Abstract: Natural killer (NK) cells are innate lymphocytes that possess traits of adaptive immunity, such as memory formation. However, the molecular mechanisms by which NK cells persist to form memory cells are not well understood. Using single cell RNA sequencing, we identified two distinct effector NK cell (NKeff) populations following mouse cytomegalovirus (MCMV) infection. Ly6C? memory precursor (MP) NK cells displayed enhanced survival during the contraction phase in a Bcl2-dependent manner, and differentiated into Ly6C+ memory NK cells. Our studies show that a NK cell-intrinsic checkpoint is controlled by the transcription factor Fli1 which limits MP NK formation by regulating early effector NK cell fitness during viral infection. Building upon this discovery, we have designed methods and materials for modulating the molecular mechanisms that regulate memory cell fate in NK cells, such as genetically modified NK cells having a deletion in the gene for the transcription factor Fli1.

Abstract: The invention provides an optimized electroporation strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of primary innate immune cells, a methodology that can, for example, produce an almost complete loss of target gene expression from a single electroporation. This methodology has been validated in human peripheral blood-derived monocyte derived macrophages, natural killer cells, and monocyte derived dendritic cells. This gene editing technology can, for example, be used to delete inhibitory molecules in natural killer cells and dendritic cells for adoptive cell therapy in cancer. It can also be used to manipulate gene expression in adoptively transferred tolorogenic dendritic cells for treatment of type 1 diabetes and other autoimmune diseases.