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 vivohistological 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 subpopulationsin vivoexhibited 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.

• Publikační typ
• časopisecké články MeSH

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.

• MeSH
• antigeny Ly imunologie   MeSH
• buňky NK imunologie   MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• ELISA MeSH
• experimentální nádory imunologie   MeSH
• lektinové receptory NK-buněk - podrodina B imunologie   MeSH
• lidský papilomavirus 16 MeSH
• MHC antigeny I. třídy imunologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• protinádorové vakcíny imunologie   MeSH
• průtoková cytometrie MeSH
• separace buněk MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• CD4-pozitivní T-lymfocyty imunologie   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• cytotoxické testy imunologické MeSH
• experimentální nádory imunologie   terapie   virologie   MeSH
• financování organizované MeSH
• genetická terapie MeSH
• ifosfamid analogy a deriváty   terapeutické užití   MeSH
• imunoenzymatické techniky MeSH
• imunoterapie MeSH
• infekce papilomavirem imunologie   terapie   virologie   MeSH
• inhibitory angiogeneze terapeutické užití   MeSH
• interleukin-12 fyziologie   MeSH
• lidé MeSH
• lidský papilomavirus 16 patogenita   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• proliferace buněk MeSH
• průtoková cytometrie MeSH
• tumor infiltrující lymfocyty imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

• Publikační typ
• abstrakt z konference MeSH

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.

• MeSH
• CD4-pozitivní T-lymfocyty metabolismus   MeSH
• cytokiny metabolismus   MeSH
• financování organizované MeSH
• imunitní systém imunologie   metabolismus   metabolismus   MeSH
• imunoterapie metody   MeSH
• interleukin-12 chemie   metabolismus   MeSH
• kinetika MeSH
• lidský papilomavirus 16 metabolismus   MeSH
• lymfatické uzliny patologie   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nádory metabolismus   virologie   MeSH
• protinádorové vakcíny MeSH
• receptor interleukinu-2 - alfa-podjednotka biosyntéza   MeSH
• regulace genové exprese u nádorů MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

Moderately immunogenic HPV 16-associated murine tumour cell line mimicking human HPV 16-associated neoplasms TC-1 (MHC class I(+)) and its variants, TC-1/P3C10 and TC-1/A9, with a marked down-regulation of MHC I molecules, were used to examine the effect of local interleukin 12 (IL-12) gene therapy for the treatment of early tumour transplants and minimal residual tumour disease obtained after cytoreductive chemotherapy (CMRTD). Experiments were designed to examine whether down-regulation of MHC class I molecules plays a role during chemotherapy and gene therapy of early tumour transplants. It was found that peritumoral administration of IL-12-producing tumour cell vaccines (single dose, day 8 after tumour cell administration) inhibited the growth of both TC-1 (MHC class I positive) tumours and their MHC class I-deficient variants. To investigate the antitumour effects in a clinically relevant setting, IL-12 gene therapy was utilised for the treatment of minimal residual tumour disease after cytoreductive chemotherapy. Intra-peritoneal treatment of tumour-bearing mice with ifosfamide derivative, CBM-4A, produced a significant tumour-inhibitory effect. This treatment was followed by peritumoral s.c. administration of genetically modified TC-1 (MHC class I positive) or MK16/I/IIIABC (MHC class I negative) vaccines producing IL-12 (single dose, day 7 after chemotherapy) or with recombinant interleukin 12 (rIL-12) in two cycles of 5 daily doses (days 8-19) after chemotherapy. This combined therapy significantly inhibited the growth of TC-1 and TC-1/A9 (MHC class I-) tumours. When the combined therapy of TC-1 (MHC class I positive) tumours was followed by peritumoral administration of bone marrow dendritic cell (BMDC) vaccines, the IL-12-mediated inhibitory effect was significantly boosted. In the next set of experiments, the impacts of chemotherapy and IL-12 adjuvant therapy on MHC class I surface expression were assessed. Chemotherapy and gene therapy of tumours led to the up-regulation of MHC I expression on MHC class I-deficient tumours (TC-1/A9 and TC-1/P3C10) and to down-regulation on MHC I-proficient tumours (TC-1). These findings indicate that the MHC I phenotype is not stable during tumour progression and treatment. Collectively, these results illustrate the efficacy of IL-12 gene therapy in combination with chemotherapy on HPV-associated tumours regardless of the level of MHC class I expression on the tumour cells.

• MeSH
• down regulace MeSH
• experimentální nádory virologie   MeSH
• financování organizované MeSH
• genetická terapie MeSH
• geny MHC třídy I MeSH
• HLA antigeny biosyntéza   MeSH
• ifosfamid analogy a deriváty   farmakologie   MeSH
• imunoterapie MeSH
• interleukin-12 fyziologie   genetika   MeSH
• lidé MeSH
• lidský papilomavirus 16 patogenita   MeSH
• modely nemocí na zvířatech MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• protinádorové vakcíny MeSH
• reziduální nádor MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH

• MeSH
• cytotoxické T-lymfocyty imunologie   MeSH
• dendritické buňky imunologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• imunoterapie metody   MeSH
• myši MeSH
• Papillomaviridae imunologie   MeSH
• peptidy imunologie   MeSH
• protinádorové vakcíny terapeutické užití   MeSH
• slezina imunologie   MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

• MeSH
• buňky NK MeSH
• cytokiny aplikace a dávkování   MeSH
• faktor stimulující granulocyto-makrofágové kolonie imunologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• genetická terapie genetika   MeSH
• interferon gama MeSH
• myši MeSH
• Papillomaviridae MeSH
• reziduální nádor chirurgie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH

• MeSH
• adjuvancia imunologická aplikace a dávkování   terapeutické užití   MeSH
• alkylační protinádorové látky terapeutické užití   MeSH
• experimentální nádory terapie   MeSH
• finanční podpora výzkumu jako téma MeSH
• ifosfamid analogy a deriváty   terapeutické užití   MeSH
• interleukin-12 terapeutické užití   MeSH
• interleukin-2 terapeutické užití   MeSH
• myši MeSH
• protinádorové vakcíny terapeutické užití   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• srovnávací studie MeSH

• MeSH
• cytokiny biosyntéza   MeSH
• geny MHC třídy I imunologie   MeSH
• interleukin-2 farmakologie   metabolismus   MeSH
• lidé MeSH
• nádorové buňky kultivované MeSH
• Papillomaviridae metabolismus   MeSH
• protinádorové látky farmakologie   MeSH
• průtoková cytometrie metody   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH