The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1βR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88fl/flNcr1Cre/+). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H+. In contrast, Ly49H- NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H+ NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells.

Natural killer (NK) cells are innate lymphocytes that play essential roles in mediating antitumor immunity. NK cells respond to various inflammatory stimuli including cytokines and stress-induced cellular ligands which activate germline-encoded activation receptors (NKRs), such as NKG2D. The signaling molecules activated downstream of NKRs are well defined; however, the mechanisms that regulate these pathways are not fully understood. IQ domain-containing GTPase-activating protein 1 (IQGAP1) is a ubiquitously expressed scaffold protein. It regulates diverse cellular signaling programs in various physiological contexts, including immune cell activation and function. Therefore, we sought to investigate the role of IQGAP1 in NK cells. Development and maturation of NK cells from mice lacking IQGAP1 (Iqgap1-/- ) were mostly intact; however, the absolute number of splenic NK cells was significantly reduced. Phenotypic and functional characterization revealed a significant reduction in the egression of NK cells from the bone marrow of Iqagp1-/- mice altering their peripheral homeostasis. Lack of IQGAP1 resulted in reduced NK cell motility and their ability to mediate antitumor immunity in vivo. Activation of Iqgap1-/- NK cells via NKRs, including NKG2D, resulted in significantly reduced levels of inflammatory cytokines compared with wild-type (WT). This reduction in Iqgap1-/- NK cells is neither due to an impaired membrane proximal signaling nor a defect in gene transcription. The levels of Ifng transcripts were comparable between WT and Iqgap1-/- , suggesting that IQGAP1-dependent regulation of cytokine production is regulated by a post-transcriptional mechanism. To this end, Iqgap1-/- NK cells failed to fully induce S6 phosphorylation and showed significantly reduced protein translation following NKG2D-mediated activation, revealing a previously undefined regulatory function of IQGAP1 via the mechanistic target of rapamycin complex 1. Together, these results implicate IQGAP1 as an essential scaffold for NK cell homeostasis and function and provide novel mechanistic insights to the post-transcriptional regulation of inflammatory cytokine production.

The intracellular spatiotemporal organization of signaling events is critical for normal cellular function. In response to environmental stimuli, cells utilize highly organized signaling pathways that are subject to multiple layers of regulation. However, the molecular mechanisms that coordinate these complex processes remain an enigma. Scaffolding proteins (scaffolins) have emerged as critical regulators of signaling pathways, many of which have well-described functions in immune cells. IQGAP1, a highly conserved cytoplasmic scaffold protein, is able to curb, compartmentalize, and coordinate multiple signaling pathways in a variety of cell types. IQGAP1 plays a central role in cell-cell interaction, cell adherence, and movement via actin/tubulin-based cytoskeletal reorganization. Evidence also implicates IQGAP1 as an essential regulator of the MAPK and Wnt/β-catenin signaling pathways. Here, we summarize the recent advances on the cellular and molecular biology of IQGAP1. We also describe how this pleiotropic scaffolin acts as a true molecular puppeteer, and highlight the significance of future research regarding the role of IQGAP1 in immune cells.