Genetic Disruption of Pdcd-1 Upstream Enhancer Boosts T Cell Function and Antitumor Responses

• Established an upstream enhancer knockout (UpEnh KO) strain using CRISPR–Cas9 genome editing to dissect Pdcd-1 regulation in T cells. • UpEnh deletion reduced PD-1 expression across multiple T cell subsets, including thymocytes, peripheral naïve Treg, and γδ T cells, as well as tumor-infiltrating exhausted CD8+, CD4+ Tconv, Treg, and γδ T cells. • Loss of UpEnh from the Pdcd-1 locus improved CD8+ T cell exhaustion state, boosted γδ T cell activity, and promoted more robust anti-tumor responses. Programmed cell death 1 (PD-1) is an inhibitory receptor that drives T cell exhaustion in tumors, limiting antitumor immunity. Current PD-1 blockade therapies have shown limited success. To uncover new strategies for modulating PD-1, we investigated an upstream enhancer (UpEnh) of the Pdcd-1 gene using a CRISPR-Cas9 knockout mouse model. Deletion of the UpEnh reduced PD-1 expression across various T cell subsets. In a tumor setting, this deletion lowered PD-1 levels on intratumoral exhausted CD8⁺, conventional CD4⁺, Treg, and γδ T cells. This resulted in improved CD8⁺ and γδ T cell function and promoted stronger antitumor immunity. Our findings establish UpEnh as a critical regulator of PD-1, presenting a potential therapeutic target.