Human NK Cell Cytotoxicity Research Articles

Mouse TIGIT inhibits NK‐cell cytotoxicity upon interaction with PVR

The activity of natural killer (NK) cells is controlled by a balance of signals derived from inhibitory and activating receptors. TIGIT is a novel inhibitory receptor, recently shown in humans to interact with two ligands: PVR and Nectin2 and to inhibit human NK-cell cytotoxicity. Whether mouse TIGIT (mTIGIT) inhibits mouse NK-cell cytotoxicity is unknown. Here we show that mTIGIT is expressed by mouse NK cells and interacts with mouse PVR. Using mouse and human Ig fusion proteins we show that while the human TIGIT (hTIGIT) cross-reacts with mouse PVR (mPVR), the binding of mTIGIT is restricted to mPVR. We further demonstrate using surface plasmon resonance (SPR) and staining with Ig fusion proteins that mTIGIT binds to mPVR with higher affinity than the co-stimulatory PVR-binding receptor mouse DNAM1 (mDNAM1). Functionally, we show that triggering of mTIGIT leads to the inhibition of NK-cell cytotoxicity, that IFN-γ secretion is enhanced when mTIGIT is blocked and that the TIGIT-mediated inhibition is dominant over the signals delivered by the PVR-binding co-stimulatory receptors. Additionally, we identify the inhibitory motif responsible for mTIGIT inhibition. In conclusion, we show that TIGIT is a powerful inhibitory receptor for mouse NK cells.

Membrane-bound interleukin-21 and CD137 ligand induce functional human natural killer cells from peripheral blood mononuclear cells through STAT-3 activation

Natural killer (NK) cell adoptive transfer is a promising approach for cancer immunotherapy; however, its development has been hindered by the lack of efficient methods to produce large numbers of functional NK cells. In this study, we engineered the leukaemia cell line K562 to express CD137 ligand (CD137L) and membrane-bound interleukin (mbIL)-21 on the cell surface, and used these cells to expand NK cells from the peripheral blood mononuclear cells. We found that purity of the NK cells (CD3(-) CD56(+) /CD16(+) ) increased from less than 30% to above 95% after a 3-week expansion and proliferation of the cells was sustained for more than 8 weeks. The surface expression of NK cell activating and inhibitory receptors, except for NKp80, was clearly increased with the expansion, and NK cell-mediated killing activity was also enhanced significantly. However, these changes in both phenotype and function were clearly reversed by JSI-124, a specific signal transducer and activator of transcription-3 (STAT-3) inhibitor. Taken together, data showed that the combination of mbIL-21 and CD137L could efficiently induce the formation of functional human NK cells from peripheral blood mononuclear cells, and STAT-3 inhibition could impair this induction. Therefore, STAT-3 activation may benefit human NK cell proliferation and cytotoxicity, and provide valuable clinical applications in NK cell immunotherapy against viral infectious diseases and cancers.

Identification of Resting and Type I IFN-Activated Human NK Cell miRNomes Reveals MicroRNA-378 and MicroRNA-30e as Negative Regulators of NK Cell Cytotoxicity

NK cells are important innate immune cells with potent cytotoxicity that can be activated by type I IFN from the host once infected. How NK cell cytotoxicity is activated by type I IFN and then tightly regulated remain to be fully elucidated. MicroRNAs (miRNAs, or miRs) are important regulators of innate immune response, but the full scale of miRNome in human NK cells remains to be determined. In this study, we reported an in-depth analysis of miRNomes in resting and IFN-α-activated human NK cells, found two abundant miRNAs, miR-378 and miR-30e, markedly decreased in activated NK cells by IFN-α, and further proved that miR-378 and miR-30e directly targeted granzyme B and perforin, respectively. Thus, IFN-α activation suppresses miR-378 and miR-30e expression to release cytolytic molecule mRNAs for their protein translation and then augments NK cell cytotoxicity. Importantly, the phenomena are also confirmed in human NK cells activated by other cytokines and even in the sorted CD16(+)CD56(dim)CD69(+) human NK cell subset. Finally, miR-378 and miR-30e were proved to be suppressors of human NK cell cytotoxicity. Taken together, our results reveal that downregulated miR-378 and miR-30e during NK cell activation are negative regulators of human NK cell cytotoxicity, providing a mechanistic explanation for regulation of NK cell function by miRNAs.

