RESUMO
Glioblastoma, isocitrate dehydrogenase-wildtype (GB), is the most common and aggressive primary brain malignancy with poor outcome. Immune checkpoint inhibitors (ICIs) have been tested in GB and, despite disappointing results, the identification of a small subgroup of responders underlies the need to improve our understanding of the tumour microenvironment (TME) immunity. This study aimed to determine whether the expression of selected immune checkpoints on tissue-resident memory T cells (Trm) may predict patient outcome. We conducted a single cohort observational study. Tumour samples were collected from 45 patients with histologically confirmed GB (WHO grade 4) and processed to obtain single-cell suspensions. Patients were assessed for the correlation of Trm phenotype with overall survival (OS) or progression-free survival (PFS) using multiparametric flow cytometry and uni/multivariate analyses. Levels of Trm expressing programmed cell death protein 1 (PD1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) were found to be linked to clinical outcome. Low frequency of Trm expressing PD1 or TIM3 or both markers defined subgroups as independent positive prognostic factors for patient survival. On multivariate analysis, low CD8+CD103+PD1+TIM3+ Trm and Karnofsky performance status (KPS) ≥70 were confirmed to be the most predictive independent factors associated with longer OS (hazard ratios-HR [95%CI]: 0.14 [0.04-0.52] p < 0.001, 0.39 [0.16-0.96] p = 0.04, respectively). The CD8+CD103+ Trm subgroups were also age-related predictors for survival in GB.
Assuntos
Glioblastoma , Receptor Celular 2 do Vírus da Hepatite A , Humanos , Receptor de Morte Celular Programada 1/metabolismo , Prognóstico , Linfócitos T CD8-Positivos , Microambiente TumoralRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, induces severe pneumonia mainly in elderly males. Epidemiological data clearly indicate sex-based differences in disease outcomes, with men accounting for about 70 % of deaths, despite similar susceptibility to infection. It is well known that females are endowed with higher capacity to produce antibodies, which correlates with viral clearance and disease resolution in the context of SARS-Cov-2 infection. Many X-linked immune genes escape X inactivation showing biallelic expression in female immune cells, particularly in plasmacytoid dendritic cells (pDCs). PDCs are more active in females and endowed with high capability to induce IFN-α-mediated B cell activation and differentiation into antibody-producing plasma cells throughout epigenetic mechanisms linked to trained immunity. Thus, we hypothesize that following SARS-CoV-2 infection, epigenetic modifications of X-linked genes involved in pDC-mediated type I IFN (IFN-I) signaling occurs more effectively in females, for inducing neutralizing antibody response as an immune correlate driving sex-biased disease outcome.
Assuntos
Formação de Anticorpos , COVID-19/diagnóstico , COVID-19/imunologia , Interferon Tipo I/fisiologia , SARS-CoV-2/imunologia , COVID-19/epidemiologia , Feminino , Humanos , Masculino , Pandemias , Prognóstico , Caracteres SexuaisRESUMO
The past decade has seen tremendous developments in novel cancer therapies through targeting immune-checkpoint molecules. However, since increasing the presentation of tumor antigens remains one of the major issues for eliciting a strong antitumor immune response, dendritic cells (DC) still hold a great potential for the development of cancer immunotherapy. A considerable body of evidence clearly demonstrates the importance of the interactions of type I IFN with the immune system for the generation of a durable antitumor response through its effects on DC. Actually, highly active DC can be rapidly generated from blood monocytes in vitro in the presence of IFN-α (IFN-DC), suitable for therapeutic vaccination of cancer patients. Here we review how type I IFN can promote the ex vivo differentiation of human DC and orientate DC functions towards the priming and expansion of protective antitumor immune responses. New epigenetic elements of control on activation of the type I IFN signal will be highlighted. We also review a few clinical trials exploiting IFN-DC in cancer vaccination and discuss how IFN-DC could be exploited for the design of effective strategies of cancer immunotherapy as a monotherapy or in combination with immune-checkpoint inhibitors or immunomodulatory drugs.
