Epigenetic plus cytokine therapy induces stem-like CD8⁺ T-cell reservoirs and intratumoral immune niches to overcome anti-PD-1 resistance

BACKGROUND: Checkpoint blockade (CPB) has limited efficacy in patients with colorectal cancer (CRC), warranting improved therapeutic strategies capable of remodeling the tumor microenvironment. Stem-like TCF1⁺CD8⁺ T cells, cytotoxic CD8⁺ T cells, and lymphoid aggregates have been associated with CPB responsiveness, either individually or as coordinated immune hubs. METHODS: We evaluated the combination of αPD-1, the class I histone deacetylase inhibitor entinostat, and the IL-15 superagonist N-803 in CPB-resistant murine CRC and breast cancer models with varying antigen presentation deficiencies. Proteomic, transcriptomic, spatial, and functional analyses were completed to identify treatment-specific immune remodeling. Translational relevance was assessed using publicly available CPB-treated cancer genomic datasets and functional assays with patient-derived peripheral blood mononuclear cells (PBMCs). RESULTS: Triple therapy increased stem-like TCF1⁺CD8⁺ T cells in tumor-draining lymph nodes and tumors, concomitant with increased intratumoral cytotoxic GZMB⁺CD8⁺ T cells. Therapeutic efficacy was associated with remodeling of the tumor microenvironment, uniquely marked by enrichment of intratumoral immune niches comprising TCF1⁺ and cytotoxic CD8⁺ T cells, type 1 conventional dendritic cells, and B cells, along with concerted cytokine and chemokine production. A transcriptional signature derived from triple therapy-induced immune niches predicted favorable clinical responses across multiple CPB-experienced cancer cohorts. These responses were functionally corroborated by increased markers of cytotoxicity in patient-derived PBMCs in vitro. CONCLUSIONS: Treatment with entinostat, N-803, and αPD-1 boosts anti-tumor intratumoral immune niches comprising TCF1⁺CD8⁺ T cells. Collectively, these findings suggest that TCF1⁺CD8⁺ T cell immune niches previously associated with αPD-1 responsiveness and lacking in non-responders, including most CRC patients, may be restored by the addition of entinostat and N-803 to αPD-1 therapy to treat αPD-1-refractory patients.