RESUMO
Standard-of-care chemo- or radio-therapy can induce, besides tumor cell death, also tumor cell senescence. While senescence is considered to be a principal barrier against tumorigenesis, senescent cells can survive in the organism for protracted periods of time and they can promote tumor development. Based on this emerging concept, we hypothesized that elimination of such potentially cancer-promoting senescent cells could offer a therapeutic benefit. To assess this possibility, here we first show that tumor growth of proliferating mouse TC-1 HPV-16-associated cancer cells in syngeneic mice becomes accelerated by co-administration of TC-1 or TRAMP-C2 prostate cancer cells made senescent by pre-treatment with the anti-cancer drug docetaxel, or lethally irradiated. Phenotypic analyses of tumor-explanted cells indicated that the observed acceleration of tumor growth was attributable to a protumorigenic environment created by the co-injected senescent and proliferating cancer cells rather than to escape of the docetaxel-treated cells from senescence. Notably, accelerated tumor growth was effectively inhibited by cell immunotherapy using irradiated TC-1 cells engineered to produce interleukin IL-12. Collectively, our data document that immunotherapy, such as the IL-12 treatment, can provide an effective strategy for elimination of the detrimental effects caused by bystander senescent tumor cells in vivo.
Assuntos
Senescência Celular/efeitos dos fármacos , Imunoterapia Adotiva/métodos , Interleucina-12/farmacologia , Neoplasias Experimentais/terapia , Taxoides/farmacologia , Carga Tumoral/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Efeito Espectador/efeitos dos fármacos , Linhagem Celular Tumoral , Terapia Combinada , Citocinas/genética , Citocinas/metabolismo , Docetaxel , Interleucina-12/biossíntese , Masculino , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Fatores de TempoRESUMO
CD4(+)CD25(+)Foxp3(+) T regulatory cells (Tregs) and CD1d-restricted invariant natural killer T (iNKT) cells are two cell types that are known to regulate immune reactions. Depletion or inactivation of Tregs using specific anti-CD25 antibodies in combination with immunostimulation is an attractive modality especially in anti-tumour immunotherapy. However, CD25 is not expressed exclusively on Tregs but also on subpopulations of activated lymphocytes. Therefore, the modulatory effects of the specific anti-CD25 antibodies can also be partially attributed to their interactions with the effector cells. Here, the effector functions of iNKT cells were analysed in combination with anti-CD25 mAb PC61. Upon PC61 administration, α-galactosylceramide (α-GalCer)-mediated activation of iNKT cells resulted in decreased IFN-γ but not IL-4 production. In order to determine whether mutual interactions between Tregs and iNKT cells take place, we compared IFNγ production after α-GalCer administration in anti-CD25-treated and "depletion of regulatory T cell" (DEREG) mice. Since no profound effects on IFNγ induction were observed in DEREG mice, deficient in FoxP3(+) Tregs, our results indicate that the anti-CD25 antibody acts directly on CD25(+) effector cells. In vivo experiments demonstrated that although both α-GalCer and PC61 administration inhibited TC-1 tumour growth in mice, no additive/synergic effects were observed when these substances were used in combination therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Galactosilceramidas/farmacologia , Interferon gama/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Células T Matadoras Naturais/efeitos dos fármacos , Neoplasias Experimentais/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Linhagem Celular Tumoral , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Galactosilceramidas/administração & dosagem , Galactosilceramidas/imunologia , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/imunologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Interleucina-4/genética , Interleucina-4/imunologia , Interleucina-4/metabolismo , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/imunologiaRESUMO
Genetically modified tumour cells producing cytokines such as interleukin 12 (IL-12) are potent activators of the antitumour immune responses and represent a promising therapeutic modality when combined with chemotherapy. The objective of this study was to examine whether IL-12-producing cellular vaccines can augment chemotherapy of human papilloma virus (HPV) 16-associated murine tumours with the cytostatic agent gemcitabine (GEM). We found that peritumoral administration of IL-12-producing tumour vaccines enhanced the effect of cytoreductive therapy with GEM both in non-metastasizing murine carcinoma TC-1 and in metastasizing murine carcinoma MK16. The percentage of mice with MK16 metastases and the number of lung metastatic nodules was substantially decreased. In another clinically relevant setting, surgical minimal residual tumour disease, the administration of IL-12-producing tumour vaccine and GEM after the MK16 tumour surgery reduced the percentage of mice with tumour recurrences; similarly, the percentage of metastasis-bearing mice and the number of metastatic nodules was decreased. Tumour inhibitory effects exerted by GEM plus IL-12 were associated with high production of interferon-γ (IFNγ) by splenocytes. Our results suggest that the IL-12-producing vaccine can enhance the effect of GEM chemotherapy in some HPV16-associated murine tumour models.
