Human NK Cell Cytotoxicity Research Articles

Antitumor effects of recombinant human prolactin in human adenocarcinoma-bearing SCID mice with human NK cell xenograft

To survey the immune regulatory function of recombinant human prolactin (rhPRL) and its potential application in adoptive immunotherapy, CB17-SCID mice were loaded with human colon adenocarcinoma HT-29 cells (5×10 5 cells/mouse, i.p.) 24 h before adoptive transfer with the purified human NK cells followed by rhPRL injection (10 μg/mouse, every other day for a total of 10 injections). Upon analysis, rhPRL did not exert any direct inhibitory effects on HT-29 cells but slightly improved the tumor cell growth both in vitro and in vivo. After SCID mice were reconstituted with human NK cells, rhPRL improved the antitumor effects of human NK cells in HT-29-bearing SCID mice, showing a prolonged survival from 70.4 to 112.1 days, and the increased survival rate from all died to 40% survival for more than 160 days. rhPRL improved the proliferation of human NK cells with or without PHA stimulation. rhPRL also directly enhanced the cytotoxicity of human NK cells against HT-29 tumor cells in 4-h coculture. The supernatant of rhPRL-stimulating NK cells inhibited the proliferation of HT-29 cells through, at least partly, the interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the supernatant. Thus, rhPRL administration in HT-29 tumor-bearing SCID mice promotes the antitumor effects of adoptively transferred NK cells.

Homophilic interaction of NTBA, a member of the CD2 molecular family: induction of cytotoxicity and cytokine release in human NK cells.

NK-T-B antigen (NTBA) is a CD2 family member that functions as a coreceptor in human NK cell activation. Several receptor/ligand interactions occur between different members of this molecular family. In this study, in order to identify the natural ligand of NTBA, we produced a chimeric protein formed by the NTBA extracellular region fused with the Fc portion of human IgG1 (termed NTBA-Fc*). NTBA-Fc* specifically binds to NTBA cell transfectants but not to cells transfected with other CD2 family members including CD2, CD48, CD84, CD150, CD229, and CD244. Moreover, NTBA-Fc* also binds to NTBA(+) but not to NTBA(-) T cell lines. Enzyme-linked immunosorbent assays, plasmon resonance analysis, as well as NTBA-Fc*-mediated down-regulation of NTBA surface expression further confirmed the occurrence of NTBA/NTBA homophilic interaction. Functionally, in NK cells, NTBA-Fc* promoted a strong production of IFN-gamma and TNF-alpha. Moreover, NTBA-transfected targets displayed increased susceptibility to NK-mediated killing as compared to untransfected cells and this effect was specifically inhibited by anti-NTBA mAb. Altogether our data indicate that NTBA is characterized by self recognition.

Determinants of antileukemia effects of allogeneic natural killer cells

In HLA-nonidentical bone marrow transplantation, we sought to determine the characteristics of donor NK cells, recipient leukemia cells, and the cytokine environment that predict the antileukemia effects of allogeneic NK cells. We found that the risk of leukemia relapse in a prospective cohort of 36 pediatric patients was best predicted by a model taking into consideration the presence of inhibitory killer-cell immunoglobulin-like receptors (KIRs) on the donor’s NK cells and the absence of corresponding KIR ligand in the recipient’s HLA repertoire (a receptor-ligand model). The risk of relapse was prognosticated less precisely by the Perugia donor-recipient KIR ligand-ligand mismatch model or by a natural cytotoxicity model. In contrast to the Perugia model, we found that the new receptor-ligand model was accurate when analysis was applied to patients with lymphoid malignancy. These findings corroborate our observations that the recipient’s KIR repertoire, which was derived from highly purified HLA-disparate CD34+ cells, always resumed a donor-specific pattern within 3 months of transplantation but did not correlate evidently with either the donor or recipient ligand repertoire. In an in vitro assay and an in vivo mouse model, human NK-cell cytotoxicity toward human leukemia cells with 11q23 chromosomal rearrangement increased with the number of receptor-ligand mismatch pairs or prestimulation with IL-12 and IL-18. These findings provide new insights into the determinants of antileukemia effects of allogeneic NK cells and therapeutic strategies.

CD59 is physically and functionally associated with natural cytotoxicity receptors and activates human NK cell-mediated cytotoxicity.

