Virus host interactions in clinical samples

Division Of Basic Sciences - Nci

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Abstract

riments. Prior studies of Kaposi sarcoma (KS) microenvironment in skin have largely examined fixed tissues with immunohistochemistry targeting selected human and KSHV proteins. More recently, investigators have begun to apply bulk and single-cell RNA-sequencing to KS lesions. Key unresolved questions remain: (1) How do the cellular constituents of normal skin differ from those in KS skin? (2) What immune landscapes characterize KS lesions? To address these questions and to define which cell types harbor KSHV along with their immune interactions, we performed spot-based spatial transcriptomics on seven KS skin tumors. This platform captured 55 µm spots across each section, allowing us to distinguish areas with or without detectable KSHV transcripts in situ and to profile latent and lytic viral gene expression programs. We derived a 75-gene human expression signature that, in linear combination, accurately classified whether a spot was KSHV-infected. Using a single-cell RNA-sequencing reference from five healthy skin donors, we applied a spatially informed deconvolution approach to estimate the relative abundance of major skin and immune cell types within each captured spot from patient tissues. Custom probe sets for five KSHV genes (ORF72, K12, PAN, ORF75, K8.1) enabled simultaneous measurement of human and viral transcripts. We also detected localized expression of several immune inhibitory factors at specific regions. Integrating spatial gene expression with inferred cell-type distributions revealed patterns consistent with mechanisms that may inhibit local immune responses to KSHV in KS lesions among people living with HIV. Comparative analysis of human transcripts in KSHV-positive versus KSHV-negative spots identified STC1 among the most strongly upregulated genes in infected regions. We previously observed elevated STC1 in bulk RNA-sequencing of KS tissue relative to matched normal skin and noted increased secretion of STC1 protein after KSHV infection of primary lymphatic endothelial cells. STC1 is a secreted glycoprotein linked to inflammation and carcinogenesis, and prior reports indicate that STC1 protein can suppress macrophage chemotaxis to monocyte chemotactic protein and other attractants. In our KS spatial dataset, high STC1 expression was negatively correlated with macrophage gene marker expression. Taken together, these findings support a working model in which KSHV infection of endothelial cells enhances STC1 secretion, thereby limiting macrophage recruitment to foci of infection within KS lesions.