RESUMO
Follicular lymphoma (FL) is a remarkably immune-responsive malignancy, which is still considered incurable. As, standard immunochemotherapy is complex, toxic and not curative, improvement in FL care is now a crucial topic in hemato-oncology. Recently, we and others have shown that dendritic cell (DC)-based therapies allow a specific immune response associated with sustained lymphoma regression in a proportion of low-tumor burden FL patients. Importantly, the rate of objective clinical response (33-50%) and of sustained remission is remarkably higher compared to similar studies in solid tumors, corroborating the assumption of the immune responsiveness of FL. Our experimental intra-tumoral strategy combined injection with rituximab and interferon-α-derived dendritic cells (IFN-DC), a novel DC population particularly efficient in biasing T-helper response toward the Th1 type and in the cross-priming of CD8 + T cells. Noteworthy, intra-tumoral injection of DC is a new therapeutic option based on the assumption that following the induction of cancer-cell immunogenic death, unloaded DC would phagocytize in vivo the tumor associated antigens and give rise to a specific immune response. This approach allows the design of easy and inexpensive schedules. On the other hand, advanced and straightforward methods to produce clinical-grade antigenic formulations are currently under development. Both unloaded DC strategies and DC-vaccines are suited for combination with radiotherapy, immune checkpoint inhibitors, immunomodulators and metronomic chemotherapy. In fact, studies in animal models have already shown impressive results, while early-phase combination trials are ongoing. Here, we summarize the recent advances and the future perspectives of DC-based therapies in the treatment of FL patients.
Assuntos
Células Dendríticas/imunologia , Imunoterapia/métodos , Linfoma Folicular/tratamento farmacológico , Linfoma Folicular/imunologia , Humanos , Linfoma Folicular/patologiaRESUMO
The perspective of combining cancer vaccines with immunomodulatory drugs is currently regarded as a highly promising approach for boosting tumor-specific T cell immunity and eradicating residual malignant cells. The efficacy of dendritic cell (DC) vaccination in combination with lenalidomide, an anticancer drug effective in several hematologic malignancies, was investigated in a follicular lymphoma (FL) model. First, we evaluated the in vitro activity of lenalidomide in modulating the immune responses of lymphocytes co-cultured with a new DC subset differentiated with IFN-α (IFN-DC) and loaded with apoptotic lymphoma cells. We next evaluated the efficacy of lenalidomide and IFN-DC-based vaccination, either alone or in combination, in hu-PBL-NOD/SCID mice bearing established human lymphoma. We found that lenalidomide reduced Treg frequency and IL-10 production in vitro, improved the formation of immune synapses of CD8 + lymphocytes with lymphoma cells and enhanced anti-lymphoma cytotoxicity. Treatment of lymphoma-bearing mice with either IFN-DC vaccination or lenalidomide led to a significant decrease in tumor growth and lymphoma cell spread. Lenalidomide treatment was shown to substantially inhibit tumor-induced neo-angiogenesis rather than to exert a direct cytotoxic effect on lymphoma cells. Notably, the combined treatment with the vaccine plus lenalidomide was more effective than either single treatment, resulting in the significant regression of established tumors and delayed tumor regrowth upon treatment discontinuation. In conclusion, our data demonstrate that IFN-DC-based vaccination plus lenalidomide exert an additive therapeutic effect in xenochimeric mice bearing established lymphoma. These results may pave the way to evaluate this combination in the clinical ground.
Assuntos
Vacinas Anticâncer/administração & dosagem , Células Dendríticas/imunologia , Sinergismo Farmacológico , Fatores Imunológicos/imunologia , Interferon-alfa/imunologia , Lenalidomida/farmacologia , Linfoma Folicular/terapia , Animais , Terapia Combinada , Feminino , Humanos , Fatores Imunológicos/farmacologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCIDRESUMO
PURPOSE: This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in combination with low doses of rituximab. PATIENTS AND METHODS: Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients. RESULTS: Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11-47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences. CONCLUSIONS: These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.
