Improved Anti-Tumour Adaptive Immunity Can Overcome the Melanoma Immunosuppressive Tumour Microenvironment

Abstract

Clinical benefits obtained from checkpoint blockade regimens demonstrate the importance of overcoming the immunosuppressive tumour microenvironment (TME) in cancer immunotherapy. Intravenous (i.v.) injection of B16 melanoma cells (H-2Kb) leads to lethal disseminated pulmonary metastasis in Balb/c recipients (H-2Kd). This lack of immune control is related to low major histocompatibility complex (MHC) expression on B16 cells which is associated with delayed and decreased anti-tumour adaptive immune responses (e.g., alloantibody formation) as: (i) other tumour types with normal H-2Kb expression are rejected with concomitant antibody production; (ii) preincubation of B16 with IFN-gamma to upregulate H-2Kb expression resulted in improved antibody production and anti-tumour activity. The delayed/decreased anti-tumour adaptive immune responses induced by B16 inoculation is not able to interrupt progression of primary metastases, while it is able to effectively eliminate secondary inoculated subcutaneously (s.c.) B16 cells from progression. This is due to the presence of an immunosuppressive TME within the primary metastases characterized by increased regulatory T cells (Tregs) and an increased T helper cells (Th) 2/1 profile. These tumour-induced immunosuppressive T cell populations are counteracted by improved adaptive immunity via active and passive immunization, resulting in effective elimination of the TME, destruction of the metastatic tumour and a reversal of Th2/1 profile in a time-sensitive manner. Thus, we here demonstrate that the TME is not irreversible and adaptive immunity is able to eradicate established solid tumour and its immunosuppressive TME. This study will help design treatments to overcome the immunosuppressive effect of the TME and improve efficacy of cancer immunotherapy.

Intratumoral immunotherapy with anti-PD-1 and TLR9 agonist induces systemic antitumor immunity without accelerating rejection of cardiac allografts

“…Normally, B16 melanoma expressing low MHC class I antigens undergo progression and regression when inoculated s.c. into allogenic Balb/c mice 15 . However, the expression of MHC class I antigens was upregulated after inoculation of B16 cells into the inflammatory environment in the allogenic recipients 15 . Balb/c mice that are immune tolerant to C57BL/6 cardiac allograft showed for B16 melanoma progression at a similar speed compared to C57BL/6 mice.…”

“…immunotherapy should provoke immune response directed to both. Normally, B16 melanoma expressing low MHC class I antigens undergo progression and regression when inoculated s.c. into allogenic Balb/c mice 15 . However, the expression of MHC class I antigens was upregulated after inoculation of B16 cells into the inflammatory environment in the allogenic recipients 15 .…”

“…After a 3‐day incubation, responder cell surface expression of activation marker CD69 was stained and analyzed by FACScan. Meanwhile, supernatant IL‐2 was quantified using flow cytometry microbeads 15 …”

“…LAK cell–mediated cytotoxicity was evaluated in antibody‐dependent cellular cytotoxicity (ADCC) assay by incubating LAK cells with testing serum–coated target B16 cells at a ratio of 20:1 for 4 hours at 37°C with 5% CO 2 . The ratio of effector‐to‐ target cells of 20:1 was selected based on pretitration 15 …”

“…To verify that i.t. immunotherapy preferentially enhances tumorspecific immunogenicity, we established a model where B16 melanoma was inoculated s.c. in Balb/c mice bearing a tolerized C57BL/6 cardiac allograft induced by the PI4Ki immunosuppressive regimen.Thus both B16 cells and cardiac allografts shared MHC antigens.B16 melanoma undergoes spontaneous progression and regression after s.c. inoculation in naïve allogenic Balb/c mice,15 whereas B16 melanoma (s.c.) progressed well in Balb/c mice bearing a tolerized C57BL/6 cardiac allograft (Figure 7A,B). Intratumoral immunotherapy inhibited B16 melanoma growth in the tolerant mice (Figure 7C,D), and this was associated with an increased incidence of acute rejection of tolerized cardiac allografts (4/10) (Figure 7E), due to the induction of immune responses directed against tumor antigens including MHC molecules, which were shared by tolerized cardiac allografts.…”