Assuntos
Antineoplásicos/farmacologia , Vacinas Anticâncer/farmacologia , Desoxicitidina/análogos & derivados , Terapia Genética/métodos , Interleucina-12/biossíntese , Neoplasias Experimentais/tratamento farmacológico , Animais , Separação Celular , Terapia Combinada , Desoxicitidina/farmacologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Papillomavirus Humano 16 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/virologia , Infecções por Papillomavirus/complicações , GencitabinaRESUMO
Loss or downregulation of MHC class I molecules on tumour cells is a common mechanism by which tumours can escape T-cell mediated immune responses. In this study, we examined the role of different immune cell lineages in the development of immunity against tumours of the same aetiology but with different MHC class I expression. In vivo depletion of CD8+ cells, but not of CD4+ or NK1.1+ cells in the immunization period resulted in complete elimination of the protective effects of immunization with irradiated TC-1 cells (MHC class I-positive cell line) against the TC-1 tumour challenge. After immunization with irradiated TC-1/A9 or with MK16 tumour cells (MHC class I-deficient sublines) a remarkable dependence on the presence of NK1.1+ cells was observed, while the tumour growth inhibition after CD4+ or CD8+ depletion was not efficient. Cytotoxic activity induced by TC-1 cell immunization was significantly abrogated in the CD8+ and CD4+ but not NK1.1+ cell-depleted mice, as compared to the immunized only controls. After MK16 or TC-1/A9 cell immunization, NK1.1+ but not CD8+ and CD4+ cell-depleted mice displayed significant reduction of specific cytotoxicity. Mice immunized with TC-1 cells showed similar percentage of IFNγ producing cells in CD8+, CD4+ and NK1.1+ cell populations. On the other hand, the highest proportion of IFNγ producing cells after immunization with TC-1/A9 or MK16 cells was concentrated into the NK1.1-positive spleen cell population. Our data demonstrate that the development of immunity against MHC class I-deficient tumours is highly dependent on the activity NK1.1+ cell population.
Assuntos
Vacinas Anticâncer/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Neoplasias Experimentais/imunologia , Animais , Antígenos Ly/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Separação Celular , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Papillomavirus Humano 16 , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologiaRESUMO
Downregulation of MHC class I expression on the cell surface is a common mechanism by which tumour cells, including cervical carcinoma, can escape the T cell-mediated anti-tumour immunity. This downregulation represents an obstacle for the efficacy of anti-tumour vaccines. In this study, we investigated the efficacy of prophylactic peptide and peptide-pulsed dendritic cell-based vaccines in a murine model of experimental MHC class I-deficient tumours (TC-1/A9), expressing E6/E7 oncogenes derived from HPV16, and compared the efficacy of particular vaccination settings to anti-tumour protection against parental MHC class I-positive TC-1 tumours. Peptide vaccine based on the 'short' peptide E749-57 harbouring solely the CTL epitope and co-administered to the C57BL/6 mice with CpG oligodeoxynucleotide (CpG ODN) 1826 was effective against MHC class I-positive but not -deficient tumours, while the 'longer' peptide E744-62 (peptide 8Q, harbouring CTL and Th epitopes)-based vaccines were also effective against MHC class I-deficient tumours. We have compared the adjuvant efficacies of two CpG ODN, CpG ODN 1826 and CpG ODN 1585. The 8Q peptide immunisation combined with CpG ODN 1585 inhibited growth of the TC-1/A9 tumours more effectively as compared to CpG ODN 1826. Further, we investigated the efficacy of cellular vaccines based on ex vivo cultured dendritic cells pulsed with either E749-57 or E744-62 peptides and matured with CpG ODN 1826. Unlike in the peptide immunisation setting, treatment with dendritic cells pulsed with a 'short' peptide resulted in the tumour growth inhibition, albeit weaker as compared to the immunisation with the longer peptide. Our data demonstrate that peptide and dendritic cell-based vaccines can be designed to elicit protective immunity against MHC class I-deficient tumours.
