Differences of tumor-recruiting myeloid cells in murine squamous cell carcinoma influence the efficacy of immunotherapy combined with a TLR7 agonist and PD-L1 blockade

Abstract

Objectives: The immune status of the tumor microenvironment has a marked impact on clinical outcomes. Here we examined the immune environments of tumor-infiltrating leukocytes (TILes) in two murine models of squamous cell carcinoma and compared the effects of immunotherapeutic agents, including a TLR7 agonist and an immune checkpoint inhibitor, and a chemotherapeutic agent, gemcitabine, in these models.

Materials and methods: TILes from NR-S1- and SCCVII-grafted mice were analyzed by flow cytometry. NR-S1-inoculated mice received resiquimod (a synthetic TLR7 agonist), an anti-PD-L1 antibody, or both, and tumor growth and TILs were examined. Gemcitabine was administered to deplete CD11b⁺ cells.

Results: More than 50% of TILes from NR-S1- and SCCVII-inoculated mice were CD11b⁺Gr-1⁺ cells. A major fraction of NR-S1 CD11b⁺ cells was Ly6G^highLy6C^low-negaF4/80^- tumor-associated neutrophils (TANs) and the majority of SCCVII CD11b⁺ cells were Ly6G^lowLy6C^-F4/80⁺ tumor-associated macrophages. NR-S1 TANs did not express MHC class II and CD86, but did express reactive oxygen species and PD-L1. Resiquimod, alone and in combination with an anti-PD-L1 antibody, did not regress NR-S1 tumors, but the combination increased the CD8/regulatory T cell-ratio, and IFN-γ and PD-1 expression in CD8⁺ TILes. Pre-administration of low-dose gemcitabine prior to the combination treatment suppressed the progression of NR-S1 tumors.

Conclusions: NR-S1 tumors with abundant recruitment of TANs were resistant to treatments with a TLR7 agonist, alone and in combination with PD-1 blockade, and required an additional gemcitabine treatment. The phenotype and status of tumor-infiltrating CD11b⁺ myeloid cells may influence the efficacy of immunotherapeutic agents.

Keywords: Anti-tumor immunity; Immune checkpoint inhibitor; Immunotherapy; Murine model; Myeloid-derived suppressor cells (MDSC); Squamous cell carcinoma (SCC); TLR7 agonist; Tumor-infiltrating lymphocytes (TIL).