RESUMO
This study demonstrates that CD8+ T cells in the tumor microenvironment display reduced functionality and hyporesponsiveness. TGF-beta contributed markedly to the tumor-infiltrating CD8+ T cells' (TILs) reduced functionality, which could be reversed using a small molecule TGF-beta inhibitor. Upon T-cell receptor (TCR) activation, the activation of ITK and ERK kinases were reduced in CD8+ TILs, as compared to splenic CD8+ T cells: TGF-beta inhibitor could reverse this phenomenon. This study demonstrates for the first time the association of the Spred-1 gene, an inhibitor of the Ras/MAPK pathway, with CD8+ TILs and TGF-beta activity. Spred-1 was upregulated in CD8+ TILs and TGF-beta enhanced the expression of Spred-1 in effector/memory CD8+ T cells and not in rested/memory CD8+ T cells. Based on these findings, this study supports the hypothesis that TGF-beta mediates an inhibitory mechanism on CD8+ TILs involving TCR-signaling blockade and the upregulation of Spred-1, thus implicating Spred-1 as a potential new target for future anti-tumor immune studies.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Linfócitos do Interstício Tumoral/fisiologia , Neoplasias Experimentais/imunologia , Receptores de Antígenos de Linfócitos T/fisiologia , Proteínas Repressoras/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Tolerância Imunológica , Interferon gama/biossíntese , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Transdução de Sinais , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
PURPOSE: To provide proper costimulation required for effective cancer T-cell immunity, Fc-GITRL fusion proteins were generated for use in immunotherapy protocols. EXPERIMENTAL DESIGN: Soluble fusion proteins consisting of the Fc fragment of immunoglobulin and the murine glucocorticoid-induced tumor necrosis factor-related receptor ligand (mGITRL) connected with different linkers were genetically engineered and tested for their potency in two BALB/c solid tumor models. RESULTS: In vivo, construct #178-14 (-5aa, -linker) showed the best activity (>90% tumor reduction) at doses ranging from 5 to 25 microg and was found to be intact by gel electrophoresis. Similar doses used with construct #175-2 (-linker) produced good but not as high tumor regression. Construct #5-1 (+linker), which was found to be relatively unstable by SDS gel electrophoresis, produced <60% tumor regression and required a higher dose (100 microg) to produce optimal results. Survival curves showed that Fc-mGITRL treatment extended the life of 80% of tumor-bearing mice to >3 months compared with controls that died by day 40. T-cell depletion studies showed that CD8(+) T cells play a major role in Fc-mGITRL immunotherapy, and tumors removed from Fc-mGITRL- and DTA-1-treated mice showed a significant influx of granzyme B(+) lymphocytes compared with controls. Finally, T regulatory (Treg) cell assays showed that, unlike other Fc fusion proteins, all three Fc-mGITRL constructs profoundly suppressed Treg activity. CONCLUSIONS: These studies suggest that a stable, intact Fc-mGITRL fusion protein can provide missing costimulation for the immunotherapy of solid tumors. In addition, Fc-mGITRL may alter Treg activity to enhance its effectiveness for tumor immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias do Colo/tratamento farmacológico , Fragmentos Fc das Imunoglobulinas/uso terapêutico , Linfócitos T Reguladores/imunologia , Fatores de Necrose Tumoral/uso terapêutico , Animais , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/imunologia , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/imunologia , Fragmentos Fc das Imunoglobulinas/metabolismo , Imunoterapia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T Reguladores/metabolismo , Fatores de Necrose Tumoral/administração & dosagem , Fatores de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismo , Fatores de Necrose Tumoral/farmacologiaRESUMO
A transgenic mouse model of autochthonous mammary carcinoma was chosen to study the impact of tumor progression on the immune system over an extended period. We found: i) that splenocyte numbers, particularly myeloid cells, increased concurrently with tumor burden; ii) the percentage of tumor-infiltrating Treg cells was similar to that in human breast cancer; iii) suppressed T cell proliferation and cytokine production and; iv) significantly elevated MCP-1 and TNF-alpha in the sera of tumor-bearing mice. The modified immune status in these tumor-bearing hosts is consistent with a "syndrome" that likely impacts the efficacy of cancer immunosurveillance and response to therapy.