Assuntos
Células Dendríticas/transplante , Imunoterapia Adotiva/métodos , Linfoma Folicular/terapia , Recidiva Local de Neoplasia/terapia , Rituximab/administração & dosagem , Adulto , Idoso , Animais , Antineoplásicos Imunológicos/administração & dosagem , Terapia Combinada , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Injeções Intralinfáticas , Interferon-alfa/farmacologia , Linfoma Folicular/imunologia , Linfoma Folicular/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/imunologia , Recidiva Local de Neoplasia/patologia , Indução de Remissão , Terapia de Salvação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Follicular lymphoma (FL) is the most common form of indolent non-Hodgkin lymphoma. This malignancy is considered virtually incurable, with high response rates to therapy but frequent relapses. We investigated the ability of monocyte-derived dendritic cells generated in the presence of IFN-α and GM-CSF (IFN-DC) and loaded with apoptotic lymphoma cells to activate immune responses against FL cells, with the ultimate goal of designing novel patient-specific vaccination strategies for the treatment of FL. In this article, we show that apoptotic tumor cell-loaded IFN-DC from FL patients, which were cultured for 2 wk with autologous lymphocytes, led to Th1 response skewing, based on significantly higher levels of IFN-γ production and a remarkable increase in CD8(+) and NK cell frequency, consistent with the detection of enhanced cytotoxic effector function toward autologous FL cells. IFN-DC were found to promote efficient NK cell activation, increased expression of cytotoxicity receptors, and extensive IFN-γ production in the virtual absence of IL-10. Moreover, direct recognition and killing of primary autologous lymphoma cells by activated NK cells from FL patients was also demonstrated. A critical role was demonstrated for MHC class I-related chain A and B and membrane-bound IL-15 in IFN-DC-mediated NK cell activation and early IFN-γ production. The overall results indicate that IFN-DC loaded with autologous apoptotic FL cells represent a valuable tool for improving the potency of therapeutic cancer vaccines through the efficient induction of NK cell activation and promotion of CD8(+) T cell antitumor immunity.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária/imunologia , Linfoma Folicular , Apoptose/imunologia , Western Blotting , Técnicas de Cocultura , Citotoxicidade Imunológica , ELISPOT , Citometria de Fluxo , Humanos , Interferon-alfa/imunologia , Células Tumorais CultivadasRESUMO
The association of Epstein-Barr virus (EBV) with plasmacytoid malignancies is now well established but how the virus influences microRNA expression in such cells is not known. We have used multiple myeloma (MM) cell lines to address this issue and find that an oncomiR, miR-21 is induced after in vitro EBV infection. The PU.1 binding site in miR-21 promoter was essential for its activation by the virus. In accordance with its noted oncogenic functions, miR-21 induction in EBV infected MM cells caused downregulation of p21 and an increase in cyclin D3 expression. EBV infected MM cells were highly tumorigenic in SCID mice. Given the importance of miR-21 in plasmacytoid malignancies, our findings that EBV could further exacerbate the disease by inducing miR-21 has interesting implications both in terms of diagnosis and future miR based therapeutical approaches for the virus associated plasmacytoid tumors.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/genética , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/genética , MicroRNAs/genética , Animais , Sítios de Ligação/genética , Ciclina D3/genética , Inibidor de Quinase Dependente de Ciclina p21/genética , Regulação para Baixo/genética , Herpesvirus Humano 4 , Camundongos , Camundongos SCID , Mieloma Múltiplo/genética , Mieloma Múltiplo/virologia , Regiões Promotoras GenéticasRESUMO
Cross-presentation allows antigen-presenting cells to present exogenous antigens to CD8(+) T cells, playing an essential role in controlling infections and tumor development. IFN-α induces the rapid differentiation of human mono-cytes into dendritic cells, known as IFN-DCs, highly efficient in mediating cross-presentation, as well as the cross-priming of CD8(+) T cells. Here, we have investigated the mechanisms underlying the cross-presentation ability of IFN-DCs by studying the intracellular sorting of soluble ovalbumin and nonstructural-3 protein of hepatitis C virus. Our results demonstrate that, independently from the route and mechanism of antigen entry, IFN-DCs are extraordinarily competent in preserving internalized proteins from early degradation and in routing antigens toward the MHC class-I processing pathway, allowing long-lasting, cross-priming capacity. In IFN-DCs, both early and recycling endosomes function as key compartments for the storage of both antigens and MHC-class I molecules and for proteasome- and transporter-associated with Ag processing-dependent auxiliary cross-presentation pathways. Because IFN-DCs closely resemble human DCs naturally occurring in vivo in response to infections and other danger signals, these findings may have important implications for the design of vaccination strategies in neoplastic or chronic infectious diseases.
