Development of a proximity ligation-imaging mass cytometry platform for spatially resolved immune checkpoint analysis

Introduction: Immune checkpoint inhibitors (ICIs) have transformed cancer therapy, yet durable clinical benefit remains limited to a subset of patients. Current biomarkers, such as PD-L1 immunohistochemistry (IHC), reflect protein abundance rather than functional receptor-ligand engagement and therefore provide only modest predictive value. To address this, we previously demonstrated that mapping PD1-PD-L1 interactions using a proximity ligation assay (PLA) outperforms PD-L1 IHC in predicting immunotherapy response in non-small cell lung cancer (NSCLC) [1]. Methods: Building on these findings, we developed a triplex PLA detecting PD1-PD-L1, PD1-PD-L2, and CD8-MHC I interactions and integrated it with Imaging Mass Cytometry (IMC). This approach enables high-plex (≈40 markers) spatial quantification of active immune signaling pathways alongside detailed immune phenotyping. The platform was applied to human tonsil tissue as a biological control and to pre-treatment NSCLC and triple-negative breast cancer (TNBC) biopsies to map checkpoint engagement, antigen recognition, and T-cell functional states within the tumor microenvironment. Results: In tonsil, PD1-PD-L1 and PD1-PD-L2 interactions were observed primarily between CD8⁺ T cells and follicular B cells, while additional PD1-PD-L1 interactions occurred between CD8⁺ T cells and CD68⁺ macrophages. These patterns suggest partly distinct cellular contexts for PD-L1 and PD-L2 engagement in lymphoid tissue. Consistent with this, preliminary tumor data indicate that PD1-PD-L2 interactions are relatively rare in TNBC compared with NSCLC, suggesting that PD-L2 involvement varies across tissue types and immune environments. Conclusion: PLA-IMC provides a functional, spatially resolved platform for screening and quantifying active immune checkpoint signaling directly in tissue. This approach holds promise to identify functional biomarkers for refined patient stratification and mapping of context-specific checkpoint activity to guide rational immunotherapy combinations in solid tumors. References: [1] Lindberg A, Muhl L, Yu H, et al. In situ detection of programmed cell death protein 1 and programmed death ligand 1 interactions as a functional predictor for response to immune checkpoint inhibition in NSCLC. J Thorac Oncol. 2025; 20:625-640.