Identification of a human natural killer cell target cell antigen with monoclonal antibodies

mAb have been derived against NK cell-sensitive target cells in an effort to identify the target cell structure involved in Ag recognition by NK cells. Several mAb were selected for further study based on their preliminary target cell binding characteristics. Flow cytometry demonstrated that each of these mAb bound to a series of NK-sensitive target cells of various origins (e.g., K562 and Molt-4) while having little or no reactivity with several NK-resistant target cell lines (e.g., SB and Daudi). Functional studies revealed that two of the mAb were able to inhibit the lysis of NK-sensitive K562 target cells by freshly isolated, endogenous NK cells in a dose-dependent fashion. Further, these mAb also could inhibit the killing of K562 target cells by both activated NK cells and cultured lymphokine-activated killer cells, as well as the cytolysis of other NK-sensitive target cells by each of these effector cell populations. Control experiments with another mAb which bound to the target cells but did not inhibit lysis implied that the effects of these mAb on NK cell function was not the result of steric hindrance. Single cell conjugate assays demonstrated that the mAb inhibited NK cell lysis via the inhibition of binding (recognition). Biochemical analysis of this target cell structure revealed that it was a molecule of approximately 42 kDa which may exist as a homodimer in its native state. Thus, it appears that the mAbs identify an unique Ag on the surface of NK cell-sensitive target cells which is involved in NK cell Ag recognition.     

Selective inhibition of natural killer activity by the monoclonal antibodies OKT10 and VEP10 at the single cell level

The monoclonal antibodies, VEP10 and OKT10, which have been shown to recognize determinants on human natural killer (NK) cells, inhibit large granular lymphocyte (LGL) NK activity against K562, MOLT4, and CEM tumor target cells in the single cell conjugate agarose assay. Inhibition of NK activity by monoclonal antibodies was expressed independently of effector-target cell binding, as inhibitory activity could be demonstrated when the monoclonal antibodies VEP10 and OKT10 were added to preformed conjugates or to the LGLs and targets prior to the binding event. In addition, this inhibition was exerted on the effector cell and not the target cell since VEP10 and OKT10 did not react with determinants on K562 target cells. Furthermore, the 4F2 monoclonal antibody, which reacted with determinants on the LGL and all of the targets used, effected no inhibition of NK activity. Inhibition of killing by OKT10 and VEP10 was specific to endogenous NK activity since the same antibodies did not inhibit antibody-dependent cellular cytotoxicity (ADCC), mixed lymphocyte-generated NK, or cytotoxic T lymphocyte (CTL) activities.     

Analysis of rat natural killer cytotoxic factor (NKCF) produced by rat NK cell lines and the production of a murine monoclonal antibody that neutralizes NKCF

Natural killer cytotoxic factor (NKCF) is produced as a result of the interaction of murine, rat, or human natural killer (NK) cells with NK-susceptible targets. This factor has been linked to the target cell lysis mediated by the NK effector cell. In the present results, culture supernatants from rat large granular lymphocyte (LGL) tumors exhibited NKCF activity which lysed the susceptible targets, MBL-2 and YAC-1. NKCF production from these rat tumor lines was spontaneous and was not significantly increased by co-incubation of the LGL tumors with target cells, target cell membranes, or by preincubation of the LGL tumor cells with interferon or interleukin 2. In addition to NKCF activity, the supernatants lysed L929, indicating the presence of tumor necrosis factor (TNF) in these preparations. The presence of this latter cytokine was verified using specific antibodies to recombinant murine TNF which neutralized the L929 activity while not affecting the NKCF activity against MBL-2 or YAC-1. Mouse monoclonal antibodies (mAb) A0287, A0462, and A0316) which significantly inhibit the NKCF cytolytic activity of these LGL-derived supernatants were also produced. These antibodies were shown to cross-react with human NKCF in a manner similar to that seen in the rat. Interestingly these same mAb demonstrated no inhibition of L929 cytotoxicity from either LGL-derived supernatants or by recombinant murine or human TNF. To examine further the specificity of these antibodies, they were chemically linked to Sepharose 4B and found to remove a significant proportion of the NKCF cytolytic activity from LGL supernatants, while not affecting the TNF reactivities in these preparations. In addition, these antibodies demonstrated significant inhibition of cell-mediated cytotoxicity by rat LGL against YAC-1 target cells. Biochemical analysis of labeled NKCF-containing supernatants indicated the major protein recognized by these anti-NKCF mAb to be approximately 12,000 m.w. The use of these mAb against NKCF should be very useful in further purification and biochemical characterization of NKCF and in studying its role in a variety of cell-mediated cytotoxicity assays.     

