Leibson, Paul J.

Abstract NKG2D is expressed on cytotoxic lymphocytes and is an important immunosurveillance receptor that recognizes stress ligands such as MICA on tumor cells or virally infected cells. Unlike traditional immune activating receptors, NKG2D couples to the non-ITAM-containing transmembrane adaptor DAP10, which recruits PI3K and Grb2 to initiate target cell lysis. Current understanding predicts an ITAM-independent mechanism of NKG2D-mediated cytotoxicity. However, we show that in primary human NK cells, the ITAM-containing adaptors DAP12, FcϵR1γ, and CD3ζ selectively regulate the Grb2-Vav1 branch of NKG2D-mediated signal transduction and cytotoxicity. Furthermore, this ITAM-adaptor regulation of NKG2D is independent of ITAM activation. Thus our data reveal a more complex interplay between NKG2D and other activating receptors than was previously imagined, and we have uncovered a novel mechanism of ITAM-adaptor regulation of cell-mediated cytotoxicity. A more thorough understanding of immunoreceptor signaling networks will lead to more precise therapies when fighting diseases such as cancer, autoimmunity, and infection.

Abstract The NK cell activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell - target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D coated beads is impaired in the absence of CrkL. Antibody-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras-family GTPase Rap1 is required for NK cell killing and is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization and granule release.

Abstract Stimulation of NK cell-mediated cytotoxicity involves the coupling of proximal Src and Syk family protein tyrosine kinases to downstream effectors. However, the mechanisms linking these second messenger pathways are incompletely understood. Here, we describe a key role for the LAT (p36) adaptor protein in human NK cell activation. LAT is tyrosine phosphorylated upon stimulation of NK cells through FcγRIII receptors and following direct contact with NK-sensitive target cells. This NK stimulation induces the association of LAT with several phosphotyrosine-containing proteins. In addition to the biochemical evidence showing LAT involvement in NK cell activation, a genetic model shows that LAT is required for FcR-dependent phosphorylation of phospholipase C-γ. Furthermore, overexpression of LAT in NK cells leads to increased Ab-dependent cell-mediated cytotoxicity and “natural cytotoxicity,” thus demonstrating a functional role for LAT in NK cells. These data suggest that LAT is an important adaptor protein for the regulation of human NK cell-mediated cytotoxicity.

Abstract Ligation of the Fc γ R on NK cells by Ab-coated target cells initiates a mode of killing referred to as antibody-dependent cell-mediated cytotoxicity (ADCC). There is clear evidence that the release from NK cells of granules containing pore-forming proteins and serine proteases can result in the rapid (within minutes) cell death of Ab-coated targets. However, little information is available as to whether NK cells can initiate subsequent killing through granule-independent mechanisms and as to the mechanisms that down-regulate NK cell-mediated responses. We demonstrate in this study that FcR stimulation of activated human NK cells not only induces granule exocytosis, but also subsequently results in the transcriptional up-regulation of Fas ligand. These FcR-stimulated NK cells can then kill targets that bear Fas (CD95/APO-1), as this cytotoxicity can be inhibited by blocking Abs to the Fas receptor. In addition, as resting NK cells become activated, their Fas receptors become competent to deliver autocrine suicide signals. We demonstrate in this work that the interaction of Fas ligand on the FcR-stimulated NK cells with their Fas receptors can result in apoptosis of the NK cells. These results suggest that the FcR-induced expression of Fas ligand on activated NK cells can critically influence the capacity of these cells to mediate paracrine and autocrine cell death.

Abstract Selective cell death plays a critical role in the development of the immune repertoire and in the elimination of target cells expressing foreign Ags. The apoptosis induced by ligation of the Fas Ag, a member of the TNFR/nerve growth factor receptor superfamily, contributes to both of these modes of cell loss. However, in spite of the molecular cloning of the Fas Ag and the identification of a specific cytoplasmic domain required for its function, it remains unclear as to which Fas-induced second messengers mediate the development of programmed cell death. We, therefore, evaluated Fas-initiated signal transduction in susceptible cell types. We determined that Fas ligation induces the rapid tyrosine phosphorylation of multiple cellular proteins. These phosphorylation events occur within 1 min and decline toward baseline by 30 min. In addition, Fas ligation increases the in vitro protein kinase activity of the tyrosine phosphorylated proteins. Pharmacologic inhibitors of protein tyrosine kinases block, in a concentration-dependent manner, Fas-induced DNA fragmentation and prolong cell survival. These results suggest that protein tyrosine kinase activation is an early and obligatory signal in Fas-induced apoptosis.

Abstract NK cell-mediated killing is inversely proportional to the amount of MHC class I expression on certain tumor targets. Two hypotheses have been proposed to explain why class I-bearing targets are more resistant to NK cell-mediated lysis: 1) the presence of class I prevents NK cell recognition of a triggering molecule on the target cell surface, or 2) class I recognition transmits a separate inhibitory signal to the NK cell. To differentiate between these potential mechanisms, we have used cloned human CD16+/CD3- NK cells, the class I-deficient cell line C1R, and C1R cells expressing high levels of transfected HLA class I gene products. If class I expression blocks NK cell recognition of the targets, then proximal cell signaling events such as phospholipase C-mediated hydrolysis of membrane phosphoinositides should be decreased in the NK cells interacting with the class I transfectants. However, we found that increases in the level of target cell class I expressions did not decrease phosphoinositide turnover or calcium signaling in NK cells. We also examined the effect of treating HLA-transfected C1R cells with mAb specific for the transfected MHC class I gene product. If class I expression has a negative regulatory influence on NK cell activation, then treating the targets with anti-HLA mAb should block the transmission of this negative signal. Consistent with this notion, addition of anti-HLA mAb (either whole Ig or F(ab’)2 fragments) led to increased lysis of the class I-transfected targets. In contrast, addition of isotype-matched mAb specific for other cell surface markers did not alter sensitivity to lysis. All of these results suggest that MHC class I expression on target cells can initiate inhibitory signals in NK cells without blocking access to target structures.

