Vav1 controls DAP10-mediated natural cytotoxicity by regulating actin and microtubule dynamics

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.     

Silencing human NKG2D, DAP10, and DAP12 reduces cytotoxicity of activated CD8+ T cells and NK cells

Human CD8+ T cells activated and expanded by TCR cross-linking and high-dose IL-2 acquire potent cytolytic ability against tumors and are a promising approach for immunotherapy of malignant diseases. We have recently reported that in vitro killing by these activated cells, which share phenotypic and functional characteristics with NK cells, is mediated principally by NKG2D. NKG2D is a surface receptor that is expressed by all NK cells and transmits an activating signal via the DAP10 adaptor molecule. Using stable RNA interference induced by lentiviral transduction, we show that NKG2D is required for cytolysis of tumor cells, including autologous tumor cells from patients with ovarian cancer. We also demonstrated that NKG2D is required for in vivo antitumor activity. Furthermore, both activated and expanded CD8+ T cells and NK cells use DAP10. In addition, direct killing was partially dependent on the DAP12 signaling pathway. This requirement by activated and expanded CD8+ T cells for DAP12, and hence stimulus from a putative DAP12-partnered activating surface receptor, persisted when assayed by anti-NKG2D Ab-mediated redirected cytolysis. These studies demonstrated the importance of NKG2D, DAP10, and DAP12 in human effector cell function.     

Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.     

DAP10 deficiency breaks the immune tolerance against transplantable syngeneic melanoma

DAP10, an activating adaptor protein, associates with the NKG2D protein to form a multisubunit receptor complex that is expressed in lymphoid and myeloid cells. The ligands for NKG2D-DAP10 receptor are expressed in both normal and tumor cells, suggesting distinct roles for this receptor in autoimmunity and cancer. In this study, we report that constitutive DAP10 activating signaling is part of regulatory mechanisms that control immunity against tumors. Mice lacking DAP10 (DAP10KO), showed enhanced immunity against melanoma malignancies due to hyperactive functioning of NK1.1+CD3+ NKT cells. DAP10 deficiency resulted in substantially increased NKT cell functions, including cytokine production and cytotoxicity, leading to efficient killing of melanoma tumors. Moreover, the antitumor phenotype of DAP10KO mice correlated with impaired activation status of CD4+CD25+ T regulatory cells (Tregs). Upon activation, DAP10KO Tregs maintained higher levels of IL-2 and produced significantly lower amounts of IL-10 and IFN-gamma cytokines when compared with wild-type Tregs. Our data suggest that DAP10 signaling is involved in adjusting the activation threshold and generation of NKT cells and Tregs to avoid autoreactivity, but also modulates antitumor mechanisms.     

A Structural basis for the association of DAP12 with mouse, but not human, NKG2D

Prior studies have revealed that alternative mRNA splicing of the mouse NKG2D gene generates receptors that associate with either the DAP10 or DAP12 transmembrane adapter signaling proteins. We report that NKG2D function is normal in human patients lacking functional DAP12, indicating that DAP10 is sufficient for human NKG2D signal transduction. Further, we show that human NKG2D is incapable of associating with DAP12 and provide evidence that structural differences in the transmembrane of mouse and human NKG2D account for the species-specific difference for this immune receptor.     

Important roles for gamma interferon and NKG2D in gammadelta T-cell-induced demyelination in T-cell receptor beta-deficient mice infected with a coronavirus

gammadelta T cells mediate demyelination in athymic (nude) mice infected with the neurotropic coronavirus mouse hepatitis virus strain JHM. Now, we show that these cells also mediate the same process in mice lacking alphabeta T cells (T-cell receptor beta-deficient [TCRbeta(-/-)] mice) and demyelination is gamma interferon (IFN-gamma) dependent. Most strikingly, our results also show a major role for NKG2D, expressed on gammadelta T cells, in the demyelinating process with in vivo blockade of NKG2D interactions resulting in a 60% reduction in demyelination. NKG2D may serve as a primary recognition receptor or as a costimulatory molecule. We show that NKG2D(+) gammadelta T cells in the JHM-infected central nervous system express the adaptor molecule DAP12 and an NKG2D isoform (NKG2D short), both required for NKG2D to serve as a primary receptor. These results are consistent with models in which gammadelta T cells mediate demyelination using the same effector cytokine, IFN-gamma, as CD8 T cells and do so without a requirement for signaling through the TCR.     