Lymphocyte function-associated antigen 1 (LFA-1) and natural killer (NK) cell activity: LFA-1 is not necessary for all killer: target cell interactions

A panel of five monoclonal antibodies detecting human lymphocyte function-associated antigen 1 (LFA-1) was generated and shown by competitive binding studies to react with at least four distinct epitopes on this molecule. The antibodies were then tested for their ability to inhibit the lytic activity of a variety of different human natural killer (NK) populations on a panel of four NK-susceptible target cells (K562, MOLT-4, HSB-2, and Jurkat). When heterogeneous NK populations derived from fresh peripheral blood and mixed-lymphocyte culture (MLC)-generated lines were used, these anti-LFA-1 monoclonal antibodies (MAbs) inhibited lysis of all four NK targets; this finding supports the notion that LFA-1 molecules play an important role in NK-mediated lysis. When tested on a cloned line of NK cells (NK 3.3), lysis of K562 was inhibited by these MAbs, but lysis of the other three targets was not affected. This represents an instance where a MAb specific for LFA-1 inhibits the lytic activity of NK cells against some but not all targets; thus the LFA-1 molecule cannot be considered under all circumstances to be an absolute requirement in NK-mediated lysis.     

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.     

Scanning electron microscopic study of natural killer cell-mediated cytotoxicity

The interaction between human natural killer (NK) cells and NK-susceptible target cells, as well as the mechanism involved in target cell lysis, were studied with scanning electron microscopy (SEM). Low density human peripheral blood lymphocytes, highly enriched with large granular lymphocytes (LGL), were used as effector cells, and K562-cells were used as NK-susceptible target cells. The surface features of LGL/NK cells were examined under SEM. In the area of interaction, NK/target-cell conjugates showed microvilli and/or filipodia, and extensive areas of intercellular contact. In addition, the effector cells in some NK/target-cell conjugates were polarized toward the target cell. Changes in target cell surface features included loss of microvilli, large surface blebs and the appearance of small pore-like lesions on the cell membrane. Our findings show that target cell lysis occurred by apoptosis and plasma membrane lesions analogous to those seen during complement-mediated cytotoxicity.     

Characteristics of human NK clones: target specificity and phenotype

Clones derived from purified human large granular lymphocytes (LGL) of three different donors were expanded in culture medium supplemented with interleukin 2 (IL 2). Their cytotoxic activity was tested in a 51Cr-release cytotoxicity assay against a panel of three to five NK-susceptible tumor cell lines. Of 196 LGL clones tested, only 44 (22.4%) displayed significant cytotoxic activity. A heterogeneous pattern of reactivity was seen; 26 clones (59%) killed all the targets within the panel tested, whereas 18 clones (41%) had a more restricted specificity. Among these 18 clones, 12 lysed only one target (K562, six clones; ADCC, three clones; Daudi, two clones; MOLT-4, one clone), whereas the other six killed two different targets (ADCC and A1ab, one clone; K562 and MOLT-4, five clones). Clones derived from LGL preselected for positive reactivity with the monoclonal antibodies (MoAb) alpha OKM1, alpha OKT10 and alpha B73.1 also demonstrated either broad or restricted patterns of cytotoxicity. The LGL reactive with MoAb alpha B73.1 gave a high percentage of cytotoxic clones. Phenotype analysis showed that clones could express both antigens associated with T cells (i.e., OKT3, OKT4, and OKT8) and antigens shared by LGL (i.e., OKM1, OKT10, and B73.1). The pattern of surface markers varied considerably among the clones; however, no clear correlation between the pattern of antigenic phenotype and cytotoxic activity was seen. These data show that clones derives from purified preparations of LGL present different functional and antigenic characteristics, and support the hypothesis that the heterogeneity of the entire NK population is attributable, at least in part, to a mixture of clones that vary substantially in their target specificities and phenotypes.     

