ABSTRACT
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.
Subject(s)
Ceramides/pharmacology , Monosaccharides/pharmacology , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/prevention & control , Neoplasm, Residual/drug therapy , Neoplasm, Residual/surgery , Papillomavirus Infections/pathology , Papillomavirus Infections/prevention & control , Animals , Human papillomavirus 16/isolation & purification , Humans , Immunotherapy , Male , Mice , Mice, Inbred C57BL , Neoplasm Recurrence, Local/immunology , Neoplasm, Residual/virology , Papillomavirus Infections/immunology , Tumor Cells, Cultured/transplantationABSTRACT
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.
Subject(s)
Angiogenesis Inhibitors/therapeutic use , Cell Proliferation , Human papillomavirus 16/pathogenicity , Interleukin-12/physiology , Lymphocytes, Tumor-Infiltrating/immunology , Neoplasms, Experimental/immunology , Papillomavirus Infections/immunology , Animals , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytotoxicity Tests, Immunologic , Flow Cytometry , Genetic Therapy , Humans , Ifosfamide/analogs & derivatives , Ifosfamide/therapeutic use , Immunoenzyme Techniques , Immunotherapy , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/therapy , Neoplasms, Experimental/virology , Papillomavirus Infections/therapy , Papillomavirus Infections/virology , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Epigenesis, Genetic/immunology , Genes, MHC Class I , Human papillomavirus 16 , Neoplasms, Experimental/immunology , Papillomavirus Infections/complications , Animals , Antigen Presentation/genetics , Antigen Presentation/immunology , Apoptosis/drug effects , Azacitidine/pharmacology , Enzyme Inhibitors/pharmacology , Histones/metabolism , Humans , Hydroxamic Acids/pharmacology , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/genetics , Neoplasms, Experimental/virology , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Cells, Cultured , Up-Regulation/drug effects , Up-Regulation/genetics , Up-Regulation/immunologyABSTRACT
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.
Subject(s)
Cytokines/metabolism , Gene Expression Regulation, Neoplastic , Human papillomavirus 16/metabolism , Immune System/immunology , Immunotherapy/methods , Interleukin-12/chemistry , Neoplasms/virology , Animals , CD4-Positive T-Lymphocytes/metabolism , Cancer Vaccines , Disease Models, Animal , Immune System/metabolism , Interleukin-12/metabolism , Interleukin-2 Receptor alpha Subunit/biosynthesis , Kinetics , Lymph Nodes/pathology , Mice , Neoplasms/metabolismABSTRACT
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.
Subject(s)
CpG Islands , Human papillomavirus 16/metabolism , Neoplasm, Residual/drug therapy , Neoplasms/genetics , Neoplasms/therapy , Neoplasms/virology , Oligonucleotides/therapeutic use , Animals , Cell Line, Tumor , Disease Models, Animal , Genes, MHC Class I , Humans , Male , Mice , Mice, Inbred C57BL , Oligonucleotides/chemistry , Recurrence , Time FactorsABSTRACT
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.
Subject(s)
Histocompatibility Antigens Class I/immunology , Human papillomavirus 16/immunology , Immunization , Neoplasms/therapy , Neoplasms/virology , Animals , Cancer Vaccines/therapeutic use , Cell Line, Tumor , Cell Proliferation , Cross Reactions , Histocompatibility Antigens Class I/analysis , Mice , Mice, Inbred C57BL , Neoplasms/immunologyABSTRACT
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.
Subject(s)
Human papillomavirus 16/isolation & purification , Immunotherapy/methods , Lung Neoplasms/immunology , Lung Neoplasms/therapy , Papillomavirus Infections/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Combined Modality Therapy , Genes, MHC Class I/genetics , Genes, MHC Class I/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Lung Neoplasms/pathology , Lung Neoplasms/virology , Male , Mice , Mice, Inbred C57BL , Neoplasm, Residual , Papillomavirus Infections/complications , Papillomavirus Infections/virologyABSTRACT
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.
Subject(s)
Cellular Senescence/drug effects , Immunotherapy, Adoptive/methods , Interleukin-12/pharmacology , Neoplasms, Experimental/therapy , Taxoids/pharmacology , Tumor Burden/drug effects , Animals , Antineoplastic Agents/pharmacology , Bystander Effect/drug effects , Cell Line, Tumor , Combined Modality Therapy , Cytokines/genetics , Cytokines/metabolism , Docetaxel , Interleukin-12/biosynthesis , Male , Mice, Inbred C57BL , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Time FactorsABSTRACT
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.
Subject(s)
Gene Expression Regulation, Neoplastic , Histocompatibility Antigens Class I/biosynthesis , Immunotherapy/methods , Neoplasms/immunology , Neoplasms/virology , Oncogene Proteins, Viral/biosynthesis , Papillomaviridae/metabolism , Papillomavirus Infections/immunology , Animals , Cell Line, Tumor , Chromium Radioisotopes , Coculture Techniques , Disease Progression , Flow Cytometry , Genes, MHC Class I , Interferon-gamma/biosynthesis , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental , Phenotype , Recurrence , Spleen/metabolism , T-Lymphocytes, Cytotoxic/metabolism , Transfection , Up-RegulationABSTRACT
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.