Syk regulation of phosphoinositide 3-kinase-dependent NK cell function

Emerging evidence suggests that NK-activatory receptors use KARAP/DAP12, CD3zeta, and FcepsilonRIgamma adaptors that contain immunoreceptor tyrosine-based activatory motifs to mediate NK direct lysis of tumor cells via Syk tyrosine kinase. NK cells may also use DAP10 to drive natural cytotoxicity through phosphoinositide 3-kinase (PI3K). In contrast to our recently identified PI3K pathway controlling NK cytotoxicity, the signaling mechanism by which Syk associates with downstream effectors to drive NK lytic function has not been clearly defined. In NK92 cells, which express DAP12 but little DAP10/NKG2D, we now show that Syk acts upstream of PI3K, subsequently leading to the specific signaling of the PI3K-->Rac1-->PAK1-->mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase-->ERK cascade that we earlier described. Tumor cell ligation stimulated DAP12 tyrosine phosphorylation and its association with Syk in NK92 cells; Syk tyrosine phosphorylation and activation were also observed. Inhibition of Syk function by kinase-deficient Syk or piceatannol blocked target cell-induced PI3K, Rac1, PAK1, mitogen-activated protein/ERK kinase, and ERK activation, perforin movement, as well as NK cytotoxicity, indicating that Syk is upstream of all these signaling events. Confirming that Syk does not act downstream of PI3K, constitutively active PI3K reactivated all the downstream effectors as well as NK cytotoxicity suppressed in Syk-impaired NK cells. Our results are the first report documenting the instrumental role of Syk in control of PI3K-dependent natural cytotoxicity.     

The zebrafish activating immune receptor Nitr9 signals via Dap12

Both inhibitory and activating forms of natural killer (NK) cell receptors are found in mammals. The activating receptors play a direct role in the recognition of virally infected or transformed cells and transduce activating signals into the cell by partnering with an adaptor protein, which contains a cytoplasmic activation motif. Activating NK receptors encoded by the mammalian leukocyte receptor complex (e.g., killer cell immunoglobulin-like receptors) and the natural killer complex (e.g., Ly49s) partner with the adaptor protein DAP12, whereas NK receptors encoded in the CD94/NKG2 complex partner with the adaptor protein DAP10. Novel immune-type receptors (NITRs) found in bony fish share several common features with immunoglobulin-type NK receptors. Nitr9 is a putative activating receptor in zebrafish that induces cytotoxicity within the context of human NK cells. One isoform of Nitr9, Nitr9L, is shown here to preferentially partner with a zebrafish ortholog of Dap12. Cross-linking the Nitr9L–Dap12 complex results in activation of the phosphytidylinositol 3-kinase→AKT→extracellular signal-regulated kinase pathway suggesting that the DAP12-based activating pathway is conserved between bony fish and mammals. Sheng Wei and Jun-min Zhou contributed equally to this work.     

TGF-β1 Down-Regulation of NKG2D/DAP10 and 2B4/SAP Expression on Human NK Cells Contributes to HBV Persistence

The mechanism underlying persistent hepatitis B virus (HBV) infection remains unclear. We investigated the role of innate immune responses to persistent HBV infection in 154 HBV-infected patients and 95 healthy controls. The expression of NKG2D- and 2B4-activating receptors on NK cells was significantly decreased, and moreover, the expression of DAP10 and SAP, the intracellular adaptor proteins of NKG2D and 2B4 (respectively), were lower, which then impaired NK cell-mediated cytotoxic capacity and interferon-γ production. Higher concentrations of transforming growth factor-beta 1 (TGF-β1) were found in sera from persistently infected HBV patients. TGF-β1 down-regulated the expression of NKG2D and 2B4 on NK cells in our in vitro study, leading to an impairment of their effector functions. Anti-TGF-β1 antibodies could restore the expression of NKG2D and 2B4 on NK cells in vitro. Furthermore, TGF-β1 induced cell-cycle arrest in NK cells by up-regulating the expression of p15 and p21 in NK cells from immunotolerant (IT) patients. We conclude that TGF-β1 may reduce the expression of NKG2D/DAP10 and 2B4/SAP, and those IT patients who are deficient in these double-activating signals have impaired NK cell function, which is correlated with persistent HBV infection.     

