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Background: Epigenetic mechanisms have important roles in the tumour escape from immune responses, such as in MHC class I downregulation or altered expression of other components involved in antigen presentation. Chemotherapy with DNA methyltransferase inhibitors (DNMTi) can thus influence the tumour cell interactions with the immune system and their sensitivity to immunotherapy.Methods: We evaluated the therapeutic effects of the DNMTi 5-azacytidine (5AC) against experimental MHC class I-deficient and -positive tumours. The 5AC therapy was combined with immunotherapy, using a murine model for HPV16-associated tumours.Results: We have demonstrated 5AC additive effects against MHC class I-positive and -deficient tumours when combined with unmethylated CpG oligodeoxynucleotides or with IL-12-producing cellular vaccine. The efficacy of the combined chemoimmunotherapy against originally MHC class I-deficient tumours was partially dependent on the CD8(+)-mediated immune responses. Increased cell surface expression of MHC class I cell molecules, associated with upregulation of the antigen-presenting machinery-related genes, as well as of genes encoding selected components of the IFNγ-signalling pathway in tumours explanted from 5AC-treated animals, were observed.Conclusion: Our data suggest that chemotherapy of MHC class I-deficient tumours with 5AC combined with immunotherapy is an attractive setting in the treatment of MHC class I-deficient tumours. [ABSTRACT FROM AUTHOR]

Crohn's disease is often purely inflammatory, but most patients develop complicated disease with strictures or fistulae. Specific etiopathogenesis of this severe disease is not definitely clear despite research efforts and learning of many pathogenetic mechanisms. Many studies have suggested that NOD2 mutations are associated with increased risk of complicated disease. Presence of NOD2 mutation itself is just one of factors contributing to development of this disease. Genetically predisposed individuals in combination with influence of environmental factors result in a disturbed innate (i.e., disturbed intestinal barrier, Paneth cell dysfunction) and adaptive (i.e., imbalance of effector and regulatory T cells and cytokines, migration and retention of leukocytes) immune response towards a diminished diversity of commensal microbiota. Data of meta-analysis made so far provide ambiguous evidence to support top-down therapy based solely on single NOD2 mutations, but suggest that targeted early-intensive therapy for high-risk patients with two NOD2 mutations might be beneficial, but more prospective trials could answer these questions.

Development of ascending aortic dilatation (AAD) in about 10 % of patients operated for aortic valve disease (AVD) is probably based on intrinsic pathology of the aortic wall. This may involve an abnormality in the process of extracellular matrix remodelling. The present study evaluated the serum levels of specific metalloproteinases (MMP-2 and MMP-9) and investigated the gene for transforming growth factor receptor 2 (TGFBR2) in 28 patients with AVD associated with AAD (mean age 60.6 years), in 29 patients (68.9 years) with AVD without AAD, and in 30 healthy controls (45.3 years). The serum levels of MMPs were determined by ELISA. Further, we focused on genetic screening of the TGFBR2 gene. Plasma MMP-2 concentrations were significantly higher in the groups of patients compared to the controls: median 1315.0 (mean 1265.2 ± SD 391.3) in AVD with AAD, 1240.0 (1327.8 ± 352.5) in AVD without AAD versus 902.5 (872.3 ± 166.2) ng/ml in the healthy controls, in both cases P < 0.001. The serum levels of MMP-9 were significantly higher in AVD with AAD patients [107.0 (202.3 ± 313.0)] and in AVD without AAD patients [107.0 (185.8 ± 264.3)] compared to the healthy controls [14.5 (21.2 ± 24.8) ng/ml], in both cases P < 0.001. No significant correlation was observed between plasma MMP-2 and MMP-9 and ascending aorta diameter. Genetic screening did not reveal any variation in the TGFBR2 gene in the patients. Measurement of MMP levels is a simple and relatively rapid laboratory test that could be used as a biochemical indicator when evaluated in combination with imaging techniques.

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 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.

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.

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.

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.

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.

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.

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.

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.

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.

