High Dimensional, Single Cell Analysis of Epstein Barr Virus (EBV)-Specific T Cells from Pediatric Transplant Recipients with EBV Dnaemia or Controlled Infection

Abstract Text: Epstein-Barr Virus (EBV) is one of the most prolific viruses in the world infecting 90-95% of the adult population. While infection is typically self-limiting in immunocompetent individuals, uncontrolled viremia and life-threatening EBV+ post-transplant lymphoproliferative disease (PTLD) can occur in immunosuppressed transplant recipients. A major gap in the field is understanding the immune determinants that distinguish transplant recipients that control EBV from those that develop EBV-associated disease. We postulate that individuals who cannot control EBV infection are deficient in a specific T cell functional and/or phenotypic signature that promotes an efficient anti-viral response. To test this hypothesis, peptides derived from latent cycle EBV antigens were used to stimulate PBMCs from pediatric transplant patients that are EBV controllers(n=11) and those with chronic EBV DNAemia (CED) (n=11), defined as have persistent (3-6 months) elevations in viral load. Single cell RNA-sequencing reveals that exhausted (LAG3/TOX/TIGIT) and cytolytic (GNYL/PRF1/GZMA) genes are predominantly co-expressed within the CD8+ cytotoxic T cell (CTL) compartment derived from the CED group, suggesting that chronic infection leads to an exhausted EBV-specific CTL subset. Flow cytometry analysis indicates that expression of CD107a in CD8+ T cells is reduced in CED samples relative to controllers. These data indicate that CD8+ T cells from the CED group degranulate less than their control counterparts, and that chronic uncontrolled infection is associated with impaired EBV-specific CTL activity. Together these findings unveil EBV-specific immune alterations that may contribute to the inability to control EBV infection in transplant recipients and could predispose to the development of EBV+ PTLD.