Triggering and mechanisms of natural killer cell mediated cytotoxicity

Abstract: Natural killer (NK) cells are innate immune cells that contribute to defense against infected and transformed cells by target cell killing and cytokine release. In addition, data suggest that NK cells contribute to immune homeostasis and reproduction. In this thesis, we assessed the contribution of individual receptors and intracellular effector molecules to the function of freshly isolated, resting human NK cells. A reductionist approach, using Drosophila cells transfected with ligands for human NK cell receptors, revealed that combinations of synergistic signals from distinct receptors were required to induce efficient NK cell cytotoxicity. Engagement of CD16 by IgG was sufficient to induce degranulation, whereas engagement of LFA-1 by ICAM-1 was sufficient to induce not only adhesion, but also granule polarization. Efficient antibodydependent cellular cytotoxicity required the combination of granule polarization induced by LFA-1 and degranulation induced by CD16. Receptors NKp46, NKG2D, 2B4, DNAM-1, and CD2 have previously been implicated in natural cytotoxicity. Unexpectedly, engagement of these receptors by specific antibodies failed to induce resting NK cell cytotoxicity. For natural cytotoxicity, co-engagement of specific pairwise combinations of activating receptors synergistically induced degranulation and cytokine production. Thus, the term co-activation receptor has been proposed to describe natural cytotoxicity receptors that function as synergistic pairs. KIR2DL4 is an evolutionary conserved member of the KIR family of receptors. Unlike other NK cell receptors, KIR2DL4 was shown to reside in intracellular vesicles. Thus, soluble, but not solid-phase agonists of KIR2DL4, including natural ligand HLA-G, induced cytokine secretion by NK cells. Without eliciting cytotoxicity, this distinctive activation has putative implications for pregnancy. Further, NK cells were assessed from patients diagnosed with familial hemophagocytic lymphohistiocytosis (FHL), an early onset, fatal immunodeficiency syndrome associated to mutations in genes implicated in cellular cytotoxicity. Analysis demonstrated a requirement for Munc13-4 and syntaxin 11 in resting NK cell degranulation. Remarkably, IL-2 stimulation partially restored degranulation and cytotoxicity by syntaxin 11 deficient NK cells. This could explain the later onset and less severe disease progression observed in FHL caused by nonsense mutations in STX11, relative to mutations in PRF1 or UNC13D. In accord, an UNC13D mutation allowing residual degranulation and cytotoxicity was also associated with later disease onset. Our data suggest that the observed defect in NK cell degranulation may contribute to the pathophysiology of FHL, that evaluation of NK cell degranulation in suspected FHL patients may facilitate diagnosis, and that these new insights may offer novel therapeutic possibilities. Our findings provide detailed insight into the molecular triggering and regulation of human NK cell function. Appreciation of the contribution of individual genetic elements to immune function promises increased understanding of disease. Of clinical relevance, new techniques facilitate improved diagnosis, whereas fundamental understanding may assist in development of better treatment.