Fatal immune dysregulation due to a gain of glycosylation mutation in lymphocyte perforin

AbstractMutations in the perforin gene (PRF1) are a common cause of the fatal immune dysregulation disorder, familial hemophagocytic lymphohistiocytosis (type 2 FHL, FHL2). Here we report a female infant born with biallelic PRF1 mutations: a novel substitution, D49N, and a previously identified in-frame deletion, K285del. We assessed the effects of each mutation on the cytotoxicity of human NK cells in which the expression of endogenous perforin was ablated with miR30-based short hairpin (sh) RNAs. Both mutations were detrimental for function, thereby explaining the clinically severe presentation and rapidly fatal outcome. We demonstrate that D49N exerts its deleterious effect by generating an additional (third) N-linked glycosylation site, resulting in protein misfolding and degradation in the killer cell. Our data provide a rationale for treating some cases of type 2 familial hemophagocytic lymphohistiocytosis, based on the pharmacologic inhibition or modification of glycosylation.

Phosphorylation of the myosin IIA tailpiece regulates single myosin IIA molecule association with lytic granules to promote NK-cell cytotoxicity

AbstractNatural killer (NK) cells are innate immune lymphocytes that provide critical defense against virally infected and transformed cells. NK-cell cytotoxicity requires the formation of an F-actin rich immunologic synapse (IS), as well as the polarization of perforin-containing lytic granules to the IS and secretion of their contents at the IS. It was reported previously that NK-cell cytotoxicity requires nonmuscle myosin IIA function and that granule-associated myosin IIA mediates the interaction of granules with F-actin at the IS. In the present study, we evaluate the nature of the association of myosin IIA with lytic granules. Using NK cells from patients with mutations in myosin IIA, we found that the nonhelical tailpiece is required for NK-cell cytotoxicity and for the phosphorylation of granule-associated myosin IIA. Ultra-resolution imaging techniques demonstrated that single myosin IIA molecules associate with NK-cell lytic granules via the nonhelical tailpiece. Phosphorylation of myosin IIA at residue serine 1943 (S1943) in the tailpiece is needed for this linkage. This defines a novel mechanism for myosin II function, in which myosin IIA can act as a single-molecule actin motor, claiming granules as cargo through tail-dependent phosphorylation for the execution of a pre-final step in human NK-cell cytotoxicity.

The PP2A inhibitor SET regulates granzyme B expression in human natural killer cells

AbstractThe ability of natural killer (NK) cells to kill malignant or infected cells depends on the integration of signals from different families of cell surface receptors, including cytokine receptors. How such signals then regulate NK-cell cytotoxicity is incompletely understood. Here we analyzed an endogenous inhibitor of protein phosphatase 2A (PP2A) activity called SET, and its role in regulating human NK-cell cytotoxicity and its mechanism of action in human NK cells. RNAi-mediated suppression of SET down-modulates NK-cell cytotoxicity, whereas ectopic overexpression of SET enhances cytotoxicity. SET knockdown inhibits both mRNA and protein granzyme B expression, as well as perforin expression, whereas SET overexpression enhances granzyme B expression. Treatment of NK cells with the PP2A activator 1,9-dideoxy-forskolin also inhibits both granzyme B expression and cytotoxicity. In addition, pretreatment with the PP2A inhibitor okadaic acid rescues declining granzyme B mRNA levels in SET knockdown cells. Down-modulation of SET expression or activation of PP2A also decreases human NK-cell antibody-dependent cellular cytotoxicity. Finally, the induction of granzyme B gene expression by interleukin-2 and interleukin-15 is inhibited by SET knockdown. These data provide evidence that granzyme B gene expression and therefore human NK-cell cytotoxicity can be regulated by the PP2A-SET interplay.