Triggering of cytotoxicity in human NK cells is induced by the combined engagement of several triggering receptors. These include primary receptors such as NKG2D and the natural cytotoxicity receptors (NCR) NKp30, NKp46 and NKp44, while other molecules, including 2B4, NTB-A and NKp80, function as co-receptors. As reported in the present study, during an attempt to identify novel NK receptors or co-receptors, we found that CD59 functions as a co-receptor in human NK cell activation; engagement of CD59 by specific mAb delivers triggering signals to human NK cells, resulting in enhancement of cytotoxicity. Similar to other NK co-receptors, the triggering function of CD59, a glycosylphosphatidylinositol (GPI)-linked protein, depends on the simultaneous engagement of primary receptors such as NCR. Accordingly, CD59-dependent triggering was virtually restricted to NK cells expressing high surface densities of NKp46, and mAb-mediated modulation of NKp46 resulted in markedly decreased responses to anti-CD59 mAb. Biochemical analysis revealed that CD59 is physically associated with NKp46 and NKp30. Moreover, engagement of CD59 resulted in tyrosine phosphorylation of CD3zeta chains associated with these NCR, but not those associated with CD16. Thus, CD59-mediated costimulation of NK cells requires direct physical interaction of this GPI-linked protein with primary triggering NK receptors.

CD66a interactions between human melanoma and NK cells: a novel class I MHC-independent inhibitory mechanism of cytotoxicity.

NK cells are able to kill virus-infected and tumor cells via a panel of lysis receptors. Cells expressing class I MHC proteins are protected from lysis primarily due to the interactions of several families of NK receptors with both classical and nonclassical class I MHC proteins. In this study we show that a class I MHC-deficient melanoma cell line (1106mel) is stained with several Ig-fused lysis receptors, suggesting the expression of the appropriate lysis ligands. Surprisingly, however, this melanoma line was not killed by CD16-negative NK clones. The lack of killing is shown to be the result of homotypic CD66a interactions between the melanoma line and the NK cells. Furthermore, 721.221 cells expressing the CD66a protein were protected from lysis by YTS cells and by NK cells expressing the CD66a protein. Redirected lysis experiments demonstrated that the strength of the inhibitory effect is correlated with the levels of CD66a expression. Finally, the expression of CD66a protein was observed on NK cells derived from patients with malignant melanoma. These findings suggest the existence of a novel class I MHC-independent inhibitory mechanism of human NK cell cytotoxicity. This may be a mechanism that is used by some of the class I MHC-negative melanoma cells to evade attack by CD66a-positive NK cells.

2B4 (CD244)-mediated activation of cytotoxicity and IFN-gamma release in human NK cells involves distinct pathways.

2B4 (CD244), a member of the CD2 subset of the Ig superfamily receptors, is expressed on all human NK cells, a subpopulation of T cells, basophils and monocytes. 2B4 activates NK cell mediated cytotoxicity, induces secretion of IFN-gamma and matrix metalloproteinases, and NK cell invasiveness. Although there have been several molecules shown to interact with 2B4, the signaling mechanism of 2B4-mediated activation of NK cells is still unknown. In this study, we found cross-linking of 2B4 on YT cells, a human NK cell line, results in the increased DNA binding activity of activator protein-1 (AP-1), an important regulator of nuclear gene expression in leukocytes. We investigated the possible role of various signaling molecules that may be involved in the activation of lytic function of YT cells via 2B4. Treatment of YT cells with various specific inhibitors indicate that 2B4-stimulation of YT cells in spontaneous and Ab-dependent cytotoxicity is Ras/Raf dependent and involves multiple MAPK signaling pathways (ERK1/2 and p38). However, only inhibitors of transcription and p38 inhibited 2B4-mediated IFN-gamma release indicating distinct pathways are involved in cytotoxicity and cytokine release. In this study we also show that 2B4 constitutively associates with the linker for activation of T cells (LAT) and that 2B4 may mediate NK cell activation via a LAT-dependent signaling pathway. These results indicate that 2B4-mediated activation of NK cells involves complex interactions involving LAT, Ras, Raf, ERK and p38 and that cytolytic function and cytokine production may be regulated by distinct pathways.

Vav-1 regulates NK T cell development and NK cell cytotoxicity.