β1 Integrin triggering affects leukemic cell line sensitivity to natural killer cells

The role of beta1 (CD29) integrins in natural killer (NK) cell-target cell conjugation and cytotoxicity has not been clearly established. Ligation of beta1 integrins in NK cells can modulate the lytic capacity in both a positive and a negative manner; however, the contribution of the beta1 integrins present on target cells remains to be evaluated. Here, we analyzed the effect of beta1 integrins expressed by potential tumor target cells on conjugation and cytotoxicity. Using normalized flow cytometry binding assays, we demonstrated that the pretreatment of MOLT-4, K562, U-937 and HL-60 human leukemia target cell lines with selected anti-beta1 monoclonal antibodies (mAb) increased conjugation to human NK cell line NKL as well as to purified NK cells. Only mAb recognizing residues 207-218 of the beta1 subunit and functionally involved in the induction of homotypic adhesion (functional epitope A1) increased conjugation of all the target cells. Moreover, mAb to adhesion molecules different from beta1 but also inducers of homotypic adhesion of the target cells, i.e. CD43 and CD50 (ICAM-3), failed to increase conjugation to NKL cells. Cytotoxicity assays demonstrated that lysis of NK-sensitive target cells (MOLT-4) also increased after pretreatment with anti-beta1 epitope A1 mAb. Importantly, pretreatment of NK-resistant target cells (U-937 and HL-60) with anti-beta1 mAb was not able to outweigh the cytotoxic inhibitory mechanisms controlled by HLA class I molecules. However, simultaneous masking of HLA class I molecules with mAb and pretreatment with anti-beta1 mAb rendered NK-resistant cells susceptible to lysis, as predicted by the missing self hypothesis. Triggering of tumor target cells through beta1 integrins may thus play a role in conjugation to NK cells as well as in co-stimulation of cell-mediated cytotoxicity.     

Preparation of target antigens specifically recognized by human natural killer cells

In an attempt to identify target cell membrane molecules recognized by natural killer (NK) cells, artificial membranes were prepared from detergent-solubilized plasma membranes of NK target cells and synthetic lipids. Such reconstituted membranes from human and rat NK target cells were shown to inhibit both human and rat NK-target cell conjugates in a species-specific fashion; these reconstituted membranes failed to inhibit NK cytotoxicity. The detergent-solubilized material from the human NK target K562 was subjected to various procedures prior to reconstitution and the conjugate inhibition assay. Conjugate inhibitory activity was lost upon trypsin digestion and incubation at 65 degrees C. This inhibition activity was absorbed to concanavalin A agarose and could subsequently be eluted with alpha-methylmannoside, resulting in approximately 20-fold purification. Gel filtration of this material on an AcA-34 column in detergent gave a broad activity peak with maximal activity in the molecular weight range of 30,000-165,000. Gel electrophoresis of purified membranes demonstrated multiple molecular weight bands in lipid membranes. The K562 membrane material, purified by concanavalin A agarose and gel filtration, inhibited conjugates between human NK cells and any of four human target cells, but not of conjugates with (1) human large granular lymphocytes and antibody-coated mouse tumor cells nor (2) rat NK cells and their target cells. Thus the purified glycoproteins from K562 retain the property of specific inhibition of human NK-target conjugates.     

Characterization by monoclonal antibodies of a target cell antigen complex recognized by nonspecific cytotoxic cells

Fish nonspecific cytotoxic cells (NCC)3 recognize and lyse a large variety of human and mouse transformed cells. In an effort to determine the Ag recognized by NCC on these targets, mAb were raised against NC-37 target cells. Four anti-NC-37 mAb were chosen for further characterization based on their effects on NCC lysis of target cells. Purified mAb 18C2 and 1E7 (IgM isotype) inhibited NCC killing of the following targets: U937, MOLT-4, K562, HL-60, DAUDI, NC-37, P815, and YAC-1. The dose-dependent inhibitory activity occurred at the target cell level and ranged from 50 to 70% at a concentration of 50 micrograms/well when compared to noninhibitory mAb 7C6 and 1D4 (IgG isotype). Similarly, mAb 18C2 protected the fish parasite Tetrahymena pyriformis from lysis by NCC when compared to mAb 7C6. Adsorption experiments demonstrated that the inhibitory effect on NC-37 lysis by NCC could be removed in a titratable fashion by incubation of mAb 1E7 with any one of the other target cell lines, but it could not be removed by incubation with effector cells. The inhibitory activity of mAb 1E7 and 18C2 was shown to be caused by the inhibition of conjugate formation between effector and NC-37 target cells. The relative membrane concentration of the antigenic determinants recognized by these mAb on the target cells was studied by flow cytometry using FITC-labeled mAb. These experiments showed that all four mAb bound to the surface of the cells tested. Biochemical analysis with Western blots and immunoprecipitation showed that mAb 18C2 and 1E7 recognize two Ag in NC-37 lysates: a larger protein of around 80 kDa and a smaller one of 42 kDa.     

