Natural killer (NK) activity of cultured S100 beta-positive T-leukemia cells

In order to clarify the function of human S100 beta-positive T-cells, S100 beta-positive T-leukemia cells (S100 beta TLC) were examined in vitro. S100 beta TLC were obtained from the peripheral blood of a patient with S100 beta-positive T-cell leukemia and enriched by an E-rosetting method. Two dimensional flow cytometric analysis indicated that the vast majority of the E-positive fraction were S100 beta TLC expressing CD3 and CD8 antigens. Although S100 beta TLC expressed CD3 antigen, they were negative for the alpha/beta and gamma/delta T-cell antigen receptor (TCR) defined by monoclonal antibodies (mabs) WT-31 and delta TCS-1, respectively. It was speculated that S100 beta TLC initially expressed alpha/beta TCR but lost it during malignant transformation. When S100 beta TLC were cultured for 24 h, they acquired cytotoxic activity towards various NK-sensitive cell lines including K-562, Molt-3 and CEM-CCLF, but did not exhibit lysing activity towards NK-resistant cell lines including Raji, Daudi and MT-1. Despite the NK-activity of cultured S100 beta TLC, they lacked the morphological features of large granular lymphocytes (LGL). S100 beta TLC did not exhibit lymphokine-activated killer (LAK) activity. When S100 beta TLC were cocultivated with NK-sensitive cells or NK-resistant cells, they selectively bound to NK-sensitive cells, indicating that they lysed target cells by cell-to-cell contact. The finding that S100 beta TLC lacked TCR molecules and their NK activity was not inhibited by mabs reactive with the CD3-TCR complex indicated that the CD3-TCR complex was not involved in their target recognition.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

Natural killer cells are deficient in the surface expression of the complement regulatory protein, decay accelerating factor (DAF)

Decay-accelerating factor (DAF) is a 75,000 m.w. membrane protein that inhibits autologous complement C3 activation at the cell surface. One-color direct immunofluorescence with anti-DAF antibody and cytofluorographic analysis indicates that normal human monocytes and granulocytes are uniform in expression of DAF, whereas 23% of peripheral blood lymphocytes are DAF deficient. A two-color indirect immunofluorescence method, used to define the phenotype(s) of the DAF-deficient lymphocytes, was less efficient in DAF detection and led to overestimation of the fraction of deficient cells. Nonetheless, the difference between DAF expression by natural killer cells, identified by the CD16 and HNK-1 antigens, was marked. DAF deficiency was intermediate in cells expressing the CD2, CD3, CD4, or CD8 markers. On the basis of the phenotypic definition of natural killer cells and their contribution to the lymphocyte population, it is concluded that a uniform deficiency of DAF on natural killer cells accounts for about one-half of the DAF-deficient lymphocytes in peripheral blood of normal donors. The finding of a complete DAF deficiency in the lymphocytes from a patient with a lymphoproliferative disorder with the predominant proliferation of CD2+, CD3+, CD8+, HNK-1+ large granular lymphocytes gives additional support for the association of DAF-deficiency with natural killer cells.     

Kinome analysis of receptor-induced phosphorylation in human natural killer cells

Background

Natural killer (NK) cells contribute to the defense against infected and transformed cells through the engagement of multiple germline-encoded activation receptors. Stimulation of the Fc receptor CD16 alone is sufficient for NK cell activation, whereas other receptors, such as 2B4 (CD244) and DNAM-1 (CD226), act synergistically. After receptor engagement, protein kinases play a major role in signaling networks controlling NK cell effector functions. However, it has not been characterized systematically which of all kinases encoded by the human genome (kinome) are involved in NK cell activation.

Results

A kinase-selective phosphoproteome approach enabled the determination of 188 kinases expressed in human NK cells. Crosslinking of CD16 as well as 2B4 and DNAM-1 revealed a total of 313 distinct kinase phosphorylation sites on 109 different kinases. Phosphorylation sites on 21 kinases were similarly regulated after engagement of either CD16 or co-engagement of 2B4 and DNAM-1. Among those, increased phosphorylation of FYN, KCC2G (CAMK2), FES, and AAK1, as well as the reduced phosphorylation of MARK2, were reproducibly observed both after engagement of CD16 and co-engagement of 2B4 and DNAM-1. Notably, only one phosphorylation on PAK4 was differentally regulated.

