Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

K562 killing by K, IL 2-responsive NK, and T cells involves different effector cell post-binding trigger mechanisms

The monoclonal antibody 13.3 specifically blocks the trigger process of the NK-K562 cytolytic sequence at a post-binding effector cell level. This antibody was used to define differences in the lytic trigger processes of NK and other mechanisms of K562 lysis. Monoclonal antibody 13.3 inhibited lysis of K562 target cells by freshly isolated peripheral blood lymphocytes (PBL) and purified large granular lymphocytes (LGL), but had no inhibitory effect on antibody-dependent cell-mediated cytotoxicity to K562 by these effectors. Lectin-dependent cellular cytotoxicity (LDCC) to this target cell was also unresponsive to 13.3. The 13.3-induced inhibition of NK-K562 lytic activity persisted when PBL were activated in culture with interleukin 2 (IL 2) for periods up to 48 hr. After 48 hr of culture, the degree of inhibition diminished progressively in medium containing fetal calf serum but not in medium containing autologous serum. This 13.3-unresponsive lytic activity in cultured PBL could be attributed to more than one cell type and was present in both the LGL and Fc gamma receptor-depleted T cell fraction. Thus, K562 lysis by freshly isolated human lymphocytes via NK, K, and LDCC mechanisms is characterized by heterogeneity of the post-binding effector cell trigger mechanism. K562 lysis by lymphocytes cultured with IL 2 is similarly heterogeneous.     

Histamine-induced suppressor factor inhibition of NK cells: reversal with interferon and interleukin 2

Culture supernatants of lymphocytes stimulated with 10(-3) to 10(-8) M histamine contain histamine-induced soluble suppressor factor (HISSF) that significantly inhibits the natural killer (NK) cell functions of allogeneic lymphocytes. Lymphocytes precultured with increasing concentrations of HISSF showed a dose-dependent suppressive effect on their NK activity. HISSF was not cytotoxic itself and produced suppressive effects on PBL, NK-enriched large granular lymphocytes (LGL), and isolated T cells. Suppression was evident throughout a range of effector:target cell ratios. Production of HISSF was specifically blocked by the H2 antagonist cimetidine, but not by the H1 antagonist clemastine fumarate. Furthermore, H1 and H2 antagonists themselves do not induce production of HISSF. Although HISSF could inhibit the cytotoxicity of LGL, LGL themselves do not produce HISSF. HISSF inhibition of NK activity could be completely reversed by treating effector lymphocytes with recombinant interferon-alpha (IFN) for 1 or 2 hr or culturing them with purified interleukin 2(IL 2) for 36 hr. Our data suggest that exogenous IFN and IL 2 may have therapeutic potential in the treatment of immunological diseases associated with histamine-induced suppressor cell activity.     

Mechanism of cell-mediated cytotoxicity at the single-cell level: VII. Trigger of the lethal hit event is distinct for NK/K and LDCC effector cells as measured in the two-target conjugate assay

Normal human peripheral blood lymphocytes (PBL) express several in vitro cytotoxic functions, among which are natural killer (NK), antibody-dependent cellular cytotoxicity (ADCC), and lectin-dependent cellular cytotoxicity (LDCC). The relationship of these various cytotoxic functions and the identity of cells involved has been a subject of controversy. Recently it was reported that NK and K for ADCC can be mediated by the same cell, suggesting that they constitute in large part a single subpopulation with multiple cytotoxic functions. The ability of this NK/K effector cell to mediate LDCC was examined here using the two target conjugate assay. The effector cells were Ficoll-Hypaque PBL or LGL-enriched fractions. The targets used were K562 or MOLT for NK, RAJI coated with antibody for ADCC, and RAJI coated with PHA or Con A or modified by NaIO₄ for LDCC. In the two-target conjugate assay, one of the targets is fluorescein labeled for identification. The results show that (a) LDCC copurifies with NK/K and is enriched in the LGL fraction, as measured in both the ⁵¹Cr-release assay and the single-cell assay for cytotoxicity; (b) single effector cells simultaneously bind to NK or ADCC and LDCC targets, revealing that single cells bear binding receptors for all targets; and (c) single lymphocytes were not able to kill both bound NK/K and LDCC targets. However, significant two-target killing was obtained when both targets were NK targets, ADCC targets, LDCC targets, or one NK and one ADCC target. These results demonstrate that the NK and LDCC effector cells are distinct subpopulations copurified in the LGL fraction. In addition, the results show that lectin is unable to trigger globally an NK effector cell to mediate cytotoxicity against a bound NK insensitive target. Thus, although both NK and LDCC effector cells are present in the LGL fraction and can bind to both types of targets, the trigger of the lethal hit event is the function of specialized effector cells.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.     

