460 Fetal natural killer cells play an essential immunoprotective role in preventing the onset of symptomatic congenital cytomegalovirus

Objectives/Goals: Congenital cytomegalovirus (cCMV) continues to be the primary infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells in response to cCMV remains largely unexplored. This study seeks to investigate how fetal NK cells respond to human cytomegalovirus (HCMV) during gestation. Methods/Study Population: Umbilical cord blood and corresponding umbilical cord tissues were collected from fetuses that had no complications during gestation. These samples, provided by the Medical College of Wisconsin Tissue Bank, were processed within 24 hours after live birth. Single-cell suspensions were prepared from the samples, and fetal NK cells were isolated and exposed to HCMV antigen peptides VMAPRTLFL, VMAPRTLIL, VMAPQSLLL, and the human self-peptide ALALVRMLI. These peptides were presented on HLA-E*01:03 BV421-conjugated tetramers produced by the National Institutes of Health Tetramer Core Facility. Additionally, fetal NK cells were also prepared for single-cell RNA sequencing (scRNA-seq), and cells were filtered and clustered based on the number of uniquely expressed genes. Results/Anticipated Results: Through unbiased clustering, our scRNA-seq analysis identified five unique fetal NK cell subsets in umbilical cord blood and four in the corresponding umbilical cord tissue. Notably, fetal NK cells exposed to HCMV during gestation were primarily mature NK cell subsets, while those from unexposed fetuses were mostly immature subsets. Additionally, HCMV-exposed fetal NK cells exhibited a strong recall response to the HCMV antigen, with a notably higher frequency and elevated production of IFN-γ. Conversely, naïve fetal NK cells from fetuses unexposed to HCMV produced significantly lower levels of IFN-γ. Finally, we identified a distinct subset of fetal NK cells that emerge following exposure to the HCMV antigen. Discussion/Significance of Impact: In this study, we show that HCMV infection can influence the formation of specific NK cell subsets and re-exposure to the HCMV antigen can trigger a recall response. These insights could pave the way for the development of innovative NK cell-based immunotherapies aimed at preventing fetuses from developing symptomatic cCMV.