Assuntos
Antígenos/metabolismo , Apresentação Cruzada/efeitos dos fármacos , Células Dendríticas/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Interferon-alfa/farmacologia , Antígenos/imunologia , Western Blotting , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endossomos/química , Endossomos/metabolismo , Citometria de Fluxo , Hepacivirus/imunologia , Hepacivirus/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Microscopia Confocal , Ovalbumina/imunologia , Ovalbumina/metabolismo , Ovalbumina/farmacocinética , Proteínas não Estruturais Virais/imunologia , Proteínas não Estruturais Virais/metabolismo , Proteínas não Estruturais Virais/farmacocinéticaRESUMO
IFN-α exerts multiple effects leading to immune protection against pathogens and cancer as well to autoimmune reactions by acting on monocytes and dendritic cells. We analyzed the versatility of human monocytes conditioned by IFN-α towards dendritic cell differentiation (IFN-DC) in shaping the autologous T-helper response. Priming of naïve CD4 T cells with autologous IFN-DC in the presence of either SEA or anti-CD3, resulted, in addition to a prominent expansion of CXCR3+ IFN-γ-producing CD4 Th1 cells, in the emergence of two distinct subsets of IL-17-producing CD4 T cells: i) a predominant Th17 population selectively producing IL-17 and expressing CCR6; ii) a minor Th1/Th17 population, producing both IL-17 and IFN-γ. After phagocytosis of apoptotic cells, IFN-DC induced Th17 cell expansion and IL-17 release. Notably, the use of neutralizing antibodies revealed that IL-23 was an essential cytokine in mediating Th17 cell development by IFN-DC. The demonstration of the IFN-DC-induced expansion of both Th1 and Th17 cell populations reveals the intrinsic plasticity of these DC in orienting the immune response and provides a mechanistic link between IFN-α and the onset of autoimmune phenomena, which have been correlated with both IL-17 production and exposure to IFN-α.
Assuntos
Interferon-alfa/farmacologia , Interleucina-12/fisiologia , Interleucina-23/fisiologia , Monócitos/efeitos dos fármacos , Células Th1/fisiologia , Células Th17/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/fisiologia , Diferenciação Celular/efeitos dos fármacos , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/fisiologia , Humanos , Interleucina-12/metabolismo , Interleucina-23/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/fisiologia , Modelos Biológicos , Monócitos/metabolismo , Monócitos/fisiologia , Células Th1/efeitos dos fármacos , Células Th17/metabolismo , Células Th17/fisiologia , Células Th17/transplanteRESUMO
Here we report a novel strategy for the induction of CD8(+) T cell adaptive immune response against viral and tumor antigens. This approach relies on high levels of incorporation in HIV-1 VLPs of a mutant of HIV-1 Nef (Nef(mut)) which can act as anchoring element for foreign proteins. By in vitro assay, we found that VLP-associated Nef(mut) is efficiently cross-presented by antigen presenting cells. Inoculation in mice of VLPs incorporating the HPV-16 E7 protein fused to Nef(mut) led to an anti-E7 CD8(+) T cell response much stronger than that elicited by E7 recombinant protein inoculated with incomplete Freund's adjuvant and correlating with well-detectable anti-E7 CTL activity. Most relevantly, mice immunized with Nef(mut)-E7 VLPs developed a protective immune response against tumors induced by E7 expressing tumor cells. These results make Nef(mut) VLPs a promising candidate for new vaccine strategies focused on the induction of CD8(+) T cell immunity.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/imunologia , Proteínas Oncogênicas Virais/imunologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/imunologia , Imunidade Adaptativa , Animais , Linhagem Celular , Apresentação Cruzada , HIV-1/imunologia , Papillomavirus Humano 16/imunologia , Humanos , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas E7 de Papillomavirus , Infecções por Papillomavirus/prevenção & controle , Vacinas contra Papillomavirus/imunologia , Proteínas Recombinantes de Fusão/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Proteínas do Envelope Viral/imunologiaRESUMO
6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) is a powerful inhibitor of the glutathione transferase P1-1 (GSTP1-1) and causes the disruption of the complex between GSTP1-1 and c-Jun N-terminal Kinase (JNK). This induces JNK activation and apoptosis in tumour cells. In the present work we assess the in vitro and in vivo effectiveness of NBDHEX on two human melanoma cell lines, Me501 and A375. NBDHEX shows IC(50) values in the low micromolar range (IC(50) of 1.2+/-0.1microM and 2.0+/-0.2 microM for Me501 and A375, respectively) and is over 100 times more cytotoxic to these cell lines than temozolomide. Apoptosis is observed in Me501 cells within 3h of the addition of NBDHEX, while in A375 cells the apoptotic event is rather late, and is preceded by a G2/M phase arrest. In both melanoma cell lines, JNK activity is required for the ability of NBDHEX to trigger apoptosis, confirming that the JNK pathway is an important therapeutic target for this tumour. NBDHEX is also both effective and well tolerated in in vivo tumour models. A tumour inhibition of 70% is observed in vivo against Me501 human melanoma and a similar result is obtained on A375 model, with 63% of tumour inhibition. These findings indicate that the activation of the JNK pathway, through a selective GSTP1-1 targeting, could prove to be a promising new strategy for treating melanoma, which responds poorly to conventional therapies.
