PI3Kδ inhibition alters CD8 T cell differentiation and reprograms the tumor microenvironment following adoptive immunotherapy.

T cell exhaustion remains a significant barrier to effective adoptive cell therapy in solid tumors. Here, we demonstrate that in vitro treatment with the PI3Kδ inhibitor CAL-101 generates T cells with enhanced stemness and metabolic fitness. These cells show increased mitochondrial dependence and spare respiratory capacity while maintaining normal basal metabolism. Under chronic antigen stimulation, CAL-101-treated T cells, including human T cells, resist terminal exhaustion and maintain stem-like properties. Using single-cell RNA sequencing and spatial transcriptomics of B16 melanoma tumors, we found that CAL-101-treated T cells preferentially differentiate into progenitor exhausted T cells within the tumor microenvironment. These cells demonstrate enhanced tumor infiltration and upregulate the Cxcl10/Cxcr3 signaling axis. The tumor microenvironment of tumors containing CAL-101-treated T cells show reduced glycolysis, oxidative phosphorylation, and proliferation, while exhibiting increased proinflammatory signaling and decreased presence of immunosuppressive tumor-associated macrophages. Single-cell analysis reveals that the CAL-10-treated T cells concurrently increase oxidative phosphorylation, proliferation, and immune signaling pathways. Mechanistically, CAL-101-treated T cells maintain high expression of stemness-associated genes (Tcf7, Slamf6) while resisting expression of genes associated with terminal exhaustion (Tim3, Mt1/2). These findings reveal the mechanisms behind how PI3Kδ inhibition generates T cells capable of establishing and maintaining an antitumor immune response, suggesting a promising strategy for improving adoptive cell therapy outcomes in solid tumors.