CD16+ lymphoblastic cell lines of crab-eating monkeys (Macaca fascicularis) shared U-5 antigen and expressed natural killer activity

The CD16+ lymphoblastic cell lines of crab-eating monkeys shared the U-5 antigen recognized by a monoclonal antibody. The CD16+U-5+ cell lines expressed high natural killer activity to K562 cells, whereas the CD16-U-5- control cell line had no significant natural killer activity. A possible involvement of the U-5 antigen in natural killer function was also suggested by reduction of the natural killer activity in peripheral blood mononuclear cells of Japanese monkeys after treatment with U-5 monoclonal antibody and complement.     

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.     

Characterization of a monoclonal antibody enhancing porcine natural killer cell activity (PNK-E)

A monoclonal antibody, termed PNK-E, that functionally enhances porcine natural killer (NK) cell activity but not antibody-dependent cellular cytotoxicity (ADCC) is investigated in this report. When PNK-E and K562 target cells were simultaneously added to effector cells, killing of target cells could be detected as early as 30 min, and a dramatic enhancement of killing activity was observed in short term 51Cr-release assays. When a panel of five NK-sensitive targets were tested, PNK-E enhanced the killing of K562, MOLT-4, and U937 cells, but not the killing of CEM and YAC-1. F(ab)'2 fragments of PNK-E did not enhance NK activity, indicating a requirement for the Fc portion of PNK-E to elicit enhancement of NK. Immunofluorescence analysis shows that PNK-E antigen is expressed on approximately 15% of peripheral blood lymphocytes with a relatively dull fluorescence staining pattern. PNK-E-positive sorted cells were enriched for large granular lymphocytes (LGL) and contained all detectable NK activity as compared to the PNK-E-negative sorted cells. When analyzed by polyacrylamide gel electrophoresis, PNK-E antibody immunoprecipitated a protein from 125I-labeled peripheral blood lymphocyte (PBL) cell lysates that resolved as a single band of approximately 205 kDa under nonreducing conditions and as two bands of approximately 50 kDa and 47 kDa under reducing conditions. The present data demonstrate a functional association between PNK-E antigen and NK cell activation.     

Natural killer cells in human colostrum

The presence of natural killer cells in human colostrum was disclosed with the use of a fluorochrome-labeled monoclonal antibody HNK-1 (Leu-7) that recognizes cells with natural killer and killer activity. Approximately 0.5% of total colostral cells were stained with this reagent. These cells were separated by the fluorescence-activated cell sorter and examined for their morphology by electron microscopy and for their cytotoxic activity against 51Cr-labeled K562 target cells. Two morphological types of natural killer cells were observed in colostrum: the first was represented by large cells with numerous vacuoles but without dense cytoplasmic granules; the second type, which occurred with lower frequency, resembled the large granular lymphocytes associated with natural killer activity in peripheral blood. The HNK-1-positive cells from colostrum displayed low cytotoxic activity against K562 target cells. Incubation of HNK-1-positive cells from peripheral blood with cell-free colostrum resulted in a dose-dependent inhibition of the cytotoxic activity. The functional changes were accompanied by morphological alterations which included degranulation and the formation of numerous vacuoles. The variances in the cytotoxic activity of peripheral blood HNK-1-positive cells suspended in different dilutions of colostrum suggest that this fluid contains humoral factors which modify morphology and function depending on their concentrations.     

Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

Immunomodulation of human leukocytes by staphylococcal enterotoxin A: augmentation of natural killer cells and induction of suppressor cells

Staphylococcal enterotoxin A (SEA), a protein isolated from culture supernatants of Staphylococcus aureus, is a potent T-cell mitogen and an inducer of interferon-gamma (IFN-gamma). We report here that SEA exhibits a number of significant in vitro immunomodulatory functions. In vitro treatment of human peripheral blood monocyte-depleted lymphocytes with SEA resulted in significant augmentation of their natural killer cytotoxicity against target cells from hemopoietic (K562, Daudi) or solid (melanoma, lung, colon) human tumor cell lines. SEA was found to be more effective than interferons-alpha (natural or Escherichia coli-derived) in augmenting natural killer (NK) cytotoxicity of peripheral blood lymphocytes. Studies on the kinetics of the augmentation revealed a significant increase of NK within 3 hr of in vitro treatment with SEA at 37 degrees C. A neutralizing monoclonal antibody specific for human IFN-gamma did not affect the augmentation of natural killer cytotoxicity by SEA, suggesting that SEA augmented natural killer cytotoxicity primarily by a mechanism not involving induction of interferon-gamma. Furthermore, in vitro treatment with SEA resulted in significant augmentation of antibody-dependent cell-mediated cytotoxicity and of natural killer-like cytotoxicity, generated in mixed lymphocyte culture, against the K562 targets. Induction of suppressor cells to proliferative responses of autologous or allogeneic mononuclear cells to phytohemagglutinin (PHA) or to allogeneic cells in mixed lymphocyte culture was observed after in vitro treatment of peripheral blood mononuclear leukocytes with SEA for 24 or 48 hr at 37 degrees C. In addition, the presence of SEA in mixed lymphocyte cultures (MLC) resulted in significant inhibition of the generation of specific T-cell-mediated cytotoxicity in MLC. These results suggest that SEA, which may be involved in S. aureus infections and in treatment with extracorporeal perfusion systems over S. aureus columns, can regulate a number of significant lymphoid functions.     

