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The present study aimed to elucidate the role of cluster of differentiation (CD)8+, CD4+, natural killer (NK), and myeloid (CD11b+) cells in the course of the growth and rejection of experimental major histocompatibility complex (MHC) class I-deficient, HPV16 E6/E7-associated TC-1/A9 tumors in mice. Stable mouse lines (F30) generated by inbreeding of Balb/c and C57BL/6 strains, which were characterized by H-2Db+d-NK1.1neg (B6-neg) and H-2Db-d+NK1.1high (Balb-high) phenotypes, were used for the present study. The novel strains spontaneously regressed tumors in 70–90% of cases. Ex vivo histological analysis of the tumor microenvironment in cryosections showed an indirect correlation between the growth of the transplanted tumor (progressor vs. regressor mice) and the proportion of immunocompetent cell infiltration in the tumors. The regressor mice exhibited a higher infiltration of tumors with CD4+ and CD8+ cells, and in Balb-high with NK cells as well, compared with the progressors. All tumor transplants also indicated a huge infiltration of CD11b+ cells, but this infiltration was not dependent on the stage of the TC-1/A9 tumor development. Depletion of individual cell subpopulations in vivo exhibited different effects on the tumor development in the two strains. Elimination of CD8-positive cells enhanced growth of TC-1/A9 tumor transplants in both hybrid stains, whereas CD4+ cell depletion affected rejection of TC-1/A9 tumors in the B6-neg mice only. Depletion of NK cells with anti-asialo GM1 antibody in the Balb-high strain led to enhancement of tumor growth, which was more pronounced after depletion of the NK1.1+ subpopulation. On the other hand, depletion of NK cells with anti-asialo GM1 in B6-neg mice did not affect the regression of TC-1/A9 tumor transplants, but increased the CD11b+ cell infiltration. In summary, these results indicate that co-operation of particular subsets of immunocompetent cells is essential for the rejection of TC-1/A9 tumor transplants. In B6-neg mice, the co-operative action of CD8+ and CD4+ cells is required, whereas in Balb-high mice, the synergy of CD8+ and NK1.1+ cells is of major importance.

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.

We have established animal models of HPV16-associated tumours with distinct levels of MHC class I expression. This model was used for examination of immune responses, production of cytokines and kinetics of immune cell subsets after IL-12 therapy of minimal residual tumour disease induced by CBA-4A (cyclophosphamide derivative) treatment. Upregulation of cytokine production was detected, compared to control animals without tumours. No differences in Th1/Th2 polarization of the immune responses after immunotherapy in animals bearing tumours with different surface expression of MHC class I molecules were observed. In the spleens of TC-1 (MHC class I+) but not of TC-1/A9 (MHC class I-) treated tumour-bearing animals, the cytotoxic CD8+ cells detectable in 51Cr microcytotoxicity assay, were found. In the spleens of TC-1/A9 but not of TC-1 tumour-treated animals, the NK activity measured as the lysis of NK-sensitive YAC-1 targets was detected. Down-regulation of the CD4+ and CD8+ subpopulations in spleens of tumour-bearing animals were not restored after therapy. The percentage of CD25+/CD4+ T regulatory (Treg) cells in lymph nodes remained unchanged. The cytoreductive chemotherapy led to strong upregulation and accumulation of immunosuppressive immature myeloid Gr-1+/CD11b+ cells (IMC) in the spleens of treated animals. The accumulation of Gr-1+/CD11b+ cells was significantly decreased after subsequent IL-12 immunotherapy. These data suggest that elimination of IMC after IL-12 immunotherapy may be responsible for the improvement of antitumour responses after adjuvant IL-12 vaccination for the treatment of CMRTD.

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.

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.

Murine carcinoma induced by MK 16 cells expressing HPV 16 E6/E7 oncogenes was utilized to examine the therapeutic effect of dendritic cell-based tumour vaccines. Mice carrying 5-day MK 16 tumours were injected peritumorally with either dendritic cells (DC) or DC pulsed with MK 16 tumour lysate. Both the unpulsed and MK 16 lysate-pulsed DC vaccines inhibited growth of the MK 16 transplants, the pulsed DC being more efficient than the unpulsed vaccines. In vitro priming of the effector cell-mediated anti-MK 16 responses by DC pulsed with MK 16 tumour lysate and a synthetic HPV 16 E7(49-57) peptide RAHYNIVTF was compared. The priming activity of the lysate was substantially higher than that of the HPV 16 E7(49-57) peptide; the priming activity was similar to that of a standard moderately immunogenic chemically-induced sarcoma. Taken collectively, these results suggest that DC vaccines pulsed with HPV 16-associated tumour lysates represent a prospective modality for treatment of HPV 16-associated carcinomas.