WF10 Stimulates NK Cell Cytotoxicity by Increasing LFA‐1‐Mediated Adhesion to Tumor Cells

The redox-active chlorite-based drug WF10 (Immunokine) was shown to have modulatory effects on both the innate and adaptive immune system in vitro and in vivo. Animal studies suggest that WF10 enhances immunity against tumors. One possible explanation for such an effect is that WF10 stimulates natural killer cell cytotoxicity against malignant cells. Here, we show that WF10 regulates human NK cell cytotoxicity in a time-dependent manner, following an S-shaped kinetic with an initial stimulation of activity followed by a decrease in activity relative to the untreated controls. WF10 does not activate NK cells on its own but co-stimulates NK cell activation mediated by different activating receptors. This is mediated by enhancing NK cell adhesion to target cells through promoting the activation of the integrin LFA-1. These data demonstrate a direct effect of WF10 on the cytotoxicity of human NK cells.

CD137 ligand mediates opposite effects in human and mouse NK cells and impairs NK-cell reactivity against human acute myeloid leukemia cells

AbstractNatural killer (NK) cells play an important role in the immunosurveillance of leukemia. Their reactivity is governed by a balance of activating and inhibitory receptors including various members of the tumor necrosis factor receptor (TNFR) family. Here we report that human NK cells acquire expression of the TNFR family member CD137 upon activation, and NK cells of acute myeloid leukemia (AML) patients display an activated phenotype with substantial CD137 expression. CD137 ligand (CD137L) was detectable on leukemic cells in 35% of 65 investigated AML patients, but not on healthy CD34+ cells, and expression was associated with monocytic differentiation. Bidirectional signaling following CD137-CD137L interaction induced the release of the immunomodulatory cytokines interleukin-10 and TNF by AML cells and directly diminished granule mobilization, cytotoxicity, and interferon-γ production of human NK cells, which was restored by blocking CD137. Cocultures of NK cells with CD137L transfectants confirmed that human CD137 inhibits NK-cell reactivity, while activating signals were transduced by its counterpart on NK cells in mice. Our data underline the necessity to study the function of seemingly analog immunoregulatory molecules in mice compared with men and demonstrate that CD137-CD137L interaction enables immune evasion of AML cells by impairing NK-cell tumor surveillance in humans.

ITAM-containing adaptors regulate NKG2D-mediated cytotoxicity in human NK cells (138.15)

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.

Consequences of 4-1BB-4-1BB Ligand Interaction for NK Cell Anti-Tumor Immunity: Opposite Effects of Seemingly Analogue Molecules in Mice and Men.

Abstract 279In mouse models, stimulation of the TNF receptor family member 4-1BB/CD137 on cytotoxic lymphocytes by agonistic antibodies potently stimulated anti-tumor immunity, which has led to the development of humanized agonistic 4-1BB mAb that are presently being evaluated in clinical phase I/II studies (www.clinicaltrials.gov, keyword: CD137). However, seemingly analogue immunoregulatory molecules may mediate different effects in mice and men, and in humans yet nothing is known regarding the role of 4-1BB in NK cell reactivity. NK cells play an important role in tumor immunosurveillance, and largely contribute to the success of therapeutic strategies like (haploidentical) stem cell transplantation (SCT) or application of monoclonal antibodies like Rituximab by mediating graft versus leukemia activity and performing antibody-dependent cellular cytotoxicity (ADCC). Here we analyzed the expression of 4-1BB in human NK cells and compared the effect of 4-1BB signaling on effector functions of NK cells in mice and men. While being absent in resting state, both human and mouse NK cells acquire expression of 4-1BB at similar levels with comparable kinetics upon activation. Next we cultured human and mouse NK cells with target cells transfected with human and mouse 4-1BB ligand (4-1BBL) and analyzed the effect of 4-1BB on NK cell effector functions. 4-1BB – 4-1BBL interaction enhanced effector functions of mouse NK cells, while degranulation, cytotoxicity and cytokine production of human NK cells were substantially impaired (all p<0.01, Student's T-test). In line, employing primary leukemia cells from patients with AML (n= 65) and CLL (n=49), which were found to express 4-1BBL in 35% and 100%, respectively, we found that blocking 4-1BB-4-1BBL interaction markedly enhanced granule mobilization, cytotoxicity and interferon-g production of allogenic and autologous human NK cells in response to leukemia cells. In CLL, this inhibitory effect of 4-1BB-4-1BBL interaction on NK cell reactivity was observed both with regard to direct and Rituximab-induced cytotoxicity and cytokine production. Together, our data implicate that one should exercise caution in applying anti-4-1BB therapeutically, underline the necessity of detailed analyses regarding the function of seemingly analogue immunoregulatory molecules in mice compared to men and demonstrate that blocking 4-1BBL-4-1BB interaction may serve to enhance NK cell reactivity in therapeutic settings like allogenic SCT and antibody-treatment of malignancies in humans. Disclosures:No relevant conflicts of interest to declare.

