The Proximity of PD-1−CD103+ Tissue-Resident CD8+ T Cells to Tumor Cells Is Correlated with Improved Clinical Outcomes in Patients with Cholangiocarcinoma

<b>Background/Objectives:</b> Cholangiocarcinoma (CCA) is characterized by a heterogeneous immune microenvironment, where the prognostic significance of CD8⁺ tissue-resident memory T (T_RM) cell activation and spatial positioning remains to be fully elucidated. This study investigated how the activation phenotypes and their spatial distribution relative to tumor cells influence anti-tumor immunosurveillance and predict clinical outcomes in CCA. <b>Methods:</b> Multiplex immunohistochemistry (mIHC) and single-cell RNA sequencing (scRNA-seq) were employed to characterize naïve (PD-1^-CD103⁺CD8⁺) and exhausted (PD-1⁺CD103⁺CD8⁺) subsets. G-cross function analysis was utilized to quantify the spatial proximity between these specific T_RM subsets and tumor cells, correlating the spatial interaction with patient overall survival. <b>Results:</b> scRNA-seq profiling revealed that PD-1^-CD103⁺CD8⁺ T_RM cells were enriched in genes associated with lymphocyte activation and cytotoxicity, while PD-1⁺CD103⁺CD8⁺ T_RM cells exhibited an exhaustion signature. Spatially, PD-1^-CD103⁺CD8⁺ T_RM cells exhibited increased interactions with tumor cells, whereas PD-1⁺CD103⁺CD8⁺ T_RM cells showed reduced engagement. Therefore, the close proximity of PD-1^-CD103⁺CD8⁺ T_RM cells to tumor cells was identified as a significant predictor of favorable clinical outcomes. <b>Conclusions:</b> The activation state of CD8⁺ T_RM cells combined with their spatial localization constitutes a critical prognostic factor in CCA. Effective anti-tumor immunosurveillance relies on the direct engagement of naïve T_RM cells with tumor cells. These findings highlight the potential of PD-1-targeted immunotherapies to remodel the spatial proximity of the tumor microenvironment, potentially promoting the redistribution of effector cells into tumor-proximal regions.