Objectives/Goals: This work seeks to elucidate the upstream and downstream circuitry of the Keap1–Nrf2 axis in natural killer (NK) cells. We aim to define how the Keap1–Nrf2 axis sets a redox license to calibrate NK function and metabolic fitness, and in turn, to translate these findings into new strategies for metabolically enhancing NK and CAR-NK-based immunotherapy. Methods/Study Population: Keap1 and Nrf2 were investigated primarily in murine models, by using NK-specific knockouts of each gene (Ncr1Cre-Nrf2fl/fl and Ncr1Cre-Keap1fl/fl) in C57BL/6 mice. Flow cytometry was used to profile knockout versus wild-type differences in lymphocyte populations and subpopulations, including receptor repertoire, maturation, and effector function. In vitro and ex vivo experiments were performed by sorting cells from the spleens of knockout mice and their wild-type littermates. Human NK cells from healthy donors were analyzed with collaborators to validate the importance of the axis for human patients. Results/Anticipated Results: Transcriptomics analysis of NK cells during murine cytomegalovirus (MCMV) infection revealed reciprocal regulation of Nrf2 and Keap1, with Nrf2 transcripts downregulated and Keap1 upregulated at day 4 post-infection, highlighting the potential contribution of this pathway to NK cell responses during viral challenge. We demonstrate that both insufficient and excessive ROS buffering impair host survival in response to MCMV and acute myeloid leukemia (AML) and compromise control of tumor metastasis in melanoma models. Importantly, both knockout models also exhibited defects in NK cell expansion, maturation, and cytotoxicity. Ongoing studies map the metabolic, epigenetic and transcriptional programs downstream of this pathway in both murine and human NK cells. Discussion/Significance of Impact: As immunotherapies are developed and optimized, a rigorous understanding of the mechanisms through which immune cells kill pathogens is critical. Collectively, our work thus far identifies the Keap1–Nrf2 axis as a critical regulator of NK cell fitness that can be used to promote cell therapeutic development.
Cato et al. (Wed,) studied this question.