A Tumor-Boundary Niche Enriched for PD-1+CD8+T Cells and BTLA+B Cells Drives Resistance to Immunotherapy in NSCLC.

CD8+T cells are central mediators of antitumor immunity, and their functional heterogeneity profoundly influences immunotherapy efficacy. We characterized the spatial distribution of CD8+T cell subsets stratified by PD-1 expression within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC). Our analyses revealed that PD-1+CD8+T cells were more prevalent in the non-central regions than in the tumor core, and higher abundance of these cells in the non-central zone was associated with poorer response to PD-1 blockade. Enrichment of PD-1+CD8+T cells diminished cancer cell apoptosis induced by anti-PD-1 mAb treatment in an in vitro co-culture system modeling in vivo conditions. The PD-1+CD8+T cell subset exhibited significantly reduced clonal diversity, as determined by TCR Vβ repertoire analysis, single-cell T cell receptor sequencing (scTCR-seq), and GLIPH2 clustering, along with impaired effector functions and distinct transcriptional programming. Mechanistically, IL-6 produced dominantly by non-central tumoral BTLA+B cells promoted PD-1 expression on CD8+T cells, mirroring the spatial pattern in tumoral PD-1+CD8+T cells. Our data further identify a spatially organized functional unit comprising PD-1+CD8+T cells and neighboring BTLA+B cells within the non-central tumor region. This specialized niche, defined by anatomical localization, functional state, TCR repertoire, and intercellular crosstalk, contributes to the establishment of resistance to immune checkpoint blockade.