Assuntos
Vacinas Anticâncer/química , Células Dendríticas/citologia , Genes MHC Classe I , Proteínas E7 de Papillomavirus/química , Animais , Ilhas de CpG , Epitopos/química , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Camundongos , Oligonucleotídeos/genética , Peptídeos/química , Radioterapia Adjuvante/métodos , Vacinas de Subunidades Antigênicas/genéticaRESUMO
Natural killer T (NKT) cells are potent modulators of antitumor immunity. Their protective effects can be achieved upon their activation by glycolipid ligands presented in the context of the CD1d molecule. These CD1d-binding glycolipid antigens have been described as potent therapeutic agents against tumors, infections, as well as autoimmune diseases. Immunoregulatory and therapeutic effects of glycolipid ligands depend on their structure and modes of administration. Therefore, more studies are needed for optimization of the particular therapeutic settings. This study was focused on the tumor-inhibitory effects of 12 carbon acyl chain beta-galactosyl ceramide (C12 beta-D-Galactosyl Ceramide; beta-GalCer(C12)) on the growth of human papillomavirus type 16 (HPV16)-associated neoplasms transplanted in syngeneic mice. Treatment of tumor-bearing mice with beta-GalCer(C12) 3-14 days after tumor cell transplantation significantly inhibited the growth of the major histocompatibility complex (MHC) Class I-positive (TC-1), as well as MHC Class I-deficient (TC-1/A9) HPV16-associated tumors. Moreover, administration of beta-GalCer(C12) after surgical removal of TC-1 tumors inhibited the growth of tumor recurrences. Similar results were obtained in the treatment of tumors after chemotherapy. beta-GalCer(C12) treatment turned out to be also synergistic with immunotherapy based on administration of IL-12-producing cellular vaccines. These results suggest that beta-GalCer(C12), whose antitumor effects have so far not been studied in detail, can be effective for the treatment of minimal residual tumor disease as well as an adjuvant for cancer immunotherapy.
Assuntos
Ceramidas/farmacologia , Monossacarídeos/farmacologia , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/prevenção & controle , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/cirurgia , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/prevenção & controle , Animais , Papillomavirus Humano 16/isolamento & purificação , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Recidiva Local de Neoplasia/imunologia , Neoplasia Residual/virologia , Infecções por Papillomavirus/imunologia , Células Tumorais Cultivadas/transplanteRESUMO
We have examined the effect of IL-12-producing cellular vaccines on the cytotoxicity and proliferative potential of CD45+ tumour-infiltrating cells (TIL) in mice carrying syngeneic TC-1 and TC-1/A9 HPV 16-associated tumours after chemotherapy with CBM-4A ifosfamide derivative. The chemotherapy resulted in the decrease of the CD4+ and CD8+ TIL, increase of the Gr-1+/CD11b+ TIL, no changes in the infiltration with CD4+/CD25+ Treg TIL, and decrease of the cytolytic and proliferative potential of the CD45+ TIL. Subsequent immunotherapy with the IL-12-producing, genetically modified TC-1 (TC-1-IL-12) cells increased tumour infiltration with CD8+ and CD4+ cells, decreased the Gr-1+/CD11b+ cells, and increased the cytolytic and proliferative potential of the CD45+ TIL. Taken together, these findings suggest that peritumoral administration of the IL-12-producing cellular vaccine can restore the cytolytic potential and inhibit immunosuppressive TIL-dependent mechanisms in the individuals bearing HPV 16-associated tumours, and explain our previously described tumour-inhibitory effects of the vaccine in mice with minimal residual disease after the tumour chemotherapy.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Proliferação de Células , Papillomavirus Humano 16/patogenicidade , Interleucina-12/fisiologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Experimentais/imunologia , Infecções por Papillomavirus/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Testes Imunológicos de Citotoxicidade , Citometria de Fluxo , Terapia Genética , Humanos , Ifosfamida/análogos & derivados , Ifosfamida/uso terapêutico , Técnicas Imunoenzimáticas , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/terapia , Neoplasias Experimentais/virologia , Infecções por Papillomavirus/terapia , Infecções por Papillomavirus/virologia , Células Tumorais CultivadasRESUMO
We have established animal models of HPV16-associated tumours with distinct levels of MHC class I expression. This model was used for examination of immune responses, production of cytokines and kinetics of immune cell subsets after IL-12 therapy of minimal residual tumour disease induced by CBA-4A (cyclophosphamide derivative) treatment. Upregulation of cytokine production was detected, compared to control animals without tumours. No differences in Th1/Th2 polarization of the immune responses after immunotherapy in animals bearing tumours with different surface expression of MHC class I molecules were observed. In the spleens of TC-1 (MHC class I+) but not of TC-1/A9 (MHC class I-) treated tumour-bearing animals, the cytotoxic CD8+ cells detectable in 51Cr microcytotoxicity assay, were found. In the spleens of TC-1/A9 but not of TC-1 tumour-treated animals, the NK activity measured as the lysis of NK-sensitive YAC-1 targets was detected. Down-regulation of the CD4+ and CD8+ subpopulations in spleens of tumour-bearing animals were not restored after therapy. The percentage of CD25+/CD4+ T regulatory (Treg) cells in lymph nodes remained unchanged. The cytoreductive chemotherapy led to strong upregulation and accumulation of immunosuppressive immature myeloid Gr-1+/CD11b+ cells (IMC) in the spleens of treated animals. The accumulation of Gr-1+/CD11b+ cells was significantly decreased after subsequent IL-12 immunotherapy. These data suggest that elimination of IMC after IL-12 immunotherapy may be responsible for the improvement of antitumour responses after adjuvant IL-12 vaccination for the treatment of CMRTD.