The role of transferrin in natural killer cell and IL-2-induced cytotoxic cell function

The growth factor transferrin (Tf) enhanced natural killer (NK) cell cytotoxicity. This enhancement was due to direct effects on NK cell function, and Tf treatment of the K562 target cell had no effect on their sensitivity. NK cells were highly enriched in the low-density large granular lymphocyte population (LGL) by Percoll gradient centrifugation. Despite the direct effect of Tf on NK cells, the number of cells expressing receptors for Tf (TfR) in NK-enriched LGL was the same as the NK-cell-depleted high-density small lymphocyte population (SL). All populations, tested without stimulation, had very few TfR+ cells. Interleukin 2 (IL-2) could induce very high NK-like activity in the LGL but not in SL. Similarly, only LGL could be induced by IL-2 to express TfR. In serum-free cultures, only limited NK-like activity could be developed which was greatly enhanced by supplementing with Tf in the cultures. The importance of Tf in NK-like development was confirmed by modulating the expression of TfR in IL-2 containing cultures with mouse monoclonal antibody OKT9 specific for TfR. OKT9 totally abrogated the induction of cytotoxic activity by IL-2 against K562 and NK-resistant target. OKT9 inhibited the induction of cytotoxicity in both lymphocytes containing active NK cells and in those predepleted of active NK cells, indicating that the development of NK-like activity from both precursor populations requires Tf. The inhibition by OKT9 was only during the induction phase. The same antibody had no effect on the cytotoxicity of fresh NK cells or the mature IL-2-induced NK-like cells. Our data therefore do not support the hypothesis of TfR as the NK recognition structure. Instead, these results indicate that Tf is important for the development of NK and NK-like activities.     

Relationship between immune system and gram-negative bacteria. II. Natural killer cytotoxicity of Salmonella minnesota Rb 345-unbound human peripheral blood lymphocytes

Spontaneous binding of human peripheral blood lymphocytes (PBL) to bacteria represents a promising approach for the characterization of lymphocyte subsets mediating different functions. In the light of previous findings on the high degree of spontaneous adherence of S. minnesota Rb cells to PBL, we have evaluated the natural killer (NK) cytotoxicity of PBL subpopulations that fail to bind to Rb bacteria. The S. minnesota Rb-unbound cell fraction exhibits higher levels of cytotoxic capacity, which is related to a more elevated frequency of active NK cells, as determined in an agarose-single cell cytotoxic assay. Moreover, the cytotoxic activity of the unbound fraction is additionally boosted by interferon-alpha pretreatment. The effector cells bear Fc gamma receptors that are involved in NK cell lysis, because a decrease of NK activity is observed after immune complex modulation of the receptors. Finally, these cells, which display a high percentage (approximately 70%) of typical large granular lymphocyte morphology, express HNK-1, T10, T8, and M1 antigens, and to a lesser extent T3 and T4 antigens. These data indicate a selective enrichment of NK cells in the S. minnesota Rb-unbound fraction.     

Natural cytotoxic effector cell activity against Shigella flexneri-infected HeLa cells

Virus and facultative intracellular bacteria both replicate within a host cell. The recognition and killing of virus-infected cells by natural killer (NK) cells is thought to be an important host immune function. However, little is known about immune recognition of bacteria-infected cells. In this report, we show for the first time that human peripheral blood lymphocytes (PBL) and large granular lymphocytes (LGL) purified from PBL have significant levels of cytotoxic activity against Shigella flexneri-infected HeLa cells. This cytotoxic activity was dependent on bacterial invasion of the HeLa cells, because HeLa cells pretreated with a noninvasive isogenic variant of S. flexneri or soluble bacterial products were not killed. Pretreatment of PBL with interleukin 2 (IL 2) or interferon-alpha greatly enhanced the cytotoxic activity of PBL against Shigella-infected HeLa cells. Cytotoxic activity present in PBL or in PBL pretreated with IL 2 was shown to be associated with both Leu-11+ and Leu-11- cell populations. These results suggest that NK cell killing of bacteria-infected cells may play an important role in host defense against facultative intracellular bacterial infections.     

