Human NK cytotoxicity against porcine cells is triggered by NKp44 and NKG2D

Pig-to-human xenotransplantation has been proposed as a means to alleviate the shortage of human organs for transplantation, but cellular rejection remains a hurdle for successful xenograft survival. NK cells have been implicated in xenograft rejection and are tightly regulated by activating and inhibitory receptors recognizing ligands on potential target cells. The aim of the present study was to analyze the role of activating NK receptors including NKp30, NKp44, NKp46, and NKG2D in human xenogeneic NK cytotoxicity against porcine endothelial cells (pEC). (51)Cr release and Ab blocking assays were performed using freshly isolated, IL-2-activated polyclonal NK cell populations as well as a panel of NK clones. Freshly isolated NK cells are NKp44 negative and lysed pEC exclusively in an NKG2D-dependent fashion. In contrast, the lysis of pEC mediated by activated human NK cells depended on both NKp44 and NKG2D, since a complete protection of pEC was achieved only by simultaneous blocking of these activating NK receptors. Using a panel of NK clones, a highly significant correlation between anti-pig NK cytotoxicity and NKp44 expression levels was revealed. Other triggering receptors such as NKp30 and NKp46 were not involved in xenogeneic NK cytotoxicity. Finally, Ab-dependent cell-mediated cytotoxicity of pEC mediated by human NK cells in the presence of xenoreactive Ab was not affected by blocking of activating NK receptors. In conclusion, strategies aimed to inhibit interactions between NKp44 and NKG2D on human NK cells and so far unknown ligands on pEC may prevent direct NK responses against xenografts but not xenogeneic Ab-dependent cell-mediated cytotoxicity.     

Human natural killer cell activity against porcine targets: modulation by control of the oxidation-reduction environment and role of adhesion molecule interactions

Xenotransplantation, especially using porcine sources, has been proposed as a means to alleviate the shortage of human organs for transplantation. NK cells appear to be important mediators of the xenogeneic immune responses, including the human anti-pig response. Having previously established the redox regulation of NK cell activity against tumor target cells, we now report that the interaction of human NK cells with porcine target cells is also regulated by redox. Thiol-deprivation strongly diminished the capacity of IL-2-activated human NK cells to kill porcine endothelial cells. This inhibition correlated with reduced proliferation and interferon (IFN)-gamma production by IL-2-activated NK cells. For fresh NK cells, pretreatment with diethyl maleate (DEM), which was used to deplete intracellular thiols, reduced lysis of porcine and human targets. Because many adhesion molecules exhibit interspecies recognition, we further investigated whether changes in expression of adhesion molecules might explain our observations. DEM treatment reduced the expression of CD11b and CD29 on fresh NK cells. Monoclonal antibody blocking studies showed that the combination of mAb to CD11b and CD18 reduced lytic activity against both PAEC as well as K562, although other qualitative differences were observed between the porcine and human target cells. These findings suggest that the oxidative stress-induced downregulation of CD18 may be important in modulating cytotoxic activity of fresh NK cells against PAEC and K562 targets through reduced formation of the CD11b/CD18 heterodimer. Thus, the appropriate manipulation of redox status may provide a means to enhance survival of non-human animal tissues in humans through modulation of adhesion molecule expression/interactions.     

Multiple receptors trigger human NK cell-mediated cytotoxicity against porcine chondrocytes

Xenotransplantation of genetically engineered porcine chondrocytes may provide a therapeutic solution for the repair of cartilage defects of various types. However, the mechanisms underlying the humoral and cellular responses that lead to rejection of xenogeneic cartilage are not well understood. In this study, we investigated the interaction between human NK cells and isolated porcine costal chondrocytes (PCC). Our data show that freshly isolated NK cells adhere weakly to PCC. Consequently, PCC were highly resistant to cytolysis mediated by freshly isolated NK cells. However, the presence of human natural Abs in the coculture was often sufficient to trigger cytotoxicity against PCC. Furthermore, IL-2 stimulation of NK cells or activation of PCC with the proinflammatory cytokines TNF-α or IL-1α resulted in increased adhesion, which was paralleled by increased NK cell-mediated lysis of PCC. NK cell adhesion to PCC could be blocked by Abs against human LFA-1 and porcine VCAM-1. NKG2D and NKp44 were involved in triggering cytotoxicity against PCC, which expressed ligands for these activating NK cell receptors. Our data further suggest that NKp30 and NKp46 may contribute to the activation of NK cells by PCC under certain conditions. Finally, comparative studies confirmed that PCC are more resistant than porcine aortic endothelial cells to human NK cell-mediated lysis. Thus, the data demonstrate that human NK cells can kill pig chondrocytes and may therefore contribute to rejection of xenogeneic cartilage. In addition, we identify potential targets for intervention to prevent the NK cell response against pig xenografts.     