Effect of interleukin 2 on the inhibition of human natural killer activity by monolayer cells

We have previously shown that human endogenous natural killer activity against K562 is inhibited by primary cultures of natural killer-resistant monolayer target cells. In this study we have analyzed the sensitivity of activated killer cells to this inhibitory effect. Interleukin-2 (IL-2), when present during an 18-hr contact of peripheral blood lymphocytes with monolayers, did not affect the inhibition of natural killer cell activity. Pretreatment of effector cells with IL-2 for 24-62 hr before the contact with monolayer cells eradicated the inhibition caused by malignant cells, benign cells remaining inhibitory. The IL-2-pretreated effector cells killed preferentially malignant target cells, although significant cytotoxicity was also detectable against benign cell cultures. The results indicate that activation of killer cells in vitro by IL-2 involves the desensitization of effector cells to the inhibitory signals of target cells, and that the selectivity of IL-2-activated killer cells toward malignant target cells involves weaker inhibition of activated killer cells by malignant cells.     

In vitro generation of human activated lymphocyte killer cells: separate precursors and modes of generation of NK-like cells and "anomalous" killer cells

The activation of human peripheral blood mononuclear cells (PBM) in culture leads to the generation of nonspecific killer cells. These cells, termed activated lymphocyte killer (ALK) cells, can kill fresh tumor cells and tumor cell lines, in addition to the natural killer (NK) cell sensitive target K562. ALK cells have features in common with both T and NK cells, but their nature and origin are unknown. In the present study, it is shown that ALK cells are in fact heterogeneous and can be generated from both large granular lymphocytes with the same phenotype as NK cells and from T cells. Cell populations enriched for NK cells, when cultured with lymphokines, rapidly acquired a T cell phenotype, enhanced cytolytic activity against K562, and the ability to lyse NK-insensitive target cells such as a melanoma cell line LiBr; these ALK cells were described as NK-like cells. On the other hand, of the cloned cells derived from PBM stimulated with irradiated B lymphoblasts and grown in lymphokines, the major proportion of cytolytic T cells (CTC) able to kill the specific stimulator lymphoblasts were also found to kill LiBr but not K562 cells. These ALK cells, which were derived from the same precursors as CTC, were designated anomalous killer (AK) cells. Consistent with this, the presence of the pan T monoclonal antibody UCHT1 from the beginning of mixed cell cultures inhibited the generation of CTC and of the AK-type of ALK cell, which killed melanoma cells, but not the NK type, which killed K562 targets. By contrast, at the effector cell level, the antibodies UCHT1 and OKT8 only blocked specific killing by CTC but did not block the killing of LiBr or of K562 targets by ALK cells. However, at the effector cell level there was additional evidence for the heterogeneity of ALK cells. Thus, monoclonal antibody 9.1C3, which blocks killing by freshly isolated NK cells, also blocked the killing of K562 targets by NK-like cells, but did not block B lymphoblast killing by CTC or melanoma cell killing by AK cells. It is concluded that after mixed lymphocyte culture, the majority of ALK cells measured by the killing of melanoma target cells arise from the same precursors and are under the same influences as classical CTC (AK cells), whereas cells killing K562 targets are derived from NK cells (NK-like cells). Once generated, the AK cells have a different mechanism of killing from both classical CTC and from NK and NK-like cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Depletion of NK by cellular immunoadsorption.

The binding of human natural killer (NK) cells to their tumor cell targets was investigated by using monolayers of sensitive target cell lines. Monolayers of K562 and HSB, a myeloid and T cell line, respectively, were prepared on poly-L-lysine-coated plastic tissue culture dishes and briefly fixed with 0.2% formaldehyde. Freshly isolated peripheral blood lymphocytes (PBL) were incubated on the monolayers. Nonadherent PBL were then removed, after gentle agitation, by decanting and gently washing the monolayer. They were tested, along with unseparated controls, for NK activity in a short-term 51Cr release assay. PBL that were nonadherent to a tested monolayer had only 20 to 60% of the control cytotoxic activity. Our results suggest that NK recognition sites on the effector lymphocytes were able to interact with reciprocal determinants on the target cell monolayers, resulting in selective loss of NK effector cells from the PBL population. The specificity of the NK effector-target interaction was investigated by testing the ability of each monolayer to remove activity against both targets. These data imply heterogeneity with regard to recognition structure within the NK effector population as well as among the target cells.     

