Epstein-Barr Virus Orchestrates Spatial Reorganization and Immunomodulation within the Classic Hodgkin Lymphoma Tumor Microenvironment

Classic Hodgkin Lymphoma (cHL) is a tumor composed of rare malignant Hodgkin and Reed-Sternberg (HRS) cells nested within a T-cell rich inflammatory immune infiltrate. cHL is associated with Epstein-Barr Virus (EBV) in ~25% of cases. The specific contributions of EBV to the pathogenesis of cHL remain largely unknown, in part due to technical barriers in dissecting the tumor microenvironment (TME) in high detail. Herein, we applied multiplexed ion beam imaging (MIBI) spatial proteomics on 6 EBV- positive and 14 EBV-negative cHL samples. We identify key TME features that distinguish between EBV-positive and EBV-negative cHL, including the relative predominance of memory CD8 T cells and increased T-cell dysfunction as a function of spatial proximity to HRS cells. Building upon a larger multi-institutional cohort of 22 EBV-positive and 24 EBV-negative cHL samples, we orthogonally validated our findings through a spatial multi-omics approach, coupling whole transcriptome capture with antibody-defined cell types for tumor and T-cell populations within the cHL TME. We delineate contrasting transcriptomic immunological signatures between EBV- positive and EBV-negative cases that differently impact HRS cell proliferation, tumor- immune interactions, and mechanisms of T-cell dysregulation and dysfunction. Our multi-modal framework enabled a comprehensive dissection of EBV-linked reorganization and immune evasion within the cHL TME, and highlighted the need to elucidate the cellular and molecular factors of virus-associated tumors, with potential for targeted therapeutic strategies. ### Competing Interest Statement S.J. is a co-founder of Elucidate Bio Inc, has received speaking honorariums from Cell Signaling Technology, and has received research support from Roche unrelated to this work. S.J.R. has received research support from Affimed, Merck, and BMS, is on the Scientific Advisory Board for Immunitas Therapeutics, and also a part of the BMS II-ON. M.A.S. has received research funding from BMS, Bayer, Abbvie, and AstraZeneca, and is on advisory boards for AstraZeneca and BMS. G.P.N. received research grants from Pfizer, Inc.; Vaxart, Inc.; Celgene, Inc.; and Juno Therapeutics, Inc. during the time of and unrelated to this work. G.P.N. and M.A. are co-founders of Ionpath Inc. G.P.N. is a co-founder of Akoya Biosciences, Inc., inventor on patent US9909167, and is a Scientific Advisory Board member for Akoya Biosciences, Inc. M.A. is a Scientific Advisory Board member for Ionpath Inc. The other authors declare no competing interests.