The isolation of natural killer (NK)-resistant variants of the K562 cell line by mutagenesis and selection with antibodies which inhibit NK cell-mediated lysis

Mechanisms involved in the lysis of tumor cells by natural killer (NK) cells were investigated by using mutagenized K562 targets resistant to the effects of NK cells. K562 cells were treated with the mutagen methyl methanesulfonate (MMS) and, to select for resistant mutants, rabbit anti-idiotypic (anti-id) antibodies were used. This anti-id was raised to a monoclonal antibody 9.1C3 which itself blocked lysis by NK cells by binding to the effector cells; the anti-id inhibited killing by binding to the K562 targets, presumably to a cell surface protein relevant to a secondary event in the NK lytic pathway. MMS-derived mutants showed a heterogeneity of staining with the anti-id, allowing the antibody to be used with flow cytometry to select a population of K562 cells relatively negative in antigen expression. The degree of reactivity of K562 cultures with the anti-id antiserum and the resistance to lysis by NK cells were inversely related. Cultures of NK-resistant K562 cells with low expression of the anti-id structure were cloned by limiting dilution: 96 clones were analyzed and one subclone, C9/2, which was six-to sevenfold less sensitive to lysis than the parental K562 cell line, was used in further studies by cold target inhibition and single cell binding assays. The increased resistance to lysis of C9/2 was not due to a reduced expression of target recognition structures, and resistance could not be overcome by prolonging the time allowed for lysis to 18 hr nor by adding exogenous recombinant leukocyte interferon. Killing of the NK-resistant variant was inhibited by mannose-6-phosphate but not by the monoclonal antibody against which the anti-id antibody was raised. It is therefore suggested that the structure on the K562 cells recognized by the anti-id antibodies is a novel secondary receptor which is important in the later stages of the NK cell cytolytic cascade.     

A monoclonal antibody-purified soluble target cell antigen inhibits nonspecific cytotoxic cell activity.

We have previously reported the characterization of mAb derived against NC-37 target cells. mAb 18C2 and 1E7 inhibit fish cytotoxicity by binding to target cells and thus preventing the formation of conjugates with fish nonspecific cytotoxic cells (NCC). It was therefore presumed that these inhibitory mAb were specific for the target cell Ag necessary for effector cell recognition. mAb 1D4 and 7C6 bind to NC-37 cells but do not inhibit fish cytotoxic activity. We now report the isolation and purification of the Ag recognized by mAb 18C2 (inhibitor) and 1D4 (noninhibitor) by affinity chromatography of solubilized NC-37 target cell extracts. The 18C2-purified soluble target Ag (STAg) caused inhibition of cytotoxicity when preincubated with fish NCC. This inhibitory activity was reversible and dose-dependent ranging from 20 to 70% inhibition with 25 to 100 micrograms 18C2 purified STAg/10(6) NCC. STAg purified by 1D4 affinity chromatography had no effect on fish cytotoxicity. mAb 18C2 and 1E7 preabsorbed with 18C2 STAg lost their inhibitory activity when tested in the fish NCC cytotoxicity assay. Preabsorption of the same mAb with 1D4 STAg had no effect on their activity.     

The role of interleukin 1 (IL 1) in tumor-NK cell interactions: correction of defective NK cell activity in cancer patients by treating target cells with IL 1