HLA-G inhibits rolling adhesion of activated human NK cells on porcine endothelial cells.

Human NK cells adhere to and lyse porcine endothelial cells (pEC) and therefore may contribute to the cell-mediated rejection of vascularized pig-to-human xenografts. Since MHC class I molecules inhibit the cytotoxic activity of NK cells, the expression of HLA genes in pEC has been proposed as a potential solution to overcome NK cell-mediated xenogeneic cytotoxicity. HLA-G, a minimally polymorphic HLA class I molecule that can inhibit a wide range of NK cells, is an especially attractive candidate for this purpose. In this study we tested whether the expression of HLA-G on pEC inhibits the molecular mechanisms that lead to adhesion of human NK cells to pEC and subsequent xenogeneic NK cytotoxicity. To this end two immortalized pEC lines (2A2 and PED) were stably transfected with HLA-G1. Rolling adhesion of activated human NK cells to pEC monolayers and xenogeneic cytotoxicity against pEC mediated by polyclonal human NK lines as well as NK clones were inhibited by the expression of HLA-G. The adhesion was partially reversed by masking HLA-G on pEC with anti-HLA mAbs or by masking the HLA-G-specific inhibitory receptor ILT-2 on NK cells with the mAb HP-F1. The inhibition of NK cytotoxicity by HLA-G was only partially mediated by ILT-2, indicating a role for other unknown NK receptors. In conclusion, transgenic expression of HLA-G may be useful to prevent human NK cell responses to porcine xenografts, but is probably not sufficient on its own. Moreover, the blocking of rolling adhesion by HLA-G provides evidence for a novel biological function of HLA molecules.     

Porcine UL16-binding protein 1 expressed on the surface of endothelial cells triggers human NK cytotoxicity through NKG2D

Cellular rejection mechanisms, including NK cells, remain a hurdle for successful pig-to-human xenotransplantation. Human anti-pig NK cytotoxicity depends on the activating receptor NKG2D. Porcine UL16-binding protein 1 (pULBP1) and porcine MHC class I chain-related protein 2 (pMIC2) are homologues of the human NKG2D ligands ULBP 1-4 and MICA and B, respectively. Although transcribed in porcine endothelial cells (pEC), it is not known whether pULBP1 and pMIC2 act as functional ligands for human NKG2D. In this study, surface protein expression of pULBP1 was demonstrated by flow cytometry using a novel pULBP1-specific polyclonal Ab and by cellular ELISA using NKG2D-Fc fusion protein. Reciprocally, pULBP1-Fc bound to primary human NK cells, whereas pMIC2-Fc did not. Transient and stable down-regulation of pULBP1 mRNA in pEC using short-interfering RNA oligonucleotide duplexes and short hairpin RNA, respectively, resulted in a partial inhibition of xenogeneic NK cytotoxicity through NKG2D in (51)Cr release assays. In contrast, down-regulation of pMIC2 mRNA did not inhibit NK cytotoxicity. Human NK cytotoxicity against pEC mediated by freshly isolated or IL-2-activated NK cells through NKG2D was completely blocked using anti-pULBP1 polyclonal Ab. In conclusion, this study suggests that pULBP1 is the predominant, if not only, functional porcine ligand for human NKG2D. Thus, the elimination of pULBP1 on porcine tissues represents an attractive target to protect porcine xenografts from human NK cytotoxicity.     

