Effectivein vivo induction of lymphokine-activated killer (LAK) cells by pretreatment with a streptococcal preparation,OK-432

Effects of a streptococcal preparation, OK-432, on precursors of lymphokine-activated killer (LAK) cells were observedin vivo. Total number of splenocytes and the ratio of asGM ₁ ⁺ cells increased gradually after i.v. administration of OK-432, reaching their peaks at 3 to 4 days. It was found that as GM ₁ ⁺ cells were nonadherent and large in size. There were little differences in the ratios of Thy-1⁺, Lyt-2⁺, and L3T4⁺ cells before and after OK-432 treatment. Mice were injected i.p. with recombinant interleukin 2 (rIL-2) at a dose of 5 × 10⁴ U per mouse 4 days after OK-432 administration and LAK activity in their splenocytes was examined using natural killer (NK) resistant EL-4 target cells. Splenocytes in mice treated with both OK-432 and rIL-2 showed higher LAK activity than those in mice treated with rIL-2 alone.In vivo treatment with anti asGM, antibody prior to rIL-2 injection abolished completely such augmentation of LAK activity in OK-432 treated mice. These results demonstrated that asGM ₁ ⁺ LAK precursor cells induced by OK-432 were effectively differentiated into LAK cells by rIL-2.     

Growth of MCF-7 human breast carcinoma in severe combined immunodeficient mice: Growth suppression by recombinant interleukin-2 treatment and role of lymphokine-activated killer cells

Summary The severe combined immunodeficient (SCID) mouse, lacking functional T and B lymphocytes, has been considered by many groups to be a prime candidate for the reconstitution of a human immune system in a laboratory animal. In addition, this immuno-deficient animal would appear to have excellent potential as a host for transplanted human cancers, thus providing an exceptional opportunity for the study of interactions between the human immune system and human cancer in a laboratory animal. However, because this animal model is very recent, few studies have been reported documenting the capability of these mice to accept human cancers, and whether or not the residual immune cells in these mice (e.g. natural killer, NK, cells; macrophages) possess antitumor activities toward human cancers. Thus, the purpose of this study was (a) to determine whether or not a human breast carcinoma cell line (MCF-7) can be successfully transplanted to SCID mice, (b) to determine whether or not chronic treatment of SCID mice with a potent lymphokine (recombinant interleukin-2, rIL-2) could alter MCF-7 carcinoma growth, and (c) to assess whether or not rIL-2-activated NK cells (LAK cells) are important modulators of growth of MCF-7 cells in SCID mice. To fulfill these objectives, female SCID mice were implanted s.c. with MCF-7 cells (5 × 10⁶ cells/mouse) at 6 weeks of age. Six weeks later, some of the mice were injected i.p. twice weekly with rIL-2 (1 × 10⁴ U mouse^–1 injection^–1). Results clearly show that MCF-7 cells can grow progressively in SCID mice; 100% of the SCID mice implanted with MCF-7 cells developed palpable measurable tumors within 5–6 weeks after tumor cell inoculation. In addition, MCF-7 tumor growth was significantly (P <0.01) suppressed by rIL-2 treatment. rIL-2 treatment was non-toxic and no effect of treatment on body weight gains was observed. For non-tumor-bearing SCID mice, splenocytes treated in vitro with rIL-2 (lymphokine-activated killer, LAK, cells) or splenocytes derived from rIL-2-treated SCID mice (LAK cells) had significant (P <0.01) cytolytic activity toward MCF-7 carcinoma cells in vitro. In contrast, splenocytes (LAK cells) derived from tumor(MCF-7)-bearing rIL-2-treated SCID mice lacked cytolytic activities toward MCF-7 cells in vitro. No significant concentration of LAK cells in MCF-7 human breast carcinomas was observed nor did rIL-2 treatment significantly alter growth of MCF-7 cells in vitro. Thus, while rIL-2 treatment significantly suppressed growth of MCF-7 breast carcinomas in SCID mice, the mechanism of this growth suppression, albeit clearly not involving T and B lymphocytes, does not appear to be mediated via a direct cytolytic activity of LAK cells toward the carcinoma cells. However, rIL-2-activated SCID mouse splenocytes (LAK cells) do possess the capability of significant cytolytic activity toward MCF-7 human breast carcinoma cells. Thus, treatment of SCID mice with a potent lymphokine (rIL-2) induces a significant antitumor host response, a response that does not involve T and B lymphocytes and appears not to involve NK/LAK cells. This host response must be considered in future studies designed to investigate the interactions of reconstituted human immune systems and human cancers within this highly promising immuno deficient experimental animal model.     

