ABSTRACT
In the majority of human tumors, downregulation of major histocompatibility complex class I (MHCI) expression contributes to the escape from the host immune system and resistance to immunotherapy. Relevant animal models are therefore needed to enhance the efficacy of cancer immunotherapy. As loss of ß2 microglobulin expression results in irreversible downregulation of surface MHCI molecules in various human tumors, the ß2 microglobulin gene (B2m) was deactivated in a mouse oncogenic TC1 cell line and a TC1/dB2m cell line that was negative for surface MHCI expression was derived. Following stimulation with interferon γ, MHCI heavy chains, particularly the H2Db molecules, were found to be expressed at low levels on the cell surface, but without ß2 microglobulin. B2m deactivation in TC1/dB2m cells led to reduced proliferation and tumor growth. These cells were insensitive to DNA vaccination and only weakly responsive to combined immunotherapy with a DNA vaccine and the ODN1826 adjuvant. In vivo depletion demonstrated that NK1.1+ cells were involved in both reduced tumor growth and an antitumor effect of immunotherapy. The number of immune cells infiltrating TC1/dB2minduced tumors was comparable with that in tumors developing from TC1/A9 cells characterized by reversible MHCI downregulation. However, the composition of the cell infiltrate was different and, most importantly, infiltration with immune cells was not increased in TC1/dB2m tumors after immunotherapy. Therefore, the TC1/dB2m cell line represents a clinically relevant tumor model that may be used for enhancement of cancer immunotherapy.
Subject(s)
Cell Line, Tumor/metabolism , Histocompatibility Antigens Class I/immunology , Neoplasms/immunology , Animals , Gene Expression Regulation, Neoplastic/genetics , Histocompatibility Antigens Class I/genetics , Humans , Immunotherapy , Interferon-gamma/immunology , Mice , Neoplasms/genetics , Neoplasms/pathology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/pathology , beta 2-Microglobulin/genetics , beta 2-Microglobulin/immunologyABSTRACT
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⺠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.
Subject(s)
Histocompatibility Antigens Class I/immunology , Immunotherapy/methods , Neoplasms, Experimental/therapy , Adjuvants, Immunologic/therapeutic use , Animals , CD8-Positive T-Lymphocytes/immunology , Cell Line, Tumor , Down-Regulation , Female , Galactosylceramides/immunology , Macrophages/immunology , Mice , Mice, Inbred C57BL , Oligodeoxyribonucleotides/immunologyABSTRACT
A bioactive O-carboxymethyl chitosan (CMCS) hydrogel crosslinked with natural phenolics with potential application in wound dressings was synthesized using a laccase from Myceliophthora thermophila (MTL). The highest degree of cross-linking (49.7%) was achieved with catechol. All the phenolic-CMCS hydrogels synthesized showed excellent anti-oxidant properties with a free radical scavenging activity up to 4-fold higher than in the absence of the phenolics, as quantified by the di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium (DPPH) assay. In addition, the hydrogels produced showed an anti-inflammatory effect as evidenced by the inhibition of enzymes [myeloperoxidase (MPO), matrix-metalloproteinase-1 (MMP-1) and human neutrophil elastase (HNE)] over-expressed in chronic wounds. Sinapyl-CMCS hydrogels showed an MMP-1 inhibition of 37%. Further, the phenolic-CMCS hydrogels did not affect the viability of the NIH 3T3 mouse fibroblast cell line and were also able to slowly release human fibroblast growth factor 2, reaching 48.3% over a period of 28days. This study thus shows the possibility of synthesizing multifunctional bioactive chitosan based hydrogels with anti-oxidant and anti-inflammatory properties using natural occurring phenolics as crosslinkers.