This study examined the importance of interleukin 1 (IL 1) in the large granular lymphocyte (LGL)-target cell interaction. K562 target cells when treated with highly purified human IL 1 for 1 hr bound greater numbers of LGL than untreated cells. LGL from patients with hepatocellular carcinoma (HCC) that bound few untreated K562 cells, attached to considerably increased numbers of IL 1-treated target cells. Cytotoxicity of LGL against target cells could similarly be increased by pulsing the latter cells with IL 1, and defective cytotoxicity of LGL from HCC patients could be corrected by treating the target K562 cells with IL 1. Lysis of PLC/PRF/5 cells, Yac-1 cells, and normal skin fibroblasts could also be increased by treatment with IL 1 for 1 hr. The enhanced binding and cytotoxicity of IL 1-treated target cells was only observed when the latter cells were preincubated with IL 1 at 37 degrees C, and was not evident at 4 degrees C. Furthermore, the IL 1-mediated effect could be abolished by treating the target cells with cycloheximide before the IL 1 pulse, or by adding rabbit anti-human IL 1 together with the IL 1. These results indicate that IL 1 affects a variety of target cells and increases their ability to bind and be lysed by enriched LGL. They demonstrate, furthermore, that defective natural cytotoxicity by the LGL of patients with advanced malignant disease can be corrected in vitro by treating the target cells with IL 1.     

Susceptibility to natural killer cell-mediated cytolysis is independent of the level of target cell class I HLA expression

To test the hypothesis that susceptibility to NK cell-mediated cytolysis varies inversely with the levels of target cell class I HLA expression, NK-susceptible K562 and MOLT-4 target cells have been transfected via electroporation with cloned human class I HLA-A2 and HLA-B7 genes. Stably transfected cells expressing varying levels of cell-surface class I HLA have been selected by fluorescent activated cell sorting and tested for susceptibility to NK-mediated cytolysis by freshly isolated peripheral blood NK cells from nine normal volunteers as well as by cloned human NK effectors and tumor cells from a patient with an NK cell lymphoproliferative disorder. Expression of class I HLA did not alter the susceptibility of K562 or MOLT-4 target cells to NK-mediated cytolysis by any of the effectors tested. In addition, the class I HLA-expressing transfectant cells were identical to mock transfected cells in their ability to compete for lysis in cold target inhibition assays. Treatment of both mock-transfected and class I HLA-transfected K562 cells with IFN-gamma resulted in decreased susceptibility to NK-mediated cytolysis which was independent of the total level of class I HLA expression. These results demonstrate that the level of target cell class I HLA expression is not sufficient to determine susceptibility or resistance to NK-mediated cytolysis of the classical NK targets K562 and MOLT-4.     

The principal tumor necrosis factor receptor in monocyte cytotoxicity is on the effector cell, not on the target cell.

Several tumor target cell lines, prototypically K562 cells, are resistant to lysis by recombinant tumor necrosis factor (TNF alpha) but are killed by monocytes expressing membrane-associated TNF, suggesting that membrane TNF could account for monocyte-mediated cytotoxicity. Formaldehyde-fixed monocytes or extracted monocyte membrane fragments are cytotoxic to K562 target cells. Treatment of monocytes with interferon-gamma (IFN-gamma) increases cytotoxicity by live and fixed cells or by extracted monocyte membranes. Both TNF and TNF receptors are detectable on monocyte membranes by FACS analysis, and the levels of each are modulated by treatment with IFN-gamma. Cytotoxicity can be inhibited by either anti-TNF or anti-TNF receptor antibodies. Incubation of effector cells with exogenous soluble TNF prior to fixation or membrane preparation increases their cytotoxicity. In contrast, incubation of the target cells with exogenous TNF neither increases nor decreases killing by effector cell membrane fragments or intact effector cells. The data suggest that the TNF receptors on the effector cell, but not on the target cell, play a crucial role in TNF-mediated cytotoxicity.     

Anti-CD3 and phorbol myristate acetate regulation of MHC unrestricted T cell cytotoxicity. Lack of a requirement for CD3/T cell receptor complex expression during tumor cell lysis