Assuntos
Citocinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Papillomavirus Humano 16/metabolismo , Sistema Imunitário/imunologia , Imunoterapia/métodos , Interleucina-12/química , Neoplasias/virologia , Animais , Linfócitos T CD4-Positivos/metabolismo , Vacinas Anticâncer , Modelos Animais de Doenças , Sistema Imunitário/metabolismo , Interleucina-12/metabolismo , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Cinética , Linfonodos/patologia , Camundongos , Neoplasias/metabolismoRESUMO
Epigenetic events play an important role in tumour progression and also contribute to escape of the tumour from immune surveillance. In this study, we investigated the up-regulation of major histocompatibility complex (MHC) class I surface expression on tumour cells by epigenetic mechanisms using a murine tumour cell line expressing human E6 and E7 human papilloma virus 16 (HPV16) oncogenes and deficient in MHC class I expression, as a result of impaired antigen-presenting machinery (APM). Treatment of the cells with the histone deacetylase inhibitor Trichostatin A, either alone or in combination with the DNA demethylating agent 5-azacytidine, induced surface re-expression of MHC class I molecules. Consequently, the treated cells became susceptible to lysis by specific cytotoxic T lymphocytes. Further analysis revealed that epigenetic induction of MHC class I surface expression was associated with the up-regulation of APM genes [transporter associated with antigen processing 1 (TAP-1), TAP-2, low-molecular-mass protein 2 (LMP-2) and LMP-7]. The results demonstrate that expression of the genes involved in APM are modulated by epigenetic mechanisms and suggest that agents modifying DNA methylation and/or histone acetylation have the potential to change the effectiveness of antitumour immune responses and therapeutically may have an impact on immunological output.
Assuntos
Epigênese Genética/imunologia , Genes MHC Classe I , Papillomavirus Humano 16 , Neoplasias Experimentais/imunologia , Infecções por Papillomavirus/complicações , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Apoptose/efeitos dos fármacos , Azacitidina/farmacologia , Inibidores Enzimáticos/farmacologia , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais/genética , Neoplasias Experimentais/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Células Tumorais Cultivadas , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Regulação para Cima/imunologiaRESUMO
Oligodeoxynucleotides containing guanine-cytidine dimers (CpG ODN) are potent inducers of anti-tumour immune responses. In this study, we analyzed the capacity of CpG ODN to inhibit the growth of both MHC class I-positive and -deficient tumours after debulking the tumour mass by chemotherapy or surgery. We employed an animal model resembling human papillomavirus (HPV) 16-associated tumours. Tumour cell lines with distinct cell surface expression of the MHC class I molecules were injected into syngeneic C57BL/6 mice, and the growing tumours were either subjected to cytoreductive chemotherapy with ifosfamide derivative, CBM-4A, or surgically removed. Subsequent treatment with synthetic CpG ODN significantly blocked the growth of the recurrent tumours. Our results indicate that the therapy with CpG ODN can be effective for the treatment of minimal residual tumour disease of the tumours that have escaped from the immune surveillance by downmodulating the MHC class I expression.
