High-Dimensional Characterization of Tertiary Lymphoid Structures Using Orion™, HALO®, and HALO AI
Tertiary lymphoid structures shape immune activation and immunotherapy response, but resolving their cellular makeup across tissues is difficult; this EACR 2026 poster pairs RareCyte's Orion 18-plex whole-slide imaging with Indica Labs' HALO and HALO AI analysis to phenotype TLS in reactive tonsil, Sjögren's salivary gland, and non-small cell lung cancer.
Presented by Indica Labs.
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- One Orion scan produced an 18-plex whole-slide image that HALO and HALO AI then analyzed. RareCyte's Orion instrument imaged FFPE sections of three human tissues — reactive tonsil, Sjögren's salivary gland, and non-small cell lung cancer — in a single scan, and Indica Labs' HALO and HALO AI segmented epithelium, stroma, and B- and T-cell zones before phenotyping cells across all three tissues.
- Supervised phenotyping and unsupervised clustering together mapped TLS beyond predefined gating. HALO's Highplex FL module drove cell segmentation and supervised phenotyping, while the High Dimensional Analysis module ran unsupervised K-means (k=7) and Phenograph clustering visualized with UMAP, surfacing phenotypic diversity within TLS that predefined gating strategies alone did not capture.
- Immune-rich and dense tumor zones within a single NSCLC carried sharply different TLS. Immune-rich tumor TLS resembled tonsil, with mature and proliferating germinal-center-like B cells alongside memory cytotoxic and helper T cells, whereas dense tumor TLS were dominated by fibroblasts and immunosuppressive M2 and PD-L1+ macrophages with more exhausted CD4+ T cells; salivary-gland TLS were separately enriched in CD4+ T cells and M1 macrophages.