Subject(s)
Genetic Therapy , Immunotherapy , Neoplasm, Residual , Neoplasms/therapy , Chemotherapy, Adjuvant , Clinical Trials as Topic , Endpoint Determination , Humans , Neoplasm Metastasis , Neoplasms/genetics , Neoplasms/immunologyABSTRACT
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.
Subject(s)
Histocompatibility Antigens Class I/metabolism , Immunologic Surveillance , Neoplasms/immunology , Tumor Escape/immunology , Down-Regulation/genetics , Gene Expression Regulation, Neoplastic , Histocompatibility Antigens Class I/genetics , Humans , Immunologic Surveillance/genetics , Tumor Escape/geneticsABSTRACT
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.
Subject(s)
Cancer Vaccines/immunology , Dendritic Cells/immunology , Neoplasms, Experimental/therapy , Oncogene Proteins, Viral/immunology , Papillomaviridae/immunology , Papillomavirus Infections/complications , Repressor Proteins/immunology , Animals , Immunophenotyping , Male , Mice , Mice, Inbred C57BL , Neoplasm, Residual , Neoplasms, Experimental/immunology , Papillomavirus E7 ProteinsABSTRACT
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.
Subject(s)
Cytokines/administration & dosage , Genetic Therapy/methods , Neoplasm, Residual/therapy , Papillomaviridae/metabolism , Repressor Proteins , Animals , Cancer Vaccines/administration & dosage , Cell Line, Tumor , Cytokines/genetics , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Interleukin-2/administration & dosage , Interleukin-2/genetics , Mice , Mice, Inbred Strains , Neoplasm Recurrence, Local/prevention & control , Neoplasm, Residual/genetics , Neoplasm, Residual/pathology , Neoplasms/drug therapy , Neoplasms/surgery , Neoplasms/virology , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins , Random Allocation , Recombinant Proteins/administration & dosage , Recombinant Proteins/genetics , Time Factors , Treatment OutcomeABSTRACT
The effectiveness of chemoimmunotherapy with ifosfamide derivative CBM-4A and recombinant IL-2, IL-12, GM-CSF, or genetically modified, cytokine-producing tumour vaccines was examined in mice carrying HPV16-associated, MHC class I+ (TC-1), and MHC class I- (MK16) tumours. Intraperitoneal treatment of TC-1 or MK16 tumour-bearing mice with CBM-4A produced a significant tumour-inhibitory effect. When the i.p. treatment of the MHC class I+ TC-1 tumour-bearing mice with CBM-4A was followed by peritumoral s.c. administration of IL-2, IL-12, or both cytokines, the growth of TC1 tumours was inhibited more vigorously than after the chemotherapy alone. In contrast, when the i.p. treatment ofEthe MHC class I- MK16 tumour-bearing mice with CBM-4A was followed by peritumoral s.c. administration of IL-2 or IL-12, the cytokine therapy had no potentiating effect. The only potentiating effect of the MK16 tumour immunotherapy was obtained when the i.p. CBM-4A pretreatment was followed by peritumoral s.c. administration of IL-2 plus IL-12. InEfurther experiments, the TC-1 and MK16 tumour-bearing mice were i.p. pretreated with CBM-4A and then injected s.c., peritumorally, with genetically modified, IL-2 or GM-CSF-producing MK16 tumour vaccines. Whereas both genetically modified tumour vaccines produced a substantial tumour-inhibitory effect in mice carrying TC-1 tumours, no effect of the vaccines was observed in mice carrying MK16 tumour inocula. The systemic effects of local cytokine treatment were examined in mice carrying s.c. MK16 neoplasms, which were pretreated i.p. with CBM-4A, and then injected peritumorally with IL-2 or GM-CSF. Peritumoral administration of GM-CSF had no antimetastatic effect, whereas peritumoral IL-2 administration produced substantial reduction of lung metastases. The systemic antimetastatic effect of IL-2 contrasted with the negligible effect of IL-2 on the s.c. MK16 tumour inoculum. Taken collectively, the results indicate that in mice carrying the MK16 (MHC class I-) tumour, the effects of the adjuvant cytokine therapy were substantially weaker than in mice carrying the TC-1 (MHC class I+) tumour inoculum.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Antigens, Neoplasm/analysis , Antineoplastic Agents, Alkylating/therapeutic use , Cancer Vaccines/therapeutic use , Granulocyte-Macrophage Colony-Stimulating Factor/therapeutic use , Histocompatibility Antigens Class I/analysis , Ifosfamide/analogs & derivatives , Ifosfamide/therapeutic use , Immunotherapy , Interleukin-12/therapeutic use , Interleukin-2/therapeutic use , Neoplasms, Experimental/therapy , Repressor Proteins , Adjuvants, Immunologic/administration & dosage , Animals , Cell Transformation, Viral , Combined Modality Therapy , Drug Screening Assays, Antitumor , Genes, MHC Class I , Genes, ras , Granulocyte-Macrophage Colony-Stimulating Factor/administration & dosage , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Injections, Subcutaneous , Interleukin-12/administration & dosage , Interleukin-12/genetics , Interleukin-2/administration & dosage , Interleukin-2/genetics , Mice , Neoplasm Metastasis , Neoplasms, Experimental/genetics , Neoplasms, Experimental/immunology , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/physiology , Papillomavirus E7 Proteins , VaccinationABSTRACT