Assuntos
Neoplasias Mamárias Experimentais/imunologia , Animais , Proliferação de Células , Citocinas/biossíntese , Citocinas/sangue , Progressão da Doença , Feminino , Citometria de Fluxo , Linfonodos/citologia , Linfonodos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Mamárias Experimentais/sangue , Camundongos , Camundongos Transgênicos , Baço/citologia , Baço/imunologia , Linfócitos T/imunologiaRESUMO
Immune responses influence the development and progression of a malignancy. The tumour can also manipulate the immune system to its own ends, often resulting in an ineffective or transient antitumour response. An appreciation of the complexity of these host-tumour interactions is therefore important for the development of more-effective cancer therapies. This article highlights some prominent mechanisms whereby tumours escape recognition and destruction by the host immune system, thus facilitating disease progression. One important consequence of tumour escape is that an antitumour immune response may unintentionally lead to the outgrowth of less immunogenic or more apoptosis-resistant tumour escape variants, which possess enhanced tumourigenic potential. Insights into the molecular mechanisms of cancer evasion and the complexity of the ever-changing interactions between host and tumour will enable a more rational design of antitumour therapies and may help not only explain disease recurrence, but also identify potential targets for therapeutic interventions. This article also offers a brief review of preclinical animal models, which are essential tools in the study of tumour immunology and cancer biology, particularly those that recapitulate the chronic nature of host-tumour interactions and help guide the development and testing of new therapies.
Assuntos
Sistema Imunitário/fisiopatologia , Imunoterapia , Neoplasias/imunologia , Neoplasias/fisiopatologia , Neoplasias/terapia , Animais , Modelos Animais de Doenças , Progressão da Doença , Humanos , Imunoterapia/efeitos adversos , Inflamação/patologia , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/patologia , Modelos Biológicos , Metástase Neoplásica , Neoplasias/patologia , Linfócitos T Reguladores/fisiologia , Evasão Tumoral/fisiologiaRESUMO
PURPOSE: To study the effect of encapsulation of recombinant adenovirus type 5 encoding Beta-galactosidase (Ad5-Betagal) in poly (D,Llactic-co-glycolic acid) (PLGA) microspheres on viral delivery to professional antigen presenting cells (APCs) in vitro, viral dissemination in vivo, and induction of protective immune responses in vivo. METHODS: PLGA microspheres containing Ad5-alphagal were prepared by a double emulsion solvent evaporation method. Encapsulation efficiency, in vitro release profile, in vitro cellular uptake and in vivo biodistribution of Ad5-alphagal loaded PLGA microspheres were determined using 125I-labeled Ad5-alphagal (125I-Ad5-alphagal). To evaluate the potential of PLGA microsphere delivery of Ad-alphagal for induction of antigen specific immune responses in vivo, Balb/c mice were immunized with the subcutaneous injection of the formulations then splenocytes of the immunized mice were assayed for cytotoxic T lymphocyte (CTL) activity against a variety of target cells in a 51Cr-release assay. Anti-alphagal antibody responses were assessed in the sera of the immunized mice by enzyme linked immunosorbent assay (ELISA). The effect of encapsulated Ad5-alphagal immunization on protection against a tumor challenge was tested in a murine artificial metastatic lung tumor model with alphagalexpressing tumor cells, CT26.CL25. RESULTS: PLGA microspheres encapsulated Ad5-alphagal with 24.8 +/- 1.4 % encapsulation efficiency and 11.4 +/- 3.6 % of the encapsulated virus retained the functional activity. In vitro release study showed slow release (15% in 11 days) of the virus from the microspheres. PLGA microsphere delivery of Ad5-alphagal resulted in enhanced uptake of the virus by APCs with an increase in the transgene expression in vitro. Administration of the virus in the encapsulated form resulted in substantially decreased viral dissemination to remote organs and tissues as compared to the free virus. Encapsulated virus were capable of eliciting antigen specific CTL as well as antibody responses against alphagal and induced protective immune responses against lethal tumor challenge at a significantly lower infectious viral dose as compared to the free virus. CONCLUSION: PLGA microsphere with Ad5-alphagal enhances the delivery of virus to APCs with reduced viral dissemination in other organs and induces protective antigen specific immune responses against viral encoded transgene.