Conclusions

The present study has identified a significant portion of the NK cell kinome and defined novel phosphorylation sites in primary lymphocytes. Regulated phosphorylations observed in the early phase of NK cell activation imply these kinases are involved in NK cell signaling. Taken together, this study suggests a largely shared signaling pathway downstream of distinct activation receptors and constitutes a valuable resource for further elucidating the regulation of NK cell effector responses.     

A serine phosphatase is involved in CD2-mediated activation of human T lymphocytes and natural killer cells.

We investigated early activation events after T cell triggering via the Ag receptor (TCR/CD3) complex as compared to activation via the CD2 surface molecule. To this end, resting peripheral human T lymphocytes were preincubated with 32P-orthophosphate and subsequently exposed to mitogenic mAb directed at either TCR/CD3 or CD2 for varying time periods. Cells were lysed and postnuclear lysates subjected to two-dimensional-gel electrophoresis (IEF and SDS-PAGE). As early as 10 min after stimulation through CD2, dephosphorylation of a cytosolic 19-kDa protein was observed. In contrast, this protein remained phosphorylated in unstimulated as well as CD3 activated T cells. Phosphoprotein (pp) 19 dephosphorylation was transient because, at later time points (2-4 h) after CD2 triggering, this protein was phosphorylated again. Phosphoaminoacid analysis indicated that pp19 is dephosphorylated on serine residues. Identical results were obtained using a CD2+ but TCR/CD3- human NK cell clone indicating that pp19 dephosphorylation occurs independent of surface expression of a TCR/CD3 complex. These data show that, in addition to protein phosphorylation events, serine dephosphorylation is involved in T cell triggering. More important, a selective signaling mechanism appears to be linked to T cell activation through the CD2 pathway.     

Immunotherapy with ligands of natural killer T cells

Natural killer T (NKT) cells are innate lymphocytes that share receptor structures and functions with conventional T cells and natural killer cells. NKT cells are specific for glycolipid antigens bound by the major histocompatibility complex class I-like protein CD1d. One striking property of NKT cells is their capacity to rapidly produce large amounts of cytokines in response to T-cell receptor engagement, suggesting that activated NKT cells can modulate adaptive immune responses. Recent pre-clinical studies have revealed significant efficacy of NKT-cell ligands such as the glycolipid alpha-galactosylceramide for treatment of metastatic cancers and infections, and for prevention of autoimmune diseases. These findings suggest that appropriate stimulation of NKT cells could be exploited for prevention or treatment of human diseases.     

Ganglioside GM3 activates ERKs in human lymphocytic cells

In this study we analyzed the signaling pathway triggered by GM3 in lymphoblastoid T-cells. In these cells, GM3 induced cPLA2 activation, arachidonic acid release, and PKC-delta translocation. In order to clarify the upstream molecular signals triggered by GM3, we analyzed the activation of extracellular signal-regulated kinase (ERK)s, a downstream effector of Ras-regulated cytoplasmic kinase cascade. Our results showed that GM3 treatment led to rapid ERK phosphorylation in lymphoblastoid T-cells, as detected by anti-phospho-p44/42 MAP kinase. Similar findings were found in human peripheral blood lymphocytes. Moreover, we showed that GM3 specifically phosphorylated ERK-2, as revealed by anti-phosphotyrosine reactivity on both cell free lysates and ERKs immunoprecipitates. The role of the CD4 cytoplasmic domain in GM3-triggered signaling pathway was investigated using A2.01/CD4-cyt399 cells, which had been transfected with a mutant form of CD4 lacking the bulk of the cytoplasmic domain. In these cells GM3 induced cPLA2 activation, arachidonic acid release, and PKC-delta translocation, but not CD4 endocytosis, indicating that the CD4 cytoplasmic domain plays a key role in GM3-triggered CD4 endocytosis and the GM3-triggered biochemical pathway is upstream of CD4 phosphorylation. These findings strongly suggest that GM3 triggers a novel signaling pathway involved in the regulation of cellular functions.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

A subset of CD16-natural killer cells without antibody-dependent cellular cytotoxicity function