Assuntos
Ilhas de CpG , Papillomavirus Humano 16/metabolismo , Neoplasia Residual/tratamento farmacológico , Neoplasias/genética , Neoplasias/terapia , Neoplasias/virologia , Oligonucleotídeos/uso terapêutico , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Genes MHC Classe I , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/química , Recidiva , Fatores de TempoRESUMO
Loss or downregulation of MHC class I molecules on tumour cells is a common mechanism by which tumours can escape from T-cell mediated immune responses. In this study we have investigated the immunologic crossreactivity between murine tumour cell lines expressing human papilloma virus (HPV) 16-derived E6/E7 oncoproteins with distinct surface expression of MHC class I molecules. The aims of this study were to demonstrate whether immune responses capable of coping with MHC class I-positive tumours can also be effective against their MHC class I-deficient derivatives and whether it is possible to induce immunity against MHC class I-deficient tumours by cellular vaccines based on MHC class I-deficient tumour cell lines. Our data showed that immunization with MHC class I-deficient but not with MHC class I positive tumour cells inhibited the growth of MHC class I-deficient tumours. In vivo depletion studies revealed that the mechanisms underlying effective immune responses against MHC class I-negative tumours in animals immunized with MHC class I-deficient tumour cells involved natural killer cells. The presented findings are of particular clinical relevance in the sense of construction of vaccines directed against a broad spectrum of HPV-associated tumours.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Papillomavirus Humano 16/imunologia , Imunização , Neoplasias/terapia , Neoplasias/virologia , Animais , Vacinas Anticâncer/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Reações Cruzadas , Antígenos de Histocompatibilidade Classe I/análise , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologiaRESUMO
It is generally accepted that T regulatory cells (T(reg) CD4(+)CD25(+)Foxp3(+)) play an important role in the suppression of tumour immunity. We examined the impact of T(reg) cell depletion with anti-CD25 antibody as adjuvant therapy in the treatment of minimal residual disease after excision of murine HPV16-associated tumours. We found that the depletion of T(reg) cells inhibited growth of the recurrences after surgery of HPV16-associated MHC class I+ as well as MHC class I-deficient tumours transplanted in syngeneic mice. These results demonstrate that depletion of CD25(+)CD4(+) T(reg) cells can be used as an efficient adjuvant treatment improving the results of surgery in the experimental systems mimicking human MHC class I+ and MHC class I-deficient, HPV16-associated neoplasms. Therefore, this therapeutic modality is worth being examined in patients with minimal residual HPV16-associated tumour disease after surgery.
Assuntos
Papillomavirus Humano 16/isolamento & purificação , Imunoterapia/métodos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/terapia , Infecções por Papillomavirus/imunologia , Linfócitos T Reguladores/imunologia , Animais , Terapia Combinada , Genes MHC Classe I/genética , Genes MHC Classe I/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasia Residual , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologiaRESUMO
Unmethylated oligodeoxynucleotides containing guanine-cytidine dimers (CpG ODN) have been described as potent inducers of selected antitumour immune responses and the immunotherapeutic efficacy of CpG ODN has been examined either alone or as a vaccine adjuvant. We hypothesized that CpG ODN therapy could be an effective tool for immunotherapy of not only conventional MHC class I(+) tumours but also of those tumours that have lost MHC class I expression during their progression. To address this hypothesis, we employed the animal model resembling MHC class I-proficient and -deficient human papilloma virus (HPV) 16-associated tumours. A cell line transformed with HPV16 E6 and E7 oncogenes, TC-1, as a prototype of MHC class I-positive line, and its MHC class I-deficient sublines TC-1/A9 and TC-1/P3C10 were injected into syngeneic C57BL/6 mice and the growing tumours were subjected to immunotherapy with CpG ODN 1826. The therapy started either 1 day after the challenge with the tumour cells or later, when the tumours had reached a palpable size. In both settings, CpG ODN 1826 significantly reduced the growth of MHC class I-proficient and -deficient tumours. Furthermore, we demonstrated that CpG ODN 1585, whose mechanism of action preferably involves indirect activation of the natural killer cells, induced regression of the MHC class I-deficient tumours TC1/A9 but not of the MHC class I-proficient tumours TC-1. This study infers that synthetic CpG ODN have a potential for the therapy of both MHC class I-proficient and -deficient tumours and thus could be also used against tumours that tend to down-regulate their MHC class I expression.