Analysis of cytostatic/cytotoxic lymphokines: relationship of natural killer cytotoxic factor to recombinant lymphotoxin, recombinant tumor necrosis factor, and leukoregulin

Previous results that were obtained by using supernatants from the co-culture of human peripheral blood lymphocytes and the natural killer susceptible cell line K562 strongly inhibited the growth of various tumor cell lines. No correlation was observed between the susceptibility of the target cell lines to growth inhibition and to lysis by natural killer cells. Rather the spectrum of cytostatic activity and the characteristics of the soluble factor were similar to those of leukoregulin (LRG), a recently described lymphokine. Because of the recent availability of recombinant tumor necrosis factor (TNF) and lymphotoxin (LT), we compare the target selectivity and mechanism of action of these (TNF, LT, LRG) factors with natural killer cytotoxic factor (NKCF). The pattern of target cell susceptibility to growth inhibition or cytolysis by the factors were quite distinct from the pattern observed when cells were exposed to NKCF. Furthermore, antibodies to rLT or rTNF had no effect on LRG cytostasis or NKCF lysis, arguing against a requirement for or synergistic interaction with low levels of LT or TNF. Some of the targets susceptible to LRG were growth inhibited but were not lysed, thereby distinguishing it from NKCF. Furthermore, LRG cytostasis was not inhibited by mannose-6-PO4 or rabbit antibodies to granule cytolysin, both of which block natural killer cytotoxic factor. Therefore, LRG appears to be a cytostatic factor produced by large granular lymphocytes in response to K562 that is distinct from NKCF, TNF, and LT. In addition, NKCF, rLT, rTNF, and LRG, although having cytotoxic/cytostatic activity, are distinct functional factors and may represent a family of lytic factors.     

Kupffer cells modulate natural killer cell activity in vitro by producing prostaglandins

The effect of Kupffer cells on natural killer (NK) cell-mediated cytotoxicity was examined. Kupffer cells prepared from rat liver suppressed NK activity against K562 cells and other tumor cell lines through a soluble factor secreted into the culture supernatant. When human peripheral blood mononuclear cells were incubated with the Kupffer cell-culture supernatant, a significant reduction of the cytotoxic activity was observed in the 6-hr chromium-release assay. This activity was dose dependent and was evident at various effector/target cell ratios. Lipopolysaccharide stimulated generation of the suppressive factor released from Kupffer cells in a dose-dependent manner. Suppression of the NK activity was observed when the Kupffer cell-culture supernatant was present in the assay system, whereas pretreatment of effector/target cells with the supernatant had minimal inhibitory effects. Autologous monocytes in human peripheral mononuclear cells were not related to this suppression. The suppressive factor in the fraction had a molecular weight below 10,000. Indomethacin, an inhibitor of prostaglandin synthesis, ameliorated the suppressive effects. These results suggest that Kupffer cells may modulate NK activity by producing PGs (E1, E2, and F2 alpha).     

Studies on the mechanism of specificity of human natural killer cells for tumor cells: correlation between target cell transferrin receptor expression and competitive activity

Previous studies to determine the nature of the specificity of natural killer (NK) cells for leukemic cells indicated that functional transferrin (Tf) receptors may be one of the determinants recognized by NK cells. To further investigate these observations, the relationship between cellular Tf receptor expression and ability to compete with a control K562 cell preparation in a standard chromium release assay was studied. K562 cells were selected at different phases of growth by removing cells from tissue culture at 1, 3, and 5 days postfeeding. Under these conditions, K562 cells, respectively, displayed relatively high, medium, and low numbers of Tf receptors and corresponding competitive activity against a control K562 cell preparation. K562 cells were modified by either trypsin, heat, or sodium butyrate (differentiation inducer) pretreatment. An NK-resistant clone was also studied. There was a good correlation between Tf receptor expression and cold competitive activity of the above K562 cell preparations (r = 0.82, P less than 0.01). The different tumor target cell lines, K562, Molt-4, Raji, HL-60, and MeWo, which would be expected to express different ranges of specificity, did not show a significant correlation between Tf receptor expression and their cold competitive activities against Cr-51-labeled K562 cells. Rabbit reticulocytes which express high numbers of Tf receptors were tested for their ability to compete with K562 cells for NK cells. These cells were able to compete with K562 cells while mature rabbit red blood cells which do not express Tf receptors did not compete well. These findings support the contention that the Tf receptor may be involved in NK cell recognition of some tumor cells.     

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.     

