NK-leukocyte chemotactic factor (NK-LCF): a large granular lymphocyte (LGL) granule-associated chemotactic factor

A chemotactic factor was identified in the supernatants of human large granular lymphocytes (LGL) activated by a glutaraldehyde-fixed NK-sensitive tumor, K562. The factor stimulated migration of human LGL, rat alveolar macrophage (RAM), and human monocytes and neutrophils (PMN). The locomotor response was chemotactic and chemokinetic on the basis of unidirectional migration in concentration gradients. The cell producing the factor was detected exclusively in LGL-rich Percoll fraction coincident with the peak of NK lytic activity and HNK-1+ cells. The monoclonal phenotype of the cell was HNK-1+, partially OKT-11+, OKM-1-, OKT-3-, OKT-4-, and OKT-8-. The factor was released by LGL within 20 min of incubation with Sr++, a cation that is able to induce LGL degranulation. A powerful chemoattractant was also detected in the granules of the rat LGL leukemia, RNK. Chemotactic activity coincided with granule enzyme beta-glucuronidase and cytolysin after RNK nitrogen cavitation and Percoll fractionation of subcellular constituents. The RNK granule chemoattractant induced unidirectional migration of human LGL and was also active against rat alveolar macrophages and human PMN. Anti-RNK granule antibody conjugated to Sepharose 4B was able to deplete the chemotactic activity from both K562-induced LGL supernatants and solubilized RNK granules. These observations indicate that a leukocyte chemotactic factor (NK-LCF) is present in NK cell granules and is probably released after tumor-induced granule exocytosis.     

Lysis of fresh human tumour cells by autologous tumour-associated lymphocytes: Two distinct types of autologous tumour killer cells induced by co-culture with autologous tumour

Summary The specific and natural killer (NK)-restricted nature of auto-tumour cytotoxicity of tumour-associated lymphocytes was studied in cancer patients with malignant pleural effusions. Large granular lymphocytes (LGL) and small T lymphocytes were isolated from carcinomatous pleural effusions by centrifugation on discontinuous Percoll gradients. Tumour cells freshly isolated from pleural effusions were classified according to their susceptibility to lysis by Percoll-purified LGL from the blood of normal donors in a 4-h ⁵¹Cr release assay. Of 12 NK-sensitive tumour samples, 11 were killed by autologous fresh effusion LGL, whereas only 2 were lysed by autologous T cells. Neither LGL nor T cells were cytotoxic to NK-resistant autologous tumour cells. T cells and LGL were each cultured in vitro with autologous tumour cells for 6 days. Effusion LGL maintained their auto-tumour killing activity in 10 of 12 autologous mixed lymphocyte-tumour cultures (MLTC) with NK-sensitive tumour, while LGL lost the activity when cultured alone. Removal of high-affinity sheep erythrocyte-rosetting cells from Percoll-purified LGL enriched effector cells. Autologous MLTC-derived LGL could also kill NK-sensitive allogeneic effusion tumour cells and K562 cells, as did fresh LGL. In autologous MLTC LGL failed to acquire lytic function to NK-resistant autologous tumour cells. In contrast, in vitro activation of effusion T cells with autologous tumour cells induced auto-tumour killer cells in 9 of 12 NK-sensitive tumour samples and 3 of 6 NK-resistant tumour cases. However, cultured T cells were incapable of killing allogeneic tumour cells and K562 cells. In the autologous MLTC effusion T cells proliferated vigorously in response to autologous tumour cells, whereas LGL showed no proliferation. The enrichment of blasts from cultured T cells on discontinuous Percoll gradients resulted in an enhancement of auto-tumour cytotoxicity, with no reactions recorded in blast-depleted, small, resting T cells. These results indicate that two distinct types of auto-tumour-recognising lymphocytes, LGL and T cells, are present in carcinomatous pleural effusions of cancer patients and that each effector type recognises different membrane moieties of autologous effusion tumour cells.     

Granules of human CD3+ large granular lymphocytes contain a macrophage regulating factor(s) that induces macrophage H2O2 production and tumoricidal activity but decreases cell surface Ia antigen expression.