Assuntos
Ilhas de CpG , Genes MHC Classe I , Imunoterapia/métodos , Neoplasias/virologia , Oligonucleotídeos , Animais , Regulação para Baixo , Papillomavirus Humano 16 , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/terapia , Infecções por PapillomavirusRESUMO
Local recurrences at the site of tumour resection as well as distant micrometastases manifested after surgery represent major problems in oncology. Adjuvant immunotherapy and gene therapy may help to cope, at least partially, with these problems. Adjuvant modalities may be more effective in treating residual tumour disease compared to bulky tumours, owing to a favourable effector/target cell ratio. The purpose of this review was to summarize, evaluate and discuss the results obtained with adjuvant immunotherapy and immunomodulatory gene therapy of surgical minimal residual tumour disease in experimental and clinical tumour systems. The prospects and limitations of adjuvant therapeutic modalities will be considered.
Assuntos
Terapia Genética , Imunoterapia , Neoplasia Residual , Neoplasias/terapia , Quimioterapia Adjuvante , Ensaios Clínicos como Assunto , Determinação de Ponto Final , Humanos , Metástase Neoplásica , Neoplasias/genética , Neoplasias/imunologiaRESUMO
Malignant transformation of somatic cells followed by selection of the transformed cell populations can give rise to tumours that display an immune escape phenotype, MHC class I deficient neoplasms. Experiments were designed to examine whether the immune escape phenotype of HPV16-associated tumours is stable or whether the MHC class I expression can change during tumour progression and therapy. It has been found that temporary growth of MHC class I- tumour MK16/1/IIIABC in syngeneic mice can lead to up-regulation of the low MHC class I expression, both in the subcutaneous tumour inocula and in their lung metastases. Mimicking this process in vitro by co-cultivation of tumour and spleen cell populations revealed that the spleen cells produce IFNgamma, which upregulates MHC class I expression on the MK16/1/IIIABC cells as well as their sensitivity to T cell-mediated cytolysis (CTLs). The up-regulation could be prevented by admixture of anti-IFNgamma antibody to the tumour/spleen cell mixtures. Similar up-regulation of the MHC class I expression was observed in HPV16-associated tumour cell lines, MK16/1/IIIABC, MK16/MET/M1, TC-1, TC-1/A9 and TC-1/P3C10 grown in vitro in the presence of IFNgamma. The up-regulation was found to be IFNgamma dose-dependent and the level of the MHC class I expression required for in vitro cytolysis of the tumour cells by CTLs could be characterized in cytofluorometry with anti-H-2 antibody. After removal of the IFNgamma from the cultivation medium or after injection of the IFNgamma-treated cells into syngeneic mice the MHC class I expression gradually dropped back to the original level or to the level observed on the tumours growing in vivo. These findings indicate that the immune escape phenotype of at least some HPV16-associated tumours is not stable and that up-regulation of the MHC class I expression can occur in vivo during progression of the MHC I- tumours, apparently due to production of IFNgamma by the immune cells in the tumour microenviroment and its vicinity. In vitro irradiation of HPV16-associated MHC class I-deficient tumour cell lines MK16/MET/M1 and TC-1/P3C10 with a dose of 150 Gy up-regulated their MHC class I expression. Similarly, substantial up-regulation of the MHC class I expression was observed in TC-1/A9 tumour recurrences after surgery. The up-regulation observed in the recurrences after surgery or after irradiation has reached the level required for in vitro cytolysis of the tumour cells by CTLs. If confirmed also with other tumour types and in human tumour systems, the up-regulation of MHC class I molecule expression during radiotherapy and in tumour recurrences after surgery may have important implications in the development of immunotherapeutic strategies.
