(D-129) Spatial Multiomics Profiling of Angioimmunoblastic T-cell Lymphoma

Introduction:: Angioimmunoblastic T-cell lymphoma (AITL) is a subtype of T-cell lymphoma that is characterized by a T follicular helper cell (TFH) phenotype. AITL presents with a highly complex tumor microenvironment with the tumor cells arranged on a scaffold of highly proliferating CD21+ follicular dendritic cells found in proximity to branching high endothelial venules (HEVs). The organized structure of the lymph node with distinct B cell and T cell zones is disrupted in the lymph node samples from patients with AITL. In this study, we utilize Deterministic Barcoding in-Tissue Sequencing (DBiT-seq) and Co-detection by indexing (CODEX) to map the transcriptomic and proteomic landscape of the lymph nodes from patients with AITL to uncover novel vulnerabilities of this tumor that would make it susceptible to immune suppression.


Materials and Methods:: In this study, 10 µm thick lymph node tissue sections on poly-L-lysine coated slides were prepared from archived FFPE tissue blocks by the Yale Pathology Tissue Services (YPTS). Hematoxylin and Eosin (H&E) staining was done on 1 slide. Next, we performed multiplexed immunofluorescence imaging (CODEX) for 12 immune-related markers: Podoplanin, CD20, CD4, CD8, CD68, CD45RO, CD3e, GranzymeB, PD1, Ki67, CD21 and CD34 using the PhenoCycler-Fusion system. Cell segmentation was performed using QuPath and downstream analysis done using the Seurat package. On an adjacent section, we used DBiT-seq, a microfluidic based method to sequence the whole transcriptome, together with a panel of 160 CITE-seq antibodies. Downstream analysis was done using Seurat.


Results, Conclusions, and Discussions:: We found 10 spatially distinct clusters differentially expressing the marker proteins. We also identified cellular neighborhoods and their composition and the cells that directly interact with the tumor cells. Using DBIT-seq, we found an average of ~670 genes per spot. UMAP showed 6 clusters and we identified a distinct cluster of B cells. We also found the expression of CXCL13, TET2, DNMT3A and PDCP1 which are markers associated with AITL. We envision that by combining multiplexed immunofluorescence and DBiT-seq will further our understanding of this cancer.


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