In vivo administration of low-dose human interleukin-2 induces lymphokine-activated killer cells for enhanced cytolysis in vitro

We have examined the effect of the intradermal administration of IL-2 on the generation of natural killer (NK) cell and lymphokine-activated killer (LAK) cell activity. Peripheral blood mononuclear cells (PBMC) obtained from borderline lepromatous (BL) and lepromatous leprosy (LL) patients and normal volunteers prior to and after IL-2 injection were stimulated in vitro with IL-2 and their cytolytic activities compared against 51Cr labeled target K562 cells, Daudi cells, and monocytes. Before IL-2 administration, PBMC obtained from BL/LL patients and normal volunteers possessed similar levels of NK cell activity indicating that the NK cell activity of the BL/LL patients was intact. LAK cell activity was induced with IL-2 in vitro in both BL/LL patients and in normal volunteers. The level of LAK cell activity in BL/LL patients was, however, suboptimal. A single intradermal dose of 25 micrograms IL-2 had no effect on the phenotype of circulating mononuclear cells in either patients or normal volunteers. However, 6-12 days after IL-2 injection and subsequent restimulation of the PBMC with IL-2 in vitro, cytolytic activity of LAK cells obtained from the BL/LL patients was enhanced while cells from normal volunteers expressed the same high levels of activity as observed before IL-2 injection.     

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.     

Differential sensitivity of cytotoxic T lymphocytes and lymphokine-activated killer cells to inhibition by L-ornithine

The selective inhibition of murine cytotoxic T lymphocyte (CTL) differentiation in C57B1/6 (B6) anti-DBA/2 mixed leukocyte cultures (MLC) by the amino acid L-ornithine (Orn) could not be reversed by addition of up to 1000 U/ml IL-2. Analysis of the effects of Orn on induction of lymphokine-activated killer (LAK cells), using dosages of IL-2 from 10-1000 U/ml and measuring cytolytic activity against two tumor targets (P815 and YAC-1) over the course of 5 days, indicated that LAK cells were not suppressed by Orn. LAK precursors and effector cells were CD8- and ASGM1+, indicating that they were derived from natural killer (NK) cells. We also found that the growth and maintenance of cloned CTL lines were not sensitive to inhibition by Orn; nor was their acquisition of nonspecific cytolytic activity in the presence of high lymphokine concentrations. Thus, induction of naive CTL shows differential susceptibility to Orn inhibition relative to LAK and LAK-like activities by NK and cloned CTL lines in response to IL-2.     

Mechanism(s) of in vitro macrophage activation with Nocardia rubra cell wall skeleton: the effects on macrophage activating factor production by lymphocytes