The hematopoietic-specific Rho-family GTP exchange factor Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells. Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, we show that Vav-1 is also expressed in murine NK cells. Vav-1(-/-) mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as wild-type mice. Unexpectedly, IL-2-activated Vav-1(-/-) NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1(-/-) NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, the results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK natural cytotoxicity. Unlike the findings for NK cells, NK T cells were dramatically diminished in Vav-1(-/-) mice and splenocytes from Vav-1 mutant mice failed to produce IL-4 in response to in vivo CD3 stimulation. These data highlight the important role of Vav-1 in NK T cell development and NK cell function.

Differential recognition of MHC class I molecules of xeno-/allo-endothelial cells by human NK cells

Using human umbilical vein endothelial cells (HUVEC) and porcine aortic endothelial cells (PAEC) as target cells, human peripheral blood NK cells (PBNK) and NK92 cells as effector cells, the differential cytotoxicities of NK cells to allo- and xeno-endothelial cells were studied. The influence of MHC class I molecules on the cytotoxicity of human NK cells was assayed using acid treatment, and blockades of MHC class I antigens, CD94 and KIR (NKB1). The results indicated that the killing of PAEC by the two kinds of NK cells is higher than that of HUVEC. After acid treatment, the cytotoxicity of the two kinds of NK cells to PAEC and HUVEC is significantly enhanced, but the magnitude of the enhancement is different. The enhancement of NK killing to acid treated HUVEC is much greater than that to PAEC. Blockade of CD94 mAb did not alter the NK cytotoxicity, while blockade of NKB1 mAb enhanced the cytotoxicity of PBNK to HUVEC and PAEC by 95% and 29% respectively. The results above suggested that the differential recognition of MHC I molecules of xeno-endothelial cells by human NK cells could be the major reason for higher NK cytotoxicity to PAEC. KIR might be the primary molecule that transduced inhibitory signals when endothelial cells were injured by NK cells.

CD69-triggered ERK activation and functions are negatively regulated by CD94 / NKG2-A inhibitory receptor.

CD69 represents a functional triggering molecule on activated NK and T cells, capable of inducing cytotoxic activity and costimulating cytokine production. It belongs to the C-lectin type superfamily, and its gene maps in the NK gene complex, close to other genes coding for NK receptors. CD94 / NKG2-A complex is the inhibitory receptor for the non classical MHC class I molecule HLA-E on human NK cells. To investigate CD69-initiated signal transduction pathways, and to evaluate CD94 / NKG2-A interference on CD69 triggering ability, we have generated transfectants expressing both receptors in the RBL cell line. Here we report that CD69 engagement leads to the activation of extracellular signal-regulated kinase (ERK) enzymes belonging to the MAPK family, and that this event is required for CD69-mediated cell degranulation. Moreover, we show that the co-engagement of CD94 / NKG2-A inhibitory receptor effectively suppresses both CD69-triggered cell degranulation in RBL transfectants, through the inhibition of ERK activation, and CD69-induced cytotoxicity in human NK cells. Thus, here we provide new information on the molecular mechanisms initiated by CD69 activation receptor, and show that CD69-initiated signaling pathways and functional activity are negatively regulated by CD94 / NKG2-A inhibitory complex.

Natural killer cell mediated cytotoxicity against VERO target cells; the suppressive effect of pentoxifylline

The K-562 cell line is widely known and used as a NK cell target. In this study we report that VERO (African green monkey kidney epithelial cell line) is an excellent target of the human NK cell cytotoxicity. Considerable cytotoxicity was observed in a 4 h 51Cr release assay with nonadherent and immunomagnetically separated CD56+ NK cells from PBMC. On the contrary, adding K-562 cells as cold target to the assay the cytotoxicity significantly decreased. Using a standard chromium-release assay the NK cell activity (NKCA) against VERO cells was investigated in a population of healthy volunteers (mean value of cytotoxicity was 26.6%) and compared with the values of cytotoxicity against K-562 target cells (32.6%). The difference was not significant (P > 0.05). The suppressive effect of PTX on in vitro NK cell activity was observed at concentration of 100 microg/ml using VERO target cells as well as K-562 cells. Our studies provide the first evidence that the NK cell activity is suppressed in vitro by PTX using VERO cells as NK target cells.

Inactivation of the a1-3 Galactosyltransferase Gene Protects Murine Hearts from Xenoantibody-dependent Human NK Cell/Monocyte Mediated Injury.

