EBV INFECTION OUTCOMES DETERMINED BY MONOCYTE AND TREG-DRIVEN IMMUNE DYNAMICS IN AN EX VIVO PBMC MODEL
Epstein-Barr virus (EBV) infects >95% of the adult population with diverse outcomes ranging from benign latency to cancers and autoimmune diseases. Immunological control of EBV infection is known to be an important determinant of EBV infection outcomes. However, species-specific viral tropism and limited infection models have impeded mechanistic insights into early host-immune control of EBV infection. Here, we use ex vivo infection of peripheral blood mononuclear cells (PBMCs), rather than routinely used B cell enriched culture systems, to study immune and viral dynamics during primary EBV infection. We combined bulk RNA sequencing, EBV transcript enrichment, and flow cytometry to characterize cellular responses across Days 1, 7-8, and 14 post-infection. Early infection triggered a monocyte-specific antiviral response marked by changes in the expression of genes associated with lipid metabolism (LIPA, lysosomal acid lipase) and chemotaxis (CCR1 and CCR2). Inhibitors of LIPA increased EBV titers during primary infection, indicating that LIPA is part of an early monocyte-driven antiviral response. At later timepoints post-infection, donor-dependent variability in lymphoblastoid cell line (LCL) outgrowth was associated with divergent immune states. Donors that failed to generate LCLs demonstrated increased frequencies of CD8+ T cells and reduced numbers of regulatory T cells (CD4CD25FOXP3). EBV transcriptomics revealed that LCL-failed donors exhibited elevated early lytic gene expression but did not establish a type III latency program. Our findings suggest that individual variations in immune cell composition and gene expression may account for differences in the immune response to EBV. These findings define temporal immune and viral signatures that predict transformation outcome and highlight intact PBMCs as a tractable model to study EBV pathogenesis in a genetically diverse, human-specific context. AUTHOR SUMMARYIndividual variation in response to Epstein-Barr virus (EBV) infection can lead to diverse pathogenic outcomes, ranging from cancers to autoimmune disease. To study this variation, we analyzed immune cell response and viral dynamics during the ex vivo primary EBV infection of peripheral blood mononuclear cells (PBMCs) from donors that either fail or succeed to generate lymphoblastoid cell lines (LCLs). Flow cytometry and RNA-seq revealed a rapid monocyte-specific antiviral response among all donors marked by genes associated with lipid metabolism (LIPA) and chemotaxis (CCR1 and CCR2). LIPA inhibition increased EBV titers during primary infection, demonstrating a functional antiviral role. At later timepoints, donor-specific differences in CD8+ T cells and Treg subsets, along with EBV gene expression, were correlated with successful LCL outgrowth. Treatment with the Treg-depleting antibody RG6292 suppressed viral transformation in donors that otherwise supported LCL outgrowth, confirming a functional role for Tregs in shaping early EBV infection outcomes. Viral transcript enrichment-seq revealed an upregulation of early lytic and failure to sustain latent gene expression correlating with failure to generate LCL. These findings highlight intact PBMCs as a tractable model to study EBV viral-host interaction in a genetically diverse, human-specific context, and that Tregs play a key determining role in viral transformation.