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Background TIGIT, an immune checkpoint molecule, has attracted attention as a therapeutic target and is being developed in combination therapy with anti-PD-1/PD-L1 antibodies. While its mechanism of action has primarily been analyzed focusing on CD8+ T cell-mediated cytotoxicity, the involvement of other immune cells remains largely unclear.Methods The mouse breast cancer cell line FM3A was subcutaneously inoculated into C3H/HeN mice. After tumor establishment, anti-PD-L1 and anti-TIGIT antibodies were administered (day 1), and tumor volume was measured to evaluate the antitumor effect. On day 4 and day 8, tumor tissues were collected, and the effect on the immune response was analyzed using flow cytometry. CD8+ cells were depleted using an anti-CD8 depletion antibody. To evaluate cytotoxic activity against tumor cells, tumor-infiltrating NK cells and macrophages were isolated using MACS and co-cultured with FM3A cells in vitro.Results In the FM3A subcutaneous inoculation model, both anti-PD-L1 and anti-TIGIT antibodies demonstrated significant antitumor effects as monotherapies, with further significant enhancement observed with combination therapy. These antitumor effects were not significantly reduced under CD8+ cell depleted conditions.On day 4, a significant increase in the proportion of GzmB+ cells among tumor-infiltrating NK cells was observed in the anti-TIGIT antibody-treated group and the combination group. NK cells isolated from anti-TIGIT antibody treated tumors exhibited enhanced cytotoxic activity against FM3A cells. Concurrently, an increase in the percentage of iNOS+ cells among macrophages was observed in both monotherapy groups and the combination group. This increase in iNOS+ macrophage population was IFN-γ dependent. Macrophages isolated from tumors exhibited enhanced cytotoxic activity against FM3A cells in both monotherapy groups and the combination group.On day 8, Th cell infiltration in tumor tissue increased in both monotherapy groups and the combination group. Furthermore, IFN-g and TNF-a producing cell among Th cells were increased in anti-TIGIT antibody treated group and the combination group.Conclusions The combination of anti-PD-L1 and anti-TIGIT antibodies demonstrated significant antitumor effects compared to each monotherapy. Our findings suggest that enhanced cytotoxic activity of NK cells and macrophages contributes to these antitumor effects. Moreover, elevated cytokine production by Th cells may contribute the activation of macrophages.Ethics Approval All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Chugai Pharmaceutical Co.Ltd., and all animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Chugai Pharmaceutical Co., Ltd.