Assuntos
Antineoplásicos/uso terapêutico , Apoptose , Glutationa S-Transferase pi/antagonistas & inibidores , Melanoma/tratamento farmacológico , Oxidiazóis/uso terapêutico , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Melanoma/metabolismo , Camundongos , Camundongos SCID , Microscopia de Fluorescência , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Dendritic cells (DCs) play a crucial role in linking innate and adaptive immunity, by virtue of their unique ability to take up and process antigens in the peripheral blood and tissues and, upon migration to draining lymph nodes, to present antigen to resting lymphocytes. Notably, these DC functions are modulated by cytokines and chemokines controlling the activation and maturation of these cells, thus shaping the response towards either immunity or tolerance.An ensemble of recent studies have emphasized an important role of type I IFNs in the DC differentiation/activation, suggesting the existence of a natural alliance between these cytokines and DCs in linking innate and adaptive immunity. Herein, we will review how type I IFNs can promote the ex vivo differentiation of human DCs and orient DC functions towards the priming and expansion of protective antitumor immune responses. We will also discuss how the knowledge on type I IFN-DC interactions could be exploited for the design of more selective and effective strategies of cancer immunotherapy.
Assuntos
Adjuvantes Imunológicos/uso terapêutico , Vacinas Anticâncer , Células Dendríticas/transplante , Imunoterapia Adotiva , Interferon-alfa/uso terapêutico , Neoplasias/terapia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Células Dendríticas/imunologia , Humanos , Imunidade Inata , Interferon-alfa/metabolismo , Neoplasias/imunologia , FenótipoRESUMO
Dendritic cells (DC) generated after a short-term exposure of monocytes to IFN-alpha and GM-CSF (IFN-DC) are highly effective in inducing cross-priming of CD8(+ )T cells against viral antigens. We have investigated the mechanisms responsible for the special attitude of these DC and compared their activity with that of reference DC. Antigen uptake and endosomal processing capabilities were similar for IFN-DC and IL-4-derived DC. Both DC types efficiently cross-presented soluble HCV NS3 protein to the specific CD8(+) T cell clone, even though IFN-DC were superior in cross-presenting low amounts of viral antigens. Moreover, when DC were pulsed with inactivated HIV-1 and injected into hu-PBL-SCID mice, the generation of virus-specific CD8(+ )T cells was markedly higher in animals immunized with IFN-DC than in mice immunized with CD40L-matured IL-4-DC. Of interest, in experiments with purified CD8(+ )T cells, IFN-DC were superior with respect to CD40L-matured IL-4-DC in inducing in vitro cross-priming of HIV-specific CD8(+ )T cells. This property correlated with enhanced potential to express the specific subunits of the IL-23 and IL-27 cytokines. These results suggest that IFN-DC are directly licensed for an efficient CD8(+) T cell priming by mechanisms likely involving enhanced antigen presentation and special attitude to produce IL-12 family cytokines.
