3191 – DELINEATING HUMAN NATURAL KILLER CELL DEVELOPMENT THROUGH AN IN VITRO CELL- DIFFERENTIATION MAP AND BARCODE-CLONAL TRACING

Abstract

Natural Killer (NK) cells represent a unique cytotoxic innate lymphoid lineage that holds great potential for anti-cancer immunotherapy. The process of human NK cell lineage specification and maturation from hematopoietic progenitors is poorly understood. While mouse and human NK cell developmental pathways converged overall in terms of molecular signatures and transcription factor requirements, inherent differences in surface marker repertoire and shared phenotypic features with other innate lymphoid subsets, highlight the need for a platform allowing direct modeling of human NK cell development. Employing an in-vitro differentiation system on OP9 stroma and multi-parameter FACS analysis, we established and characterized a NK cell developmental trajectory that recapitulates multiple diverse maturation stages proposed in a current model of human NK cell development ex-vivo in the bone marrow and tonsils. Differentiation kinetics, expression of transcription factors (Tbet, Eomes) and effector molecules (Perforin, Granzyme-B) confirmed that in-vitro NK cell maturation progressed through this developmental trajectory in a sequential manner. Functional assays including IFNg secretion, release of cytotoxic granules and direct killing of tumor cells supported the increased effector functions as differentiating NK cells progressed from the early to the final maturation stages. We also further characterized NK cell differentiation steps using transcriptomic profiles from single cell RNA-sequencing. Finally, we employ DNA-barcode-clonal tracing to study developmental relationship between different stages in our in vitro NK cell differentiation model. In summary, we established a framework that allows further understanding of human NK cell development and provides a platform for studies on identifying regulatory networks controlling this process. Natural Killer (NK) cells represent a unique cytotoxic innate lymphoid lineage that holds great potential for anti-cancer immunotherapy. The process of human NK cell lineage specification and maturation from hematopoietic progenitors is poorly understood. While mouse and human NK cell developmental pathways converged overall in terms of molecular signatures and transcription factor requirements, inherent differences in surface marker repertoire and shared phenotypic features with other innate lymphoid subsets, highlight the need for a platform allowing direct modeling of human NK cell development. Employing an in-vitro differentiation system on OP9 stroma and multi-parameter FACS analysis, we established and characterized a NK cell developmental trajectory that recapitulates multiple diverse maturation stages proposed in a current model of human NK cell development ex-vivo in the bone marrow and tonsils. Differentiation kinetics, expression of transcription factors (Tbet, Eomes) and effector molecules (Perforin, Granzyme-B) confirmed that in-vitro NK cell maturation progressed through this developmental trajectory in a sequential manner. Functional assays including IFNg secretion, release of cytotoxic granules and direct killing of tumor cells supported the increased effector functions as differentiating NK cells progressed from the early to the final maturation stages. We also further characterized NK cell differentiation steps using transcriptomic profiles from single cell RNA-sequencing. Finally, we employ DNA-barcode-clonal tracing to study developmental relationship between different stages in our in vitro NK cell differentiation model. In summary, we established a framework that allows further understanding of human NK cell development and provides a platform for studies on identifying regulatory networks controlling this process.