Immunotherapy augments the effect of 5-azacytidine on HPV16-associated tumours with different MHC class I-expression status.

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.

Effects of 5-azacytidine and trichostatin A on dendritic cell maturation.

Maturation of dendritic cells (DC) towards functional antigen-presenting cells is a complex process, the regulation of which may also involve epigenetic mechanisms. Thus, it is of interest to investigate how gene expression changes during DC maturation can be influenced with epigenetic agents, such as DNA methyltransferase or histone deacetylase inhibitors. Here, we document the effects of DNA methyltransferase inhibitor 5-azacytidine (5AC) and histone deacetylase inhibitor trichostatin A (TSA) on the murine bone marrow-derived, as well as on the human monocyte-derived DC maturation. The major impact of 5AC and TSA on the DC maturation process consisted in the inhibition of unmethylated CpG oligodeoxynucleotide (CpG ODN) 1826 or LPS-induced activation of pro- and anti-inflammatory cytokine gene expression activation. In the in vitro studies, TSA but not 5AC significantly reduced the capacity of the peptide-pulsed DC to induce total spleen as well as CD8(+) or CD4(+) cell proliferation. IFNγ production by the specific CD4(+) spleen cells co-cultured with TSA- but not with 5AC-treated DC was lower, as compared to the cytokine production after co-cultivation with untreated mature DC. Collectively, these results demonstrate the potential of epigenetic agents, which are under intensive investigation as promising anti-tumour agents, to hamper the immune response induction through their inhibitory effects on DC.

Immunotherapeutic efficacy of vaccines generated by fusion of dendritic cells and HPV16-associated tumour cells.

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.

Immunogenicity of dendritic cell-based HPV16 E6/E7 peptide vaccines: CTL activation and protective effects.

We have investigated the capacity of cellular vaccines based on dendritic cells loaded with human HPV16 E6/E7 oncoprotein-derived peptides to induce immune responses in vitro and to elicit protective immunity in a murine experimental model mimicking human HPV16-associated carcinomas. Dendritic cells loaded with the HPV16 E6/E7 peptides exhibiting CTL or Th epitopes (E6(41-50), E6(81-90), E6(98-107), E6(130-137), E7(49-57), and E7(44-62)) were able to stimulate in vitro DNA synthesis in syngeneic H-2b spleen cells. The priming capacity of peptide-loaded BMDC and peptide-loaded dendritic cell lines DC2.4 and JAWS II was compared. It has been found that both peptide-loaded BMDC and established dendritic cell lines can activate the syngeneic responder cells, but the priming capacity of BMDC was substantially higher. In the second set of experiments, we have examined the cytolytic activity of syngeneic spleen cells after repeated activation in vitro with BMDC loaded with HPV16 synthetic peptides containing CTL epitopes. Significant cytotoxic responses against HPV16 E6/E7 antigen-expressing TC-1 targets have been found after repeated in vitro activation with all peptides containing the CTL epitopes. In contrast, peptide E7(44-62) harbouring both Th and CTL epitopes induced significant cytotoxic responses already after single in vitro activation. This cytotoxic effect could be enhanced with admixture of the E7(49-57) peptide. Experiments with MHC class I proficient (TC-1, MK16-IFNgamma) and deficient (MK16) target cells revealed that the dendritic cells loaded with the E6/E7 HPV16 peptides activated CTLs in vitro, but not the other cytolytic effector mechanisms. The effectiveness of the peptide-loaded BMDC-based cellular vaccines was also investigated in vivo. C57BL/6 (H-2b) mice were immunized with various peptide-loaded BMDC and subsequently challenged with TC-1 cells. The strongest protective effect was achieved with the BMDC loaded with the peptide E7(44-62) harbouring both CTL and Th epitopes. Mice immunized with the E7(44-62) peptide remained tumour-free after s.c. transplantation of the TC-1 cells and exhibited long-lasting protective immunity, whereas the mice immunized with E6(81-90) and E7(49-57) peptides did not remain tumour-free and exhibited only partial inhibition of tumour growth detectable as depression of the tumour growth curves.

The role of NK1.1+ cells in the protection against MHC class I+ HPV16-associated tumours.

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.

Immunotherapy of HPV 16-associated tumours with tumour cell line/dendritic cell line (TC-1/DC2.4) hybrid vaccines.

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.

Local IFN-gamma therapy of HPV16-associated tumours.

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.

Adjuvant cytokine treatment of minimal residual disease after surgical therapy in mice carrying HPV16-associated tumours: cytolytic activity of spleen cells from tumour regressors.

It has been found previously that IL-2, IFNgamma and GM-CSF were capable of reducing the recurrence rate of HPV 16-associated tumours in mice with SMRTD. We were interested whether the therapeutic effect of the surgery and adjuvant cytokine treatment was accompanied by cytolytic activity of spleen cells and whether the activity of the spleen cells was different in mice that had rejected tumour residua after surgery and adjuvant therapy with cytokines (tumour regressors) as compared to those that had not rejected the tumour residua (tumour progressors). We have examined the cytolytic activity of spleen cells from MHC class I+ TC-1 tumour regressors and progressors after treatment of TC-1 SMRTD with GM-CSF, and the activity of spleen cells from MHC class I- MK16 tumour regressors and progressors after treatment of MK16 SMRTD with IL-2 and IFNgamma. It has been found that irrespective of the tumour type and adjuvant treatment, the spleen cells from tumour regressors after surgery were regularly more cytolytic when allowed to react with target cells from HPV 16-associated tumours than the spleen cells from tumour progressors. No substantial differences between the cytolytic activity of spleen cells from the operated-only and operated plus cytokine (GM-CSF, IL-2, IFNgamma) adjuvant treated groups were observed. The cytolytic activity of spleen cells from mice with SMRTD allowed to react with MHC class I+ , MHC class I-, NK-sensitive and NK-resistant targets is compatible with the interpretation that in the mice with MHC class I+ TC-1 tumours, primarily cytotoxic T lymphocytes (CTL) were efficient, whereas in the mice with MHC class I- MK16 tumours, both NK and non-lymphocytic effector cells were involved.

MHC class I+ and class I- HPV16-associated tumours expressing the E7 oncoprotein do not cross-react in immunization/challenge experiments.

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.

Tumour-inhibitory effects of dendritic cells administered at the site of HPV 16-induced neoplasms.

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.

Freezing and thawing of murine bone marrow-derived dendritic cells does not alter their immunophenotype and antigen presentation characteristics.

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.

Chemoimmunotherapy of cancer: potentiated effectiveness of granulocyte-macrophage colony-stimulating factor and ifosfamide derivative CBM-4A.

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.