Assuntos
Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/biossíntese , Imunoterapia/métodos , Neoplasias/imunologia , Neoplasias/virologia , Proteínas Oncogênicas Virais/biossíntese , Papillomaviridae/metabolismo , Infecções por Papillomavirus/imunologia , Animais , Linhagem Celular Tumoral , Radioisótopos de Cromo , Técnicas de Cocultura , Progressão da Doença , Citometria de Fluxo , Genes MHC Classe I , Interferon gama/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Fenótipo , Recidiva , Baço/metabolismo , Linfócitos T Citotóxicos/metabolismo , Transfecção , Regulação para CimaRESUMO
Dendritic cell (DC)-based vaccines are being intensively investigated for the treatment of a variety of human neoplasms. However, little attention has until now been paid to the use of DC-based vaccines for immunotherapy of tumour residua after surgery. In this communication, an animal model mimicking human HPV16-associated neoplasms was employed to examine the effect of DC-based vaccines for the treatment of surgical minimal residual tumour disease. Mice were subcutaneously inoculated with syngeneic TC-1 tumour cells of HPV16 origin. When the tumours reached approximately 1 cm in diameter, they were surgically removed and the operated mice were injected into the site of the operation with bone marrow-derived DC, which were either pulsed with TC-1 cell lysates or co-cultured with irradiated TC-1 cells. It has been found that the growth of TC-1 tumour recurrences in the mice treated with these vaccines was substantially suppressed, as compared to the operated-only controls. The phenotypic analysis of the spleen cells has shown that the percentage of CD3+ cells was diminished in the operated-only and vaccinated mice carrying recurrent tumours, in comparison with healthy control mice and with operated tumour-free mice. Moreover, accumulation of immature myeloid cells (CD11b+/Gr-1+) was observed in spleens of the tumour-bearing mice. These findings indicate that the immune system of the tumour-bearing individuals was compromised, as compared to that of normal individuals or tumour regressors. To our knowledge, this is the first report that has demonstrated the positive effect of local administration of the DC-based, HPV16 E6/E7 oncoprotein-containing, tumour lysate-loaded vaccines in the treatment of surgical minimal residual tumour disease.
Assuntos
Vacinas Anticâncer/imunologia , Células Dendríticas/imunologia , Neoplasias Experimentais/terapia , Proteínas Oncogênicas Virais/imunologia , Papillomaviridae/imunologia , Infecções por Papillomavirus/complicações , Proteínas Repressoras/imunologia , Animais , Imunofenotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasia Residual , Neoplasias Experimentais/imunologia , Proteínas E7 de PapillomavirusRESUMO
Malignant conversion and subsequent in vivo selection can give rise to the cell populations that show stable expression of an immune escape phenotype, MHC class I deficient neoplasms. Deficiencies associated with the MHC class I down-regulation are either irreversible, such as beta2 microglobulin and class I heavy chain gene disabling mutations, or reversible. The reversible MHC class I deficiencies involve all levels of the MHC class I-restricted antigen presentation machinery. They can be repaired, at least partially and in vitro, by cytokines (IFNgamma, TNFalpha) or by DNA demethylation/histone hyperacetylation procedures. The reduced levels of MHC class I antigens result in decreased sensitivity to MHC class I-restricted, cytotoxic T lymphocyte-mediated lysis, the major component of the tumour rejection reaction. MHC class I down-regulation helps tumour cells evade the classical T cell-dependent immune responses but simultaneously imposes another, the NK cell-mediated, surveillance stimulated by the 'missing self' signals. The innate and adaptive antitumour immunity may be under some conditions interconnected: primary activation of the MHC class I-unrestricted surveillance mechanisms may lead to the production of IFNgamma by the activated NK/gammadelta T cells; the in situ produced IFNgamma may then up-regulate the MHC class I molecule expression on the tumour cell surface and in this way it may stimulate the more efficient, MHC class I-restricted, adaptive immunity. If we accept that the MHC class I down-regulation can, under some conditions, indeed be a mechanism of the tumour escape from the immune defence, the problem arises how to cope efficiently with this escape. Either therapeutic procedures aiming at up-regulation of MHC class I expression, or enhancement of MHC class I-unrestricted (CD4+, NK, NKT, gammadelta T) tumour defence effector mechanisms by dendritic cell-based therapeutic vaccines, by cytokines (IL-2, IL-12, IFNgamma, GM-CSF), or by the cytokine gene-based, genetically modified tumour vaccines should be considered.
