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Natural killer (NK) cells have important functions in cancer immunosurveillance, BM allograft rejection, fighting infections, tissue homeostasis, and reproduction. NK cell–based therapies are promising treatments for blood cancers. Overcoming their currently limited efficacy requires a better understanding of the molecular mechanisms controlling NK cell development and dampening their effector functions. NK cells recognize the loss of self-antigens or up-regulation of stress-induced ligands on pathogen-infected or tumor cells through invariant NK cell receptors (NKRs), and then kill such stressed cells. Two second-messenger pathways downstream of NKRs are required for NK cell maturation and effector responses: PIP3 generation by PI3K and generation of diacylglycerol and IP3 by phospholipase-Cγ (PLCγ). In the present study, we identify a novel role for the phosphorylated IP3 metabolite inositol (1,3,4,5)tetrakisphosphate (IP4) in NK cells. IP4 promotes NK cell terminal differentiation and acquisition of a mature NKR repertoire. However, in mature NK cells, IP4 limits NKR-induced IFNγ secretion, granule exocytosis, and target-cell killing, in part by inhibiting the PIP3 effector-kinase Akt. This identifies IP4 as an important novel regulator of NK cell development and function and expands our understanding of the therapeutically important mechanisms dampening NK cell responses. Our results further suggest that PI3K regulation by soluble IP4 is a broadly important signaling paradigm.

In MHC class I–deficient hosts, natural killer (NK) cells are hyporesponsive to cross-linking of activation receptors. Functional competence requires engagement of a self–major histocompatability complex (MHC) class I–specific inhibitory receptor, a process referred to as “licensing.” We previously suggested that licensing is developmentally determined in the bone marrow. In this study, we find that unlicensed mature MHC class I–deficient splenic NK cells show gain-of-function and acquire a licensed phenotype after adoptive transfer into wild-type (WT) hosts. Transferred NK cells produce WT levels of interferon-γ after engagement of multiple activation receptors, and degranulate at levels equivalent to WT NK cells upon coincubation with target cells. Only NK cells expressing an inhibitory Ly49 receptor specific for a cognate host MHC class I molecule show this gain-of-function. Therefore, these findings, which may be relevant to clinical bone marrow transplantation, suggest that neither exposure to MHC class I ligands during NK development in the BM nor endogenous MHC class I expression by NK cells themselves is absolutely required for licensing.

We have previously demonstrated that the NKR repertoire is profoundly disrupted by SHIP deficiency. This repertoire disruption is characterized by receptor dominance where inhibitory signals from 2B4 repress killing of complex targets expressing MHC class I and activating ligands. In this study, we examine the molecular basis of receptor dominance in SHIP−/− NK cells. In this study, we show that in SHIP−/− NK cells there is a pronounced bias toward the 2B4 long isoform. We have also characterized signaling molecules recruited to 2B4 in SHIP−/− NK cells. Interestingly, we find that ∼10- to 16-fold more Src homology region 2 domain-containing phosphatase 1 (SHP1) is recruited to 2B4 in SHIP−/− NK cells when compared with wild type. Consistent with SHP1 overrecruitment, treatment with sodium orthovanadate or a novel inhibitor with micromolar activity against SHP1 restores the ability of SHIP−/− NK cells to kill Rae1+ RMA and M157+ targets. These findings define the molecular basis for hyporesponsiveness by SHIP-deficient NK cells.

Inhibitory receptors that recognize MHC class I molecules regulate NK cell responses and self-tolerance. Recent evidence indicates that self-ligands not present in the MHC locus also can modulate NK function. In this study, we show that an inhibitory receptor that recognizes an MHC-independent ligand is over expressed in SHIP−/− mice at all stages of NK development and differentiation. Overexpression of this receptor compromises key cytolytic NK functions, including killing of allogeneic, tumor, and viral targets. These results further demonstrate the critical role that SHIP plays in regulation of the NK receptor repertoire and show that regulation of MHC-independent inhibitory receptors is crucial for NK recognition and cytolysis of complex targets.

The changes in signal transduction associated with the acquisition of specific cell fates remain poorly understood. We performed massive parallel assessment of kinase signatures of the radiations of the hematopoietic system, including long-term repopulating hematopoietic stem cells (LT-HSC), short-term repopulating HSC (ST-HSC), immature natural killer (iNK) cells, NK cells, B cells, T cells and myeloid cells. The LT-HSC kinome is characterised by non-canonical Wnt, Ca2+ and classical protein kinase C (PKC)-driven signalling, which is lost upon the transition to ST-HSC, whose kinome signature prominently features receptor tyrosine kinase (RTK) activation of the Ras/MAPK signalling cassette. Further differentiation to iNK maintains signalling through this cassette but simultaneously leads to activation of a PI3K/PKB/Rac signalling, which becomes the dominant trait in the kinase signature following full differentiation towards NK cells. Differentiation along the myeloid and B cell lineages is accompanied by hyperactivation of both the Ras/MAPK and PI3K/PKB/Rac signalling cassette. T cells, however, deactivate signalling and only display residual G protein-coupled pathways. Thus, differentiation along the hematopoietic lineage is associated with major remodelling of cellular kinase signature.