Konrad Krzewski

Many NK cell receptors activate ERK2 and JNK1 to trigger microtubule organizing center and granule polarization and cytotoxicity

Natural killer (NK) cells are components of the innate immune system that recognize and kill tumor or virus-infected target cells through specific NK activating receptor/ligand interactions. Lymphocyte function-associated antigen (LFA)-1 and its ligand ICAM-1 are also required to initiate conjugation and actin cytoskeletal remodeling. The NK activating receptors, many of which are expressed on a single NK cell, signal the polarization of the microtubule organizing center (MTOC) together with cytolytic granules to the synapse with target cells. After ligation of any one of these receptors, Src family kinases initiate activation of two signal pathways, the phosphoinositide-3 kinase → ERK2 and the phospholipase Cγ → JNK1 pathways. Both are required for polarization of the MTOC and cytolytic granules, a prerequisite for killing the targets. Crosslinking of CD28, NKG2D, NKp30, NKp46, NKG2C/CD94, or 2B4 leads to the phosphorylation of both ERK2 and JNK1, although they use different proximal signaling modules. Thus, many, if not all, activating receptors stimulate these two distal pathways, independent of the proximal signaling module used. By contrast, CD2, DNAM-1, and β1-integrin crosslinking do not activate either pathway; they may be costimulatory molecules or have another function in the synapse.

CD28-stimulated ERK2 phosphorylation is required for polarization of the microtubule organizing center and granules in YTS NK cells

Activation of natural killer (NK) cell cytotoxicity requires adhesion and formation of a conjugate with a susceptible target cell, followed by actin polymerization, and polarization of the microtubule organizing center (MTOC) and cytolytic granules to the NK cell immune synapse. Here, by using the YTS NK cell line as a model, CD28 is shown to be an activating receptor. It signals cytotoxicity in a process dependent on phosphoinositide-3 kinase activation, leading to sustained extracellular signal-regulated kinase 2 (ERK2) phosphorylation. ERK and phospho-ERK localize to microtubule filaments. Neither conjugation with targets nor actin polymerization is affected by blocking ERK2 activation. However, both polarization of the MTOC and cytolytic granules to the synaptic region and NK cell cytotoxicity are strongly reduced by blocking ERK2 activation. A role for the CD28/CD80 interaction in cytotoxicity of human peripheral NK cells also was established. By contrast, lymphocyte function-associated antigen 1 (LFA-1) ligation transduces only a transient ERK2 activation and fails to induce killing in YTS cells. Thus, in YTS cells, a CD28 signal is used to polarize the MTOC and cytolytic granules to the NK cell immune synapse by stimulating sustained ERK2 activation.

Formation of a WIP-, WASp-, actin-, and myosin IIA-containing multiprotein complex in activated NK cells and its alteration by KIR inhibitory signaling.

The tumor natural killer (NK) cell line YTS was used to examine the cytoskeletal rearrangements required for cytolysis. A multiprotein complex weighing ∼1.3 mD and consisting of WASp-interacting protein (WIP), Wiskott-Aldrich syndrome protein (WASp), actin, and myosin IIA that formed during NK cell activation was identified. After induction of an inhibitory signal, the recruitment of actin and myosin IIA to a constitutive WIP–WASp complex was greatly decreased. Both actin and myosin IIA were recruited to WIP in the absence of WASp. This recruitment correlated with increased WIP phosphorylation, which was mediated by PKCθ. Furthermore, the disruption of WIP expression by WIP RNA interference prevented the formation of this protein complex and led to almost complete inhibition of cytotoxic activity. Thus, the multiprotein complex is important for NK cell function, killer cell immunoglobulin-like receptor inhibitory signaling affects proteins involved in cytoskeletal rearrangements, and WIP plays a central role in the formation of the complex and in the regulation of NK cell activity.

Human NK cell lytic granules and regulation of their exocytosis.

Natural killer (NK) cells form a subset of lymphocytes that play a key role in immuno-surveillance and host defense against cancer and viral infections. They recognize stressed cells through a variety of germline-encoded activating cell surface receptors and utilize their cytotoxic ability to eliminate abnormal cells. Killing of target cells is a complex, multi-stage process that concludes in the directed secretion of lytic granules, containing perforin and granzymes, at the immunological synapse. Upon delivery to a target cell, perforin mediates generation of pores in membranes of target cells, allowing granzymes to access target cell cytoplasm and induce apoptosis. Therefore, lytic granules of NK cells are indispensable for normal NK cell cytolytic function. Indeed, defects in lytic granule secretion lead or are related to serious and often fatal diseases, such as familial hemophagocytic lymphohistiocytosis (FHL) type 2–5 or Griscelli syndrome type 2. A number of reports highlight the role of several proteins involved in lytic granule release and NK cell-mediated killing of tumor cells. This review focuses on lytic granules of human NK cells and the advancements in understanding the mechanisms controlling their exocytosis.

The killer's kiss: the many functions of NK cell immunological synapses.

