Poster #6: Mason Dao

Spatial proteomics identifies virus-associated CD8-centered immune neighborhoods in congenital placental infection

Mason Dao¹˒²; Abin Abraham, MD PhD²; James Garifallou, MS²; Dušan Veličković, PhD³˒⁴; Rebecca M. Harris, MD²˒⁵; Rebecca L. Linn, MD²˒⁵; Rita Leite, MD⁵; Daniel Martinez, BS²; Samuel Parry, MD⁵; Lauren E. Schwartz, MD⁵; and Scott M. Gordon, MD, PhD²˒⁵ ¹ University of Pennsylvania, Philadelphia, Pennsylvania ² Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania ³ Pacific Northwest National Laboratory, Richland, Washington ⁴ Environmental Molecular Sciences Laboratory, Richland, Washington ⁵ Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania

Congenital viral infections are associated with placental inflammation and injury, but their local immune organization remains incompletely characterized. We used single-cell spatial proteomics, complementing histopathologic and transcriptomic approaches, to examine virus-associated protein states and CD8+ T-cell microenvironments. Methods: Multiplexed CODEX imaging was performed on eight third-trimester control, cytomegalovirus (CMV), and lymphocytic choriomeningitis virus (LCMV) placentas using a 36-protein panel. Approximately 3.2 million cells were assigned to 16 populations and 11 whole-tissue neighborhoods. The 10 nearest cells surrounding each CD8+ T cell were grouped into 15 CD8-centered cellular neighborhoods (CD8-CNs). We evaluated cell composition, protein states, 50-µm immune-diversity hotspots, and complementary ligand–receptor predictions. Results: Placental viral infection was organized into discrete immune microenvironments rather than diffuse inflammation. In CMV, CD8+ T cells increased from 1.5% of whole tissue to 7.1% within immune-diversity hotspots (4.6-fold enrichment). APC-like cells increased from 1.3% to 4.7% (3.6-fold enrichment), while CD4+ T cells showed 5.2-fold enrichment. These villous regions exhibited elevated HLA-DR, cytotoxic protein activity, and checkpoint engagement. CD8-CNs further resolved trophoblast–macrophage, endothelial–stromal, macrophage–CD8, CD8-dominant, APC–T-cell, and decidual/EVT-associated contexts. LCMV displayed the highest mean tissue diversity (LCMV, 0.97; CMV, 0.83; control, 0.78) and basal-plate inflammatory and stromal–vascular neighborhoods characterized by innate immune infiltration, IFN-γ-associated activation, increased IDO1, and altered trophoblast–stromal–vascular organization. Hofbauer/macrophage-rich neighborhoods formed a shared resident immune scaffold, with CMV associated with villous adaptive cytotoxic immunity and LCMV associated with basal-plate innate inflammation and tissue remodeling. Ligand–receptor inference identified LRP1-linked signaling as a candidate interface program connecting immune clearance, trophoblast stress, and stromal remodeling. Conclusions: Together, these findings support a model in which a shared Hofbauer/macrophage scaffold is differentially engaged: CMV links antigen presentation and checkpoint activity with villous CD8+ cytotoxicity, whereas LCMV links IFN-γ/IDO1-associated activation with basal-plate stromal–vascular remodeling. Candidate LRP1-linked signaling may coordinate immune clearance, trophoblast stress, and matrix remodeling.