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Ácido Láctico/administração & dosagem , Ácido Poliglicólico/administração & dosagem , Polímeros/administração & dosagem , beta-Galactosidase/administração & dosagem , Adenoviridae/imunologia , Animais , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Células Cultivadas , Feminino , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Radioisótopos do Iodo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologia , Transgenes , Vacinação , beta-Galactosidase/genética , beta-Galactosidase/imunologia , beta-Galactosidase/metabolismoRESUMO
The type of immune response developed against the hepatitis B virus (HBV) is crucial in determining the outcome of the disease. The protective effects of vaccine-induced antibody responses against subsequent exposure to HBV are well-established. After the establishment of chronic HBV infection, cell-mediated immune response is curative while humoral response is detrimental. A therapeutic vaccine that could switch the type of response could lead to disease resolution. Hepatitis B core antigen (HBcAg)(129-140) has been identified as a Th2-biased peptide in H-2(b) mice when it is administered along with complete Freund's adjuvant (CFA). We formulated HBcAg(129-140) along with monophosphoryl lipid A in poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles. Naive mice immunized with the nanoparticle formulation developed a strong Th1-type response while mice immunized with the control formulation of CFA and peptide did not. We then primed mice with CFA and peptide to establish a Th2-type immune response before administering the nanoparticle formulation. Mice receiving the nanoparticle formulation being primed with CFA still developed a strong Th1-type response, while mice that received incomplete Freund's adjuvant and peptide instead of nanoparticles did not. The ability of PLGA nanoparticles to alter the type of immune response elicited by a peptide, even in the context of an ongoing immune response, makes PLGA nanoparticles a strong candidate for the formulation of therapeutic vaccines.
Assuntos
Antígenos do Núcleo do Vírus da Hepatite B/administração & dosagem , Fragmentos de Peptídeos/imunologia , Células Th1/imunologia , Sequência de Aminoácidos , Animais , Feminino , Imunização , Interferon gama/biossíntese , Ácido Láctico/administração & dosagem , Lipídeo A/administração & dosagem , Lipídeo A/análogos & derivados , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Nanoestruturas , Ácido Poliglicólico/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros/administração & dosagem , Linfócitos T/imunologia , Células Th2/imunologiaRESUMO
PURPOSE: To evaluate several methods for extraction of peptides from liposomal formulations as a first step in their quantification, and to determine the encapsulation efficiency for a panel of 8 peptides. METHODS: Eight peptides were chosen due to their importance in the field of vaccine development. Three different extraction media were examined: 25% ethanol, 98% ethanol, and 100% methanol. After extraction from liposomes, peptide content was measured using reverse phase HPLC. RESULTS: The effectiveness of the extraction media for peptide recovery varied considerably for the different peptides studied. In general, more hydrophilic peptides were recovered to a greater extent using 25% ethanol while more hydrophobic peptides were more thoroughly recovered using 98% ethanol. Encapsulation efficiencies (EE) ranged from 1% to 99% for the different peptides. No strong correlation was found between the average hydrophobicity values for the peptides and their EE. CONCLUSIONS: The most effective solvent for the extraction of a peptide from liposomes depends on the physicochemical properties of the peptide. Although the peptide sequence characteristics may provide guidance on the choice of the extraction media, only peptide recovery experiments will be able identify the optimal medium for extraction.
