Tumor-associated macrophages (TAMs) plentifully infiltrate the tumor microenvironment (TME), but their role in anti-tumor immunity is controversial. Depending on the acquired polarization, they can either support tumor growth or participate in the elimination of neoplastic cells. In this study, we analyzed the TME by RNA-seq and flow cytometry and examined TAMs after ex vivo activation. Tumors with normal and either reversibly or irreversibly decreased expression of major histocompatibility complex class I (MHC-I) molecules were induced with TC-1, TC-1/A9, and TC-1/dB2m cells, respectively. We found that combined immunotherapy (IT), composed of DNA immunization and the CpG oligodeoxynucleotide (ODN) ODN1826, evoked immune reactions in the TME of TC-1- and TC-1/A9-induced tumors, while the TME of TC-1/dB2m tumors was mostly immunologically unresponsive. TAMs infiltrated both tumor types with MHC-I downregulation, but only TAMs from TC-1/A9 tumors acquired the M1 phenotype upon IT and were cytotoxic in in vitro assay. The anti-tumor effect of combined IT was markedly enhanced by a blockade of the colony-stimulating factor-1 receptor (CSF-1R), but only against TC-1/A9 tumors. Overall, TAMs from tumors with irreversible MHC-I downregulation were resistant to the stimulation of cytotoxic activity. These data suggest the dissimilarity of TAMs from different tumor types, which should be considered when utilizing TAMs in cancer IT.

• Keywords
• colony-stimulating factor-1, immunotherapy, macrophages, major histocompatibility complex, repolarization, tumor,
• Publication type
• Journal Article MeSH

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8⁺ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

• Keywords
• CpG ODN, DNA immunization, MHC-I, cancer immunotherapy, tumor-associated macrophages, α-galactosylceramide,
• MeSH
• Adjuvants, Immunologic therapeutic use   MeSH
• CD8-Positive T-Lymphocytes immunology   MeSH
• Down-Regulation MeSH
• Neoplasms, Experimental therapy   MeSH
• Galactosylceramides immunology   MeSH
• Immunotherapy methods   MeSH
• Macrophages immunology   MeSH
• Histocompatibility Antigens Class I immunology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Cell Line, Tumor MeSH
• Oligodeoxyribonucleotides immunology   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Adjuvants, Immunologic MeSH
• Galactosylceramides MeSH
• Histocompatibility Antigens Class I MeSH
• Oligodeoxyribonucleotides MeSH

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.

• MeSH
• Azacitidine pharmacology   MeSH
• DNA Primers MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Neoplasms, Experimental drug therapy   immunology   therapy   virology   MeSH
• Immunotherapy * MeSH
• Real-Time Polymerase Chain Reaction MeSH
• Human papillomavirus 16 isolation & purification   MeSH
• Methylation MeSH
• Histocompatibility Antigens Class I immunology   MeSH
• Mice, Inbred C57BL MeSH
• Mice MeSH
• Antimetabolites, Antineoplastic pharmacology   MeSH
• Base Sequence MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Azacitidine MeSH
• DNA Primers MeSH
• Histocompatibility Antigens Class I MeSH
• Antimetabolites, Antineoplastic MeSH