Natural killer (NK) cells comprise a subset of lymphocytes involved in protection against microbial pathogens and tumors. NK cells recognize host cells that are missing MHC class I molecules and eliminate them through localized delivery of lytic granules. The majority of NK cell effector functions require direct cell-to-cell contact. Binding to a target cell is accompanied by creation of complex structures at the cell-cell interface known as immunological synapses. Recent studies have contributed immensely to the characterization of several types of NK cell immunological synapses and understanding of the variety of processes originating at this intriguing place. The emerging picture illustrates NK cell immune synapses as the sites of highly complex regulation of NK cell activity.

Cutting Edge: Mouse CD300f (CMRF-35–Like Molecule-1) Recognizes Outer Membrane-Exposed Phosphatidylserine and Can Promote Phagocytosis

Reportedly, CD300f negatively regulates interactions between dendritic and T cells and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines, an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, cosedimentation, and surface plasmon resonance using phospholipid-containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.

Complex regulation of human NKG2D-DAP10 cell surface expression: opposing roles of the γc cytokines and TGF-β1

Natural killer (NK) cells help protect the host against viral infections and tumors. NKG2D is a vital activating receptor, also expressed on subsets of T cells, whose ligands are up-regulated by cells in stress. Ligation of NKG2D leads to phosphorylation of the associated DAP10 adaptor protein, thereby activating immune cells. Understanding how the expression of NKG2D-DAP10 is regulated has implications for immunotherapy. We show that IL-2 and TGF-β1 oppositely regulate NKG2D-DAP10 expression by NK cells. IL-2 stimulation increases NKG2D surface expression despite a decrease in NKG2D mRNA levels. Stimulation with IL-2 results in a small increase of DAP10 mRNA and a large up-regulation of DAP10 protein synthesis, indicating that IL-2-mediated effects are mostly posttranscriptional. Newly synthesized DAP10 undergoes glycosylation that is required for DAP10 association with NKG2D and stabilization of NKG2D expression. TGF-β1 has an opposite and dominant effect to IL-2. TGF-β1 treatment decreases DAP10, as its presence inhibits the association of RNA polymerase II with the DAP10 promoter, but not NKG2D mRNA levels. This leads to the down-regulation of DAP10 expression and, as a consequence, NKG2D protein as well. Finally, we show that other γ(c) cytokines act similarly to IL-2 in up-regulating DAP10 expression and NKG2D-DAP10 surface expression.

WIP is essential for lytic granule polarization and NK cell cytotoxicity

Natural killer (NK) cells play important roles in host immunity by killing virus-infected and tumor cells. Killing of the target cell is achieved by formation of an immune synapse and localized secretion of lytic granules containing perforin and granzymes. Here, we demonstrate that Wiskott–Aldrich syndrome protein (WASp)-interacting protein (WIP), important in generation of a large complex of proteins involved in actin cytoskeleton rearrangements, is indispensable for NK cell cytotoxicity. WIP knockdown completely inhibited cytotoxicity, whereas overexpression of WIP enhanced NK cell cytolytic ability. WIP was found to colocalize with lytic granules. WIP segregated to the lysosomal fraction, where granzyme B activity was also found, and the interaction between WIP and granules was independent of WASp. Importantly, WIP knockdown inhibited polarization of lytic granules to the immune synapse, but not conjugate formation. These results indicate that WIP is involved in lytic granule transport and is essential for regulation of NK cell cytotoxic function.

LAMP1/CD107a is required for efficient perforin delivery to lytic granules and NK cell cytotoxicity

Secretory lysosomes of natural killer (NK) cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network-derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity.

Dendritic cell and natural killer cell cross-talk: a pivotal role of CX3CL1 in NK cytoskeleton organization and activation

Initial molecular events leading to natural killer lymphocyte (NK) and dendritic cell (DC) interactions are largely unknown. Here, the role of CX3CL1 (fractalkine), a chemokine expressed on mature dendritic cells (mDCs) has been investigated. We show that CX3CL1 promotes NK activation by mDCs. After blocking of CX3CL1 by antibody, no activation occurred but major histocompatibility complex (MHC) class I neutralization restored DC-mediated NK activation, suggesting an interaction between CX3CL1 signaling and the functioning of inhibitory KIR. Then the YTS NK cell line, in which the inhibitory receptor KIR2DL1 had been introduced, was used. The presence of KIR2DL1 did not decrease YTS activation by HLA-Cw4 DC when CX3CL1 was functional. In contrast, CX3CL1 neutralization led to killer cell immunoglobulin-like receptor (KIR) phosphorylation and SHP-1 recruitment in YTS(KIR2DL1) cultured with HLA-Cw4 mDCs. Moreover, CX3CL1 neutralization promoted dispersion of lipid rafts and the formation of a multiprotein complex required for cytoskeletal rearrangements in YTS NK cells. These findings point to a pivotal role of CX3CL1 in the activation of resting NK cells by mature DCs.