Assuntos
Lipoproteínas/metabolismo , Lipossomos/análise , Peptídeos/metabolismo , Sequência de Aminoácidos , Colágeno Tipo IV/química , Colágeno Tipo IV/isolamento & purificação , Etanol/metabolismo , Antígenos do Núcleo do Vírus da Hepatite B/química , Antígenos do Núcleo do Vírus da Hepatite B/isolamento & purificação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas/química , Lipoproteínas/isolamento & purificação , Lipossomos/metabolismo , Metanol/metabolismo , Dados de Sequência Molecular , Mucina-1/química , Mucina-1/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/química , Peptídeos/isolamento & purificação , Solubilidade , Solventes/análise , Proteínas Virais/química , Proteínas Virais/isolamento & purificaçãoRESUMO
This study demonstrates, for the first time, that murine regulatory T (Treg) cells in the tumor microenvironment display both enhanced proliferation and reduced functionality. This enhanced proliferation, combined with decreased apoptosis, leads to an intratumoral accumulation of Treg cells with a unique phenotype: CD4(+)CD25(+)FoxP3(+)GITR(high)CD27(low)CD62L(-). The loss of functionality is associated with down-regulation of the TCR signaling complex, including IL-2-inducible T cell kinase. It is also demonstrated that tumor-infiltrating Treg cells have impaired TCR-mediated signaling and calcium influx. Based on these findings, this study supports the hypothesis that 1) tumor-infiltrating Treg cells lose functionality due to their diminished ability to become effectively activated and 2) intratumoral accumulation of Treg cells may compensate for the impaired functionality, thus maintaining immune tolerance to the tumor.
Assuntos
Apoptose/imunologia , Sinalização do Cálcio/imunologia , Proliferação de Células , Neoplasias Experimentais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Regulação para Baixo/imunologia , Feminino , Tolerância Imunológica , Ativação Linfocitária , Camundongos , Camundongos Knockout , Neoplasias Experimentais/enzimologia , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Reguladores/enzimologiaRESUMO
IL-2 is well described as a cytokine with two markedly distinct functionalities: as a necessary signal during CD4(+) and CD8(+) T cell activation/expansion and as an essential cytokine for the maintenance of CD4(+)CD25(+)FoxP3(+) T cells (regulatory T (T(REG)) cells) during homeostasis. In this study we demonstrate for the first time that, compared with the use of IL-2 alone, a complex of IL-2 and anti-IL-2 Ab (IL-2 complex) enhances the effectiveness of a viral vaccine in a mouse model with known Ag specificity. IL-2 complex led to an increase in the number of Ag-specific effector/memory CD8(+) T cells, cytokine production, and CTL lysis following Ag-specific restimulation in a vaccination setting. Our results further demonstrate that this effect is temporary and declines over the course of a few days after the IL-2 complex treatment cycle. Moreover, in contrast to the use of IL-2 alone, IL-2 complex greatly increased the ratio of effector/memory CD8(+) T cells to T(REG) cells. This phenomenon can thus potentially be used in the enhancement of immune responses to vaccination.
