Investigating functional responses of unique CD16 Fc receptor complexes in human natural killer cells 3218

Abstract Description Natural killer (NK) cells are innate lymphocytes that act as first responders to harmful cells capable of effectively killing them without a need for priming. They are regulated by a variety of stimulatory and inhibitory surface cell receptors that bind to different ligands on target cells. In human NK cells, CD16 is an activating receptor that recognizes the Fc portion of IgG antibodies and relies on two adaptor molecules (CD3ζ and Fc?) for association to form stable complexes. These adaptors contain immunoreceptor tyrosine-based activation motifs (ITAMs) for signaling: CD3ζ contains three ITAMs and Fc? contains one ITAM per chain. CD16 can form homodimers or heterodimers of CD3ζ and Fc?; however, which complex provides optimal signal is unknown. Our goal is to study different CD16 receptor-adaptor complexes in a human NK cell line (NK92s) in order to determine which complex leads to the best surface cell signaling and activation. To tightly control expression of our desired molecules and because NK92s express CD3ζ, we performed CRISPR-KO at the endogenous CD3ζ locus while delivering an adeno-associated virus (AAV)-mediated template of different Fc-receptor complexes to achieve knock-in of: CD16:Fc?, CD16:CD3ζ, and CD16:Fc?:CD3ζ. These cells were sorted and validated to express our desired FcR complexes and then used in functional assays including platebound stimulations and killing assays. Funding Sources NIH Grant AI146581 Topic Categories Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)