CTL (cytotoxic T lymphocytes) and LGL (large granular lymphocytes) exocytose cytoplasmic granules on activation after recognition of their target, releasing granule-associated molecules. We have previously suggested that this process could release immunoregulatory molecules. In this study we investigated whether normal human LGL granules contained a factor regulating different macrophage activity. Human CD3+ LGL cells were generated by activating peripheral blood lymphocytes (PBL) for 10-12 days with recombinant human IL-2 (rhIL-2), and granules were isolated from disrupted cell homogenate by Percoll gradient fractionation. Solubilized granules were tested for macrophage-activating factor (MAF) activity in three different macrophage assays. When M-CSF-differentiated murine bone marrow-derived macrophages were incubated 9 hr with human LGL granules, they were fully activated to lyse the TNF-resistant P815 tumor cells. The granule-MAF showed a synergistic effect with rhIL-1 beta, rmTNF-alpha, and rmIFN-tau in the cytolytic assay. In addition, proteose-peptone-elicited murine peritoneal macrophages profoundly increased H2O2 production after activation with human LGL granules. However, unlike IFN-tau, no increase in peritoneal macrophage Ia antigen expression was detected after incubation with granules. Moreover, granule-MAF suppressed Ia induction by IFN-tau. These results confirm that human CD3+ LGL granules contain a molecule(s) capable of regulating macrophage function.     

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.     

Protective effects of differentiation inducers on natural killer sensitivity of K 562 cells: Analysis at a single-cell level

K 562 cells induced to differentiate by sodium butyrate (SB) or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were studied for their capacities to be bound and killed by large granular lymphocytes (LGL) in a single-cell cytotoxicity assay in agarose. After SB treatment, K 562 cells were less efficient in binding to LGL, whereas the frequency of killer cells among bound LGL was unaffected. When TPA was used to induce K 562 differentiation, the binding of LGL to their target and the lytic efficiency of the bound LGL were both diminished when compared to control K 562 cells. It has been demonstrated that the expression of structures involved in the binding of natural killer (NK) effectors to their targets could be correlated with the target-differentiation stage. It is shown that phorbol-ester treatment can also affect NK target structures involved in the killing step.     

Activation of rat and human alveolar macrophage intracellular microbicidal activity by a preformed LGL cytokine

An alveolar macrophage-activating factor was released from Percoll fractionated large granular lymphocytes (LGL) within minutes of contact with either the natural killer (NK)-sensitive K562 tumor or heat-killed Staphylococcus aureus. The factor enhanced the intracellular killing of S. aureus without altering the rate of phagocytosis. Factor release was blocked by treatment of LGL with monensin, a carboxylic ionophore that inhibits vesicular traffic, but was unaffected by actinomycin D and cycloheximide pretreatment, suggesting that the cytokine was performed. The cell producing the factor was found only in Percoll fractions containing high concentrations of lytic NK cells and LGL, and the phenotypes of the LGL were HNK-1+ and E rosette-. The macrophage activating factor was a small protein of 10,000 to 20,000 daltons, as determined by gel fractionation, and was sensitive to proteolytic enzymes and heat and pH labile. Active supernatants were devoid of antiviral (interferon; IFN) or interleukin 2 (IL 2) activity, and IFN-beta, IFN-gamma, IL 2, and interleukin 1 were unable to activate staphylococcidal activity, suggesting that the LGL macrophage activating factor was distinguishable from these cytokines.     

Bacterial activation of human natural killer cells. Characteristics of the activation process and identification of the effector cell

We showed previously that contact of human peripheral blood lymphocytes with glutaraldehyde-fixed Salmonella bacteria augmented their cytotoxic capacity against NK-sensitive targets. We have now analyzed the characteristics of the activation and also identified the subsets of lymphocytes responding to bacterial contact. Blocking of protein synthesis with cyclohexamide totally abrogated bacterial induction of activated killing (AK), whereas inhibition of DNA synthesis with mitomycin C did not significantly affect the capacity of lymphocytes to respond to bacterial contact. Both the induction and the effector phase of AK were radioresistant. The AK cells exhibited efficient lytic activity, comparable to that induced by recombinant IL 2 (rIL 2), against NK-resistant targets (including both hematopoietic and solid tumor cell lines). All inducible cytotoxic activity was contained within the subset of lymphocytes expressing Leu-19 (NKH-1) antigen. Leu-19- lymphocytes exhibited no significant NK activity and could not be further stimulated by bacterial contact, rIL 2, or IFN-alpha. Within the Leu-19+ lymphocyte subset, two distinct cell types were present; CD3-, Leu-19+ NK cells and CD3+. Leu-19+ T cells. The CD3+, Leu-19+, T cells mediated low levels of non-MHC-restricted cytotoxicity against K562, but did not respond to bacterial contact, even though rIL 2 could augment their lytic activity slightly. However, the cytotoxic activity of CD3-, Leu-19+ NK cells was significantly augmented by bacterial contact. Within the CD3-, Leu-19+ NK cell population both CD16+ and CD16- cells responded to bacterial activation. The CD3-, CD16-, Leu-19+ cells constituted 1 to 4% of the Percoll-fractionated low buoyant density lymphocytes and accounted for the activation seen within the CD16- lymphocyte population. Thus bacterial stimulation of NK activity seems to be mediated for the most part via CD16+, Leu-19+ cells, and a minor overall contribution is mediated via CD3-, CD16-, Leu-19+ cells. No apparent involvement of T cells was seen in the lytic response of lymphocytes to bacterial contact.     