Feline cytotoxic large granular lymphocytes induced by recombinant human IL-2

Large granular lymphocytes (LGL) have been characterized phenotypically and functionally as cytotoxic T lymphocytes, NK cells or lymphokine-activated killer cells. The most prominent morphologic feature of LGL is large cytoplasmic granules that are thought to contain the molecules responsible for cell lysis. In this study, we describe the morphologic and functional characteristics of IL-2-dependent cytotoxic lymphocytes derived from feline PBL. Stimulation of feline PBL with Con A followed by culturing in 50 U of gibbon monkey IL-2 human rIL-2 induced long term lymphocyte cultures. These lymphocytes are cytotoxic for the feline leukemia virus-induced T cell lymphoma (FL74), in a 4-h 51Cr release assay. All cell lines are either constitutively cytotoxic for FL74 cells, or cytotoxic in a lectin-dependent cell cytotoxic assay, the latter being a characteristic of low passage cultures. In contrast, no cell lines express self lysis or lysis for other lines. [3H]TdR uptake showed that 1 U of human rIL-2 produces a 50% maximal proliferative response by feline lymphocytes suggesting a high degree of homology between the ligand binding sites of feline and human IL-2R. Feline cytotoxic lymphocytes possess abundant cytoplasm containing large azurophilic granules characteristic of LGL. These granules are bound by a bilipid membrane and contain numerous smaller membrane-bound vesicles 50 to 60 nm in diameter. A model is proposed, whereby subsequent to binding of LGL to target cell the large granules fuse to the LGL plasma membrane and release the small vesicles into the binding pocket. The vesicles then transport the lytic molecules directly and selectively to the target cell membrane.     

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.     

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.     

An exoglycosidase-sensitive triggering site on NK cells which is coupled to transmethylation of membrane phospholipids

Glycosidic enzymes were used as probes to analyze the mechanism of NK cell-mediated cytotoxicity. Pretreatment of nylon wool-enriched CBA/J spleen cells, a murine NK clone, or human peripheral blood lymphocytes (PBL) with alpha-mannosidase, an exoglycosidase, led to a marked dose-dependent inhibition of NK lytic activity against YAC-1.2 or K562 tumor cells. Maximal inhibition occurred after a 60-min pretreatment of murine effectors at 37 degrees C, and the kinetics of NK inhibition by alpha-mannosidase was similar to the reported kinetics for enzymatic activity. Released hexose was detected chemically in the supernatant of mouse spleen cells treated with NK inhibitory dose of alpha-mannosidase, and inactivation of enzymatic function with EDTA reversed the NK inhibitory effect. These results suggest that alpha-mannosidase inhibited NK function by virtue of its enzymatic action. Culture of human PBL for 20-hr after treatment with this enzyme led to a greater than 70% recovery in NK lytic function. Recovery was blocked by incorporating tunicamycin, a glycosylation inhibitor of asparagine-linked glycoproteins, into the culture medium. These results suggest that the alpha-mannosidase-sensitive site may be de novo synthesized glycoprotein. Neuraminidase, beta-galactosidase, endo-beta-N-acetylglucosaminidase-D and H, and peptide-N-glycosidase treatments did not inhibit human NK cell lysis of K562 cells. Pretreatment of nylon wool-enriched CBA/J spleen cells or Percoll-enriched human LGL with alpha-mannosidase did not influence their capacity to bind YAC 1.2 target cells or K562 target cells, respectively, Ca++ pulse experiments revealed that the alpha-mannosidase-sensitive site on the NK cells was involved after target-effector binding but before the Ca++ influx. Pretreatment of effector cells with this enzyme which normally occurs after effector-target cell interaction. These results suggest that the phospholipid methylation reaction is coupled to the alpha-mannosidase-sensitive site on the NK cells. By analogy to other physiologic systems, such as histamine release in mast cells, the triggering of phospholipid methylation in the NK cells may serve as a mechanism for signal transduction across the plasma membrane.     