Utilization of vaccines generated by fusion of dendritic cells and tumour cells is a promising approach to tumour immunotherapy. We have examined the therapeutic efficacy of vaccines generated by fusion of HPV16-associated tumour cells TC-1 with syngeneic and allogeneic dendritic cells. Locally administered hybrid cells generated by fusion of MHC class I+ TC-1 cells and syngeneic DC inhibited the growth of MHC class I+ TC-1 tumours, but not the growth of MHC class I- TC-1/A9-derived tumours. The growth of TC-1 tumours was also inhibited by hybrids generated by fusion of TC-1 cells and allogeneic DC. The therapeutic efficacy was enhanced by co-administration of the vaccine with synthetic immunostimulatory ODN CpG 1826.

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.

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.

Depletion of NK1.1+ cells by repeated i.p. injections of PK136 antibody significantly enhanced growth of MHC class I+ tumours in syngeneic mice. Depletion starting before tumour transplantation or on the day of transplantation substantially accelerated tumour growth; depletion starting on day 7 or 14 after tumour transplantation was without any effect. These results indicate that the NK1.1+ cells play an important inhibitory role during the early phase of the growth of some MHC class I+ tumours. Since the relevant target for NK cells is a "missing self" signal, absence of the MHC class I molecules, the NK cells cannot be expected to directly inhibit the growth of the MHC class I+ tumours. The results indicate that the effects of non-NK cells or indirect effects mediated by NK cell interactions and release of cytokines were responsible for the results.

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.

Moderately immunogenic HPV 16-associated tumours TC-1 (MHC class I(+), HPV 16 E6/E7(+), G12V Ha-ras(+)) and MK16/1/III ABC (MHC class I(-), HPV 16 E6/E7(+), G12V Ha-ras(+)), both of the H-2(b) haplotype and transplanted in syngeneic mice, were used to examine the adjuvant effects of IL-2 and dendritic cells for surgical therapy. Mice were inoculated s.c. with the respective tumour cells, and when the tumours reached 8-12 mm in diameter, they were extirpated. Three days after surgery, the experimental mice were treated with IL-2, IL-2 gene-modified tumour vaccines, or dendritic cells, injected s.c. to the site of previous surgery. It has been found in both, MHC class I(+) and MHC class I(-) tumours that the recombinant IL-2 and IL-2 gene-modified vaccines substantially reduced the tumour recurrence rate and inhibited growth of tumour recurrences. The dendritic cells were significantly effective only in mice with surgical minimal residual TC-1 (MHC class I(+)) tumour disease and when injected before they have reached the terminal stage of their differentiation.

It has been demonstrated repeatedly that a high proportion of tumours derived from MHC class I+ precursors are MHC class I-. Since a major task in immunotherapy strategies for treatment of malignancies is to develop polyvalent tumour vaccines efficient against a broad spectrum of tumours, we have examined whether MHC class I+ cell-based tumour vaccines can cross-protect against homologous MHC class I- tumour challenge and vice versa. For these purposes, we have used two oncogenic cell lines induced independently by co-transfection of murine H-2b cells with E61E7 HPV16 and activated Ha-ras oncogenes, the tumours TC-1 (MHC class I+, HPV16 E7+) and E7+). Surprisingly, it was found that these two tumours do not cross-react, although both of them contain the crucial HPV16-coded tumour rejection antigen E7. Preimmunization with the MHC class I+ tumour did not protect against a subsequent challenge with the MHC class I- tumour and vice versa; however, immunization with the TC-1 tumour could protect syngeneic mice against the TC-1 tumour challenge and, similarly, immunization with the MK16/1/IIIABC tumour could protect mice against the MK16/1/IIIABC tumour challenge. If this finding can also be confirmed as a more general phenomenon with other MHC class I+ and class 1- tumours, it could have serious implications for design of immunotherapeutic vaccines and protocols.