Large granular lymphocytes are central cells in the interleukin-2-dependent differentiation pathway of natural killer cells

Peripheral blood low-density cells were sorted, with respect to their ability to accumulate the lysosomotropic agent mepacrine (Mep), into lysosome-rich (Mep+) and lysosome-poor (Mep-) cell populations. Cells of large granular lymphocyte (LGL) morphology and phenotype were found in the Mep+ but not in the Mep- cell population. The latter cells lacked any natural killer (NK) activity. Cultures of the Mep- cells resulted in the appearance of cells showing K-562 lytic activity, LGL morphology and CD16 and/or Leu-7 positivity. This process was facilitated by the supplementation of the culture with recombinant human interleukin-2 (rIL-2). Mep+ cells retested after 7 days of culture showed a decline in the fraction of granular (LGL and Mep+) cells. This decrease was less pronounced but also seen in rIL-2-supplemented cultures. In spite of the lower number of typical LGL, Mep+ cells cultured with rIL-2 were mostly large but scarcely granular; rIL-2-activated K-562 killing (rIL-2 AK) of originally Mep+ cells was much higher than K-562 lytic activity of these cells at the beginning of the culture, and as compared to rIL-2 AK of Mep- cells. From this finding it is apparent that the most active rIL-2 AK cells originate from low-density granular (Mep+) cells (LGL) and, therefore, we propose to call them 'giant' NK cells. Furthermore, in the presence of rIL-2, LGL differentiate from agranular (Mep-) low-density cells. In view of these data, LGL appear to be resting cells on the differentiation pathway of NK cells.     

Target cell directed NK inactivation. Concomitant loss of NK and antibody-dependent cellular cytotoxicity activities

We investigated the inactivation of human NK cells, a population of large granular lymphocytes (LGL), with K562, an NK-sensitive target cell (TC) and KLCL, an NK-resistant TC, but which can be lysed by NK cells via antibody (Ab)-dependent cellular cytotoxicity. NK-enriched effector cells (ECc) were first treated with either K562 or Ab-coated KLCL (Ab-KLCL). After incubation, ECc were separated from their TC then examined for residual NK and ADCC activities, phenotypic changes, and changes in LGL morphology. K562-treated ECc and Ab-KLCL-treated ECc, when retested against the inactivating TC, respectively, lost greater than 90% of their lytic activities. However, K562-treated ECc lost 60 to 70% of their activity against Ab-KLCL, whereas Ab-KLCL-treated ECc lost less than 10% of their activity against K562. In contrast to what we observed with K562-treated ECc, we detected significant reductions in plasma membrane expression of Leu-11a and Leu-11b on Ab-KLCL-treated ECc. Although the proportion of OKM1+ cells remained unchanged after the inactivation process, the density of OKM1 on both K562-treated ECc and Ab-KLCL-treated ECc increased significantly. Morphologic analysis revealed no apparent differences in the percentages of LGL before and after treatment with K562 or Ab-KLCL. Finally, IL-2 restored lytic potential to both K562-treated ECc and Ab-KLCL-treated ECc and, in addition, IL-2-induced enhancement of Ab-KLCL-treated ECc was accompanied by a partial reexpression of Leu-11a. These data support the hypothesis that NK-cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity may result from a common lytic mechanism, although the initiation steps and regulation of the pathway are distinct.     

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.     

Lipids are a constitutive component of cytolytic granules

Cytolytic granules are specific organelles of activated cytotoxic lymphocytes mediating storage and regulated excretion of lytic molecules for killing of target cells. A variety of the other granule components may also participate in granule-mediated cytotoxicity. In this study, the subcellular localization of lipids in the granules of human decidual CD56+ natural killer-like cells was determined by staining with malachite green aldehyde and imidazole-buffered osmium tetroxide. Lipids were shown, for the first time, to be a constitutive component of cytolytic granules. Lipids formed an additional structural microdomain, located between the granule-limiting membrane and the granule core. Images of the granules on serial sections suggested that intragranular lipids wrap the core. We speculate that granule lipids participate in packing of lytic molecules inside the granules, in autocrine signaling ending granule secretion, and in the killing process.     

