Rationale: An immunosuppressive tumor microenvironment (TME) is a major obstacle in tumor immunotherapy. Stimulator of interferon genes (STING) agonists trigger an inflammatory innate immune response to potentially overcome tumor immunosuppression. While STING agonists may hold promise as potential cancer therapy agents, tumor resistance to STING monotherapy has emerged in clinical trials, and the mechanisms remain unclear. Methods: The in vivo anti-tumor immunity of STING agonist ADU-S100 (S100), plus anti-T cell immunoglobulin and mucin-domain containing-3 antibody (αTim-3) were measured using murine tumor models. Tumor-specific T cell activation and alterations in the TME were detected using flow cytometry. The maturation and function of dendritic cells (DC) were also measured using flow cytometry, and the importance of CD4⁺ T cells in combination therapy was measured by blocking antibodies. Additionally, the effect of S100 on CD4⁺ T was verified via in vitro assays. Lastly, the impact of conventional dendritic cells (cDC) 2 with a high expression of Tim-3 on survival or therapeutic outcomes was further evaluated in human tumor samples. Results: S100 boosted CD8⁺ T by activating cDC1 but failed to initiate cDC2. Mechanistically, the administration of S100 results in an upregulation of Tim-3 expressed in cDC2 (Tim-3⁺cDC2) in both mice and humans, which is immunosuppressive. Tim-3⁺cDC2 restrained CD4⁺ T and attenuated the CD4⁺ T-driven anti-tumor response. Combining S100 with αTim-3 effectively promoted cDC2 maturation and antigen presentation, releasing CD4⁺ T cells, thus reducing tumor burden while prolonging survival. Furthermore, high percentages of Tim-3⁺cDC2 in the human TME predicted poor prognosis, whereas the abundance of Tim-3⁺cDC2 may act as a biomarker for CD4⁺ T quality and a contributing indicator for responsiveness to immunotherapy. Conclusion: This research demonstrated that blocking Tim-3 could enhance the anti-tumor immunity of STING agonist ADU-S100 by releasing CD4⁺ T cells through regulating cDC2. It also revealed an intrinsic barrier to ADU-S100 monotherapy, besides providing a combinatorial strategy for overcoming immunosuppression in tumors.

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