Assuntos
Antígenos de Histocompatibilidade Classe I/metabolismo , Vigilância Imunológica , Neoplasias/imunologia , Evasão Tumoral/imunologia , Regulação para Baixo/genética , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Vigilância Imunológica/genética , Evasão Tumoral/genéticaRESUMO
Moderately immunogenic HPV16-associated tumours TC-1 (MHC class I+, HPV16 E6/E7+, G12V Ha-ras+) and MK16/1/IIIABC (MK16, MHC class I-, HPV16 E6/E7+, G12V Ha-ras+), both of the H-2b haplotype and transplanted in syngeneic mice, were used to examine the effects of local IL-2 and GM-CSF cytokine or gene therapy in the treatment of minimal residual tumour disease. The mice carrying MHC class I+ TC-1 tumour residua after surgery were injected into the site of the surgery either with irradiated, IL-2 gene-modified MK16 tumour cells, or with recombinant IL-2. It has been found that both, the recombinant IL-2 and the IL-2 gene-modified tumour vaccine substantially reduced the percentage of tumour recurrences in the operated mice. Similarly, when the mice carrying TC-1 tumour residua after surgery were injected with recombinant GM-CSF, the recombinant GM-CSF inhibited growth of the tumour residua in the operated mice. Gene therapy with irradiated, GM-CSF secreting MK16 cells did not produce any tumour-inhibitory effect. In further experiments, mice bearing s.c. TC-1 tumours were injected i.p. with ifosfamide derivative CBM-4A and 8 days later, peritumourally, either with IL-2 gene-modified and IL-2-producing MK16 cells, or with recombinant IL-2. It has been found that both, the recombinant IL-2 and the IL-2 gene therapy substantially reduced the percentage of tumour-bearing mice. When the mice bearing s.c. TC-1 tumours were injected i.p. with ifosfamide derivative CBM-4A and then, peritumourally, either with irradiated, GM-CSF gene-modified and GM-CSF-producing MK16 cells, or with recombinant GM-CSF, it was found that both, the recombinant GM-CSF and GM-CSF gene therapy inhibited growth of tumour residua. Comparative experiments were performed with the MHC class I-, metastasizing tumour MK16. It has been found that both, recombinant IL-2 and GM-CSF, can inhibit growth of the tumour residua after surgery or chemotherapy. The lung metastases in mice with surgical minimal residual tumour disease or in mice with tumour residua after chemotherapy were inhibited by IL-2 but not by GM-CSF. The MK16 tumour vaccine producing IL-2 inhibited growth of tumour residua after chemotherapy, but not the tumour residua after surgery. The GM-CSF-producing vaccine was without significant effect in both, surgically- and chemotherapeutically-induced minimal residual MK16 tumour disease. In conclusion, the MHC class I+ and MHC class I-, HPV16-associated tumours were found to be sensitive to IL-2 and GM-CSF therapy after surgery or after cytoreductive chemotherapy. It is yet to be addressed if this is more general case with HPV16-associated experimental tumours. If so, it would be of interest to further investigate whether such adjuvant therapy can also help to eradicate the residua after surgery and chemotherapy in patients carrying HPV16-associated neoplasms.
Assuntos
Citocinas/administração & dosagem , Terapia Genética/métodos , Neoplasia Residual/terapia , Papillomaviridae/metabolismo , Proteínas Repressoras , Animais , Vacinas Anticâncer/administração & dosagem , Linhagem Celular Tumoral , Citocinas/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/administração & dosagem , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Interleucina-2/administração & dosagem , Interleucina-2/genética , Camundongos , Camundongos Endogâmicos , Recidiva Local de Neoplasia/prevenção & controle , Neoplasia Residual/genética , Neoplasia Residual/patologia , Neoplasias/tratamento farmacológico , Neoplasias/cirurgia , Neoplasias/virologia , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus , Distribuição Aleatória , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Fatores de Tempo , Resultado do TratamentoRESUMO
MHC class I downregulation is an important mechanism of tumour escape from T cell-mediated immune responses. Approximately 40-90% of human tumours derived from various MHC class I+ tissues were reported to be MHC class I deficient. Decreased or absent MHC class I expression is frequently associated with the invasive and metastatic tumour phenotype. Altered MHC class I antigen expression involves total loss, loss of haplotype, locus downregulation, allelic loss or downregulation, and combinations. Description of partial or complete losses of MHC class I molecules in tumour cells as mechanisms of immune escape often fails to consider an increased susceptibility to NK cell-mediated lysis, which is a direct consequence of such losses. A low MHC class I level favours NK cells as effectors, whereas a high level of MHC class I favours T cells as effectors. The microheterogeneity of MHC class I expression in tumour cell populations, the balance of the MHC-restricted and MHC-unrestricted defence as well as the selective pressure of antigen-specific (CTL) and antigen non-specific (NK) effector mechanisms decide the final outcome of the MHC class I expression in the primary tumour and its metastases as well as the final outcome of the tumour defence reaction. Despite the MHC class I molecule deficiency and the resulting absence of the CD8+ T cell-mediated immunity, the tumour hosts were found to be capable of being immunized against MHC class I- tumours. The purpose of this review is to discuss the positive results of MHC class I- tumour treatment obtained with immunomodulatory cytokines and tumour vaccines, as well as the prospects and limitations of such therapy.