Abstract Antibody-dependent cellular cytotoxicity is initiated when low affinity Fc receptors (Fc gamma R type III/CD16) on NK cells bind to sensitized (i.e., antibody coated) target cells. Fc gamma R cross-linkage induces the activation of phospholipase C (PLC), which hydrolyses membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. However, the mechanism that couples Fc gamma R stimulation to PLC activation remains unknown. In this study, we investigated whether the Fc gamma R is coupled to PLC via a guanine nucleotide-binding (G) protein or an alternative pathway. Stimulation of electropermeabilized human NK cells with GTP gamma S induced inositol phosphate (IP) release, indicating the presence of a G protein-linked PLC activity in these cells. However, stimulation with both anti-Fc gamma R mAb and GTP gamma S provoked additive rather than synergistic increases in IP formation. Furthermore, exogenous GDP strongly inhibited GTP gamma S-stimulated IP release, but failed to inhibit the response to anti-Fc gamma R mAb stimulation. These results suggested GTP gamma S and anti-Fc gamma R mAb activated PLC through distinct regulatory mechanisms, and that Fc gamma R was not linked to PLC via a G protein. Hence, an alternative transduction mechanism for Fc gamma R-PLC coupling was considered. Antibody-mediated Fc gamma R cross-linkage was shown to rapidly stimulate tyrosine phosphorylation of multiple proteins in NK cells. Pretreatment with the tyrosine kinase inhibitor, herbimycin A, inhibited these phosphorylation events and disrupted the coupling between Fc gamma R ligation and PLC activation. These observations suggest that Fc gamma R in NK cell is coupled to PLC via a G protein-independent, but tyrosine kinase-dependent pathway.

Abstract IL-5 production in vivo plays a unique role in the production, activation, and localization of eosinophils in a variety of allergic conditions. The current paradigm suggests that allergen-specific Th2 cells are the main source for the IL-5 production. The experiments outlined in this work, however, suggest that in vivo production of IL-5 by NK cells can separately influence eosinophil-associated inflammatory responses. Specifically, a mouse model of allergic inflammation was used in which C57BL/6 mice were immunized and challenged with a short ragweed Ag extract, known to induce a selective eosinophilia within the peritoneal cavity. Peritoneal lavage fluids from these mice also contained increased numbers of T cells and NK cells, as well as significantly elevated levels of IL-4, IL-5, and IFN-γ. Flow-cytometric analysis of cytokine-producing cells in peritoneal lavage fluid revealed increased numbers of IL-5-producing cells in both T cell and NK cell populations following allergen exposure. Depletion of NK cells by treatment with NK1.1 Abs selectively reduced the number of infiltrating eosinophils by more than 50%. Moreover, the inhibition of the infiltration of eosinophils was accompanied by a complete loss of IL-5-producing NK cells and significantly reduced levels of peritoneal lavage fluid IL-5, whereas the number of IL-5-producing T cells was not affected. Thus, the results presented in this study provide clear evidence for a novel immunoregulatory function of NK cells in vivo, promoting allergen-induced eosinophilic inflammatory responses by the production of IL-5.

Abstract Activation-induced cell death (AICD) is initiated by the TCR-dependent up-regulation of Fas ligand (FasL) mRNA. The subsequently generated soluble or cell-associated FasL gene products bind Fas, leading to apoptosis of the T cells. Although TCR stimulation is essential to initiate AICD, little is known about which TCR-initiated second messengers are required for FasL expression. We provide evidence in this work that T cells lacking the tyrosine kinase ZAP-70 are unable to up-regulate FasL and undergo AICD. Transfection of wild-type ZAP-70 into the ZAP-70-deficient T cells restores their sensitivity to TCR-induced apoptosis, whereas transfection of catalytically inactive ZAP-70 does not. These results provide clear evidence that ZAP-70 tyrosine kinase is essential in up-regulating FasL for TCR-induced apoptosis.

Abstract Ligation of MHC class I-recognizing receptors on NK cells dramatically modulates their secretory and cytotoxic function. This study focuses on characterizing key signaling events regulating these activities after ligation of the C-type lectin superfamily member, CD94. We isolated separate clonal populations of human NK cells in which ligation of CD94 (kp43) either triggered cell-mediated cytotoxicity (group A clones) or potently inhibited NK cell activation (group B clones). We then evaluated the proximal signaling events that regulate these alternative responses. CD94 stimulation of group A clones induced the rapid activation of intracellular protein tyrosine kinases (i.e., lck and ZAP-70), phospholipase C, and phosphatidylinositol 3-kinase. In contrast, CD94 ligation on group B clones had none of the above noted effects and instead inhibited the FcR-induced tyrosine phosphorylations of ZAP-70 and phospholipase C-gamma 2, the formation of phospho-zeta/ZAP-70 complexes, and the release of inositol phosphates. These results define distinct proximal signaling events initiated after CD94 ligation and suggest that clonotypic differences in signaling generate fundamentally different NK cell-mediated responses.