Experimental Combined Immunotherapy of Tumours with Major Histocompatibility Complex Class I Downregulation.

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.

Establishment and characterization of a mouse tumor cell line with irreversible downregulation of MHC class I molecules.

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.

Anti-inflammatory and anti-oxidant properties of laccase-synthesized phenolic-O-carboxymethyl chitosan hydrogels.

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.