RESUMO
Prostate cancer is the second most common cancer in men, for which there are no reliable biomarkers or targeted therapies. Here we demonstrate that elevated levels of Δ133TP53ß isoform characterize prostate cancers with immune cell infiltration, particularly T cells and CD163+ macrophages. These cancers are associated with shorter progression-free survival, Gleason scores ≥ 7, and an immunosuppressive environment defined by a higher proportion of PD-1, PD-L1 and colony-stimulating factor 1 receptor (CSF1R) positive cells. Consistent with this, RNA-seq of tumours showed enrichment for pathways associated with immune signalling and cell migration. We further show a role for hypoxia and wild-type p53 in upregulating Δ133TP53 levels. Finally, AUC analysis showed that Δ133TP53ß expression level alone predicted aggressive disease with 88% accuracy. Our data identify Δ133TP53ß as a highly accurate prognostic factor for aggressive prostate cancer.
Assuntos
Neoplasias da Próstata/imunologia , Proteína Supressora de Tumor p53/imunologia , Células A549 , Biomarcadores Tumorais/imunologia , Linhagem Celular Tumoral , Humanos , Células MCF-7 , Macrófagos/imunologia , Masculino , Células PC-3 , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Isoformas de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/imunologia , Linfócitos T/imunologia , Microambiente Tumoral/imunologia , Proteína Supressora de Tumor p53/genéticaRESUMO
Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.
Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Melanoma/terapia , Proteínas Virais/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Técnicas de Cultura de Células , Epitopos de Linfócito T/imunologia , Imunoterapia/métodos , Lectinas Tipo C/metabolismo , Ativação Linfocitária , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Superfície Celular/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Proteínas Virais/administração & dosagemRESUMO
∆122p53 mice (a model of ∆133p53 isoform) are tumour-prone, have extensive inflammation and elevated serum IL-6. To investigate the role of IL-6 we crossed ∆122p53 mice with IL-6 null mice. Here we show that loss of IL-6 reduced JAK-STAT signalling, tumour incidence and metastasis. We also show that ∆122p53 activates RhoA-ROCK signalling leading to tumour cell invasion, which is IL-6-dependent and can be reduced by inhibition of JAK-STAT and RhoA-ROCK pathways. Similarly, we show that Δ133p53 activates these pathways, resulting in invasive and migratory phenotypes in colorectal cancer cells. Gene expression analysis of colorectal tumours showed enrichment of GPCR signalling associated with ∆133TP53 mRNA. Patients with elevated ∆133TP53 mRNA levels had a shorter disease-free survival. Our results suggest that ∆133p53 promotes tumour invasion by activation of the JAK-STAT and RhoA-ROCK pathways, and that patients whose tumours have high ∆133TP53 may benefit from therapies targeting these pathways.
Assuntos
Neoplasias Colorretais/metabolismo , Interleucina-6/metabolismo , Janus Quinases/metabolismo , Fatores de Transcrição STAT/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Intervalo Livre de Doença , Feminino , Células HCT116 , Humanos , Masculino , Camundongos , Invasividade Neoplásica , Metástase Neoplásica , Prognóstico , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
The results of adoptive T-cell therapies (ACTs) are very encouraging and show clinical evidence that ACT can provide a cure for patients with metastatic disease. However, various response rates and long-term cancer remission have been observed in different ACT trials. The types of T cells, prior treatment with chemotherapy and co-administration of other immune-target therapies have been found to influence the efficacy of ACT. In this study, we investigate the ability of ACT using CD4+ T helper 1 (Th1) cells and CD8+ cytotoxic T lymphocytes (CTLs) to reject the growth of established B16-ovalbumin (OVA) melanoma. CD8+ CTLs were found to be the main effector T cells that mediated tumour regression. However, low tumour-free survival rates were observed in ACT with CD8+ CTLs only. Co-transferring CD4+ Th1 cells and CD8+ CTLs has been observed to induce a synergistic antitumour response, resulting in complete regression in 80% of the tumour-bearing mice. We also examined a prior Dacarbazine (DTIC) and after virus-like particle (VLP)-OVA vaccine treatment to enhance ACT, but no therapeutic benefit was observed during primary B16-OVA tumour growth. Nevertheless, the ACT-mediated antitumour response was able to generate memory responses to both B16-OVA and B16-gp33 tumours. VLP-OVA vaccination following ACT enhances the memory responses to tumours that express a heterogenic population of both B16-OVA and B16-gp33 cells; however, it abolished the memory response to tumours consisting of only gp33-expressing cells. These findings provide important information for designing therapeutic treatments for patients with metastatic disease and cancer relapse to achieve durable cancer remission.
