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Background INCB177054 is a selective inhibitor of diacylglycerol kinase (DGK)α and DGKζ. In preclinical tumor models, DGK inhibition combined with anti-PD1 therapy led to significant tumor growth inhibition and durable immune memory. To assess the immunomodulatory effects of DGK inhibition, we tested INCB177054 and an earlier compound, INCB171138, in CT26 and MC38 syngeneic tumor mouse models.Methods To examine the impact of DGK inhibition on immune cells in the tumor microenvironment (TME), we profiled CD45 + cells from CT26 tumors using single cell RNA-sequencing (scRNA-seq) after INCB171138 ± anti-PD1. Then, to further investigate such impact on T cells, we analyzed T cells from the tumor and spleen in the MC38 model treated with INCB177054 ± anti-PD1 using paired scRNA-seq and single cell T-cell receptor sequencing (scTCR-seq).Results Although the immune cell composition in CT26 tumors was not significantly altered, DGK inhibition induced profound shifts in activation states across multiple cell types. In macrophages, INCB171138 ± anti-PD1 triggered both interferon responses and TNF-α signaling. In natural killer cells, INCB171138 ± anti-PD1 upregulated gene signatures indicative of maturation and enhanced cytotoxicity. In CD8 + T cells, INCB171138 increased signatures of terminal exhaustion and cytotoxicity, similar to anti-PD1.When focusing on CD8+ T cells following INCB177054 treatment in the MC38 model, we observed a decrease in naïve or naïve-like T cells, and an increase in T cells with an exhaustion signature. Notably, INCB177054 + anti-PD1 increased a subset of T cells characterized by high expression of Pdcd1, Havcr2, and Entpd1, but low Tox expression. Within the tumor, most CD8+ T cells with the exhaustion signature exhibited high clonal sharing, consistent with derivation from progenitor exhausted T cells. A distinct cluster with low expression of exhaustion markers (Pdcd1, Havcr2, Entpd1, and Tox) showed increased clonal sharing with other CD8+ T cell subsets in the INCB177054 + anti-PD1 group, suggesting a potentially unique differentiation trajectory. As expected, splenic clonal sharing was limited. Complementary bulk TCR sequencing confirmed that INCB177054 + anti-PD1 enhanced clonal expansion of intratumoral T cells, with a trend toward increased expansion in the spleen.Conclusions Inhibition of DGK induced widespread immune cell activation in the TME directly or indirectly, including altered activation states in CT26 tumors and shifts in CD8 + T cell phenotypes in MC38 tumors. These findings support the immunostimulatory potential of DGK inhibition and the rationale for clinical evaluation. A phase 1/2 study of INCB177054 in select tumors (NCT06873789) is underway.