Integrated Single‐Cell and TCR Profiling Reveals Protection‐Associated CD8+ T Cell Subsets Linked to Viral Control in PRRSV
ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major threat to global swine industry, yet the immune mechanisms underlying protective vaccination are incompletely understood. Here, we applied integrated single‐cell RNA sequencing and T cell receptor (TCR) profiling to characterize immune responses in a PRRSV vaccination–challenge model spanning complete, partial, and non‐protection outcomes. We identified distinct CD8+ T cell subsets that were selectively enriched in protected animals vaccinated with modified live vaccine (MLV) and marked by clonal expansion, strong cytotoxic transcriptional programs, and enhanced functional activity, which correlated with rapid control of viremia after challenge. In contrast, non‐protected animals accumulated dysfunctional CD8+ T cells expressing exhaustion‐associated markers such as CTLA4 despite partial cytotoxic signatures. Mechanistically, the protection‐associated responses were primarily driven by viral structural proteins (SP). Replacing the SP‐coding region of a heterologous strain reshaped the CD8+ T cell landscape from a mixed cytotoxic/exhausted profile toward a protective program, accompanied by improved clinical outcomes. Further, optimal CD8+ T cell activation required macrophages/monocytes‐derived innate signaling, including TLR4 and TLR8 pathways, and was enhanced by CD4+ T cell help. Together, our findings define protection‐associated CD8+ T cell states linked to viral control and provide insights for rational PRRSV vaccine design.