RESUMO
The dendritic cell (DC) is the foremost antigen-presenting cell (APC) for ex vivo expansion of tumour-specific patient T cells. Despite marked responses in some patients following reinfusion of DC-activated autologous or HLA-matched donor T cells, overall response rates remain modest in solid tumours. Furthermore, most studies aim to generate immune responses against defined tumour-associated antigens (TAA), however, meta-analysis reveals that those approaches have less clinical success than those using whole tumour cells or their components. Tumour lysate (TL) is used as a source of tumour antigen in clinical trials and potentially represents the full range of TAAs in an undefined state. Little is known about how different APCs cooperate to present TL antigens. We examined the effect of oxidised whole-cell lysate (ox-L) versus soluble fraction freeze-thaw lysate (s-L) on bone marrow-derived DCs and macrophages, and magnetic bead-isolated splenic B cells. The APCs were used individually, or in combination, to prime T cells. CD8+ T cells produced interferon (IFN)-γ in response to both s-L and ox-L, but only proliferated in response to ox-L. IFN-γ production and proliferation was enhanced by priming with the DC+B cell combination. Compared to DC alone, a trend toward greater interleukin (IL)-12 production was observed when DC+B cell were loaded with s-L and ox-L antigens. CD8+ T-cell specific lysis in vivo was greatest in ox-L-primed groups and DC+B cell priming significantly increased in vivo cytotoxicity compared to DC alone. These improved T-cell responses with two APCs and stressed cell lysate has implications for APC-based adoptive cell therapies.
RESUMO
Accumulating evidence suggests tumor protein 53 (p53) promotes correct cellular differentiation. Thus, mutant TP53 may be more frequent in tumors with irregular differentiation. This study investigated whether TP53 mutations were more frequent in diffuse large B cell lymphoma (DLBCL) that lacked the B cell lineage marker CD19. Sixteen CD19 negative and 78 CD19 positive DLBCL were sequenced for TP53 mutations. Twenty nine tumors had TP53 mutations and were associated with poorer survival. Mutant TP53 was more frequent in CD19 negative lymphomas (81% versus 21%, p < 0.0001). Analysis of other B cell markers revealed a lack of paired box 5 (PAX5) in CD19 positive lymphomas with mutant TP53 (50%), which was more frequent compared to tumors with wild-type TP53 (15%, p = 0.002). In summary, DLBCL lacking CD19 or PAX5 expression were more likely to have mutant TP53, suggesting irregular B cell marker phenotypes are associated with TP53 mutation.
Assuntos
Antígenos CD19/metabolismo , Biomarcadores Tumorais/metabolismo , Linfoma Difuso de Grandes Células B/genética , Mutação/genética , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Sequência de Bases , Diferenciação Celular/genética , Demografia , Feminino , Humanos , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , Proteína Supressora de Tumor p53/químicaRESUMO
Adoptive cell therapies (ACTs) using tumor-reactive T cells have shown clinical benefit and potential for cancer treatment. While the majority of the current ACT are focused on using CD8(+) cytotoxic T lymphocytes (CTL), others have shown that the presence of tumor-reactive CD4(+) T helper (Th) cells can greatly enhance the anti-tumor activity of CD8(+) CTL. However, difficulties in obtaining adequate numbers of CD4(+) Th cells through in vitro expansion can limit the application of CD4 Th cells in ACT. This study aims to optimize the culture conditions for mouse CD4 T cells to provide basic information for animal studies of ACT using CD4 T cells. Taking advantage of the antigen-specificity of CD4(+) Th cells from OT-II transgenic mice, we examined different methodologies for generating antigen-specific CD4(+) Th1 cells in vitro. We found that cells grown in complete advanced-DMEM/F12 medium supplemented with low-dose IL-2 and IL-7 induced substantial cell expansion. These Th cells were Th1-like, as they expressed multiple Th1-cytokines and exhibited antigen-specific cytotoxicity. In addition co-transfer of these CD4(+) Th1-like cells with CD8(+) CTL significantly enhanced tumor regression, leading to complete cure in 80% of mice bearing established B16-OVA. These observations indicate that the CD4(+) Th1-like cells generated using the method we optimized are functionally active to eliminate their target cells, and can also assist CD8(+) CTL to enhance tumor regression. The findings of this study provide valuable data for further research into in vitro expansion of CD4(+) Th1-like cells, with potential applications to cancer treatment involving ACT.