Antibody-dependent cellular cytotoxicity mediated by murine lymphocytes activated in recombinant interleukin 2

The incubation of murine splenocytes in recombinant interleukin 2 (RIL 2) gives rise to lymphokine-activated killer (LAK) cells that can lyse fresh, NK-resistant tumor cells but not normal cells in 4-hr 51Cr-release assays. Lysis by this IL 2-activated cell population was enhanced up to 100-fold by prior reaction of target cells with specific antisera reactive with antigens on the target cells. This antibody-dependent cellular cytotoxicity (ADCC) also resulted in lysis of fresh normal target cells, which are not usually susceptible to LAK lysis. The ADCC was evident after 24 hr of incubation of splenocytes in RIL 2, but peak lytic activity was reached after 3 to 4 days of incubation. The concentrations of RIL 2 needed for the in vitro activation of the effectors in order to attain maximal ADCC was between 100 and 3000 U/ml and parallel the IL 2 concentrations required to generate LAK cells. ADCC mediated by IL 2-activated splenocytes was completely blocked by anti-FcR monoclonal antibodies. Although antisera directed against MHC antigens were used in most experiments, anti-B16 monoclonal antibodies have also shown the ability to induce ADCC mediated by RIL 2-activated syngeneic and allogeneic cells. Treatment of the precursor splenocyte populations with anti-asialo GM1 and complement eliminated the direct LAK activity and the antibody-dependent cytotoxicity, suggesting that both direct and indirect tumor cell lysis may be caused by the same effector cell. ADCC mediated by LAK cell populations represents another possible mechanism for the in vivo therapeutic effects of these cells.     

HLA-E and HLA-G expression on porcine endothelial cells inhibit xenoreactive human NK cells through CD94/NKG2-dependent and -independent pathways

Human NK cells contribute a significant role to host defense as well as xenogeneic cytotoxicity. Previous studies using human 721.221 cell line have shown that peptides derived from the leader sequence of the HLA-G binds and up-regulates the surface expression of HLA-E molecules, which was considered to consequently provide negative signals to human NK cells. However, the direct role of HLA-G in inhibiting human NK cells remains controversial. In this study, we showed that the expression of HLA-G or HLA-E in porcine endothelial cells directly protected sensitive porcine cells from human NK cell-mediated xenogeneic cytotoxicity. Ab blocking assays using F(ab')2 of the HLA class I-specific mAb PA2.6 indicated that the protection was directly mediated by the expression of HLA-G and HLA-E on the porcine cells. The HLA-E-mediated protection was blocked by anti-human CD94 Ab. In addition, the engagement of HLA-E lead to the phosphorylation of the CD94/NKG2 complex and the recruitment of SH2 domain-containing protein phosphatase 1 (SHP-1) to the complex. Therefore, HLA-E protected porcine cells from xenoreactive human NK cells through a CD94/NKG2-dependent pathway. In contrast, HLA-G inhibited human NK cells in the absence of CD94/NKG2 phosphorylation or SHP-1 recruitment, and the inhibition was not blocked by anti-CD94 Ab. Therefore, HLA-G protected porcine cells from human NK cells through a CD94/NKG2-independent pathway. These results demonstrated that both HLA-E and HLA-G could directly inhibit human NK cells in the absence of other endogenous HLA class I molecules. These results also have practical implications in preventing xenograft rejection mediated by human NK cells.     

Ultrastructural study of biochemically modulated ADCC in HSV-1 infected and uninfected Chang liver cells

The effect of cytochalasin B, ouabain and 25-OH cholesterol on specific lysis due to antibody dependent cellular cytotoxicity (ADCC) in allogeneic and xenogeneic systems was studied using Herpes simplex I infected Chang liver cells. Cytochalasin B reduced both cytotoxicity and lymphocyte/target (LT) binding in the allogeneic system whereas cytotoxicity but not LT binding was reduced in the xenogeneic system. Ouabain inhibited ADCC in both systems as well as LT binding in the allogeneic system; however, binding in the xenogeneic system was not significantly reduced. The 25-OH cholesterol produced a marked decrease in ADCC in both systems but had no significant effect on LT binding in either system. The biochemical and ultrastructural data suggest that the modulators act at different stages in the ADCC response and that there may be more than one mechanism of ADCC to handle different types of target antigens.     