The low affinity IgG receptor, CD16 (Fc gamma RIII), is expressed on almost all peripheral blood natural killer (NK) cells. A small subset of CD3- CD16- CD56+ NK cells, representing less than 1% of peripheral blood lymphocytes, expands during in vivo IL-2 treatment. To analyze this CD16- NK cell subset in more detail, NK clones have been generated. One of them (TNK2) has been used to study the function of these cells in more detail. It is demonstrated that TNK2 exerts normal NK activity and displays large granular lymphocyte morphology. Since this clone lacks CD16 expression, antibody-dependent cellular cytotoxicity cannot be exerted. CD16 monoclonal antibodies fail to induce cytotoxic activity against NK-resistant target cells. These studies reveal that the lack of CD16 detection is not due to the modulation or the stage of activation of these NK cells. TNK2 is representative of this small subset of peripheral blood NK cells, expanded during IL-2 treatment, which does not express Fc gamma RIII and therefore cannot perform antibody-dependent cellular cytotoxicity.     

Leu-Leu-OMe sensitivity of human activated killer cells: delineation of a distinct class of cytotoxic T lymphocytes capable of lysing tumor targets

Sensitivity to L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) was used to characterize the phenotype of human activated killer cells. Natural killer cells (NK) and the precursors of both the alloantigen-specific cytotoxic T lymphocytes (CTL) and the NK-like activated killer cells generated after stimulation with allogeneic cells were deleted from human peripheral blood lymphocytes by preincubation with Leu-Leu-OMe. It was noted, however, that cytotoxic lymphocytes could be generated from Leu-Leu-OMe-treated lymphocyte precursors after 2 to 6 days of culture with the nonspecific mitogen, phytohemagglutinin (PHA). The characteristics of these killer cells indicated that they were a unique population that could be distinguished from other cytotoxic cells. Killing by these cells exhibited slow kinetics in that 18 hr cytotoxicity assays were required to detect full cytotoxic potential. When 18 hr assays were used, PHA-stimulated cytotoxic cells generated from Leu-Leu-OMe-treated lymphocytes were able to kill both NK-sensitive K562 cells and the relatively NK-resistant renal cell carcinoma cell line, Cur. These cytotoxic lymphocytes were HNK-1, Leu-11b (CD16), and OKM1 (CR3)-negative at both the precursor and effector stage of activation. Furthermore, these cells were derived from a CD3-positive precursor. Finally, killing by activated effectors was inhibited by OKT3. Unlike activation of Leu-Leu-OMe-sensitive large granular lymphocytes, generation of these cytotoxic T cells was totally prevented by treatment with mitomycin c before stimulation. Thus, a unique class of tumoricidal T cells can be characterized by resistance of lymphocyte precursors to a concentration of Leu-Leu-OMe, which has been shown to ablate NK, mixed lymphocyte culture-activated NK-like cytotoxic precursors, and the precursors of alloantigen-specific CTL.     

Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes

Though the nicotinic acetylcholine receptor (nAChR) subunits alpha9 and alpha 10 have been thoroughly characterized within hair cells of the organ of Corti in the inner ear, prior studies have shown that they are also expressed in lymphocytes. In this report, we sought to more definitively characterize the nAChR subunits alpha9 and alpha10 within various populations of human lymphocytes. Using a combination of techniques, including RT-PCR, single-cell RT-PCR, Northern and western blot analysis, and immunofluorescence, expression of both alpha9 and alpha 10 was demonstrated in purified populations of T-cells (CD3+, CD4+, CD8+ and the Jurkat, MT2 and CEM T-cell lines) and B-cells (CD19+, CD80+ and EBV-immortalized B-cells). Single-lymphocyte recording techniques failed to identify an ionic current in response to applied acetylcholine in either T-cells or B-cells. These results clearly demonstrate the presence of these nicotinic receptor subunits within several populations of human lymphocytes, implicating their role in the immune response. However, a lack of demonstrated response to applied acetylcholine using standard single-cell recording techniques suggests a physiology different than that seen in hair cells of the inner ear.     

Involvement of beta 2-integrins in the migration of human natural killer cells.