RESUMO
Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c+ APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.
RESUMO
Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.
Assuntos
Apresentação de Antígeno/imunologia , Células Apresentadoras de Antígenos/imunologia , Manose/imunologia , Animais , Antígenos/imunologia , Linfócitos B/imunologia , Células Dendríticas/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Humanos , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Coelhos , Receptores de Superfície Celular/imunologiaRESUMO
Noroviruses are an emerging threat to public health, causing large health and economic costs, including at least 200,000 deaths annually. The inability to replicate in cell culture or small animal models has limited the understanding of the interaction between human noroviruses and their hosts. However, an alternative strategy to gain insights into norovirus pathogenesis is to study murine norovirus (MNV-1) that replicates in cultured macrophages. While the innate immune response is central to the resolution of norovirus disease, the adaptive immune response is required for viral clearance. The specific responses of macrophages and dendritic cells to infection drive the adaptive immune response, with chemokines playing an important role. In this study, we have conducted microarray analysis of RAW264.7 macrophages infected with MNV-1 and examined the changes in chemokine transcriptional expression during infection. While the majority of chemokines showed no change, there was specific up-regulation in chemokines reflective of a bias toward a Th1 response, specifically CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL10 and CXCL11. These changes in gene expression were reflected in protein levels as determined by ELISA assay. This virus-induced chemokine response will affect the resolution of infection and may limit the humoral response to norovirus infection.
Assuntos
Quimiocinas/metabolismo , Norovirus/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Quimiocinas/genética , Análise por Conglomerados , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interferons/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Regulação para Cima , Replicação ViralRESUMO
Theileria annulata, an intracellular parasite of bovine lymphoid cells, induces substantial phenotypic alterations to its host cell including continuous proliferation, cytoskeletal changes and resistance to apoptosis. While parasite induced modulation of host cell signal transduction pathways and NFκB activation are established, there remains considerable speculation on the complexities of the parasite directed control mechanisms that govern these radical changes to the host cell. Our objectives in this study were to provide a comprehensive analysis of the global changes to host cell gene expression with emphasis on those that result from direct intervention by the parasite. By using comparative microarray analysis of an uninfected bovine cell line and its Theileria infected counterpart, in conjunction with use of the specific parasitacidal agent, buparvaquone, we have identified a large number of host cell gene expression changes that result from parasite infection. Our results indicate that the viable parasite can irreversibly modify the transformed phenotype of a bovine cell line. Fifty percent of genes with altered expression failed to show a reversible response to parasite death, a possible contributing factor to initiation of host cell apoptosis. The genes that did show an early predicted response to loss of parasite viability highlighted a sub-group of genes that are likely to be under direct control by parasite infection. Network and pathway analysis demonstrated that this sub-group is significantly enriched for genes involved in regulation of chromatin modification and gene expression. The results provide evidence that the Theileria parasite has the regulatory capacity to generate widespread change to host cell gene expression in a complex and largely irreversible manner.
Assuntos
Regulação da Expressão Gênica , Interações Hospedeiro-Parasita/genética , Linfoma não Hodgkin/patologia , Theileria annulata/fisiologia , Animais , Bovinos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Leucócitos/parasitologia , Naftoquinonas/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Cervical cancer is caused by high-risk, cancer-causing human papillomaviruses (HPV) and is the second highest cause of cancer deaths in women globally. The majority of cervical cancers express well-characterized HPV oncogenes, which are potential targets for immunotherapeutic vaccination. Here we develop a rabbit haemorrhagic disease virus (RHDV) virus-like particle (VLP)-based vaccine designed for immunotherapy against HPV16 positive tumours. An RHDV-VLP, modified to contain the universal helper T cell epitope PADRE and decorated with an MHC I-restricted peptide (aa 48-57) from the HPV16 E6, was tested for its immunotherapeutic efficacy against the TC-1 HPV16 E6 and E7-expressing tumour in mice. The E6-RHDV-VLP-PADRE was administered therapeutically for the treatment of a pre-existing TC-1 tumour and was delivered with antibodies either to deplete regulatory T cells (anti-CD25) or to block T cell suppression mediated through CTLA-4. As a result, the tumour burden was reduced by around 50% and the median survival time of mice to the humane endpoint was almost doubled the compared to controls. The incorporation of PADRE into the RHDV-VLP was necessary for an E6-specific enhancement of the anti-tumour response and the co-administration of the immune modifying antibodies contributed to the overall efficacy of the immunotherapy. The E6-RHDV-VLP-PADRE shows immunotherapeutic efficacy, prolonging survival for HPV tumour-bearing mice. This was enhanced by the systemic administration of immune-modifying antibodies that are commercially available for use in humans. There is potential to further modify these particles for even greater efficacy in the path to development of an immunotherapeutic treatment for HPV precancerous and cancer stages.