We have examined whether peritumoral administration of IFN-gamma can inhibit growth of HPV16-associated, MHC class I- tumour MK16/1/IIIABC (MK16) transplanted in syngeneic mice. It has been found that peritumoral administration of recombinant IFN-gamma performed on days 0-11 after tumour challenge inhibited growth of MK16 s.c. tumour transplants. If the therapy with IFN-gamma was started when the tumours had already reached a palpable size, the IFN-gamma administration was without any effect. To investigate the antitumour effects of IFN-gamma in a clinically more relevant setting, surgical minimal residual tumour disease was utilized. Subcutaneously growing MK16 carcinomas, 8-12 mm in diameter, were removed and the operated mice were injected with IFN-gamma on days 3-14 after the operation at the site of surgery. Treatment with IFN-gamma resulted in a moderate, reproducible, but statistically insignificant inhibition of tumour recurrences. In the next experiments we have addressed the question whether the tumour-inhibitory effect of IFN-gamma was due to the upregulation of MHC class I molecule expression on MK16 tumour cells. IFN-gamma-treated and control mice were sacrificed, their tumours were explanted, and the expression of MHC class I molecules on the MK16 tumour cells was examined. As presumed, the MHC class I expression on the cells of IFN-gamma-treated tumours, as well as on their lung metastases, was upregulated. However, an unexpected moderate upregulation of the MHC class I expression was also observed on MK16 tumours from the control, exogenous IFN-gamma-uninjected mice. Cytofluorometric analysis of the in vivo transplanted MK16 tumours from both groups has excluded that the increased percentage of the MHC class I molecules on the tumour cell populations could be due to the infiltration of the tumours with MHC class I+ leukocytes, since no expression of MHC class II, CD11b, CD80/CD86, and CD11c molecules in the MK16 cell population was observed.

Hybridization of established dendritic cell lines with tumour cells represents a prospective technology for the construction of antitumour vaccines. Experiments were designed to examine whether administration of cell populations prepared by fusion of HPV 16-associated tumour TC-1 cells with dendritic cell line DC2.4 could be used for treatment of TC-1 tumours growing in syngeneic mice. The therapeutic potency of TC-1/DC2.4 fusion vaccine administered 24 h after fusion and that of TC-1/DC2.4 hybrid cells selected for 3 weeks in HAT-containing medium was tested. It has been found that administration of both types of fusion vaccines at the site of growing TC-1 tumour transplants significantly inhibited tumour growth with regard to the percentage of tumour-bearing mice and to the size of the transplanted tumours. Peritumoral administration of the DC2.4 cells alone also reduced the size of growing TC-1 tumours, but not the percentage of the tumour-bearing mice. Although in the groups of mice treated with fusion vaccines the size of the tumours was reproducibly smaller than that in the mice treated with parental DC2.4 cells, the difference was not statistically significant.

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.

Experiments were designed to examine whether administration of APC at the site of HPV 16-associated tumours can inhibit tumour growth and whether the efficacy of established dendritic cell lines is comparable to that of fresh BMDC populations. Mice were inoculated s.c. with APC, either bone marrow-derived dendritic cells differentiated in medium supplemented with GM-CSF and IL-4 (BMDC), or with established dendritic cell lines DC2.4 or JAWS II. The pretreated mice, together with untreated controls, were challenged with syngeneic HPV 16-transformed cells MK16 at the site of APC administration. It has been found that both BMDC and dendritic cell lines can inhibit tumour growth and that the efficacy of the established dendritic cell lines DC2.4 and JAWS II was comparable to that of fresh BMDC populations. In vitro induction of proliferative spleen cell responses by co-cultivation with MK16 antigen-pulsed BMDC or MK16 antigen-pulsed dendritic cell lines revealed that both types of APC populations can prime immune reactions directed against syngeneic HPV 16-associated neoplasms. Taken together, the results suggest that local increase in the number of dendritic cells at the site of HPV 16-associated tumours can inhibit progression of the tumours and that the dendritic cell lines which are efficient in this respect can be considered and should be tested in both, preclinical and human systems for delivery of therapeutic vaccines against HPV 16-associated neoplasms.

The aim of this paper was to assess whether the BMDC after freezing and thawing are capable to retain the immunophenotype and antigen-presenting capacity. BMDC were generated from bone marrow precursor cells as described previously by culturing the cells in medium containing GM-CSF and IL-4. Afterwards, the cells were harvested, counted and used for phenotyping and priming of syngeneic spleen cells. For cryopreservation, the BMDC were frozen in the presence of 10% of dimethylsulphoxide (DMSO) and 90% foetal calf serum. Forty to fifty percent of both samples, frozen/thawed as well as fresh BMDC, exhibited characteristic DC morphology, and the DC obtained from the frozen/thawed samples expressed a similar level of MHC class I-, MHC class II-, CD80-, CD86-, CD11c-, CD11b-, CD54- and CD205-molecule as fresh DC. To examine the in vitro priming effect of cryopreserved BMDC on syngeneic non-adherent murine C57BL/6 (B6) spleen cells, the BMDC were thawed, pulsed with the lysate prepared from HPV 16-associated tumour MK16 and used for 3H-thymidine assay. The findings of the experiments indicate that fresh as well as cryopreserved murine BMDC preparations pulsed with tumour lysate were efficient to prime the mitogenic activity of syngeneic non-adherent splenocytes. Taken together, the results suggest that frozen/thawed BMDC are morphologically, phenotypically and functionally comparable with fresh BMDC and can be used for construction of dendritic cell-based tumour vaccines.