The Traffic of the NKG2D/Dap10 Receptor Complex during Natural Killer (NK) Cell Activation

NKG2D is an important activating receptor for triggering the NK cell cytotoxic activity, although chronic engagement of specific ligands by NKG2D is also known to provoke decreased cell surface expression of the receptor and compromised NK cell function. We have studied the dynamics of surface NKG2D expression and how exposure to the specific ligand major histocompatibility complex class I chain-related molecule B (MICB) affects receptor traffic and fate. While in the NKL cell line and “resting” NK cells NKG2D was found principally at the cell surface, in activated primary NK cells an intracellular pool of receptor could also be found recycling to the plasma membrane. Exposure of NK cells to targets expressing MICB resulted in degradation of ∼50% of total NKG2D protein and lysosomal degradation of the DAP10 adaptor molecule. Consistent with these observations, confocal microscopy experiments demonstrated that DAP10 trafficked to secretory lysosomes in both transfected NKL cells and in activated primary NK cells upon interaction with MICB-expressing target cells. Interestingly, polarization to the synapse of secretory lysosomes containing DAP10 was also observed. The implications of the intracellular traffic of the NKG2D/DAP10 receptor complex for NK cell activation are discussed. We propose that the rapid degradation of NKG2D/DAP10 observed coincident with recruitment of the receptor to the cytotoxic immune synapse may explain the loss of NKG2D receptor expression after chronic exposure to NKG2D ligands.The killer cell lectin-like receptor NKG2D is one of the best characterized NK³ cell-activating receptors. Signaling via NKG2D depends on its association with DAP10, a transmembrane adaptor molecule containing the sequence YINM, which signals via recruitment of phosphatidylinositol 3-kinase and Grb2 (growth factor receptor-bound protein 2) (1, 2). Effector cell activation mediated by NKG2D has been described as immune recognition of the “induced self,” because the cellular ligands for NKG2D (NKG2D-L): the polymorphic MHC class I chain-related molecules (MIC) A and MICB and the UL16-binding proteins are not normally expressed but instead are up-regulated on target cells after pathogen infection or tumor transformation to render these cells susceptible to NK cell lysis (3). Strikingly, however, although induced expression of NKG2D-L acts as a danger signal to provoke an immune response, a number of studies performed in mouse models have shown that chronic exposure to NKG2D-L can also lead to down-modulation of the surface expression of NKG2D and impaired NK cell cytotoxic function (4–6).In humans, a common feature of patients with multiple different tumors is the presence in the serum of high levels of soluble MICA and -B or UL16-binding proteins, released by tumor cells, that are associated with an impairment of CTL and NK cell cytotoxic function (7–10). These observations have been interpreted as suggesting that the release of soluble NKG2D-L is a strategy of tumor immune evasion (11). However, recent data show that receptor interaction with cell membrane-anchored MICB can also lead to impaired NKG2D function. We have shown that brief cytotoxic interactions between NK cells and MICB-expressing target cells trigger a synaptic interchange of NKG2D and MICB as well as a rapid down-modulation of surface NKG2D and compromised NK cell cytotoxicity suggesting that NKG2D traffic is rapidly altered upon recognition of MICB expressed on target cell (12).The surface level of a receptor is dictated by the relative rates of synthesis and transport to the plasma membrane and endocytosis, recycling, and degradation. The loss of cell surface NKG2D observed after NKG2D-L binding (7–10, 12) raises the question of what is the intracellular fate of the receptor on interaction with NKG2D-L. However, the traffic of this receptor has not been previously studied. Here we describe the dynamics of surface NKG2D expression and examine how cytotoxic interactions between NK cells and the MHC class I- 721.221 (here called 221) cells that express MICB (here called 221B) affect the traffic and fate of the NKG2D/DAP10 receptor complex. In NKL cells and resting primary NK cells NKG2D is mainly expressed at the cell surface; however, in activated primary NK cells an intracellular pool of receptor recycling to the cell surface is detected. During cytotoxic interactions the recognition of MICB expressed on target cells results in a rapid degradation of NKG2D/DAP10 that is associated with the traffic of DAP10 to secretory lysosomes (SLs) (13, 14). Our data provide new insights into the dynamics of NKG2D receptor expression in NK cells and suggest a plausible model to explain how chronic exposure to NKG2D-L could lead to NKG2D down-modulation and compromised NK cell function.     