Assuntos
Vacinas Anticâncer/uso terapêutico , Vírus da Doença Hemorrágica de Coelhos/genética , Papillomavirus Humano 16/imunologia , Infecções por Papillomavirus/terapia , Vacinas contra Papillomavirus/uso terapêutico , Neoplasias do Colo do Útero/terapia , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Vírus da Doença Hemorrágica de Coelhos/imunologia , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Humanos , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Proteínas Oncogênicas Virais/imunologia , Infecções por Papillomavirus/imunologia , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Proteínas Repressoras/imunologia , Carga Tumoral , Neoplasias do Colo do Útero/imunologia , Neoplasias do Colo do Útero/patologia , Neoplasias do Colo do Útero/virologia , Vacinas Sintéticas/uso terapêutico , Proteínas Estruturais Virais/metabolismo , Vírion/genética , Vírion/imunologia , Vírion/isolamento & purificaçãoRESUMO
Virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) can be used as a scaffold to facilitate the delivery of antigens to induce cell-mediated immune responses. In this study, we investigated the immune response to lymphocytic choriomeningitis virus-derived peptide antigen (gp33) delivered by RHDV VLP. The gp33 peptides were incorporated into the VLP in 2 different forms, either recombinantly expressed inside the VLP (VLP-gp33r) or chemically coupled to the surface of the VLP (VLP-gp33c). We showed that VLP-gp33r induced a greater level of cytotoxicity than VLP-gp33c against gp33-coated target cells in vivo. Both VLP, when delivered as prophylactic vaccines, inhibited the growth of Lewis' lung carcinoma tumors expressing gp33 (LL-LCMV) in mice to a similar degree. Studies to investigate the mechanism induced by these VLP showed that 2 CD11c DC subsets, CD8α and CD8α, acquired VLP in vivo and in vitro, and VLP-gp33r were cross-presented by both these subsets to prime CD8 T cells through a TAP-independent, endosomal recycling pathway. Depletion of Langerin DC in vivo before and after vaccination with VLP-gp33r, lead to reduced cytotoxicity implicating these cells in the induction of cytotoxic effector cells. These results suggest that recombinant VLP expressing tumor peptides targeted to Langerin DC may have clinical application. Finally we found that VLP-gp33r were more effective antitumor vaccines than VLP-gp33c when delivered therapeutically. The findings of this study suggest the potential of VLP as a platform for delivery of tumor-associate antigen and elicit protective immunity against tumors.
Assuntos
Antígenos Virais/imunologia , Vacinas Anticâncer/administração & dosagem , Carcinoma Pulmonar de Lewis/terapia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Vírus da Coriomeningite Linfocítica/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Lewis/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Células Dendríticas/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/imunologiaRESUMO
Virus-like particles (VLP) are effective vehicles for delivery of heterologous antigen to antigen-presenting cells. However VLP alone are insufficiently stimulatory to generate the signals required to facilitate effective priming of naïve T cells. We show that the VLP derived from rabbit hemorrhagic disease virus can bind the galactose-containing adjuvant α-galactosylceramide to form a composite particle for co-delivery of antigen and adjuvant to the same antigen-presenting cell. Vaccination with VLP and α-galactosylceramide activated splenic iNKT cells to produce IFN-γ and IL-4, led to the generation of antigen-specific T cells that protected prophylactically against subcutaneous tumor challenge, and was more effective at generating anti-tumor immune responses than either component individually. These data demonstrate a novel method for immunopotentiating VLP to increase their efficacy in the generation of anti-tumor responses via the innate ligand recognition properties of calicivirus-derived nanoparticles.