Comparison of in vitro antibody-targeted cytotoxicity using mouse, rat and human effectors

Antibodies can direct tumor cell lysis by activating complement-mediated and cell-mediated cytoxicities (antibody-dependent cell-mediated cytotoxicity, ADCC). Clinical translation of these effects into successful cancer therapy has been slow. Choosing an appropriate animal model to test new therapeutic strategies is difficult because of species differences in immunological effector functions. In previous work, we found that an unmodified anti-ganglioside mouse IgG3 monoclonal antibody (mAb), 3F8, could successfully treat clinical tumors in humans and experimental tumors in rats but not experimental tumors in mice. We explored the reasons for this species difference by performing in vitro antibody-dependent cytotoxicity assays comparing the potency of polymorphonuclear neutrophils (PMN), natural killer (NK) cells and complement from the three species: mouse, rat and human. 3F8-dependent complement-mediated cytotoxicity produced more than 70% specific release when human and rat sera were used and only 20% with mouse serum. PMN-mediated ADCC was 35%–70% with human effectors, 25%–60% with rat and undetectable with mouse. Human eosinophils did not contribute to this ADCC. Cytotoxicity utilizing interleukin-2-activated NK cells was antibody-independent in all three species but the specific release was 60%–70% with human and rat NK cells and 10% with mouse NK cells. These data suggest that, for mouse IgG3, the rat may provide a more relevant rodent model than the mouse for testing the in vivo antitumor effects of monoclonal antibodies. Received: 20 January 2000 / Accepted: 24 March 2000     

Neonatal porcine Sertoli cells inhibit human natural antibody-mediated lysis

Sertoli cells protect cotransplanted cells from allogeneic and xenogeneic rejection. Additionally, neonatal porcine Sertoli cells (NPSCs) survive long-term as xenografts in nonimmunosuppressed rodents. This has led to the hypothesis that NPSCs could be used to prevent cellular rejection in clinical transplantation, thereby eliminating the need for chronic immunosuppression. Prior to transplantation of NPSCs in humans it is necessary to determine whether they are also protected from humoral-mediated xenograft rejection. The presence of Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (alphaGal epitope) as well as binding of human immunoglobulin G (IgG) and IgM to NPSCs was examined by immunocytochemical and fluorescence-activated cell sorter analysis. alphaGal was detected on 88.5% +/- 3.0% of NPSCs. Consistent with this, 71.7% +/- 1.0% and 65.4% +/- 5.2% of NPSCs were bound by IgG and IgM, respectively. When cultured NPSCs underwent an in vitro cytotoxicity assay by incubation with human AB serum plus complement, no increase in cellular lysis was observed, while controls--porcine aorta endothelial cells--were shown to contain > 60% dead cells. Finally, activation of the complement cascade was examined by immunohistochemistry. C3 and C4 were deposited on the surface of the NPSC membrane, indicating activation of complement. Although the complement cascade was activated, the membrane attack complex (MAC) was not formed. These data demonstrate that despite expression of alphaGal, binding of xenoreactive antibodies, and the activation of complement, NPSCs survive human antibody and complement-mediated lysis by preventing MAC formation. This suggests that NPSCs may be able to survive humoral-mediated rejection in a clinical situation.     

Histone deacetylase inhibitors suppress natural killer cell cytolytic activity

Treatment of transformed cells from leukemia or solid tumors with histone deacetylase inhibitors (HDACi) was shown to increase their sensitivity to NK cell lysis. In this study, treatment of IL-2-activated NK cells with HDACi including suberoylanilide hydroxamic acid and valproic acid was studied. Both drugs at therapeutic concentrations inhibited NK cell cytotoxicity on human leukemic cells. This inhibition was associated with decreased expression and function of NK cell activating receptors NKp46 and NKp30 as well as impaired granule exocytosis. NFkappaB activation in IL-2-activated NK cells was inhibited by both HDACi. Pharmacologic inhibition of NFkappaB activity resulted in similar effects on NK cell activity like those observed for HDACi. These results demonstrate for the first time that HDACi prevent NK cytotoxicity by downregulation of NK cell activating receptors probably through the inhibition of NFkappaB activation.     