Oncogenic, moderately immunogenic, MHC class I- and class II-, B7-, MK16/1/III ABC (MK16) cells were previously established by co-transfection of HPV16 E6/E7 and activated H-ras oncogene DNA into C57BL/6 kidney cells. Subcutaneous transplantation of these cells produced progressively growing local neoplasms which metastasized spontaneously to lungs and lymph nodes. The MK16 cells were implanted into syngeneic mice and used to examine whether the tumour lacking the signal molecules required for the induction of and sensitivity to T cell immunity is susceptible to local IL-2 treatment and IL-2 gene therapy. Peritumoural administration of human rIL-2 or murine IL-2 gene-modified MK16 tumour vaccine inhibited growth of subcutaneous MK16 tumour transplants and reduced the number of their lung metastases. Spleen cells from MK16 tumour-immunized mice were not cytolytic when allowed to react with the MK16 target cells, although they efficiently lysed the MHC class I+ malignant TC1 cells, obtained from C57BL/6 lung cell cultures after transfection with the same plasmids as those used for the derivation of the MK16 cells. However, when the MK16 cells were cultivated in vitro in the presence of IFNgamma, they acquired, together with the expression of MHC class I molecules, the sensitivity to the cytolytic effect of spleen cells from the MK16 tumour-immunized mice. These results indicate that experimental tumours which are MHC class I- and mimick in this respect a high proportion of human HPV16-associated carcinomas are suitable for IL-2 treatment.
Subject(s)
Antineoplastic Agents/pharmacology , Genes, MHC Class I/genetics , Interleukin-2/pharmacology , Papillomaviridae/metabolism , Animals , Cytokines/biosynthesis , Down-Regulation , Flow Cytometry , Humans , Interleukin-2/metabolism , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , Neoplasm Transplantation , Time Factors , Tumor Cells, CulturedABSTRACT
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.
Subject(s)
Down-Regulation , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class I/immunology , Immunotherapy/methods , Neoplasms/immunology , Neoplasms/therapy , Alleles , CD8-Positive T-Lymphocytes/metabolism , Haplotypes , Humans , Killer Cells, Natural/cytology , Neoplasm Metastasis , PhenotypeABSTRACT
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.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Galactosylceramides/pharmacology , Interferon-gamma/immunology , Interleukin-2 Receptor alpha Subunit/immunology , Natural Killer T-Cells/drug effects , Neoplasms, Experimental/drug therapy , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antigens, CD1d/immunology , Antigens, CD1d/metabolism , Cell Line, Tumor , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Forkhead Transcription Factors/immunology , Forkhead Transcription Factors/metabolism , Galactosylceramides/administration & dosage , Galactosylceramides/immunology , Gene Expression/drug effects , Gene Expression/immunology , Heparin-binding EGF-like Growth Factor , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/immunology , Intercellular Signaling Peptides and Proteins/metabolism , Interferon-gamma/genetics , Interferon-gamma/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-4/genetics , Interleukin-4/immunology , Interleukin-4/metabolism , Kaplan-Meier Estimate , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Natural Killer T-Cells/immunology , Natural Killer T-Cells/metabolism , Neoplasms, Experimental/immunology , Neoplasms, Experimental/pathology , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes, Regulatory/drug effects , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , Tumor Burden/drug effects , Tumor Burden/immunologyABSTRACT
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.
Subject(s)
Cancer Vaccines/immunology , Histocompatibility Antigens Class I/immunology , Killer Cells, Natural/immunology , Neoplasms, Experimental/immunology , Animals , Antigens, Ly/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cell Separation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Human papillomavirus 16 , Male , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily B/immunologyABSTRACT
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.
Subject(s)
Antineoplastic Agents/pharmacology , Cancer Vaccines/pharmacology , Deoxycytidine/analogs & derivatives , Genetic Therapy/methods , Interleukin-12/biosynthesis , Neoplasms, Experimental/drug therapy , Animals , Cell Separation , Combined Modality Therapy , Deoxycytidine/pharmacology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Human papillomavirus 16 , Male , Mice , Mice, Inbred C57BL , Neoplasms, Experimental/virology , Papillomavirus Infections/complications , GemcitabineABSTRACT
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.