Assuntos
Adjuvantes Imunológicos , Vacinas Anticâncer/imunologia , Galactosilceramidas/imunologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/uso terapêutico , Animais , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Galactosilceramidas/administração & dosagem , Galactosilceramidas/uso terapêutico , Vírus da Doença Hemorrágica de Coelhos/genética , Masculino , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Linfócitos T/imunologia , Vírion/genética , Vírion/imunologiaRESUMO
Autoimmune diseases are characterized by the immune system mounting a response against self. The exact etiology of autoimmune diseases and autoimmunity remain unclear. Here, we demonstrate that Δ133p53, an isoform of the tumor suppressor protein p53, is involved in the development of autoimmunity. We have previously generated a mouse model of Δ133p53 (Δ122p53). Δ122p53 mice develop an autoimmune/ inflammation-like phenotype that includes the production of autoantibodies, elevated levels of pro-inflammatory cytokines and lymphocyte aggregations in various organs. Microarray analysis reveals that expression of Δ122p53 induces a number of pro-inflammatory genes, including the STAT1 pathway and interferon-related transcription profile. Comparative genetic signatures have been observed in human SLE (systemic lupus erythematosus) patients, and we show that Δ133p53 regulates STAT1 in human cells. Our findings provide the first evidence of a role for p53 isoforms in the development of autoimmune disease.
Assuntos
Doenças Autoimunes/metabolismo , Inflamação/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Autoanticorpos/sangue , Doenças Autoimunes/genética , Autoimunidade/genética , Linhagem Celular Tumoral , Citocinas/metabolismo , Perfilação da Expressão Gênica , Humanos , Inflamação/genética , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/patologia , Linfócitos/imunologia , Linfócitos/metabolismo , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologiaRESUMO
The p53 protein is a pivotal tumor suppressor that is frequently mutated in many human cancers, although precisely how p53 prevents tumors is still unclear. To add to its complexity, several isoforms of human p53 have now been reported. The Δ133p53 isoform is generated from an alternative transcription initiation site in intron 4 of the p53 gene (Tp53) and lacks the N-terminus. Elevated levels of Δ133p53 have been observed in a variety of tumors. To explore the functions of Δ133p53, we created a mouse expressing an N-terminal deletion mutant of p53 (Δ122p53) that corresponds to Δ133p53. Δ122p53 mice show decreased survival and a different and more aggressive tumor spectrum compared with p53 null mice, implying that Δ122p53 is a dominant oncogene. Consistent with this, Δ122p53 also confers a marked proliferative advantage on cells and reduced apoptosis. In addition to tumor development, Δ122p53 mice show a profound proinflammatory phenotype having increased serum concentrations of interleukin-6 and other proinflammatory cytokines and lymphocyte aggregates in the lung and liver as well as other pathologies. Based on these observations, we propose that human Δ133p53 also functions to promote cell proliferation and inflammation, one or both of which contribute to tumor development.
Assuntos
Proliferação de Células , Inflamação/genética , Neoplasias Experimentais/genética , Proteína Supressora de Tumor p53/genética , Sequência de Aminoácidos , Animais , Western Blotting , Imunofluorescência , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução GenéticaRESUMO
Effective vaccines and immunotherapies against cancer require professional antigen-presenting cells to cross-present exogenous antigen to initiate cytotoxic T-cell responses to destroy tumors. Virus-like particles (VLPs), containing tumor antigens, which can immunize against cancers, are cross-presented by dendritic cell (DC) but the mechanism by which this occurs is not fully understood. Here, we used VLPs, derived from rabbit hemorrhagic disease virus (RHDV) with both murine and human DCs, to elucidate these pathways. We have employed inhibitors to demonstrate that these VLPs are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. VLP-derived peptides are loaded onto major histocompatibility complex I that have been recycled from the cell surface. Antigen-coupled VLPs and murine ovalbumin-specific and human melanoma-associated antigen recognized by T cells (MART-1)-specific CD8(+) T cells were used to demonstrate cross-presentation via this alternate, receptor recycling pathway, which operated independently of the proteasome and the transporter-associated with antigen presentation. Finally, we found that cross-presentation of VLPs in vivo was not confined to CD8α(+) DC subsets. These data define the cross-presentation pathway for RHDV VLPs and may lead to improved cancer immunotherapies.
