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

Abstract

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.

Distinct Responsiveness of Tumor-Associated Macrophages to Immunotherapy of Tumors with Different Mechanisms of Major Histocompatibility Complex Class I Downregulation

“…Due to the variability in immunogenicity of the used cell lines and the growth of induced tumors, different experimental conditions were used for each cell line (see Materials and Methods). Treated tumors were collected either 2 days after IT termination when tumors induced with TC-1/A9 cells were usually partially regressed, or 9 days after IT when the growth of TC-1/A9-induced tumors was restored [ 15 ].…”

“…Total RNA for next-generation sequencing (NGS) was isolated from mouse tumor samples (3 per group) with the NucleoSpin RNA kit (Macherey Nagel, Düren, Germany), according to the manufacturer’s protocol, as described previously [ 15 ]. The integrity of the RNA was determined by the Experion RNA StdSens assay (Bio-Rad Laboratories), with an RNA integrity number of higher than 9.3 for all samples.…”

“…The NO concentration was measured in the form of NO 2 − , which is easily detected with Griess reagent, composed of 0.2% naphthylethylenediamine dihydrochloride (Sigma-Aldrich, 222488) and 2% sulphanilamide (Sigma-Aldrich, S9251) in 5% phosphoric acid solution, as previously described [ 15 ]. Briefly, equal volumes of the cell culture supernatant and Griess reagent were mixed, and the absorbance was measured at 540 nm using a microplate reader (Tecan).…”

“…Moreover, a better understanding of tumor complexity has shown that CTL efficacy is impaired by the immunosuppressive character of the tumor microenvironment (TME) [ 12 ]. In this context, IT involving cells of innate immunity, such as macrophages, which abundantly infiltrate the TME and show a high capacity to eliminate neoplastic cells when adequately stimulated, appears as an attractive and complementary strategy [ 13 ], especially in the treatment of weakly immunogenic tumors [ 14 , 15 ]. These macrophages are classified as M1 or classically activated macrophages and can exert an anti-tumor effect through the production of high levels of proinflammatory cytokines, such as interleukin (IL)-12 and tumor necrosis factor (TNF)-α, as well as radical oxygen and nitrogen species, such as nitric oxide (NO), which is produced by the enzyme inducible nitric oxide synthase (iNOS).…”

Anti-tumour effects of a xenogeneic fibroblast activation protein-based whole cell tumour vaccine in murine tumour models

“…the Fas ligand) and increased cytokine (e.g. TGF-b and interleukin-10) delivery [30,31], cancerous cells can inhibit antigen presentation by selectively losing tumour-specific antigens and downregulating MHC-I molecule expression, thus directly avoiding T cell recognition [32,33] or by upregulating ligands, such as PD-L1, that inhibit T cell receptors, which leads to the transient activation or depletion of limited antigen-specific T cell populations and attenuates specific cytotoxicity during the immune response [10,11,34]; 2. The infiltration of various cell subpopulations in tumours blocks the cytotoxicity of CTL or NK/T cells to abnormal cells [35]; 3.…”