No abstract

The activation of phosphoinositide metabolism represents a critical step in the signaling pathways leading to the activation of cytolytic machinery, but its regulation is partially understood. We report here that the stimulation of the lowaffinity receptor for immunoglobulin G (IgG) (Fc␥RIIIA, CD16) on primary human natural killer (NK) cells induces a phosphatidylinositol 3-kinase (PI3K)-dependent activation of the small G protein Arf6. We first demonstrate a functional role for Arf6-dependent signals in the activation of the antibodydependent cellular cytotoxicity (ADCC) attributable to the control of secretion of lytic granule content. We also show that Arf6 couples CD16 to the lipidmodifying enzymes phosphatidylino- IntroductionCD16, the low-affinity receptor for the Fc fragment of immunoglobulin G (IgG), is a major surface structure on natural killer (NK) cells capable of triggering the antibody-dependent cellular cytotoxicity (ADCC) and the expression of genes encoding surface activation molecules and cytokines upon engagement by IgGopsonized targets. 1,2 CD16 represents the prototype of the so-called immunoreceptor tyrosine-based activation motif (ITAM)-containing NK receptors whose clustering results in a cascade of signaling events responsible for cytolytic granule-mediated target cell killing. 2 This process involves several steps, including the formation of a cytolytic synapse between NK and target cells, the rapid reorientation of the Golgi complex and the microtubule-organizing center (MTOC), the polarization of lytic granules toward the target contact site followed by granule docking and fusion at specialized secretory domains within the cytolytic synapse leading to the release of granule content. 3 Most of the signaling components controlling secretion of the granule content after their polarization have yet to be identified.The activation of the lipid-modifying enzymes phospholipase C gamma (PLC␥) and phosphatidylinositol 3-kinase (PI3K) plays a central role in the activation of cytolytic machinery 2,4 ; notably, they share the common substrate phosphatidylinositol4,5-bisphosphate (PIP2). 5 Furthermore, PIP2 is also required for the latest steps of the secretory pathway by marking membrane microdomains for specific structural and functional modifications required for granule docking and fusion and for the disassembly of cortical actin barrier. [5][6][7] Little is known about how PIP2 levels are regulated in cells of the immune system. PIP2 is mainly synthesized by the phosphorylation of phosphatidylinositol4-phosphate (PI4P) at the D-5 position of the inositol ring by phosphatidylinositol4phosphate 5-kinase type I (PI5KI). 5,7 Three isoforms of PI5KI have been described, PI5KI␣, PI5KI␤, and PI5KI␥, with distinct subcellular localization and functions. PI5KI␥ targets to focal adhesion and nerve terminals where PIP2 is required for focal adhesion assembly and for recycling of synaptic vesicle membrane, respectively 8,9 ; PI5KI␤ is present in the vesicular perinuclear region 7 and is required for co...

Although membrane phospholipid phosphatidylinositol-4,5bisphosphate (PIP2) plays a key role as signaling intermediate and coordinator of actin dynamics and vesicle trafficking, it remains completely unknown its involvement in the activation of cytolytic machinery. By live confocal imaging of primary human natural killer (NK) cells expressing the chimeric protein GFP-PH, we observed, during effector-target cell interaction, the consumption of a preexisting PIP2 pool, which is critically required for the activation of cytolytic machinery. We identified type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) ␣ and ␥ isoforms as the enzymes responsible for PIP2 synthesis in NK cells. By hRNA-driven gene silencing, we observed that both enzymes are required for the proper activation of NK cytotoxicity and for inositol-1,4,5-trisphosphate (IP3) generation on receptor stimulation. In an attempt to elucidate the specific step controlled by PI5KIs, we found that lytic granule secretion but not polarization resulted in impaired PI5KI␣-and PI5KI␥-silenced cells. Our findings delineate a novel mechanism implicating PI5KI␣ and PI5KI␥ isoforms in the synthesis of PIP2 pools critically required for IP3-dependent Ca 2؉ IntroductionNatural killer (NK) cells and cytotoxic T lymphocytes are critical effectors in the defense against tumor and viral infections 1 ; they exert cytotoxic function through the polarized secretion of granules containing proteolytic molecules, such as perforin and granzymes. This process involves several steps, including the formation of a cytolytic synapse between cytolytic effector and target cell, the rapid reorientation of the microtubule-organizing center along with lytic granules toward the target contact area followed by granule docking and fusion at specialized secretory domains within the cytolytic synapse. 2,3 Several structurally distinct receptors have been implicated in the activation of NK-cell cytolytic machinery: when cross-linked by the corresponding ligands on target cell, they trigger multiple and intersecting signaling pathways responsible for functional activation. 4 A vast array of activating NK receptors belonging to different families are coupled to the lipid modifying enzymes phosphatidylinositol3-kinase (PI3K) and phospholipase C␥ (PLC␥), which provide signals critically required for the activation of the cytolytic machinery [5][6][7][8] ; notably, both enzymes use the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) as common substrate. 9 Besides its role as signaling intermediate, PIP2 also acts as critical regulator of various cellular processes, including actin remodeling, membrane and vesicle trafficking, adhesion, and ion transport. 10,11 Surprisingly, the role of PIP2 and its regulatory mechanisms in lymphocyte-mediated cytotoxicity remain completely undefined.The main cellular source of PIP2 are type I phosphatidylinositol-4-phosphate-5-kinase (PI5KI) family members which phosphorylate PI4P on the D5 position of the inositol ring. Three major PI5KI is...

Increased expression of CD94/NKG2A in peritoneal NK cells may mediate the resistance of endometriotic tissue to NK cell-mediated lysis, thus contributing to the progression of the disease.

The CD94/NKG2 complex is expressed on T and NK lymphocytes. CD94 molecules covalently associate to activating or inhibitory NKG2 molecules, and their expression finely tunes cell responses. Human γδ T cells express several NKRs. Expression of these receptors is confined to the cytolytic Vδ2 subset, which coexpresses the FcγRIII CD16 and CD45RA and has been defined as Vγ9Vδ2 T(EMRA) cells. We show that the CD94/NKG2C complex, associated with KARAP/DAP12, is fully functional in γδ T cells, as determined by measuring IFN-γ production, T cell proliferation, and cytolytic activity by γδ lymphocytes. In contrast, NKG2A expression was found on all γδ T cell memory subsets, suggesting a crucial role of the inhibitory signal provided by this receptor on γδ T cell responses. Moreover, we found Vγ9Vδ2 T(EMRA), NK, and CD8+ αβ T cells coexpressing NKG2A and NKG2C receptors. Functional experiments showed that the inhibitory signal mediated by the NKG2A receptor prevails when double-positive cells are activated. Finally, NKG2A expression on γδ LDGL correlates with asymptomatic pathology, even in the presence of NKG2C coexpression, whereas in symptomatic patients affected by severe disease, the inhibitory NKG2A receptor is absent, and a variety of activatory NKRs was found. We propose that the silent behavior of γδ cells in LDGL patients is a result of effective inhibitory HLA class I receptors.