Two genes, the gene coding for IL-2 and the gene encoding the CD80 molecule, were inserted into murine sarcoma MC12 cells. Tumorigenicity of a variety of cell clones with different expression of the inserted genes was assessed. Most of the genetically manipulated MC12 cell clones were less tumorigenic than the parental MC12 cell population. Tumorigenicity of the clones declined with increasing production of IL-2 as well as with the increasing expression of the CD80 molecule. When the tumorigenicity of the clones carrying an inserted IL-2 gene was compared with that of the clones carrying an inserted CD80 gene, it was found that the insertion of the IL-2 gene suppresses tumorigenicity more efficiently than insertion of the CD80 gene. Admixture of the IL-2-producing MC12 clones to the tumorigenic CD80(+) MC12 cell doses could completely inhibit the tumorigenicity of the CD80(+) cells. Insertion of the CD80 gene into sarcoma cells substantially enhanced the adhesive interaction between the MC12 sarcoma and syngeneic T lymphocytes.

The present study was designed to examine the kinetics and function of peritoneal exudate cells (PEC) during local interleukin 2 (IL-2) gene therapy of the X63-Ag8.653 plasmacytoma growing in the peritoneal cavity. BALB/c mice were inoculated i.p. on day 0 with a tumorigenic dose of the syngeneic plasmacytoma and on day 1 with non-tumorigenic plasmacytoma cells carrying an inserted IL-2 gene and producing constitutively IL-2. At regular time intervals the experimental mice were sacrificed and their peritoneal exudate cells were used for phenotypic analysis and Cr-51 microcytotoxicity assay. On the first day after i.p. inoculation of the genetically modified plasmacytoma cells the percentage of Thy 1.2+, CD3+, TCR alphabeta+ T lymphocytes and NK+ cells in the peritoneal fluid dramatically increased. The levels of the positive cells continually decreased until day 11, when the values of normal, healthy mice were obtained. The percentage of Thy 1.2+ and CD3+ cells remained at these, or slightly lower values, until the end of the observation period. A similar, though more slowly descending kinetics was seen in the CD5+ cell population, whereas the CD8+ cells, compared to the controls, exhibited only a short-term peak between days 3 and 5, and the values of TCR alphabeta+ and NK+ cells exhibited a second peak between days 25 and 48. The percentage of TCR gammadelta+ cells showed a permanent, moderately elevated plateau from day 1 till the end of the observation period. In control, untreated mice, inoculated i.p. with the X63-Ag8.653 plasmacytoma, the kinetics of peritoneal exudate cells was different. A moderate, permanent elevation of all of the T and NK cell subsets examined occured during the observation period. In addition, the percentage of TCR alphabeta+, TCR gammadelta+ and NK+ cells further increased continually from day 11 till the end of the observation period. The cytolytic activity of the peritoneal exudate cells was examined in vitro concurrently with FACS phenotyping. Free tumour-specific killer cells generated in the peritoneal fluid due to the local IL-2 gene therapy were found only on day 6, and these cells were cytolytic for both, the parental X63-Ag8.653 and the genetically modified X63-m-IL-2 plasmacytoma cells.

We have examined the effect of IL-12-producing cellular vaccines on the cytotoxicity and proliferative potential of CD45+ tumour-infiltrating cells (TIL) in mice carrying syngeneic TC-1 and TC-1/A9 HPV 16-associated tumours after chemotherapy with CBM-4A ifosfamide derivative. The chemotherapy resulted in the decrease of the CD4+ and CD8+ TIL, increase of the Gr-1+/CD11b+ TIL, no changes in the infiltration with CD4+/CD25+ Treg TIL, and decrease of the cytolytic and proliferative potential of the CD45+ TIL. Subsequent immunotherapy with the IL-12-producing, genetically modified TC-1 (TC-1-IL-12) cells increased tumour infiltration with CD8+ and CD4+ cells, decreased the Gr-1+/CD11b+ cells, and increased the cytolytic and proliferative potential of the CD45+ TIL. Taken together, these findings suggest that peritumoral administration of the IL-12-producing cellular vaccine can restore the cytolytic potential and inhibit immunosuppressive TIL-dependent mechanisms in the individuals bearing HPV 16-associated tumours, and explain our previously described tumour-inhibitory effects of the vaccine in mice with minimal residual disease after the tumour chemotherapy.