Cytolytic activity of human natural killer cell subpopulations isolated by four-color immunofluorescence flow cytometric cell sorting

The natural killer activity and phenotypic properties of six different subpopulations of normal human peripheral blood lymphocytes obtained by four-color immunofluorescence cell sorting were examined. Phycoerythrin-conjugated CD16 and CD56 were used simultaneously to identify (CD16 + CD56+) NK cells. The cells most effective in mediating NK cytolysis against K562 target cells were CD3-(16 + 56+)57 -8+. Although most of the K562 killing was found in the CD3-(16 + 56 +) groups of cells, a substantial degree of NK activity was detected in the CD3+(16 + 56 +) subpopulations of some individuals. The level of expression of CD57 and CD8 was significantly higher on CD3+(16 + 56 +) than on CD3-(16 + 56 +) cells.     

Interferon-alpha induction of lymphocytes containing parallel tubular structures

The induction by IFN-alpha in peripheral blood lymphocytes of parallel tubular structures (PTS) and/or electron-dense granules occurring in a minority of peripheral blood lymphocytes was examined. IFN reportedly augments natural killer (NK) cell activity of large granular lymphocytes (LGL); these cells contain PTS and/or electron-dense granules. Normal peripheral blood mononuclear cells were incubated with IFN-alpha and surface antigen expression was measured by means of indirect immunofluorescence and, at the ultrastructural level, using gold labelled monoclonal antibodies. Surface antigen reactivity with the monoclonal antibodies OKT 3, 4, 8 and Anti-Leu-7 (HNK-1) showed no difference between the IFN-alpha incubation and non-IFN-alpha groups. However, electron microscope investigation revealed significant absolute increases in the percentage of OKT 8+ and Anti-Leu-7+ cells which were PTS-positive after IFN-alpha treatment compared with the control groups. The cytotoxicity assay using the K562 cell line showed enhanced lytic activity. Our results suggest that cells coexpressing the OKT 8 and Leu-7 antigens may be responsible for a minor proportion of the increase in PTS but that IFN-alpha mainly induces PTS and/or associated structures in cells which express the OKT 8+ antigen. These PTS+/OKT 8+ cells may contribute to enhanced cell cytotoxicity.     

A C9 related channel forming protein in the cytoplasmic granules of human large granular lymphocytes

Large granular lymphocytes (LGL) from human blood main-tained in culture for 2 to 6 weeks with IL-2 were found positive in the K562 cell killing assay. The cytoplasmic granules of the LGL were isolated, lysed and the soluble proteins were passed over a Sepharose-anti-C9 column. The retained protein was eluted with NaC1 and found to consist by SDS polyacrylamide gel electrophoresis of essentially one component with a molecular weight of approximately 70 000. The protein did not give a positive precipitation test with anti-human C9 by Ouchterlony analysis, but it reacted reproducibly with anti-human C9 by Western blot analysis. By ELISA the cross reaction with human C9 was less than 1%. The C9 related lymphocyte protein lacked C9 hemolytic activity, but it formed functional pores in liposomes in presence of Ca⁺⁺ . These results suggest that the cytoplasmic granules of human LGL that are capable of killing NK target cells contain C9 related protein which is involved in the cellular cytotoxicity reaction.     

Cytotoxic activity against trophoblast and choriocarcinoma cells of large granular lymphocytes from human early pregnancy decidua

Large granular lymphocytes (LGL) are the most abundant cell type in first trimester human pregnancy decidua. We have shown previously that CD56-positive decidual LGL have cytotoxic activity against the natural killer (NK) target K562, and that this cytotoxicity is augmented by pretreatment with interleukin-2 (IL-2). We now report that flow cytometrically purified populations of CD56-positive decidual LGL have no cytotoxic activity against either the BeWo choriocarcinoma cell line or freshly isolated term trophoblast. Incubation of unfractionated decidual cells with IL-2 induced cytotoxicity against BeWo, but term trophoblast remained resistant to lysis. Both BeWo and trophoblast showed much lower binding frequencies to decidual or peripheral blood cells than K56 targets, and excess trophoblast did not inhibit cytotoxic activity against K562. This suggests that the resistance of trophoblast to lysis by either decidual or peripheral blood LGL is due to the lack of accessible NK target structures on the surface of trophoblast.     