174 NK cells and monocytes are the predominant cell type in rejecting vascularized discordant xenografts in which hyperacute rejection has been prevented by complement inhibition i.e. in delayed xenograft rejection. a-Gal is the major porcine xenoantigen and anti-Gal Fab fragments inhibit human NK cell adhesion and cytotoxicity to porcine endothelial cells in vitro. In an attempt to further define the relationship between endothelial expression of a-Gal and NK cell-mediated cardiac rejection we studied a-Gal deficient mice in an ex-vivo perfusion model. A modified Langendorff apparatus was used to study hearts from mice with a targeted deletion in the Gal transferase gene (Gal KO) and size matched non-transgenic control mice. Cardiac work was determined by a pressure transducer affixed to the ventricle. Freshly isolated human PBMC (peripheral blood mononuclear cells) were obtained from healthy volunteers by density gradient sedimentation of whole blood. Hearts were perfused with buffer alone, human IgG, PBMC or the combination of human IgG and PBMC on the same day. A minimum of 4 perfusions were performed in each treatment group. After 70 minutes perfusions, hearts were snap frozen for histological analysis. Perfusion of control hearts with PBMC or IgG did not significantly alter the cardiac work over 70 minutes compared to buffer alone. When control hearts were perfused with PBMC+IgG there was a prompt and significant fall in cardiac function within 20 minutes which continued to decline throughout the perfusion period. In contrast to control hearts, there was no significant difference in performance between Gal KO hearts perfused with human IgG alone or PBMC+IgG. Histological examination of control hearts perfused with PBMC+IgG showed marked tissue disruption of non-transgenic hearts whereas identically perfused Gal KO mouse hearts showed minimal damage. This study shows that anti-Gal xenoantibody facilitates PBMC mediated cardiac dysfunction and morphological damage in this ex vivo model and that removal of Gal epitope by gene targeting abrogates the influence of IgG xenoantibody. Thus genetic manipulations which reduce or remove the a-Gal epitope may in addition to inhibiting hyperacute rejection, have an important role in inhibiting the cell-mediated component of delayed xenograft rejection.

Inactivation of the al-3 Galactosyltransferase Gene Protects Murine Hearts from Xenoantibody-dependent Human NK Cell / Monocyte Mediated Injury.

174 NK cells and monocytes are the predominant cell type in rejecting vascularized discordant xenografts in which hyperacute rejection has been prevented by complement inhibition i.e. in delayed xenograft rejection. a-Gal is the major porcine xenoantigen and anti-Gal Fab fragments inhibit human NK cell adhesion and cytotoxicity to porcine endothelial cells in vitro. In an attempt to further define the relationship between endothelial expression of a-Gal and NK cell-mediated cardiac rejection we studied a-Gal deficient mice in an ex-vivo perfusion model. A modified Langendorff apparatus was used to study hearts from mice with a targeted deletion in the Gal transferase gene (Gal KO) and size matched non-transgenic control mice. Cardiac work was determined by a pressure transducer affixed to the ventricle. Freshly isolated human PBMC (peripheral blood mononuclear cells) were obtained from healthy volunteers by density gradient sedimentation of whole blood. Hearts were perfused with buffer alone, human IgG, PBMC or the combination of human IgG and PBMC on the same day. A minimum of 4 perfusions were performed in each treatment group. After 70 minutes perfusion, hearts were snap frozen for histological analysis. Perfusion of control hearts with PBMC or IgG did not significantly alter the cardiac work over 70 minutes compared to buffer alone. When control hearts were perfused with PBMC+IgG there was a prompt and significant fall in cardiac function within 20 minutes which continued to decline throughout the perfusion period. In contrast to control hearts, there was no significant difference in performance between Gal KO hearts, perfused with human IgG alone or PBMC+IgG. Histological examination of control hearts perfused with PBMC+IgG showed marked tissue disruption of non-transgenic hearts whereas identically perfused Gal KO mouse hearts showed minimal damage. This study shows that anti-Gal xenoantibody facilitates PBMC mediated cardiac dysfunction and morphological damage in this ex vivo model and that removal of Gal epitope by gene targeting abrogates the influence of IgG xenoantibody. Thus genetic manipulations which reduce or remove the a-Gal epitope may in addition to inhibiting hyperacute rejection, have an important role in inhibiting the cell-mediated component of delayed xenograft rejection.