The interaction of TIGIT with PVR and PVRL2 inhibits human NK cell cytotoxicity

NK cell cytotoxicity is controlled by numerous NK inhibitory and activating receptors. Most of the inhibitory receptors bind MHC class I proteins and are expressed in a variegated fashion. It was recently shown that TIGIT, a new protein expressed by T and NK cells binds to PVR and PVR-like receptors and inhibits T cell activity indirectly through the manipulation of DC activity. Here, we show that TIGIT is expressed by all human NK cells, that it binds PVR and PVRL2 but not PVRL3 and that it inhibits NK cytotoxicity directly through its ITIM. Finally, we show that TIGIT counter inhibits the NK-mediated killing of tumor cells and protects normal cells from NK-mediated cytotoxicity thus providing an "alternative self" mechanism for MHC class I inhibition.

Demethylating treatment suppresses natural killer cell cytolytic activity

NK cells as a component of the innate immune system provide a first line of defense against viral infections and malignancies. Killer immunoglobulin-like receptors (KIRs) are a polymorphic gene family expressed on NK cells. The crucial role of DNA methylation in determining the variegated expression pattern of KIRs has recently been reported. In this study the direct effect of DNA demethylating treatment on human NK cell cytotoxicity was analyzed. In a human NK cell line NK-92MI, inhibitory KIR genes exhibited characteristic epigenetic repression, whose promoter regions are densely methylated. Treatment of NK-92MI cells with methyltransferase inhibitor 5-azacytidine significantly increased the expression levels of inhibitory KIRs and strongly suppressed their cytolytic activity against human K562 leukemic cells. Cytolytic activity of human polyclonal NK cells was also suppressed upon 5-azacytidine-treatment. The suppression of NK cell cytotoxicity was associated with 5-azacytidine-induced overexpression of inhibitory KIRs and impaired granzyme B and perforin release by these cells. The results demonstrate for the first time that demethylating treatment can suppress NK cell cytolytic activity. The aberrant methylation patterns of KIR genes during NK cell differentiation and maturation may have importance for its abnormal function.

CLEC2A Is a Novel Skin-Specific, Stimulatory Ligand of Human NK Cells (39.8)

Abstract The Natural Killer Gene Complex (NKC) encodes for numerous C-type lectin-like receptors (CTLR) variably expressed on lymphocytes and myeloid cells. Well-known representatives of NKC-encoded CTLR are NKG2D, CD69, and Ly49 molecules. However, the NKC also contains a considerable number of genes which are barely characterized. Recently, we described the CLEC2A gene encoding for a new member of the human CLEC2 family of NKC-encoded CTLR. CLEC2A, in contrast to other human CLEC2 family members such as CD69/CLEC2C and AICL/CLEC2B, is virtually not expressed by peripheral leukocytes, but its expression appears restricted to skin. We now report that CLEC2A encodes for a non-disulfide-linked, homodimeric surface CTLR which is expressed on freshly isolated skin cells. Addressing a potential immune-associated function, we find that CLEC2A stimulates cytotoxicity and cytokine release of human NK cells. However, we failed to ascertain CLEC2A engagement by known activating NK receptors. Instead, we identified a hitherto undescribed NKC-encoded CTLR (NKp65) that specifically interacts with CLEC2A and activates NK cytotoxicity. In summary, we define CLEC2A as a skin-specific and NK cell-stimulating CTLR engaging NKp65, a novel activating CTLR which may allow for dedicated immunosurveillance of human skin. This work was supported by funds (SFB 685/A1) of the German Research Foundation (DFG).

