Spatial mapping of PD1-PD-L2 interactions suggests an additional checkpoint axis in non-small cell lung cancer

Background: Immune checkpoint inhibitors have transformed non-small cell lung cancer (NSCLC) treatment, yet durable benefit remains limited. Current biomarkers, such as PD-L1 immunohistochemistry (IHC), measure protein abundance rather than functional PD1-ligand engagement and therefore provide limited predictive value. The second PD1 ligand, PD-L2, has often been ignored, but may influence immune regulation in the cellular context. Therefore, the activation of the PD1-PD-L1 axis is not sufficient to explain clinically relevant immune mechanisms. Methods: We analyzed a tissue microarray of early-stage, surgically resected NSCLC (N=345), a cohort previously extensively immunoprofiled, including PD-L1 IHC and PD1-PD-L1 proximity ligation assay (PLA) [1]. We extended this work using fluorescent PLA (co-stained for CD3 and pan-cytokeratin) to visualize PD1-PD-L2 interactions across tumor, stromal, and T-cell compartments, alongside PD-L2 protein expression by IHC. Image analysis was performed in QuPath. Results: In normal lung, PD-L2 was primarily expressed on macrophages and occasional granulocytes. Among tumors, squamous cell carcinomas (SqCC) showed higher PD-L2 expression than adenocarcinomas (AD) (p=0.003) and increased PD1-PD-L2 interactions (p=0.047), consistent with greater immune infiltration. Notably, amplification of 9p24.1, including the PD-L1 and PD-L2 locus, was also observed more frequent in SqCC. PD1-PD-L2 interactions displayed a distinct immune contexture from PD1-PD-L1. Within tumor cell compartment, PD1-PD-L2 correlated only weakly with CD4⁺, CD8⁺, CD163⁺, FoxP3⁺, PD1, and PD-L1, while PD1-PD-L1 showed strong associations with these immune populations, indicating partly distinct mechanisms of immune regulation. In the stroma, both ligands showed more similar immune-association patterns. Clinically, high PD1-PD-L1 interaction was generally associated with better prognosis. In contrast, PD1-PD-L2 interaction in SqCC showed only a trend toward better survival (p=0.061), whereas in AD it demonstrated a trend toward shorter survival (p=0.092). Also, compartment-specific PD1-PD-L2 interactions in the tumor stroma were associated with poor prognosis, particularly in SqCC with T cell-rich stroma (p=0.033). Conclusion: Our findings suggest that PD1-PD-L2 represents a spatially and biologically distinct checkpoint axis in NSCLC, differing from PD1-PD-L1 in immune phenotypes and clinical associations. Including PD-L2-specific interaction analyses may enhance understanding of checkpoint biology and support refined patient stratification, particularly in SqCC, where PD-L2 co-amplification and upregulation appear to be more common. References: [1] Lindberg A, Muhl L, Yu H, et al. J Thorac Oncol. 2025;20:625-640.