The effectiveness of combined chemoimmunotherapy with ifosfamide derivative CBM-4A and granulocyte-macrophage colony-stimulating factor (GM-CSF) was investigated in two experimental tumor models, 3MC-induced MHC class I+ sarcoma Mc12 and HPV16 E6/E7 oncogene-induced MHC class I- carcinoma MK16, transplanted in syngeneic mice. Treatment of Mc12 and MK16 tumor-bearing mice with GM-CSF or CBM-4A alone produced moderate anti-tumor effects. However, when the tumor-bearing mice were first treated i.p. with a single dose of CBM-4A (150 mg/kg) and three days later peritumorally with five daily doses of GM-CSF (100 ng/day), substantially stronger tumor-inhibitory effects were observed. The results indicate that in both, MHC class I+ and MHC class I- tumors, the combined chemoimmunotherapy can inhibit tumor progression more effectively than GM-CSF therapy or chemotherapy alone, and they suggest that GM-CSF should be considered as adjuvant to chemotherapy in clinical trials with HPV 16-associated neoplasms.

In the endeavour to develop a model for studying gene therapy of cancers associated with human papillomaviruses (HPVs), mouse cells were transformed with the HPV type 16 (HPV16) and activated H-ras oncogenes. This was done by cotransfection of plasmid p16HHMo, carrying the HPV16 E6/E7 oncogenes, and plasmid pEJ6.6, carrying the gene coding for human H-ras oncoprotein activated by the G12V mutation, into secondary C57BL/6 mouse kidney cells. An oncogenic cell line, designated MK16/1/IIIABC, was derived. The epithelial origin of the cells was confirmed by their expression of cytokeratins. No MHC class I and class II molecules were detected on the surface of MK16/1/IIIABC cells. Spontaneous metastases were observed in lymphatic nodes and lungs after prolonged growth of MK16/1/IIIABC-induced subcutaneous tumours. Lethally irradiated MK16/1/IIIABC cells induced protection against challenge with 10(5) homologous cells, but not against a higher cell dose (5 x 10(5)). Plasmids p16HHMo and pEJ6.6 were also used for preventive immunization of mice. In comparison with a control group injected with pBR322, they exhibited moderate protection, in terms of prolonged survival, against MK16/1/IIIABC challenge (P < 0.03). These data suggest that MK16/1/IIIABC cells may serve as a model for studying immune reactions against HPV16-associated human tumours.

Experiments were designed to examine whether local cytokine therapy of subcutaneous (s.c.) tumours results in inhibition of their lung metastases. Moderately immunogenic, major histocompatibility complex (MHC) class I and II negative. B7 negative, metastasizing murine carcinoma MK16 transplantable in syngeneic mice was obtained by co-transfection of human papilloma virus type 16 (HPV 16) E6/E7 and activated H-ras oncogene plasmid DNA into C57BL/6 kidney cells. After s.c. transplantation of the malignantly converted MK16 cells, the majority of the transplanted mice developed lung metastases; the number and size of the lung metastases increased with the increasing size of the s.c. tumour. Therapy of 5-day MK16 tumours by peritumoral administration of recombinant interleukin-2 (IL-2) and recombinant interleukin-12 (IL-12) inhibited growth of the s.c. MK16 tumour transplants and reduced the number of MK16 lung metastases. To investigate the antimetastatic effect of IL-2 and IL- 12 in a clinically more relevant setting, surgical minimal residual tumour disease was utilized. Subcutaneously growing MK16 carcinomas, 8-12 mm in diameter, were removed on day 30 and the operated mice were injected with IL-2 or IL- 12 on days 35-39 and 42-46 at the site of the operation. Treatment with IL-2 significantly reduced the percentage of MK16 tumour recurrences as well as the number of lung metastases, whereas the effect of IL- 12 was substantially weaker and statistically insignificant.