Human large granular lymphocytes with the NK cell phenotype (CD16+ or CD56+CD3-) were greatly enriched among the cells which migrated spontaneously through untreated or albumin-coated, 3-microns pore size polycarbonate filters for 1 to 8 h. Three days of rIL-2 treatment (300 IU/ml) and 3 to 5 wk of rIL-2 treatment (100 IU/ml) generated a 2.7 +/- 0.9-fold and 5.6 +/- 0.8-fold increase in cell migration, respectively. The adhesion and subsequent migration of freshly isolated NK cells was mainly mediated by CD11b/CD18, because migration could be inhibited by 80 +/- 8% anti-CD11b (Mac-1) antibodies but not with antibodies against CD11a (LFA-1) or CD11c (p150,95), the other alpha-chains of the beta 2-integrins. After rIL-2 activation, however, CD11a/CD18 was the major receptor utilized in migration, inasmuch as anti-CD11a antibody caused a 69 +/- 8% reduction in the number of migrated cells. Anti-CD11b antibody decreased migration by 43 +/- 12%, and together these antibodies inhibited migration by 82 +/- 7%. Anti-CD11a alone did not have any effect on adhesion, but CD11a/CD18 cooperated in the adhesion because anti-CD11b decreased adhesion by 40 +/- 11% and together these antibodies inhibited adhesion by 74 +/- 6%. The ability of large granular lymphocytes to rapidly utilize beta 2-integrins and unidentified ubiquitous ligands for binding and migration may be significant for their capacity to function in the first line of immune defense under highly variable conditions.     

Intracellular and intercellular signal transduction pathways and age alterations of proliferative activity and apoptosis intensity in cells of immune system

The paper considers age-associated alterations of intracellular and intercellular cascades of transduction of proliferative, differentiating, pro- and antiapoptotic signals, their interaction and influence on proliferative activity, differentiation and apoptosis of the immune system cells. One of initial causes of these alterations is accumulation with age of a growing number of antigens exposed on the surface of antigen-presenting cells. As a result of chronic antigenic stimulation, caused by this factor, an insufficient quantity or a slowed down appearance of growth factor receptors (in particular, IL-2 receptor) and costimulation molecules, primarily CD28, on T-cells membrane is observed. Because of this proliferative and antiapoptotic signals, received by T-cells, have a smaller intensity that predetermine reduction of their proliferative activity, and also activity of telomerase, and a greater susceptibility to apoptosis. Permanent activation of immune system is also reflected in age-related increase of expression of CD95 and type I tumour necrosis factor receptor by lymphocytes (that aggravates their susceptibility to apoptosis), and in intensification of proinflammatory cytokine synthesis. The second main cause of alterations in the immune system is an age-related decrease in the synthesis of growth factors that are necessary for cell survival and proliferation. In particular, because of the lack of IL-7, apoptosis intensity of maturing T-cells increases in thymus. Thymic stromal cells remain without contact signals and growth factors generated by lymphocytes, and also undergo apoptosis that causes further reduction of T-lymphopoiesis. Similar events also occur in bone marrow that predetermines age-related decrease in B-lymphopoiesis and in telomerase activity of haemopoietic stem cells, and also their proliferative potential reduction.     

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.     

Natural killer cells and natural killer T cells in Lyme arthritis

Introduction

Natural killer (NK) and natural killer T (NKT) cells provide a first line of defense against infection. However, these cells have not yet been examined in patients with Lyme arthritis, a late disease manifestation. Lyme arthritis usually resolves with antibiotic treatment. However, some patients have persistent arthritis after spirochetal killing, which may result from excessive inflammation, immune dysregulation and infection-induced autoimmunity.

Methods

We determined the frequencies and phenotypes of NK cells and invariant NKT (iNKT) cells in paired peripheral blood (PB) and synovial fluid (SF) samples from eight patients with antibiotic-responsive arthritis and fifteen patients with antibiotic-refractory arthritis using flow cytometry and cytokine analyses.

Results

In antibiotic-responsive patients, who were seen during active infection, high frequencies of CD56bright NK cells were found in SF, the inflammatory site, compared with PB (P <0.001); at both sites, a high percentage of cells expressed the activation receptor NKG2D and the chaperone CD94, a low percentage expressed inhibitory killer immunoglobulin-like receptors (KIR), and a high percentage produced IFN-γ. In antibiotic-refractory patients, who were usually evaluated near the conclusion of antibiotics when few if any live spirochetes remained, the phenotype of CD56bright cells in SF was similar to that in patients with antibiotic-responsive arthritis, but the frequency of these cells was significantly less (P = 0.05), and the frequencies of CD56dim NK cells tended to be higher. However, unlike typical NKdim cells, these cells produced large amounts of IFN-γ, suggesting that they were not serving a cytotoxic function. Lastly, iNKT cell frequencies in the SF of antibiotic-responsive patients were significantly greater compared with that of antibiotic-refractory patients where these cells were often absent (P = 0.003).

