OBJECTIVES/GOALS: Congenital cytomegalovirus (cCMV) remains to be the leading infectious cause of fetal anomalies. The role of fetal natural killer (NK) cells during cCMV remains largely unknown. The objective of this study is to define the transcriptomes of fetal NK cells exposed to human cytomegalovirus (HCMV) infection during gestation. METHODS/STUDY POPULATION: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV seropositive (HCMV+) and two HCMV seronegative (HCMV-) fetuses that did not experience any complications during gestation. These samples were provided by the Medical College of Wisconsin Tissue Bank and were processed within 24 hours following live birth. CD7+ CD3e-CD14-CD19-CD20- fetal NK cells were isolated, using the BD FACSAria sorter. Following cell sorting, single-cell RNA sequencing (scRNA-seq) was performed, and cDNA libraries were constructed and sequenced via NextSeq 550. Cell Ranger was then used to algin the cDNA reads and the Seurat R package was used to analyze the transcriptional data. Cells were filtered and clustered based on the number of uniquely expressed genes. RESULTS/ANTICIPATED RESULTS: Four sets of umbilical cord blood and matching umbilical cord tissues were collected from two HCMV+ and two HCMV- fetuses. We were able to successfully sort and capture fetal NK cells and perform scRNA-seq on these samples. Following unbiased clustering, we observed and characterized five distinct fetal NK cell subsets in the umbilical cord blood and four fetal NK cell subsets in the corresponding umbilical cord tissue. Our findings revealed that HCMV+ fetal NK cells primarily consisted of mature NK cell subsets, while HCMV- fetal NK cells constituted the majority of the immature subsets. Importantly, we identified a unique subset of NKG2CHi fetal NK cells that were significantly elevated in the HCMV+ fetuses. Finally, we defined a group of transcription factors involved in the formation of antiviral fetal NK. DISCUSSION/SIGNIFICANCE: Here, we demonstrate that HCMV infection can induce the formation of distinct NK cell subsets and drive their unique transcriptional profiles. These findings have the potential to guide the development of an innovative NK cell immunotherapy that could help prevent fetuses from developing symptomatic cCMV.