Investigating the lysis of small-cell lung cancer cell lines by activated natural killer (NK) cells with a fluorometric assay for NK-cell-mediated cytotoxicity

Activation of natural killer (NK) cells with interleukin-2 (IL-2) and IL-12 leads to an enhanced lysis of tumour cells. We investigated the ability of NK cells, with or without prior activation, to lyse a variety of small-cell lung cancer (SCLC) target cells. Specific lysis was measured with a fluorometric assay for NK-cell-mediated cytotoxicity: target cells were labelled with 3,3′-dioctadecyloxacarbocyanine, a green membrane dye. After co-incubation with NK cells, dead target cells were stained with propidium iodide, a red DNA dye that only penetrates dead cells. Of all eight SCLC cell lines tested, three were susceptible to lysis by non-activated NK cells, three were only susceptible to lysis by NK cells activated with IL-2 and IL-12 and two were not even susceptible to lysis by activated NK cells. The differences in target cell susceptibility showed no correlation with the expression of MHC-I on the surface of the target cells or with the expression of the adhesion molecules CD50, CD54, CD58 or CD102. Comparing the kinetics of the lysis of one SCLC cell line sensitive to non-activated NK cells and one sensitive only to activated NK cells, we found that maximum lysis of the former was obtained after 1 h, whereas significant lysis of the latter was only obtained after 4 h of incubation. This might be due to different mechanisms engaged in target cell lysis. Received: 23 December 1998 / Accepted: 8 April 1999     

A short synthetic peptide fragment of human interleukin-1 beta increases both human and murine natural killer activity

The effect of a short synthetic fragment of human interleukin-1 beta (hu IL-1 beta) on natural killer (NK) activity was examined. Peripheral-blood mononuclear cells (PBMC) from normal donors showed a significant increase in NK activity against K562 leukemia cells after preincubation for 18 h with the IL-1 peptide. A similar augmentation was not observed after culturing the cells in the presence of hu IL-1 beta. The increase in tumor cell lysis could not be ascribed to a cytolytic activity of the synthetic fragment on target cells, since the peptide caused no direct lysis of various tumor cell lines. Although the peptide enhanced NK cytotoxicity of PBMC, highly purified large granular lymphocytes were not susceptible to its stimulatory effect. The addition to the cultures of antibodies to human interleukin-2 (hu IL-2) completely blocked the peptide-induced boost of NK cytotoxicity, suggesting that IL-2 is mainly involved in the activation process. The ability of the IL-1 peptide to increase NK activity was further confirmed in vivo in the mouse. Cytotoxicity against YAC-1 lymphoma cells, which was very low in the spleen of untreated BALB/c mice, was in fact significantly increased after a single inoculation of the peptide. These data thus indicate that a short synthetic peptide fragment of hu IL-1 beta is able to increase both human and murine NK activity.     

Human NK cells lyse organ-specific endothelial cells: analysis of adhesion and cytotoxic mechanisms

Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3), mesenteric lymph node (HMLNEC), brain (HBrMEC), and lung (HLMEC) and skin (HSkMEC.2) ECs, they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated, purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing, we demonstrated using different chelators that the necessary adhesion step was governed by an Mg(2+)-dependent, but Ca(2+)-independent, mechanism as opposed to the subsequent Ca(2+)-dependent killing. To identify the cytotoxic pathway used by NKL2 cells, the involvement of the classical and alternate pathways was examined. Blocking of the Ca(2+)-dependent cytotoxicity pathway by EGTA/MgCl(2) significantly inhibited endothelial target cell killing, suggesting a predominant role for the perforin/granzyme pathway. Furthermore, using confocal microscopy, we demonstrated that the interaction between NKL2 effectors and ECs induced cytochrome c release and Bid translocation in target cells, indicating an involvement of the mitochondrial pathway in NKL2-induced EC death. In addition, although all tested cells were sensitive to the cytotoxic action of TNF, no susceptibility to TRAIL or anti-Fas mAb was observed. The present studies emphasize that human NK cell cytotoxicity toward ECs may be a potential target to block vascular injury.     