Conclusions

In patients with antibiotic-responsive arthritis, the high percentage of activated, IFN-γ-producing CD56bright NK cells in SF and the presence of iNKT cells suggest that these cells still have a role in spirochetal killing late in the illness. In patients with antibiotic-refractory arthritis, the frequencies of IFN-γ-producing CD56bright and CD56dim NK cells remained high in SF, even after spirochetal killing, suggesting that these cells contribute to excessive inflammation and immune dysregulation in joints, and iNKT cells, which may have immunomodulatory effects, were often absent.     

Immunocytochemical characterization of S-100 beta-positive human T-lymphocytes by a double immunostaining method

The antigenic properties of S-100 beta-positive human T-lymphocytes (S-100 beta+ T-cells) were investigated by a double immunostaining technique employing an indirect immunoperoxidase method for cytoplasmic S-100 beta subunit and an immunoalkaline phosphatase method for cell surface antigens detected by various monoclonal antibodies to human lymphocytes. S-100 beta+ T-cells recognized by their diffuse intracytoplasmic immunoperoxidase reaction, also expressed CD2, CD3, CD8 antigens demonstrated by surface blue alkaline phosphatase reactivity, but not CD4, CD1, CD25 (interleukin-2 receptor), or HLA-DR antigens. However, they displayed a blastic change to T-cell mitogens, such as Concanavalin A(Con-A) and PHA, followed by the expression of CD25 and HLA-DR antigens. Under normal conditions, S-100 beta+ T-cells comprised approximately 5-22.8% of CD8+ cells amongst human peripheral blood mononuclear cells.     

Phenotypic characterization of lymphokine-activated killer cells from human lymph node lymphocytes

We previously reported that lymphokine-activated killer (LAK) activity can be generated in human lymph node lymphocytes (LNL) at the same level as that in peripheral blood lymphocytes (PBL), despite the absence of active natural killer (NK) cells. In the present study, we investigated the surface phenotype of LNL-LAK cells by fractionation of lymphocytes, using a panning method. LNL isolated from lung cancer patients were cultured in the presence of recombinant interleukin 2 for 8 days and separated into T cells and non-T cells according to the expression of CD3 antigen. LAK effectors were enriched in the CD3- non-T cells. However, the CD3+ cells also mediated a low but substantial level of LAK activity, which was attributed to a CD8+ T-cell subset. Further investigation of the CD3- cells revealed that most of the CD3- effector cells expressed neither B-cell (CD20) nor NK-cell (CD16) markers. Precursors of this CD3-CD20-CD16- (null) population appeared to be also CD3-, CD20-, and CD16-. From these results, we would stress the significant contribution of CD3-CD20-CD16- null cells to the LAK phenomenon, which has not been focused on in PBL.     

Tyrosine phosphorylation of the Fc gamma RIII(CD16): zeta complex in human natural killer cells. Induction by antibody-dependent cytotoxicity but not by natural killing.

NK cells are large granular lymphocytes capable of killing certain tumor cells and virally infected cells in a non-MHC-restricted manner. NK cells can also effect an antibody dependent cytotoxicity that is triggered by CD16, an FcR for IgG. In NK cells, CD16 is expressed in association with zeta, a signal transducing subunit of the TCR complex. Here we show that, just as T cell activation via the TCR complex results in tyrosine phosphorylation of zeta TCR, NK cell activation via CD16 results in tyrosine phosphorylation of zeta NK. Whereas antibody-dependent cytotoxicity also results in tyrosine phosphorylation of zeta, natural cytotoxicity does not. Our results indicate that zeta functions as a transducing element for antibody dependent, but not antibody independent killing by NK cells. Consequently, NK cells are likely to express at least two distinct receptor complexes capable of triggering cytolytic effector function.