Tumor growth accelerated by chemotherapy-induced senescent cells is suppressed by treatment with IL-12 producing cellular vaccines.

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.

Dendritic cells pulsed with tumor cells killed by high hydrostatic pressure induce strong immune responses and display therapeutic effects both in murine TC-1 and TRAMP-C2 tumors when combined with docetaxel chemotherapy.

High hydrostatic pressure (HHP) has been shown to induce immunogenic cell death of cancer cells, facilitating their uptake by dendritic cells (DC) and subsequent presentation of tumor antigens. In the present study, we demonstrated immunogenicity of the HHP-treated tumor cells in mice. HHP was able to induce immunogenic cell death of both TC-1 and TRAMP-C2 tumor cells, representing murine models for human papilloma virus-associated tumors and prostate cancer, respectively. HHP-treated cells induced stronger immune responses in mice immunized with these tumor cells, documented by higher spleen cell cytotoxicity and increased IFNγ production as compared to irradiated tumor cells, accompanied by suppression of tumor growth in vivo in the case of TC-1 tumors, but not TRAMP-C2 tumors. Furthermore, HHP-treated cells were used for DC-based vaccine antigen pulsing. DC co-cultured with HHP-treated tumor cells and matured by a TLR 9 agonist exhibited higher cell surface expression of maturation markers and production of IL-12 and other cytokines, as compared to the DC pulsed with irradiated tumor cells. Immunization with DC cell-based vaccines pulsed with HHP-treated tumor cells induced high immune responses, detected by increased spleen cell cytotoxicity and elevated IFNγ production. The DC-based vaccine pulsed with HHP-treated tumor cells combined with docetaxel chemotherapy significantly inhibited growth of both TC-1 and TRAMP-C2 tumors. Our results indicate that DC-based vaccines pulsed with HHP-inactivated tumor cells can be a suitable tool for chemoimmunotherapy, particularly with regard to the findings that poorly immunogenic TRAMP-C2 tumors were susceptible to this treatment modality.

Epigenetic regulations in the IFNγ signalling pathway: IFNγ-mediated MHC class I upregulation on tumour cells is associated with DNA demethylation of antigen-presenting machinery genes.

Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.

DNA demethylating agent 5-azacytidine inhibits myeloid-derived suppressor cells induced by tumor growth and cyclophosphamide treatment.

MDSCs represent one of the key players mediating immunosuppression. These cells accumulate in the TME, lymphoid organs, and blood during tumor growth. Their mobilization was also reported after CY therapy. DNMTi 5AC has been intensively studied as an antitumor agent. In this study, we examined, using two different murine tumor models, the modulatory effects of 5AC on TU-MDSCs and CY-MDSCs tumor growth and CY therapy. Indeed, the percentage of MDSCs in the TME and spleens of 5AC-treated mice bearing TRAMP-C2 or TC-1/A9 tumors was found decreased. The changes in the MDSC percentage were accompanied by a decrease in the Arg-1 gene expression, both in the TME and spleens. CY treatment of the tumors resulted in additional MDSC accumulation in the TME and spleens. This accumulation was subsequently inhibited by 5AC treatment. A combination of CY with 5AC led to the highest tumor growth inhibition. Furthermore, in vitro cultivation of spleen MDSCs in the presence of 5AC reduced the percentage of MDSCs. This reduction was associated with an increased percentage of CD11c+ and CD86+/MHCII+ cells. The observed modulatory effect on MDSCs correlated with a reduction of the Arg-1 gene expression, VEGF production, and loss of suppressive capacity. Similar, albeit weaker effects were observed when MDSCs from the spleens of tumor-bearing animals were cultivated with 5AC. Our findings indicate that beside the direct antitumor effect, 5AC can reduce the percentage of MDSCs accumulating in the TME and spleens during tumor growth and CY chemotherapy, which can be beneficial for the outcome of cancer therapy.

Administration of anti-CD25 mAb leads to impaired α-galactosylceramide-mediated induction of IFN-γ production in a murine model.

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.

Cyclophosphamide-induced myeloid-derived suppressor cell population is immunosuppressive but not identical to myeloid-derived suppressor cells induced by growing TC-1 tumors.

