of pre-existing immunity to BV in humans and the lack of viral gene expression in mammalian cells, make BV a candidate for vaccination.