Human natural cytotoxic lymphocytes: definition by a monoclonal antibody of a subset which kills an anchorage-dependent target cell line but not the K-562 cell line

A monoclonal mouse antibody (HNC-1A3) which defines a subset of human lymphocytes with natural cytotoxic activity was produced and studied. HNC-1A3+ cells represent 12 +/- 3% of peripheral blood mononuclear leukocytes. When sorted out using a fluorescence-activated cell sorter, they consist of 60% small lymphocytes, 35% large (predominantly agranular) lymphocytes, and 5% monocytes. They contain 30 +/- 6% E-rosette-forming cells, 6 +/- 1% OKT4+ cells, 17 +/- 6% OKT8+ cells and less than 2% OKT10+ or Leu-7 (HNK-1)+ cells. They are responsible for most of the natural cytotoxic activity against the MA-160 prostatic adenoma cell line but mediate an insignificant amount of cytotoxicity against the NK prototype target K562 cell line. Conversely, Leu-7+ cells which mediate most NK activity against K562 are weakly active against MA-160. Our data suggest a heterogeneity among leukocytes mediating natural cytotoxicity, with restricted specificities for the recognition sites on target cells.     

Analysis of cellular immune response to EBV by using cloned T cell lines

Eight cloned T cell lines specific for Epstein Barr virus-transformed B lymphocytes were derived. In the presence of the autologous virus-infected B cells, the T cell lines show HLA-restricted cytotoxic activity and also secrete alpha-interferon in sufficient amounts to inhibit infection and transformation. Four of these clones showed restriction to a single HLA locus (two for A3, and two for B7) and three showed exquisite self-restriction lysing only autologous targets. These seven clones expressed the classical cell surface phenotype of cytotoxic T cells being T3, 8, 11, and la-positive and T4-negative. An eighth clone that lacked the T8 surface marker appeared to recognize both B7 and BW51. HLA restriction was confirmed: 1) by the ability of a monoclonal antibody against an HLA-A,B,C framework antigen (W6-32) to block the cytotoxicity; 2) the failure of the clones to lyse Daudi, an EBV-positive, HLA-A,B, C-negative cell line; and 3) successful competition of the cytotoxicity by autologous but not allogeneic cold targets. The cloned T cells do not kill EBV-negative targets such as autologous pokeweed mitogen blasts and cell lines including CEM and the natural killer cell target K562. The results suggest T cell clones may be generated against an EBV-associated membrane antigen on transformed B cells, perhaps equivalent to the lymphocyte-determined membrane antigen, and that the recognition is restricted by a single HLA determinant. We propose that single T cells can play multiple roles in controlling EBV infection in vitro and in vivo including the elimination of transformed cells by cytotoxicity and the prevention by secreted interferon of further re-infection and transformation.     

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.     

Human T cell clones use a CD3-associated surface antigen recognition structure to exhibit both NK-like and allogeneic cytotoxic reactivity

In the present study, we developed human non-MHC-restricted CTL clones from human peripheral blood mononuclear cells activated in vitro with recombinant IL 2 and subsequently expended with PHA. The CD3/Ti+ clones were selected for their ability to exhibit non-MHC-restricted CTL reactivity by killing various tumor cell lines in culture, including the line K562 which does not express MHC antigens. We report that, at least for some of the NK-like T cell clones, it is possible to establish an allo-CTL activity, and that the CD3-associated surface antigen recognition structure might be involved in both reactivities.     

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides

The present study examines and compares the cytolysis of K-562 and MOLT-4 cells mediated by human natural killer (NK) cells from fresh peripheral blood and lymphotoxins (LT) derived from human lymphoid cell populations after lectin stimulation in vitro. Lymphotoxins were obtained from 5-hr concanavalin A (Con A)-restimulated human peripheral blood lymphocytes (PBL) which were precultured for 5 days in medium and fetal calf serum or with allogeneic human B-lymphoid cell lines. Two classes of probes were employed in both direct (cell) and indirect (supernatant) induced target-cell lysis: (a) various saccharides and (b) antibodies reactive with human LT forms. Two sugars, N-acetylglucosamine and α-methylmannoside, were able to inhibit direct cell lysis of both MOLT-4 and K-562 target cells. However, saccharide inhibition was distinct for each type of target even when effector cells were obtained from the same donor. These same saccharides were also able to inhibit 20–30% of the total LT activity in a supernatant for L-929 cells and 50–90% of the lytic activity on MOLT-4 cells. Anti-human F(ab′)₂ (IgG) and rabbit anti-α₂ LT sera blocked direct cell lysis of MOLT-4 and K-562 targets in 50% of the experiments. The anti-α₂ LT serum only recognizes a portion of the LT forms in these supernatants. These results reveal that, while both direct and indirect cell lysis are complex phenomena, they may both occur in some cases by a common mechanism(s).     

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.