T cells gene-engineered with DAP12 mediate effector function in an NKG2D-dependent and major histocompatibility complex-independent manner

NKG2D is an important activating/co-stimulatory receptor harnessed by NK and T cells in immune surveillance. In contrast to NK cells, T cells fail to express the activation-signaling molecule DAP12 even when activated and, therefore, ligation of NKG2D alone is insufficient to induce T cell cytolytic function. To test whether we could endow T cells with NK cell-like effector function, we have engineered DAP12 into T cells by retroviral transduction (T-DAP12). T-DAP12 cells were demonstrated to specifically secrete interferon-gamma following receptor ligation and to mediate potent and specific lysis of the NKG2D ligand (NKG2D-L) (Rae-1beta) expressing MHC class I-deficient and class I-sufficient tumors. To circumvent the inability of T-DAP12 cells to proliferate following NKG2D ligation by Rae-1beta expressing tumors, DAP12 was engineered into OT-1 cells with an endogenous T cell receptor specific for chicken ovalbumin peptide (amino acids 257-264). Importantly, following a period of proliferation through endogenous T cell receptor ligation, OT-1-DAP12 cells retained specificity against NKG2D-L expressing major histocompatibility complex class I-deficient tumor. In adoptive transfer experiments, T-DAP12 cells enhanced the survival of NK cell-depleted RAG-1-deficient mice inoculated with RMA-S-Rae-1beta but not parental RMA-S tumors. Overall, this study demonstrated the significant potential of suppressing tumors and other cellular targets expressing NKG2D-L by endowing T cells with innate NK cell-like function.     

The isoforms of phospholipase C-gamma are differentially used by distinct human NK activating receptors

The two isoforms of phospholipase C (PLC)-gamma couple immune recognition receptors to important calcium- and protein kinase C-dependent cellular functions. It has been assumed that PLC-gamma1 and PLC-gamma2 have redundant functions and that the receptors can use whichever PLC-gamma isoform is preferentially expressed in a cell of a given hemopoietic lineage. In this study, we demonstrate that ITAM-containing immune recognition receptors can use either PLC-gamma1 or PLC-gamma2, whereas the novel NK cell-activating receptor NKG2D preferentially couples to PLC-gamma2. Experimental models evaluating signals from either endogenous receptors (FcR vs NKG2D-DAP10) or ectopically expressed chimeric receptors (with ITAM-containing cytoplasmic tails vs DAP10-containing cytoplasmic tails) demonstrate that PLC-gamma1 and PLC-gamma2 both regulate the functions of ITAM-containing receptors, whereas only PLC-gamma2 regulates the function of DAP10-coupled receptors. These data suggest that specific immune recognition receptors can differentially couple to the two isoforms of PLC-gamma. More broadly, these observations reveal a basis for selectively targeting the functions initiated by distinct immune recognition receptors.     