Assuntos
Antígenos Virais/imunologia , Linfócitos T CD8-Positivos/imunologia , Apresentação Cruzada/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Interferon-alfa/imunologia , Animais , Formação de Anticorpos/imunologia , Apresentação de Antígeno , Linfócitos T CD4-Positivos/imunologia , Ligante de CD40/imunologia , Diferenciação Celular , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/citologia , Endossomos/imunologia , HIV-1/imunologia , Humanos , Interferon-alfa/genética , Interleucinas/metabolismo , Camundongos , Fenótipo , Solubilidade , Fatores de TempoRESUMO
Critical changes occurring in Rac-1 molecule, a cytoskeleton organizing small GTPase associated with cell ruffling, have been analyzed in dendritic cells (DCs) derived from monocytes cultured with granulocyte-macrophage colony-stimulating factor and IFN-alpha or IL-4. Although with different kinetics, both agents induced activation of Rac-1 molecule and, more importantly, an upregulation of both protein expression and mRNA transcription. These findings strengthen the role of Rac-1 molecule in the induction of DC differentiation and suggest that, besides its activation, the upregulation of Rac-1 molecule might also play a role in the acquisition of DC mature phenotype.
Assuntos
Células Dendríticas/citologia , Monócitos/citologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Diferenciação Celular , Citocinas/farmacologia , Citoesqueleto/ultraestrutura , Células Dendríticas/enzimologia , Ativação Enzimática , Humanos , Monócitos/efeitos dos fármacos , Monócitos/enzimologia , RNA Mensageiro/metabolismo , Transcrição Gênica , Regulação para Cima , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Recent studies have revealed that type I interferons (IFNs) are powerful inducers of the differentiation and activation of dendritic cells (DCs). These findings emphasize the importance of these cytokines in linking innate and adaptive immunity, suggesting that effects of type I IFN on DCs can play a role in the antitumor and antiviral activity observed in some IFN-treated patients. Thus, the evaluation of the effects of IFN on the differentiation/activation of DCs has become an important approach for testing novel biologically important IFN activities, and the description of some reference methods are urgently needed. In this chapter, we describe some methods for testing the effects of IFNs on the differentiation and activation of human DCs from the peripheral blood monocytes and for the characterization of the DCs generated after IFN treatment.
Assuntos
Diferenciação Celular/fisiologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/fisiologia , Interferon Tipo I/farmacologia , Animais , Antígenos CD/metabolismo , Biomarcadores , Movimento Celular/fisiologia , Células Cultivadas , Meios de Cultura/química , Citocinas/metabolismo , Células Dendríticas/citologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Interferon Tipo I/metabolismo , Camundongos , Camundongos SCID , Monócitos/citologia , Monócitos/fisiologiaRESUMO
Bordetella pertussis toxin B-oligomer (PTX-B) has been shown to inhibit HIV infection and replication in vitro. The potential anti-viral effect of PTX-B was tested here in an in vivo surrogate model of HIV infection, i.e. SCID mice reconstituted with human peripheral blood leukocytes (PBL) (hu-PBL-SCID) and infected with a CCR5-dependent (R5) HIV-1 strain. SCID mice inoculated intra-peritoneal (i.p.) with PTX-B and then infected with the R5 strain SF-162 were sacrificed 7 days later and analyzed for human PBL (hu-PBL) lymphoid tissue reconstitution, infection of hu-PBL, plasma viremia and viral rescue from ex vivo-cultivated i.p. hu-PBL. Unlike mice treated with 500 ng per animal of PTX-B showing no evidence of viral inhibition, daily administration of PTX-B (50 ng per mouse) strongly inhibited virus infection and replication, as determined by undetectable viremia, absence of infected hu-PBL and lack of rescue of infectious HIV in most animals. Furthermore, PTX-B injection 2 h before and twice after infection prevented HIV-1 infection and replication in all (10/10) tested animals. Thus, PTX-B potently inhibited virus infection and replication in hu-PBL-SCID mice, supporting the hypothesis that it may represent a new pharmacological agent against HIV-1 infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Toxina Pertussis/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Humanos , Leucócitos/virologia , Camundongos , Camundongos SCIDRESUMO
A major challenge of AIDS research is the development of therapeutic vaccine strategies capable of inducing the humoral and cellular arms of the immune responses against HIV-1. In this work, we evaluated the capability of DCs pulsed with aldrithiol-2-inactivated HIV-1 in inducing a protective antiviral human immune response in SCID mice reconstituted with human PBL (hu-PBL-SCID mice). Immunization of hu-PBL-SCID mice with DCs generated after exposure of monocytes to GM-CSF/IFN-alpha (IFN-DCs) and pulsed with inactivated HIV-1 resulted in a marked induction of human anti-HIV-1 antibodies, which was associated with the detection of anti-HIV neutralizing activity in the serum. This vaccination schedule also promoted the generation of a human CD8+ T cell response against HIV-1, as measured by IFN-gamma Elispot analysis. Notably, when the hu-PBL-SCID mice immunized with antigen-pulsed IFN-DCs were infected with HIV-1, inhibition of virus infection was observed as compared with control animals. These results suggest that IFN-DCs pulsed with inactivated HIV-1 can represent a valuable approach of immune intervention in HIV-1-infected patients.