The effects of anti-CD3 mAb on MHC-unrestricted cytotoxic activity of NK depleted PHA-activated human T cells were examined. Anti-CD3 mAb had variable effects on killing of K562 or Daudi targets. Whereas lower concentrations of OKT3 often inhibited lysis of either target, higher concentrations (greater than 1 micrograms/ml) frequently increased K562 killing and always augmented Daudi lysis. However, lysis of the renal cell carcinoma, Cur, was consistently inhibited by OKT3 over a broad concentration range. Such variable effects were not related to differential regulation of heterogeneous subsets of effector cells, as similar patterns of OKT3-mediated modulation of tumor cell lysis by T cell clones was also observed. Another IgG2a anti-CD3 mAb, 64.1, and either F(ab')2 fragments of OKT3 or intact OKT3 in the presence of aggregated human Ig were found to inhibit lysis of Cur, K562, and Daudi targets consistently. Additional experiments were carried out to determine whether modulation of CD3 accounted for the inhibitory effects of the anti-CD3 mAb. PMA was noted to cause modulation of CD3 from the surface of PHA or alloantigen-activated T cells, and the combination of anti-CD3 and PMA caused even more marked modulation of CD3. Whereas preincubation with PMA and/or anti-CD3 decreased alloantigen-specific cytotoxic T cell function in relative proportion to the loss of CD3 expression, no consistent relationship between CD3 expression and the capacity of PHA-activated T cells to kill Cur targets was noted. PMA alone caused no consistent alteration of Cur lysis. Moreover, in the presence of PMA, anti-CD3 mAb caused no significant inhibitory effect on Cur lysis, in spite of increased modulation and in some cases virtual total loss of surface CD3 expression. These findings indicate that when FcR interactions are prevented, anti-CD3 mAb consistently inhibit MHC-unrestricted cytotoxicity by PHA-activated T cells. Despite this, the data support the conclusion that CD3/TCR complex interactions with target cells are not required for either target cell recognition or triggering of lysis by MHC-unrestricted cytotoxic T cells.     

The low affinity 40,000 Fc gamma receptor and the transferrin receptor can be alternative or simultaneous target structures on cells sensitive for natural killing

The role of the low avidity 40,000 dalton receptor for IgG (Fc gamma R) present on K562 and U937 cells in sensitivity to natural killing (NK) was studied by using a murine monoclonal antibody (mAb) specific for the 40,000 dalton Fc gamma R (alpha Fc gamma R mAb). Pretreatment of K562 target cells with intact alpha Fc gamma R mAb or its Fab fragment or anti-transferrin receptor (alpha TFR) mAb partially blocked in a dose-dependent manner, NK activity to K562 cells. However, combined pretreatment with alpha Fc gamma R and alpha TFR mAb completely blocked NK activity against K562 targets. As compared with K562 cells, lower levels of NK were elicited against Molt-4, U937, HL-60, and Daudi targets. Although NK activity to Molt-4 targets was not affected by alpha Fc gamma R mAb, it was fully prevented by pretreatment with alpha TFR mAb. In contrast, NK to U937 cells was not influenced by alpha TFR mAb, but it was strongly inhibited by alpha Fc gamma R mAb. The resistance of 3H-TdR-prelabeled adherent HEp-2 cells to natural cell-mediated cytotoxicity was not affected by either mAb. Lectin-dependent cell-mediated cytotoxicity (LDCC) against HEp-2 cells due to the presence of concanavalin A, and was completely abrogated by pretreatment of the targets with alpha TFR mAb, but was unaffected by alpha Fc gamma R mAb. By use of the flow cytometer, a significant correlation was detected between the relative expression of 40,000 dalton Fc gamma R and the susceptibility to NK, whereas the expression of TFR was discordant from NK sensitivity. As determined in the single cell cytotoxicity assay alpha Fc gamma R mAb reduced the frequency of target binding effector cells without affecting the number of dead bound targets. This pattern of inhibition was found against both K562 and U937 targets. Alternatively, alpha TFR mAb inhibited both binding and killing of K562 and Molt-4 targets. Because pretreatment of HEp-2 cells with alpha TFR mAb did not influence conjugate formation, the blocking of LDCC to HEp-2 cells by alpha TFR mAb can be related to post-binding events. These data show that although both the 40,000 dalton Fc gamma R and the TFR can be target structures for NK cell recognition, the TFR may also play an important role in the post-binding events.     

Natural killer cell-target interactions: the role of 4F2 antigen in the human system

The manner by which natural killer cells discriminate between target and nontarget cells is a subject of intense investigation. Recently, the antigen recognized by the 4F2 monoclonal antibody has been implicated as the target recognition molecule of cloned human natural killer cells. Here we report the results of our studies on the possible role of 4F2 antigen in target recognition by natural killer cells present in the peripheral blood. In our hands, the 4F2 antibody only weakly blocked the killing of K562 leukemia cells by human natural killer cells. Furthermore, no correlation was observed between the level of cell surface 4F2 antigen and the natural killer susceptibility of several tumor cell lines, the ability of these cells to bind to natural killer cells, or the ability of these cell lines to compete with K562 cells in a natural killer assay. Therefore, the 4F2 antigen does not appear to be the target recognition molecule of most peripheral blood natural killer cells.     