NK Cell Mediated Tumor Cytotoxicity is enhanced by Mild Thermal Stress through Heat Shock Factor 1 (50.24)

Abstract Natural killer (NK) cells directly kill tumor cells and regulate downstream adaptive immune responses. Previous studies in our lab suggest that mild thermal stress (39.5 C) significantly enhances the cytotoxicity of human peripheral blood NK cells compared to normothermic conditions (37 C) against human colon tumor cells. This enhancement was not seen when syngeneic human PBMC were used as targets, nor when the IL-2 dependent human NK cell lines NKL or NK92MI, or IL-2 activated primary NK cells were used as effectors. Thus, we hypothesized that resting NK cell cytotoxic activity is regulated by mild thermal stress. Blocking of the activating receptor NKG2D and its stress-induced ligand MICA with blocking antibodies suggested the involvement of these molecules in the thermal regulation of NK cytotoxicity. We found that inhibitory receptor KIR2DL1 levels on NK cells were not changed with thermal stress. Increased clustering of NKG2D on NK cells was observed with thermal stress, suggesting the potential for enhanced efficiency of activating signals. However prevention of lipid raft clustering with methyl B-cyclodextrin on NK cells shows no significant affect on thermally enhanced cytotoxicity. On target cells, MICA expression appeared to be upregulated by mild thermal stress in a manner that is dependent on the transcription factor HSF-1. Overall, these data suggest that thermal therapy might be clinically utilized to help enhance NK mediated tumor cell killing. Supported by NIH P01 CA94045, R01 CA71599, Komen Foundation DISS0402487 and Fulbright Program.

P110δ is required for innate immunity to transplantable lymphomas

NK cell (natural killer cells) are lymphocytes of innate immunity that kill tumour cells and respond to infections, without prior stimulation. A balance of activating and inhibitory signals regulates NK cell cytotoxicity, but the molecular mechanisms are not fully understood. General inhibitors of PI3K (phosphoinositide 3-kinase) suppress cytotoxicity in human and mouse NK cells. However, which isoforms and how they regulate NK cell activation is unknown, and no data have been published on mice carrying PI3K mutations. p110delta expression is restricted to leucocytes, where it plays central roles in lymphocyte development and signalling. We have used mice carrying a catalytically inactive mutant form of p110delta in order to determine its role in NK cell biology. We show here that p110delta is not required to kill tumour cells, but unexpectedly p110delta mutant mice failed to fully reject transplanted lymphomas. Our results show for the first time a critical role for p110delta in NK cell biology in vivo.

Lipophilic statins suppress cytotoxicity by freshly isolated natural killer cells through modulation of granule exocytosis

NK cells, a component of the innate immune system, provide a first line of defense against viral infections and malignancies, interact with the adaptive immune system and have a role in rejection of allogeneic bone marrow transplants and solid allo- and xenotransplants. Immunoregulatory activity by the anti-hypercholesterolemia agents, 3-hydroxy-3-methyl-glutaryl Coenzyme A (HMG-CoA) reductase inhibitors, known as statins, has recently been reported. We analyzed the effects of three statins on human NK cell cytotoxicity. Two lipophilic statins (simvastatin and fluvastatin) suppressed the cytotoxic activity of fresh and IL-2-stimulated NK cells, while pravastatin, a hydrophilic statin, did not. Suppression was not associated with changes in intracellular perforin, granzyme A or granzyme B levels, or with changes in expression of leukocyte function-associated antigen-1, an integrin known to regulate NK activity and reported to be altered by statin treatment. Decreased cytotoxicity was associated with decreased CD107a surface expression, indicating that the exocytosis pathway was compromised by simvastatin and fluvastatin but not by pravastatin. Mevalonate, the immediate downstream product of HMG-CoA reductase, partially reversed the effect of lipophilic statins on cytotoxicity and CD107a expression. Lipophilic statins also suppressed the release of the granule component, granzyme B, by IL-2-activated NK cells following stimulation with K562. That lipophilic statins suppress NK cell activity through inhibition of the exocytosis pathway suggest an additional potential role for statins in inhibition of transplantation responses.