Interleukin-12 administered in vivo decreases human NK cell cytotoxicity and antibody-dependent cellular cytotoxicity to human immunodeficiency virus-infected cells.

Persons infected with human immunodeficiency virus (HIV) have cellular cytotoxicity defects. Interleukin (IL)-12 is a potent stimulator of cytotoxicity. Fifteen HIV-infected patients were studied in a phase 1, single-dose escalation trial of human recombinant IL-12. One day after subjects received an IL-12 dose of 300 or 1000 ng/kg, they had a reduction in absolute lymphocyte count and peripheral blood mononuclear cell recovery. In evaluable patients 24 h after IL-12 administration, there was a 31% reduction overall in NK cell cytotoxicity (NKC) to HIV-infected cells at all doses and a 52% reduction in antibody-dependent cellular cytotoxicity (ADCC) at doses of 300 and 1000 ng/kg. In vitro incubation of patients' cells with IL-12 (before IL-12 administration) for 24 h increased NKC but had no effect on ADCC. The paradoxic acute reduction in cell number and cytotoxicity in vivo may be due to NK cell trafficking or regulatory cytokine mechanisms not apparent in vitro.

Tumor therapy by immune recruitment with bispecific antibodies.

Many strategies for experimental immunotherapy of cancers aim at inducing and expanding tumor-specific cytotoxic T-lymphocytes. One of the most promising approaches uses bispecific monoclonal antibodies (Bi-MAbs) which are able to accumulate and activate human effector cells at the tumor site. Human resting peripheral NK- or T cells targeted by appropriate Bi-MAbs to tumor cells expressing a tumor-associated antigen display multiple signs of activation including proliferation, cytokine secretion, upregulation of cytotoxic peptides and enzymes and induce an efficient tumor cell lysis in vivo. Moreover, tumor-bearing SCID which were treated by effector cell-triggering Bi-MAbs and human peripheral blood lymphocytes had complete regressions of established tumors and most or all animals were cured by human NK-cell or T-cell cytotoxicity, respectively. Local tumor site-specific activation of T-cells was demonstrated, and enhanced granzyme and perforin expression together with the results of inhibition experiments suggest both mechanisms as the major contributors to the cytolytic machinery of Bi-MAb-mediated T-cell cytotoxicity in vivo. The encouraging results of this approach, which is able to cure animals with even advanced disseminated tumors, together with the local site-specific effector cell activation, which suggests minimal side effects, warrant the clinical evaluation of this Bi-MAb approach. As lymphocytes from tumor patients can be adequately activated by the respective Bi-MAbs, the clinical application of Bi-MAbs promises to become a safe, efficient and simple approach which should be readily applicable to the treatment of human malignancies that cannot be cured by standard regimens.

Effect of somatostatin on human lymphocyte proliferation and NK-cell cytotoxicity

Effect of somatostatin on human lymphocyte proliferation and NK-cell cytotoxicity

Chemical specificity of effector cell/tumor cell bridging by a Viscum album rhamnogalacturonan enhancing cytotoxicity of human NK cells

The component in Viscum album extract Iscador-M® enhancing the NK cytotoxicity of human CD56+CD3− NK cells (87–95% enrichment) in cocultures with K 562 tumor cells and increasing the formation of NK cell/tumor cell conjugates was identified as a rhamnogalacturonan. Both activities were abolished by treatment of V. album extract with poly-α-d-galacturonidase and α-rhamnosidase and both activities were inhibited in the presence of galacturonic acid and acetylated rhamnose (6-deoxymannose). Inhibition was also observed in the presence of structurally related derivatives such as acetylated mannose or acetylated mannonic acid γ-lactone, the latter exhibiting a 5–10-fold higher inhibitory potential. The rapid formation of NK cell/tumor cell conjugates in the presence of V. album extract was based on the bridging of NK cells with tumor cells by rhamnogalacturonan. Using a specially adapted agglutination assay, the saccharide residues of the rhamnogalacturonan interacting with NK cells and tumor cells could be identified by the formation of homologous cell conjugates induced by acetylated rhamnose or acetylated mannose conjugated to dextran and by polygalacturonic acid: terminal acetylated rhamnose or acetylated mannose bound only to NK cells in a dose-dependent manner but not to K562 tumor cells, and terminal galacturonic acid only to K562 tumor cells but not to NK cells. This type of bridging represents a novel mechanism of enhancement of NK cytotoxicity.

