Antitumor synergy of CHO-chemotherapy and αCD40/CpG-immunotherapy involves activation of tumor-infiltrating Mϕ

2081

We recently reported on T cell-/NK cell-independent MΦ-mediated antitumor effects induced in SCID/beige mice by synergistic treatment with anti-CD40 mAb + CpG-ODN immunotherapy (αCD40/CpG-IT). In this study we tested if αCD40/CpG will activate MΦ in mice immunosuppressed following chemotherapy (CT). C57BL/6 mice received CT according to the CHO protocol (Cyclophosphamide 100 mg/kg, i.p., Doxorubicin, 3.3 mg/kg, i.p., Vincristine, 0.5 mg/kg, i.v., all given at once). In naïve mice, this treatment suppressed both the ability of T cells to respond to in vitro stimulation via CD3, and the ability of IL-2-activated NK cells to lyse YAC-1 tumor cells. In contrast, CHO-CT was found to prime peritoneal MΦ to in vitro stimulation with CpG, resulting in augmented nitric oxide (NO) production and suppression of tumor cell proliferation. The effect of CHO-CT on MΦ also involved upregulation of endogenous IFN-γ and IL-12. αCD40/CpG-IT given 3 days after CHO-CT partially restored responsiveness of T cells to in vitro CD3-stimulation, but not the ability of IL-2 stimulated NK cells to lyse YAC-1 cells. αCD40/CpG-IT synergized with CHO-CT in activating MΦ to secrete NO and suppress tumor cell proliferation, even without additional in vitro stimulation. In addition, CHO-CT + αCD40/CpG-IT - treatment resulted in synergistic upregulation of endogenous IFN-γ and IL-12 in peritoneal MΦ. Notably, similar induction of cytokines was observed in subcutaneous tumor-associated MΦ, which comprised the major population of B16 tumor-infiltrating leukocytes. Combining CHO-CT with αCD40/CpG-IT led to strong antitumor effects in C57BL/6 mice with established (~8-10 mm) subcutaneous B16 tumors. Hence, we conclude that CHO-CT and αCD40/CpG-IT synergize in activation of MΦ, including tumor-associated MΦ, and induction of antitumor effects in vivo.