Myeloid-derived suppressor cells (MDSC) play an important role in tumor escape from antitumor immunity. MDSC accumulate in the lymphoid organs and blood during tumor growth and their mobilization was also reported after cyclophosphamide (CY) administration. In this communication, spleen MDSC accumulating after CY therapy (CY-MDSC) were compared with those expanded in mice bearing human papilloma viruses 16-associated TC-1 carcinoma (TU-MDSC). Although both CY-MDSC and TU-MDSC accelerated growth of TC-1 tumors in vivo, their phenotype and immunosuppressive function differed. CY-MDSC consisted of higher percentage of monocyte-like subpopulation and this was accompanied by lower relative expression of immunosuppressive genes and lower suppression of T-cell proliferation. After interferon-γ stimulation, the expression of immunosuppressive genes increased, but the suppressive ability of CY-MDSC did not reach that of TU-MDSC. The phenotype and function of MDSC obtained from mice bearing TC-1 tumors treated with CY was, in general, found to lie between CY-MDSC and TU-MDSC. After in vitro cultivation of MDSC in the presence of interleukin 12 (IL-12), the percentage of CD11b+/Gr-1+ cells decreased and was accompanied by an increase in the percentage of CD86+/MHCII+ cells. The strongest modulatory effect was noticed in the group of CY-MDSC. The susceptibility of CY-MDSC to all-trans-retinoic acid (ATRA) was also evaluated. In vitro cultivation with ATRA resulted in MDSC differentiation, and ATRA inhibited MDSC accumulation induced by CY administration. Our findings identified differences between CY-MDSC and TU-MDSC and supported the rationale for utilization of ATRA or IL-12 to alter MDSC accumulation after CY chemotherapy with the aim to improve its antitumor effect.

Genetically modified tumour vaccines producing IL-12 augment chemotherapy of HPV16-associated tumours with gemcitabine.

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.

NK1.1+ cells are important for the development of protective immunity against MHC I-deficient, HPV16-associated tumours.

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.

Induction of protective immunity against MHC class I-deficient, HPV16-associated tumours with peptide and dendritic cell-based vaccines.

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.

Therapy for minimal residual tumor disease: beta-galactosylceramide inhibits the growth of recurrent HPV16-associated neoplasms after surgery and chemotherapy.

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.

New fibrillin gene mutation - possible cause of ascending aortic dilation in patients with aortic valve disease: Preliminary results.

BACKGROUND: Approximately 10% of patients who undergo surgery for aortic valve disease (stenosis or regurgitation) suffer from ascending aortic dilation (AAD). A possible genetic etiology of AAD associated with aortic valve disease has been repeatedly mentioned in the literature, but a specific responsible gene mutation has not been described. METHODS: In the present study, two groups of patients were compared, all of whom underwent surgery for aortic valve disease. Group A was a cohort of 27 patients who suffered from aortic valve disease associated with AAD. Group B was a cohort of 29 patients with structural aortic valve disease, but without concomitant AAD (control group). Genomic DNA was extracted from the white blood cells of peripheral blood samples and was amplified using primers specific for chosen exons of the fibrillin-1 gene, including their intron/exon boundaries. Exons 26 and 27 were selected for analysis. RESULTS: Analysis of the intronic part situated close to exon 27 showed insertion of cytosine between nucleotide 37 682 and 37 683 of query sequence. This insertions was classified as IVS 37 682 and 37 683insC. This mutation was found in all 27 patients from group A (patients with structural aortic valve disease accompanied by significant AAD). The abovementioned mutation was not found in any of the 29 patients from group B. CONCLUSIONS: This finding has potential implications for risk stratification and therapeutic targeting not only for patients with existing disease, but also for the general population. Future studies are needed to determine the clinical utility of the finding; however, the present hypothesis needs to be verified by further molecular studies.

Induction of MHC class I molecule cell surface expression and epigenetic activation of antigen-processing machinery components in a murine model for human papilloma virus 16-associated tumours.

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.

Immunization with MHC class I-negative but not -positive HPV16-associated tumour cells inhibits growth of MHC class I-negative tumours.

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.

CpG oligodeoxynucleotides are effective in therapy of minimal residual tumour disease after chemotherapy or surgery in a murine model of MHC class I-deficient, HPV16-associated tumours.

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.

Depletion of T(reg) cells inhibits minimal residual disease after surgery of HPV16-associated tumours.

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.

Inhibitory effects of unmethylated CpG oligodeoxynucleotides on MHC class I-deficient and -proficient HPV16-associated tumours.

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.

Minimal residual disease as the target for immunotherapy and gene therapy of cancer (review).

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.

Immune escape phenotype of HPV16-associated tumours: MHC class I expression changes during progression and therapy.

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.

HPV16-associated tumours: therapy of surgical minimal residual disease with dendritic cell-based vaccines.

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.

Treatment of minimal residual disease after surgery or chemotherapy in mice carrying HPV16-associated tumours: Cytokine and gene therapy with IL-2 and GM-CSF.

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.