Identification of agonist combinations that stimulate macrophages to induce anti-tumor T cells and overcome immunosuppression

Macrophages are the major immune cell population in most human solid cancers. Therefore, we hypothesized that appropriate activation of macrophages in tumors could convert an immunosuppressive into a proinflammatory tumor microenvironment and, thus, enable T cell-mediated killing of tumor cells. To identify appropriate activating receptors, we selected putative agonists from literature and used them for stimulation of primary monocyte-derived human macrophages as single agents or in combination. Stimulated macrophages were co-cultured with autologous T cells to test their ability to reactivate antigen-specific T cells. To mimic tumor-specific clustering, we expressed selected agonistic antibodies on the cell surface of tumor cells and used them in coculture assays to determine macrophage stimulation and T cell activation. For the most promising agonists and combinations, we also tested their ability to repolarize M2 to M1 macrophages and determined the induced cytokine and chemokine profile. The most effective agonist combinations were: CD40 + TLR4, CD40 + TLR7/8, CD40 + Clec5a, CD40 + CSF1R, CD40 + CSF2RB and TLR4 + TLR7/8. Macrophages that were stimulated with these combinations activated autologous antigen-specific T cells that were cytotoxic towards tumor cells. These combinations were also able to overcome TGF-beta-mediated immunosuppression in vitro. Tumor-specific activation of macrophages with these agonist combinations may represent a promising approach to enhance the anti-tumor immune response and increase survival of patients with macrophage-rich solid tumors.