CD11a-c/CD18 and GP84 (LB-2) adhesion molecules on human large granular lymphocytes and their participation in natural killing.

Effect of mAb against the CD11a-c, CD18, and GP84 adhesion molecules on the binding and cytotoxicity of human NK cells was studied. The target cells were K562, MOLT-4, Raji, and fresh uncultured autologous endometrial carcinoma cells. Antibodies against adhesion relevant epitopes of CD11a(TA-1/LFA-1), CD11b(Mol/OKM1/Mac1), or CD11c (Leu-M5) did not inhibit NK function. The mAb 60.3 against CD18, the common beta-chain associated to CD11a-c, strongly inhibited both the binding and cytotoxicity of large granular lymphocytes (LGL) against all the target cells tested. Also the antibody LB-2 against the GP84 adhesion molecule inhibited NK function to some degree. 60.3 and LB-2 antibodies exerted an additive effect in the inhibition of both binding and cytotoxicity. However, even this antibody combination did not completely block NK activity, suggesting a heterogeneity of adhesion structures in the NK system. According to both FACS analyses and immunoprecipitation studies, all the tested antibodies recognized either a subpopulation or all of LGL. On the other hand, antibodies against CD11b, CD11c, and LB-2 showed only marginal reactivity with highly purified LGL-free T cells.

Suppression of human NK cell cytotoxicity by an MLC-Generated cell population.

A suppressor cell generated in late MLC was capable of inhibiting the cytotoxic activity of fresh NK cells and MLC-generated NK-like cells. Maximum suppression was obtained by using the cells from a 12-day MLC and resulted in a 47% inhibition in fresh NK activity as measured by 51Cr release. The inhibition was also apparent in the single-cell cytotoxicity assay, where suppression was manifested at the level of target recognition. This was reflected in the number of target-binding conjugates (TBC), with a decreased number of TBC consistently found when day 12 MLC cells were added to fresh NK cells or MLC-generated NK-like cells. A soluble suppressive factor generated in MLC did not appear to be the mechanism underlying the suppression, because the addition of the supernatant from a 12-day MLC had no effect on the target binding or cytotoxic activity of fresh NK cells. An adherent cell population was not involved, as the removal of G-10 adherent cells from the day 12 MLC did not alter suppression of NK cytotoxicity. The phenotype of the regulatory cell in the day 12 MLC was Fcmu+ and HNK-1+ (Leu-7). These suppressor cells do not bear antigens detected by monoclonal antibodies to T cells (Leu-4), suppressor T cells (Leu-2a), HLA-DR, or the Fc gamma receptor on NK cells (B73.1). The manner in which the HNK-1+, Fcmu+ suppressor cells exerted their inhibitory effect was by binding directly to Fc gamma +, HNK-1+ fresh NK cells. In turn, the Fc gamma +, HNK-1+ NK cells were rendered incapable of binding to target cells. These results suggest that NK cells themselves can function as immunoregulators, controlling their own cytotoxic activity.

Behcet's Syndrome and Vitamin K

Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. In this study, we describe an experimental system that circumvents some of the difficulties. Adhesion through β₂ integrin LFA-1 is a common requirement of CTLs and NK cells for efficient lysis of target cells. However, the contribution of LFA-1 to activation signals for NK cell cytotoxicity, besides its role in adhesion, is unclear. The role of LFA-1 was evaluated by exposing NK cells to human ICAM-1 that was either expressed on a <i>Drosophila</i> insect cell line, or directly coupled to beads. Expression of ICAM-1 on insect cells was sufficient to induce lysis by NK cells through LFA-1. Coexpression of peptide-loaded HLA-C with ICAM-1 on insect cells blocked the LFA-1-dependent cytotoxicity of NK cells that expressed HLA-C-specific inhibitory receptors. Polarization of cytotoxic granules in NK cells toward ICAM-1- and ICAM-2-coated beads showed that engagement of LFA-1 alone is sufficient to initiate activation signals in NK cells. Thus, in contrast to T cells, in which even adhesion through LFA-1 is dependent on signals from other receptors, NK cells receive early activation signals directly through LFA-1.