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.     

Interleukin-2 receptor monoclonal antibodies have no effect on anti-CD3 monoclonal antibody-mediated cytotoxicity in CD3+ leukemic large granular lymphocytes

We studied the role of Fc receptors and interleukin-2 (IL-2) receptor subunits in anti-CD3 monoclonal antibody (MAb)-mediated cytotoxicity of CD3+ leukemic large granular lymphocytes (LGL). Peripheral blood mononuclear cells from four patients with CD3+ LGL leukemia were cultured with 1 microgram/ml of anti-CD3 MAb. Anti-CD3 MAb-mediated cytotoxicity was not inhibited when K562 target cells were preincubated with heat-aggregated human IgG, suggesting that binding of the effector cell-bound anti-CD3 MAb to Fc receptors of target was not involved in cytotoxicity. Induction of cytotoxicity was not blocked by the addition of either anti-p55 or anti-p75 IL-2 receptor MAbs. These results show that the induction of cytotoxicity by anti-CD3 MAb is not mediated through IL-2 receptor subunits in CD3+ leukemic LGL.     

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.     

Human trophoblast and JEG choriocarcinoma cells are sensitive to lysis by IL-2-stimulated decidual NK cells.

Freshly isolated decidual large granular lymphocytes (LGL) show natural killer (NK) activity against K562 cells but not against normal human trophoblast. We now show that these decidual LGL proliferate in vitro in response to recombinant interleukin-2 (rIL-2) and that these rIL-2-stimulated cells acquire a broad cytolytic potential that is characteristic of lymphokine-activated killer (LAK) cells. Both fetal fibroblasts and JEG-3 choriocarcinoma cells are resistant to lysis by freshly isolated decidual effectors but are readily killed by IL-2-stimulated decidual LGL. The ability to kill these target cells is acquired after only 18 hr exposure to rIL-2. rIL-2-activated decidual LGL also kill cultured normal trophoblast cells but much lower levels of cytolysis were seen even after the effectors had been stimulated with rIL-2 for 4-6 days. The preferential killing of malignant over normal human trophoblast cells raises questions about the potential role of IL-2-activated decidual LGL in the control of unduly invasive or malignant trophoblast populations in vivo.     

Intrapleural administration of OK432 in cancer patients: Augmentation of autologous tumor killing activity of tumor-associated large granular lymphocytes

Summary Ten patients with carcinomatous pleural effusions were treated with single intrapleural (i.pl.) injections of the streptococcal preparation OK432 on day 0 and the effects of i.pl. OK432 on the lysis of fresh or cryopreserved autologous tumor cells isolated from the pleural effusions were observed on day 7. In eight patients tumor cells in the effusions had decreased or disappeared by day 7. The other two patients, however, had no clinical evidence of therapeutic benefit from i.pl. OK432. Effusion tumor cells were relatively resistant to lysis by autologous lymphocytes when tested in a 4-h ⁵¹Cr-release assay. Positive reactions were recorded for blood and effusion lymphocytes in two of ten untreated patients. Injection of OK432 i.pl. resulted in an induction or augmentation of cytotoxicity against autologous tumor cells and K562 in the effusions of seven of ten subjects by day 7. In contrast, autologous tumor killing activity of blood lymphocytes was not always modified by i.pl. OK432. Purification of large granular lymphocytes (LGL) by discontinuous Percoll gradient centrifugation enriched autologous tumor killing activity, with no reactivity in LGL-depleted, small T lymphocytes. Significant lysis of autologous tumor cells was observed with effusion LGL from seven of ten untreated patients. Seven days after i.pl. OK432 injection, effusion LGL expressed enhanced cytotoxicity against autologous effusion tumor cells, whereas T cells were still not cytotoxic to autologous tumor cells on day 7. The frequency of LGL among effusion lymphocytes was not altered by i. pl. OK432. Adherent effusion cells were not involved in lysis of autologous effusion tumor cells in either untreated or OK432-treated patients. In vitro treatment of blood and effusion lymphocytes with OK432 induced an enhancement of autologous tumor-killing activity in patients who subsequently responded to i.pl. OK432 treatment. OK432 augmented in vitro autologous tumor killing activity of LGL, whereas T cells failed to lyse autologous tumor cells even after in vitro activation with OK432. These results indicate that i.pl. administration of OK432 to cancer patients will result in an augmentation of autologous tumor killing activity of LGL in the pleural effusions, and that this could be responsible for the antitumor activity of i.pl. OK432 therapy.