Natural killer-sensitive targets stimulate production of TNF-alpha but not TNF-beta (lymphotoxin) by highly purified human peripheral blood large granular lymphocytes

Highly purified populations of large granular lymphocytes (LGL) have been shown to mediate natural killer (NK) cell activity. The mechanism of target cell killing by NK cells is as yet undefined; however, it has been postulated that such killing may involve soluble cytotoxic factors produced and secreted by NK cells. The data presented show that NK-sensitive, but not NK-resistant, tumor cell lines induce highly purified populations of human LGL to produce factors with cytotoxic and/or cytostatic activities. We have identified one of these factors as tumor necrosis factor-alpha (TNF-alpha), and have shown that production of this factor is enhanced by recombinant human interferon-gamma (rHuIFN-gamma). We have also examined the role of TNF-alpha in the cytotoxic function of NK cells. The data show that although highly purified LGL populations produce low levels of TNF-alpha, the cytotoxic/cytostatic activity of this lymphokine on tumor target cells does not correlate with the cytotoxic activity of highly purified populations of LGL on tumor target cells. Furthermore, NK cell-mediated cytotoxicity is not reliably inhibited by antibodies directed against various epitopes of recombinant human TNF-alpha and/or recombinant TNF-beta (lymphotoxin) or rHuIFN-gamma. These data show that although TNF-alpha is produced by highly purified NK-containing LGL cell populations, this factor does not appear to be responsible for NK cell cytotoxicity against classical NK target cells such as Molt-4 or K562. We suggest that NK function can be attributed to a combination of factors rather than to a single factor alone, and that at least two major phenomena are involved in LGL function: the rapid cytotoxic events which lead to the cell lysis measured in classical in vitro NK assays such as against K562; and the release of factors such as TNF-alpha with cytotoxic/cytostatic activities which would inhibit the growth of invading tumor cells in vivo.     

Apparent sensitivity of human K lymphocytes to the spatial orientation and organization of target cell-bound antibodies as measured by the efficiency of antibody-dependent cellular cytotoxicity (ADCC)

Although monoclonal antibodies (mAb) can elicit potent ADCC by human K lymphocytes, different mAb, even of the same antibody subclass or even of the same target antigen specificity, vary considerably as to their efficiency in eliciting ADCC. The extensive variability in ADCC efficiencies of murine IgG2a mAb is analyzed here. In cold-target inhibition experiments it was found that only cells coated with "ADCC-efficient" IgG2a mAb, and not "ADCC-inefficient" IgG2a mAb, inhibit K effector cell lysis of radiolabeled target cells by ADCC. This result indicates that the spatial orientation of the antibodies on the target cell membrane influences the net efficiency of ADCC reactions by affecting the efficiency of interaction between antibody and the Fc receptors (FcR) of K cells. It is proposed that a "favorable" orientation of antibodies on the target cell membrane is required for efficient ADCC reactions. This proposal is directly supported by the observation that one IgG2a mAb (20.8.4), which cross-reacts with several different H-2 alloantigens, was found to elicit efficient ADCC only when bound to certain of its possible target cell antigens. It was also observed in these studies that the organization of antibodies on a target cell membrane influences the net efficiency of ADCC reactions. It is proposed that a "favorable" antibody organization on the target cell membrane is also required for efficient ADCC reactions. This proposal is supported by the observation that certain antihuman beta 2m (anti-Hu beta 2m) IgG2a mAb, which elicit efficient ADCC lysis of human target cells, fail to elicit the lysis of murine cells having Hu beta 2m molecules coupled randomly to their external membrane surfaces. The differences in the way the Hu beta 2m was organized on the surfaces of the human cells and the murine-Hu beta 2m cell conjugates presumably caused differences in the way the bound antibodies were organized on the cell surfaces, which in turn resulted in the ADCC efficiency differences observed for the same mAb with the different target cell types. Because ADCC reactions appear to be sensitive to both the orientation and the organization of cell surface-bound antibodies, certain types of structural alterations or variations in the membrane molecules (relative to other neighboring structures on the target cell membrane) are potentially detectable by quantitative differences or variations in ADCC reactions.