Human epidermal cells and squamous carcinoma cells synthesize a cytokine that augments natural killer cell activity

Normal as well as transformed epidermal cells (EC) have recently been reported to produce a cytokine--EC-derived thymocyte-activating factor (ETAF), which according to its biologic as well as biochemical properties is indistinguishable from macrophage-derived interleukin 1 (IL 1). In the present study, the effect of supernatants (SN) derived from normal EC and a human squamous carcinoma cell (SCC) line were tested for their effects on natural killer (NK) cell activity. EC- as well as SCC-derived SN were able to augment in vitro NK cell activity of peripheral blood lymphocytes against K 562 cells. In contrast, adherent cell-derived, IL 1-containing SN did not affect NK cell activity. Upon high-pressure liquid chromatography (HPLC) gel filtration, ETAF and the EC-derived NK cell activity-augmenting factor (ENKAF) exhibited a similar m.w. However, by using reverse-phase HPLC, ETAF and ENKAF eluted as distinct peaks of activity, indicating that SCC cell-derived ENKAF is different from ETAF. Furthermore, ENKAF does not contain interleukin 2 (IL 2) or interferon (IFN) activity. The enhancement of NK cell activity was dose dependent and evident after 20 hr of preincubation of effector cells. Pretreatment of target cells with ENKAF did not affect the susceptibility of the target cells. The NK activity of large granular lymphocytes (LGL) purified by discontinuous Percoll gradient centrifugation and further depleted of high-affinity sheep erythrocyte rosetting cells was enhanced by ENKAF. In contrast, no NK cell activity was expressed by LGL-depleted T cell populations before or after treatment with ENKAF. In a single cell cytotoxicity assay in agarose, the number of lymphocyte binding to K 562 was not affected by ENKAF, but the frequency of dead conjugated target cells and presumably of active killer cells was increased by pretreatment with ENKAF. Additional incubation of LGL with ETAF did not further increase ENKAF-mediated augmentation of NK activity. In contrast to ETAF, ENKAF was not chemotactic for polymorphonuclear leukocytes. These results indicate that normal as well as transformed EC release a unique cytokine--ENKAF--which augments NK cell activity of LGL but is distinct from ETAF, IL 2, and IFN.     

Specific effect of anti-transferrin antibodies on natural killer cells directed against tumor cells. Evidence for the transferrin receptor being one of the target structures recognized by NK cells

Treatment of PBL or Percoll-isolated LGL with anti-transferrin antibodies plus complement reduced their natural killing activity against K-562 cells between 30 and 70%. The same antibodies inhibited natural cytotoxicity when added directly to the assay. Similar depletion or inhibition of NK cytotoxicity was observed when using HeLa cells as targets. The decrease or inhibition by transferrin antibodies was less marked when IFN-treated PBL or LGL as effector cells were used. The inhibition of anti-transferrin antibodies seems to be located at the level of the effector cell population. When PBL but not target K-562 cells were pretreated with anti-transferrin antibodies and were washed before use in the assay, cytotoxicity was decreased by 50%. In addition, about 80% of the LGL positively selected on anti-transferrin plates stained with Leu-11. Furthermore, no reduction by anti-transferrin antibodies plus complement treatment of PBL or LGL, or inhibition by antibodies alone, was observed when the cells were tested against HSV-1-infected cells. Membrane extracts from LGL inhibited NK cytotoxicity against K-562 or HeLa cells. Moreover, the inhibitory component of this extract was removed by anti-transferrin IgG but not by control IgG. These results are in agreement with the recent hypothesis that NK cells recognize the transferrin receptor in tumor target cells, because both the transferrin receptor and anti-transferrin antibodies may share a similar structure that interacts with the NK cells.     