Assuntos
Anticorpos/imunologia , Antígenos/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Interleucina-2/imunologia , Linfócitos T Reguladores/imunologia , Vacinas/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/imunologia , Interferon gama/biossíntese , Células Matadoras Naturais/imunologia , Camundongos , Fatores de TempoRESUMO
OX40L is a member of the tumor necrosis factor superfamily that provides a costimulatory signal to CD4+ and CD8+ T cells while inhibiting the effects of suppressive CD4+ CD25+ regulatory T cells. Because of this dual activity, OX40L may provide significant antitumor immunity in tumor-bearing mice. To study its clinical potential, a fusion protein consisting of mOX40L linked to the C-terminus of the Fc fragment of immunoglobulin was genetically engineered. After demonstrating its potency in vitro, several assays were performed to evaluate its antitumor effect in comparison to the OX40 agonist antibody OX86. Dosing studies in Colon 26-bearing and renal cell carcinoma (RENCA)-bearing mice showed that although OX86 produced modest tumor regression, Fc-mOX40L produced complete remission in both tumor models. Survival studies confirmed these results and showed that Fc-mOX40L treatment produced lasting responses throughout the 5-month observation period. Flow cytometric analysis of treated and untreated tumors and tumor-draining lymph nodes identified a qualitative difference in the activity of Fc-mOX40L compared with OX86 treatment as evidenced by differences in lymphoid and macrophage populations. These studies reflect the profound therapeutic potential of Fc-mOX40L, which substantially exceeds the agonist antibody OX86 in ability to produce complete tumor remissions and promote long-term survival in solid tumor models.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Neoplasias do Colo/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Ligante OX40 , Receptores Fc , Proteínas Recombinantes de Fusão , Animais , Linfócitos T CD8-Positivos/imunologia , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Neoplasias do Colo/patologia , Intervalo Livre de Doença , Feminino , Humanos , Imunofenotipagem , Neoplasias Renais/imunologia , Neoplasias Renais/patologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/patologia , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Ligante OX40/genética , Ligante OX40/imunologia , Ligante OX40/uso terapêutico , Receptores Fc/genética , Receptores Fc/imunologia , Receptores Fc/uso terapêutico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Indução de Remissão , Transdução de SinaisRESUMO
Regulatory T cells (T(REGs)) control the key aspects of tolerance and play a role in the lack of antitumor immune responses. Cyclophosphamide (CY) is a chemotherapeutic agent with a dose-dependent, bimodal effect on the immune system. Although a previous study demonstrated that CY reduces the number of T(REGs), the mechanism involved in this process has yet to be defined. In this report, it is established that low-dose CY not only decreases cell number but leads to decreased functionality of T(REGs). CY treatment enhances apoptosis and decreases homeostatic proliferation of these cells. Expression of GITR and FoxP3, which are involved in the suppressive activity of T(REGs), is down-regulated after CY administration, though the level of expression varies depending on the time studied. This is the first report demonstrating that CY, in addition to decreasing cell number, inhibits the suppressive capability of T(REGs). The relevance of the loss of suppressor functionality and the changes in gene expression are further discussed.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Ciclofosfamida/farmacologia , Imunossupressores/farmacologia , Animais , Antígenos de Superfície/genética , Antígenos de Superfície/imunologia , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Linfócitos B/citologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Proteínas de Ligação a DNA/genética , Feminino , Fatores de Transcrição Forkhead , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Proteína Relacionada a TNFR Induzida por Glucocorticoide , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Interleucina-2/metabolismo , Receptores de Fator de Crescimento Neural/genética , Receptores do Fator de Necrose Tumoral/genéticaRESUMO
PURPOSE: The purpose of this study was to demonstrate and characterize phagocytosis of poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres by human dendritic cells (DCs). METHODS: Parallel cultures of DCs and macrophages (Mphi) were established from peripheral blood leukocytes using media supplemented with granulocyte-macrophage colony stimulator factor and interleukin-4 (for DC) or granulocyte-macrophage colony stimulator factor alone (for Mphi). PLGA nanospheres containing tetramethylrhodamine-labeled dextran with or without an adjuvant, monophosphoryl lipid A, were prepared using a water/oil/water solvent evaporation technique. Cells were incubated with the nanospheres for 24 h. Confocal laser scanning microscopy was used to determine the intracellular location of the nanospheres and flow cytometry to measure the fraction of phagocytic cells in the culture and the amount of uptake per cell. After phagocytosis, cells were stained for MHC class II molecules, CD14, CD80, and CD86 to identify the phagocytic population. RESULTS: DCs phagocytosed PLGA nanospheres as efficiently as Mphi. Cell-surface marker expression conclusively established that the phagocytic cells were DC. CONCLUSIONS: DCs can take up PLGA nanospheres. Because DCs are the key professional antigen-presenting cells capable of stimulating naive T cells, our data suggest that PLGA nanospheres can be used as an efficient delivery system for vaccines designed to activate T cell-mediated immune responses.