Assuntos
Apresentação de Antígeno/imunologia , Apresentação Cruzada/imunologia , Epitopos/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Vacinas de Partículas Semelhantes a Vírus/imunologia , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células da Medula Óssea/imunologia , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Endocitose/imunologia , Feminino , Vírus da Doença Hemorrágica de Coelhos/imunologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fagocitose/imunologia , Pinocitose/imunologia , Proteoma/imunologia , Coelhos , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Baço/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
OBJECTIVE: Ileocolitis is a recognized feature of ankylosing spondylitis (AS) and is likely to play a role in the pathogenesis of AS, in conjunction with the normal intestinal microbiota. In order to investigate the host immune response in AS, we measured cytokines in tissue culture following exposure of peripheral blood mononuclear cells (PBMC) to autologous colonic bacteria. METHODS: Twenty-one patients with AS and 21 matched controls were recruited. Subjects in the AS group were assessed clinically. Bacteroides species belonging to the B. fragilis group were selectively cultured from stool samples and paired with blood samples from each participant. Ten cultures of autologous Bacteroides were randomly selected from cultures grown from the fecal specimens of each of the 21 patients with AS and 21 controls. These were then tested for reactivity with PBMC and the cytokines produced by proliferating lymphocytes [interleukin 10 (IL-10), IL-17, interferon-gamma, tumor necrosis factor-alpha] were measured in cell culture supernatants. Differences between groups were analyzed using censored normal regression analysis. RESULTS: The patients with AS had severe active AS with Bath AS Disease Activity Index 5.5 (+/-1.6) and C-reactive protein (mg/l) 13.8 (+/-12.2) (mean+/-standard deviation). IL-10 concentrations in ex vivo assay supernatants were lower in the AS group compared with controls (p=0.047). There were no statistically significant differences between the groups for other cytokines. CONCLUSION: In AS, reduced IL-10 production in response to stimulation with autologous Bacteroides cultures may represent a mechanism by which intestinal inflammation develops and persists, a situation analogous to inflammatory bowel disease.
Assuntos
Bacteroides/imunologia , Fenômenos do Sistema Imunitário/fisiologia , Espondilite Anquilosante , Adolescente , Adulto , Idoso , Animais , Citocinas/imunologia , Fezes/microbiologia , Trato Gastrointestinal/microbiologia , Humanos , Interleucina-10/metabolismo , Leucócitos Mononucleares/metabolismo , Masculino , Pessoa de Meia-Idade , Espondilite Anquilosante/imunologia , Espondilite Anquilosante/microbiologia , Adulto JovemRESUMO
Recombinant virus-like particles (VLP) expressing heterologous tumor antigens have recently been investigated for use as vaccines. We have chemically conjugated ovalbumin (OVA) or OVA-derived CD4 (OTII) and CD8 (OTI) epitopes, to rabbit hemorrhagic disease virus (RHDV) VLP. VLP conjugated with OVA were able to cross-prime CD8+ cells from OT1 mice transgenic for the OVA T cell receptor. VLP.OTI was able to induce higher antigen-specific cytotoxicity in vivo than VLP mixed with either the protein or the peptide. Furthermore we have shown that the growth of the aggressive B16.OVA melanoma in mice was significantly delayed in those animals that had been vaccinated with VLP.OVA or with VLP coupled with both OTI and OTII peptides prior to the introduction of the tumor. Neither VLP.OTI nor VLP.OTII alone were capable of inhibiting tumor growth. This work suggests that RHDV VLP offer a versatile scaffold for multiple vaccine epitopes, enabling cross-presentation of the antigen to elicit potent cell-mediated and anti-tumor responses.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vacinas Anticâncer/uso terapêutico , Vírus da Doença Hemorrágica de Coelhos/imunologia , Ovalbumina/uso terapêutico , Animais , Apresentação de Antígeno/imunologia , Antígenos de Neoplasias/imunologia , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Vacinas Anticâncer/imunologia , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/uso terapêutico , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina/imunologiaRESUMO
Baculoviruses deliver strong activation signals to dendritic cells and can promote potent immune responses. These properties can be harnessed to use baculovirus as an adjuvant and carrier particle for immunogenic peptides. In this study we use a chemical linker to couple peptides to the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Intranasal delivery of baculovirus coupled with immunogenic peptides to mice elicited antigen-specific IgG1 and IgG2a antibody. Furthermore, antigen-specific IgA was detected in the lung, and an IFN-gamma response was observed upon re-stimulation with antigen. We show that chemical coupling enables the rapid modification of AcMNPV, allowing multiple epitopes to be delivered simultaneously on a self-adjuvanting carrier particle.