Fractionation, morphological and functional characterization of effector cells responsible for human natural killer activity against cell-line targets

Human natural killer cells cytotoxic against cell-line target cells (NK-CLT) were isolated and characterized by utilizing adsorption-elution of the effector cells from the K-562 target cells. The cell associated with the cytotoxicity was a large lymphocyte with pale and characteristically granular cytoplasm. Thus, its morphology was identical with that of the large granular lymphocyte (LGL) previously shown to be the principal cytotoxic NK cell against fetal fibroblasts (NK-FF). The association of LGL with natural killer activity was verified with contact analysis from mixtures of unfractionated effector cells and target cells, which revealed that the number of contact of LGL with K-562 was correlated to the level of the individually expressed intensity of natural cytotoxicity. The ANAE-staining distribution of LGL was intensively positive with granular or diffuse staining pattern. In direct surface marker analysis LGL were E-rosette forming but, in contrast to NK-FF, heterogenous in regard to the Fc receptors. During in vitro incubation after elution from the target cells, the cytotoxic activity of LGL increased several fold. Also, the presence of K-562 among unfractionated effector cells caused an augmentation of cytotoxicity. This phenomenon was not observed as a result of effector cell-fetal fibroblast coculturing. Evidence from fetal fibroblast adsorption-elution and aggregated IgG blocking experiments suggested that the LGL with strong expression of Fc receptors were initially cytotoxic “mature” NK-cells, whereas the LGL with a weak expression of Fc receptors were initially noncytotoxic, but contact with K-562 “augmented” or “recruited” them to nonselective cytotoxicity.     

Natural cytotoxicity of lymphocytes from lymph nodes draining breast carcinoma and its augmentation by interferon and OK432

Summary Lymphocytes isolated from axillary lymph nodes draining breast carcinoma were tested for natural killer (NK) activity against K562 in a 4-h ⁵¹Cr-release assay, and the in vitro effects of interferon (IFN) and OK432 (a streptococcal preparation) on their cytotoxicity were examined in comparison with NK activity of autologous peripheral blood lymphocytes (PBL). The levels of NK activity were lower in lymph node lymphocytes (LNL) than in PBL of the same patients. Significant levels of LNL-mediated lysis were recorded in 14 of 42 (33%) lymph node samples and in nine of 14 (64%) patients. Purification of large granular lymphocytes (LGL) from lymph node cells by discontinuous Percoll density gradient centrifugation resulted in an induction or enhancement of cytotoxic activity, with no reactivity in LGL-depleted, small T-lymphocyte populations. Positive reactions were observed with 10 of 13 (77%) LGL samples. The low reactivity of LNL was not attributable to coexistent suppressor cells for NK function, since lymph node cells failed to suppress NK activity of normal PBL. Partially purified human IFN and OK432 augmented NK activity of patients' PBL in approximately 70% and 90% of the cases, respectively, while LNL-mediated lysis was augmented in only 7% and 36% of the lymph node samples by IFN and OK432, respectively. These results indicate that K562-reactive NK cells and/or their precursors may frequently be present at subthreshold levels in the lymph nodes draining breast carcinoma, and that the augmentation of LNL-mediated cytotoxicity by OK432 might provide a local potentiation of natural immune function at the host-tumor interface rather than IFN.     

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.     

Modulation of human natural killer cell cytotoxic activity, lymphokine production, and interleukin 2 receptor expression by thymic hormones

Thymic hormone preparations have been shown to modulate natural killer (NK) activity in vivo in mice. We have investigated the effects of thymosin fraction 5 (TF5) on the in vitro NK cell activity of highly purified human large granular lymphocytes (LGL). The results indicate that TF5 but not kidney fraction 5 (a preparation used as control) is able to enhance the spontaneous NK activity of normal LGL. In addition, TF5 exhibited additive effects with recombinant interferon-alpha in enhancing NK activity in vitro. TF5 also enhanced interleukin 2 production and interleukin 2 receptor expression as well as interferon-gamma production in mitogen-stimulated LGL. Thymosin-alpha 1, a synthetic polypeptide originally isolated in its native form from TF5, also exhibited enhancing effects on LGL activities, suggesting that it is the active species in TF5. These results indicate that thymic hormones might regulate NK activity through the induction of lymphokine production and receptor expression by LGL.     

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.