CD1d1 displayed on cell size beads identifies and enriches an NK cell population negatively regulated by CD1d1

NK cells destroy microbe-infected cells while sparing healthy cells, and are controlled, in part, by inhibitory receptors specific for class I Ag-presenting molecules. CD1d1, a beta(2)-microglobulin-associated class I-like molecule, binds glycolipids and stimulates NKT cells. We previously demonstrated that target cell lysis by IL-2-activated mouse NK cells is inhibited by target cell expression of CD1d1, suggesting that IL-2-activated NK cells may express a CD1d1-specific inhibitory receptor. We now report that a significant subset of mouse IL-2-activated NK cells specifically binds cell size beads displaying either naturally expressed or recombinant CD1d1. In contrast, although tetramers of soluble recombinant CD1d1 loaded with alpha-galactosylceramide identify NKT cells, binding of this reagent to resting or IL-2-activated NK cells was undetectable, even with activated NK cells sorted with CD1d1 beads. Cytotoxicity by the CD1d1 bead-separated NK subset was strongly inhibited by CD1d1, compared with the NK cell subset not bound to CD1d1 beads. An Ab that blocks NKT cell recognition of CD1d1 also reverses CD1d1 inhibition of NK lysis, suggesting that TCRs of NKT cells and NK inhibitory receptor(s) may interact with a similar site on CD1d1. These results provide direct evidence for a physical interaction of NK cells with CD1d1, mediated by a functional, CD1d1-specific low-affinity inhibitory NK receptor. Display of ligands on cell size beads to maximize multivalent interaction may offer an alternative approach to examine NK cell receptor-ligand interactions, particularly those of lower expression and/or lower affinity/avidity that may go undetected using tetrameric reagents.     

Stimulation of murine natural killer (NK) cells by a monoclonal antibody specific for the NK1.1 antigen. IL-2-activated NK cells possess additional specific stimulation pathways

NK cells lyse certain tumor cell targets but the effector cell surface molecules responsible for this reactivity remain uncertain. The allotypic NK1.1 Ag is the most specific serologic marker on murine cells that display non-MHC-restricted cytolysis of tumor cell targets, but no function has been previously ascribed to this Ag. In this report, we demonstrate that, in the presence of a mAb specific for the NK1.1 Ag (mAb PK136), freshly isolated and IL-2-activated NK cells from C57BL/6 mice can be induced to lyse an otherwise resistant target cell, Daudi. This phenomenon is effector and mAb specific because NK cells derived from BALB/c mice do not express the NK1.1 Ag and cannot be triggered by mAb PK136. We demonstrate that IL-2 activated but not freshly isolated NK cells express the Ly-6 and VEA Ag, originally described as T cell activation Ag. Moreover, mAb specific for Ly-6 and VEA induce target cell lysis by IL-2 activated but not freshly isolated NK cells. These mAb effects are specific, concentration dependent, and display kinetics that are similar to spontaneous cytolysis of NK-sensitive targets. The Fc portion of the activating antibodies and only FcR bearing target cells participate in mAb-induced activation, consistent with the mechanism of redirected lysis. Finally, analysis of Daudi cells transfected with beta 2-microglobulin gene demonstrate that the expression of MHC class I Ag by the target cell does not affect its sensitivity to mAb-induced lysis by NK cells. These data demonstrate that the NK1.1 Ag is functionally active on both freshly isolated and IL-2-activated NK cells and that IL-2-activated NK cells possess additional pathways of specific stimulation.     

IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury

NK cells promptly disappear from the circulation of patients treated with high dose i.v. rIL-2. To further study this process, we evaluated the effects of IL-2 (1000 U/ml) on normal donor PBMC incubated for 1 h on cultured human saphenous vein endothelial cells (EC). Although the NK activity of non-adherent PBMC recovered from flasks coated only with fibronectin increased in the presence of supplemental IL-2, the activity of cells recovered from flasks coated with EC decreased when IL-2 was added to the medium. The percentage of NK (CD16+) cells among the EC-non-adherent PBMC was reduced relative to that of the input cells when IL-2 was added. The percentage of CD16+ cells in the EC-adherent PBMC, as well as their NK activity, increased in the presence of added IL-2. Although EC had no effect on the lysis of labeled K-562 cells by unstimulated PBMC in cold target competition experiments, they were able to compete in cytolytic assays using PBMC previously activated by exposure to IL-2 for 1 h. EC were not lysed by these briefly activated PBMC in 3-h cytotoxicity assays but were lysed by these effectors in 18-h assays and in 3-h assays using PBMC pre-activated by more prolonged culture with IL-2. The ability of IL-2 to induce NK cell adhesion to EC was not blocked by a mixture of neutralizing antisera raised against rTNF-alpha, rIL-1 alpha, and rIL-1 beta, factors known to promote leukocyte adhesion to EC. We conclude that IL-2 rapidly induces NK cell adhesion to EC and propose that this effect accounts for the disappearance of circulating NK cells after the infusion of high doses of IL-2. In addition, these results suggest that NK cells activated by IL-2 in vivo may injure the endothelium and contribute to the extravasation of plasma and the retention of fluid characteristic of IL-2 treatment.     

Murine trophoblast can be killed by lymphokine-activated killer cells

The ability of fetal trophoblast cells in the placenta to resist cell-mediated lysis may be important for successful pregnancy. Previous studies in this laboratory demonstrated that cultured midterm mouse trophoblast cells are not susceptible to allospecific CTL generated by standard in vitro protocols, to antibody-dependent cell-mediated cytotoxicity, or to naive or IFN-activated NK cells, despite expressing the requisite target structures. However, we now report that murine trophoblast can be killed, in a non-MHC-specific manner, by LAK cells. Normal mouse spleen cells cultured for 4 days in IL-2-containing lymphokine preparations characteristically killed both NK-sensitive (YAC-1) and NK-resistant (EL4, P815) target cells, and mediated significant lysis of both cultured and freshly isolated trophoblast cells (35 to 55%, E/T 100/1). Pretreatment of the LAK cells with anti-ASGM1 antibody and C markedly reduced the lysis of trophoblast and YAC-1 targets, suggesting that the responsible cells belonged to the NK lineage. The ability of IL-2-activated NK cells to kill midterm murine trophoblast cells was confirmed using a population of highly lytic NK cells generated by culturing spleen cells from severe combined immunodeficiency mice in 500 U/ml rIL-2 for 5 days. These effector cells killed YAC-1, EL4 and P815 target cells at much lower E/T ratios than was achieved with the normal splenic LAK cells, and mediated significant lysis of both freshly isolated (45 to 50%, E/T 20/1) and cultured trophoblast cells (68 to 76%, E/T 20/1). The susceptibility of trophoblast to LAK cells and IL-2-activated NK cells supports the need for suppressor mechanisms regulating IL-2 activity at the maternal-fetal interface.     

Blocking NK cell inhibitory self-recognition promotes antibody-dependent cellular cytotoxicity in a model of anti-lymphoma therapy

Human NK cells lyse Ab-coated target cells through the process of Ab-dependent cellular cytotoxicity (ADCC). Improving ADCC responses is desirable because it is thought to be an important antitumor mechanism for some Abs. NK cell inhibitory receptors, such as killer cell Ig-like receptors, engage with MHC class I molecules on self-cells to block NK cell activation. Accordingly, we enhanced ADCC responses by blocking NK cell inhibitory receptors, thus perturbing induction of the self-recognition signal. In a cell line model of anti-lymphoma therapy, the combination of rituximab with an Ab that blocks inhibitory self-recognition yielded increased NK cell-mediated target cell lysis when compared with rituximab alone. To validate this proof-of-concept, we then used a more representative approach in which an individual's fresh primary NK cells encountered autologous, EBV-transformed B cells. In this system, rituximab and a combination of Abs that block NK cell inhibitory receptors yielded improved NK cell-mediated lysis over rituximab alone. The results show, for the first time, that disruption of inhibitory self-recognition can efficiently promote ADCC in a human model, applying an autologous system in which physiologic checkpoints are in place. This method provides an alternative approach to potentiate the therapeutic benefit of antitumor Abs that mediate ADCC.