Assuntos
Vacinas contra a AIDS/uso terapêutico , Síndrome da Imunodeficiência Adquirida/imunologia , Células Dendríticas/imunologia , HIV-1/imunologia , Interferon-alfa/imunologia , Linfócitos/imunologia , Vacinas de Produtos Inativados/uso terapêutico , Síndrome da Imunodeficiência Adquirida/prevenção & controle , Animais , Células Dendríticas/transplante , Células Dendríticas/virologia , Humanos , Separação Imunomagnética/métodos , Transfusão de Linfócitos , Linfócitos/citologia , Camundongos , Camundongos SCID , Transplante Heterólogo/imunologiaRESUMO
Infection by HIV-1 causes persistent, long-term high virus production in macrophages. Major evidence, both in humans and in primate models, shows the crucial role of macrophages in sustaining virus production and in mediating a cytopathic effect on bystander CD4+ T lymphocytes and neuronal cells. In the present study, we used severe combined immunodeficient (SCID) mice engrafted with human peripheral blood lymphocytes (hu-PBL-SCID mice) to investigate the in vivo effect of HIV-1-infected macrophages on virus spread and CD4+ T lymphocyte depletion, and the ability of a mAb against nerve growth factor (NGF, a neurokine essential for the survival of HIV-1-infected macrophages) to suppress the pathogenetic events mediated by infected macrophages. Injection of mice with as few as 500 HIV-exposed macrophages causes (i) complete depletion of several millions of autologous CD4+ T lymphocytes, (ii) sustained HIV viremia, and (iii) spreading of HIV-1 DNA in mouse lymphoid organs. In contrast, in vivo treatment with an anti-NGF Ab completely abrogates all effects mediated by HIV-infected macrophages. Taken together, the results demonstrate the remarkable power of macrophages in sustaining in vivo HIV-1 infection, and that such a phenomenon can be specifically abrogated by an anti-NGF Ab. This may open new perspectives of experimental approaches aimed at selectively eliminating persistently infected macrophages from the bodies of HIV-infected patients.
Assuntos
Infecções por HIV/prevenção & controle , Fator de Crescimento Neural/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/etiologia , Infecções por HIV/imunologia , HIV-1 , Humanos , Transfusão de Linfócitos , Linfopenia/prevenção & controle , Macrófagos/imunologia , Macrófagos/virologia , Camundongos , Camundongos SCID , Fator de Crescimento Neural/imunologia , Testes de Neutralização , Transplante Heterólogo , Viremia/etiologia , Viremia/imunologia , Viremia/prevenção & controleRESUMO
Since denditric cells (DC) represent the main players linking innate and adaptive immunity, their prompt generation from blood cells would be instrumental for an efficient immune response to infections. Consistent with this, CD2+ monocytes were found to express the DC maturation marker CD83, along with acquisition of high antigen-presenting activity, after a surprisingly short time in culture. This rapid process is associated with expression of IFN-alpha/beta genes and secretion of low levels of pro-inflammatory cytokines. Exposure of monocytes to IFN-alpha, but not to IL-4, induced persistence of CD2+/CD83+ cells, which were fully competent in stimulating primary responses by naive T cells. These results unravel the natural pathway by which infection-induced signals rapidly transform pre-armed monocytes into active DC.