Phosphatidylinositol 3-kinase activation is required to form the NKG2D immunological synapse

The receptor NKG2D allows natural killer (NK) cells to detect virally infected, stressed, and tumor cells. In human cells, NKG2D signaling is mediated through the associated DAP10 adapter. Here we show that engagement of NKG2D by itself is sufficient to stimulate the formation of the NK immunological synapse (NKIS), with recruitment of NKG2D to the center synapse. Mutagenesis studies of DAP10 revealed that the phosphatidylinositol 3-kinase binding site, but not the Grb2 binding site, was required and sufficient for recruitment of DAP10 to the NKIS. Surprisingly, we found that in the absence of the Grb2 binding site, Grb2 was still recruited to the NKIS. Since the recruitment of Grb2 was dependent on phosphatidylinositol-(3,4,5)-trisphosphate (PIP₃), we explored the possibility that recruitment to the NKIS is mediated by a pleckstrin homology (PH) domain-containing binding partner for Grb2. We found that the PH domain of SOS1, but not that of Vav1, was able to be recruited by PIP₃. These results provide new insights into the mechanism of immunological synapse formation and also demonstrate how multiple mechanisms can be used to recruit the same signaling proteins to the plasma membrane.     

Generation, affinity maturation, and characterization of a human anti-human NKG2D monoclonal antibody with dual antagonistic and agonistic activity

In humans, NKG2D is an activating receptor on natural killer (NK) cells and a costimulatory receptor on certain T cells and plays a central role in mediating immune responses in autoimmune diseases, infectious diseases, and cancer. Monoclonal antibodies that antagonize or agonize immune responses mediated by human NKG2D are considered to be of broad and potent therapeutic utility. Nonetheless, monoclonal antibodies to NKG2D that are suitable for clinical investigations have not been published yet. Here, we describe the generation, affinity maturation, and characterization of a fully human monoclonal antibody to human NKG2D. Using phage display technology based on a newly generated naïve human Fab library in phage display vector pC3C followed by a tandem chain shuffling process designed for minimal deviation from natural human antibody sequences, we selected a human Fab, designated KYK-2.0, with high specificity and affinity to human NKG2D. KYK-2.0 Fab blocked the binding of the natural human NKG2D ligands MICA, MICB, and ULBP2 as potently as a commercially available mouse anti-human NKG2D monoclonal antibody in immunoglobulin G (IgG) format. Conversion of KYK-2.0 Fab to IgG1 resulted in subnanomolar avidity for human NKG2D. KYK-2.0 IgG1 was found to selectively recognize defined subpopulations of human lymphocytes known to express NKG2D, that is, the majority of human CD8+, CD16+, and CD56+ cells as well as a small fraction of human CD4+ cells. In solution, KYK-2.0 IgG1 interfered with the cytolytic activity of ex vivo expanded human NK cells. By contrast, immobilized KYK-2.0 IgG1 was found to strongly induce human NK cell activation. The dual antagonistic and agonistic activity promises a wide range of therapeutic applications for KYK-2.0 IgG1 and its derivatives.     

Activation of V gamma 9V delta 2 T cells by NKG2D

Human Vgamma9 Vdelta2 T cells recognize phosphorylated nonpeptide Ags (so called phosphoantigens), certain tumor cells, and cells treated with aminobisphosphonates. NKG2D, an activating receptor for NK cells, has been described as a potent costimulatory receptor in the Ag-specific activation of gammadelta and CD8 T cells. This study provides evidence that Vgamma9 Vdelta2 T cells may also be directly activated by NKG2D. Culture of PBMC with immobilized NKG2D-specific mAb or NKG2D ligand MHC class I related protein A (MICA) induces the up-regulation of CD69 and CD25 in NK and Vgamma9 Vdelta2 but not in CD8 T cells. Furthermore, NKG2D triggers the production of TNF-alpha but not of IFN-gamma, as well as the release of cytolytic granules by Vgamma9 Vdelta2 T cells. Purified Vgamma9 Vdelta2 T cells kill MICA-transfected RMA mouse cells but not control cells. Finally, DAP10, which mediates NKG2D signaling in human NK cells, was detected in resting and activated Vgamma9 Vdelta2 T cells. These remarkable similarities in NKG2D function in NK and Vgamma9 Vdelta2 T cells may open new perspectives for Vgamma9 Vdelta2 T cell-based immunotherapy, e.g., by Ag-independent killing of NKG2D ligand-expressing tumors.