The direct effects of Toll-like receptor ligands on human NK cell cytokine production and cytotoxicity

Toll-like receptor (TLR) ligands are potent inducers of the innate immune system, of which NK and NKT cells play an important role. We examined the direct activation of highly purified human NK and/or NKT cells with known TLR ligands. NK/NKT cells were positive for all known TLR mRNA (TLR1–10). Ligands for TLR2–5 induced production of significant amounts of IFN-γ by purified NK cells. However, a TLR9 ligand failed to induce significant levels of the cytokine. NK cells were depleted from PBMCs to confirm that they were the main source of IFN-γ following treatment with TLR ligands, which resulted in a significant decrease in cytokines. The direct effects of TLR ligands on NK cytotoxicity were determined using 51Cr-labeled K562 target cells. Ligands for TLR2–5 were potent inducers of NK cell cytotoxicity, a TLR9 ligand was not. Our results suggest that TLR ligands can directly stimulate and enhance NK cell cytokine production and induce cytotoxic activities.

Expression of Human Ecto-5'-Nucleotidase in Pig Endothelium Increases Adenosine Production and Protects from NK Cell-Mediated Lysis

Ecto-5'-nucleotidase (E5'N) is an endothelial surface enzyme that controls conversion of extracellular nucleotides into immunosuppressive adenosine. We evaluated whether expression of human E5'N on pig endothelial cells (EC) attenuates human NK cell-mediated cytotoxicity. A pig EC line was stably transfected with human E5'N and human NK cell adhesion and cytotoxicity toward pig EC cultures was measured by flow cytometry and intracellular enzyme release. E5'N activity in pig EC lysates increased from 0.68 +/- 0.07 to 1013 +/- 293 nmol/min/mg protein, whilst the rate of AMP to adenosine metabolism by intact cells increased from 0.37 +/- 0.05 to >300 nmol/min/mg protein in non-transfected and transfected cells, respectively. The rate of adenosine production in transfected cells increased also with ATP as the extracellular substrate. Cytotoxicity of human NK cells was reduced from 10.7 +/- 0.4% and 11.1 +/- 1.1% with non-transfected pig EC to 5.2 +/- 0.2% and 5.0 +/- 0.2% in transfected cells with 50 microM and 250 microM AMP, respectively. Reduction of cytotoxicity in E5'N-transfected EC was abolished by the E5'N inhibitor and was mimicked in non-transfected EC by the addition of adenosine, demonstrating the key role of adenosine produced by E5'N in inhibiting NK cell cytotoxicity. We suggest that overexpression of E5'N in EC of transgenic pigs is a possible strategy to ameliorate rejection after xenotransplantation.

Endothelial Cells Derived from Pigs Lacking Gal??(1,3)Gal: No Reduction of Human Leukocyte Adhesion and Natural Killer Cell Cytotoxicity

The expression of galactose-alpha(1,3)galactose (Gal) on porcine cells represents a major barrier to xenotransplantation. The generation of Gal-/- pigs to overcome this barrier redirected the focus of research to other rejection mechanisms, including cellular immunity. The present in vitro study investigated (1) the adhesive interactions between human leukocyte subsets and primary endothelial cells derived from inbred Gal-/- and Gal+/+ pigs, and (2) the susceptibility of such Gal-/- porcine endothelial cells to human natural killer (NK) cell cytotoxicity. Primary porcine aortic endothelial cells (PAEC) were isolated from Gal-/- (PAEC-Gal-/-) and Gal (PAEC-Gal+/+) pigs. Human peripheral blood mononuclear cells (PBMC), polymorphonuclear neutrophils (PMN), and NK cells were isolated from healthy volunteers and tested in functional adhesion and cytotoxicity assays. Adhesion of human PBMC, PMN, or purified NK cells on PAEC-Gal-/- cells was not different from that on PAEC-Gal+/+ cells. Comparing the different leukocyte subsets of PBMC, a preferential adhesion of NK and B cells on both PAEC-Gal-/- and PAEC-Gal+/+ was detected. Tumor-necrosis factor-alpha stimulation of PAEC-Gal-/- and PAEC-Gal+/+ induced an increase of CD62E and CD106 expression and increased cellular adhesion, in particular, of PMN. The lack of Gal-/- expression on PAEC-Gal cells did not prevent xenogeneic human NK-cell cytotoxicity mediated by freshly isolated or interleukin-2-activated NK cells. Neither human leukocyte adhesion nor xenogeneic NK-cell cytotoxicity against PAEC are impaired by the lack of Gal, indicating that Gal is not a dominant target of cellular rejection.

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.