Summary BALB/c mouse peritoneal macrophages prepared from WPC which had been treated with N. CWS demonstrated potent cytostatic activity against syngeneic Meth A fibrosarcoma cells. The maximum cytostatic activity developed in the macrophages when WPC were incubated with 25 g/ml N. CWS for 3 days. NAPC from BALB/c mice given an i. p. injection with 100 g N. CWS 7 days previously (N. CWS-NAPC) or supernatants from N. CWS-NAPC also activated peritoneal macrophages in vitro. However, when peritoneal macrophages were incubated with N. CWS in the absence of NAPC, or when T cells were depleted from WPC by treatment with anti-Thy 1.2 antibody and complement, N. CWS failed to enhance the cytostatic activity of the macrophages. Furthermore, thioglycollate-elicited peritoneal macrophages from C3H/HeN mice increased their cytolytic properties by incubation with supernatant fluids from N. CWS-treated spleen cells. These findings suggest that in vitro macrophage activation with N. CWS depends on MAF secreted from T lymphocytes. Abbreviations used: N. CWS, Nocardia rubra cell-wall skeleton; BRM, biological response modifier; MAF, macrophage activating factor; IL-1, interleukin 1; IL-2, interleukin 2; IFN-, interferon gamma; PCCM, peritoneal cell culture medium; SCCM, spleen cell culture medium; TCM, tumor culture medium; HI-FCS, heat-inactivated fetal calf serum; Con A, concanavalin A; PC, peritoneal cells; PEC, peritoneal exudate cells; WPC, whole peritoneal cells; APC, adherent peritoneal cells; TGC-APC, thioglycollateelicited adherent peritoneal cells; NAPC, nonadherent peritoneal cells; SN, supernatants; NK cells, natural killer cells; LAK cells, lymphokine activated killer cells E:T ratio, effector: target cell ratio; WSA, water soluble adjuvant; LPS, lipopolysaccharide; MDP, N-acetyl-muramyl-L-alanyl-D-isoglutamine     

Generation of lymphokine-activated killer cell activity from human thymocytes

We have generated lymphokine-activated killer (LAK) cells from human thymocytes in order to assess the relationship between LAK cells and T cells. Fresh thymocytes lack natural cytotoxic activity, and cytotoxicity cannot be stimulated by short term (1 hr) incubation with interferon or recombinant interleukin 2 (rIL-2). In addition, thymocytes are phenotypically devoid of cells bearing the natural killer (NK)-associated markers cluster designation (CD) 16 and NKH-1. After culture for 5 to 8 days with rIL-2, thymocytes display high levels of cytotoxic activity against both NK-sensitive and NK-resistant targets. Thymocytes require slightly more IL-2 than do peripheral blood lymphocytes to generate LAK activity. We have examined the phenotype of the thymocyte LAK precursor and effector cells. Thymocyte LAK precursors are of low to medium density, CD1-negative, and predominantly CD3-negative. Although CD3-positive cells proliferate in response to rIL-2, they are low in cytolytic capabilities. The effector cells, like the LAK precursors, are low to medium density lymphocytes. The cytotoxic cells are predominantly CD3-negative, and cytotoxic activity cannot be blocked with the use of anti-CD3 monoclonal antibodies. The effector cells also lack most NK-associated markers (HNK-1, and the CD16 markers Leu-11b and B73.1) but possess the NK-associated marker NKH-1 (N901). The responsive cell appears to be at a very early stage of thymic development, and it does not appear to either require or express the CD3-T cell receptor complex.     

Heterogeneous lymphokine-activated killer cell precursor populations

Summary We developed a monoclonal antibody (mAb) 211, which recognizes the precursors in peripheral blood of lymphokine-activated killer cells (LAK) induced by recombinant interleukin-2 (rIL-2). In conjunction with complement mAb 211 also eliminates natural killer cells (NK) and a majority of the cytotoxic T lymphocytes. B cells and monocytes do not express the 211 antigen. Since mAb 211 recognized such a large percentage of peripheral blood lymphocytes we examined which 211⁺ subpopulation was the predominant precursor of rIL-2-induced LAK cells using two-color fluoresence-activated cell sorting (fluorescein-conjugated 211 mAb plus phycoerythrin-CD11b). This method identified the 211⁺/ CD11b⁺ population as the predominant phenotype of the rIL-2-induced LAK precursor. In addition, we directly compared the phenotype of the LAK precursor induced by delectinated T-cell growth factor (TCGF) to that induced by rIL-2. The 211-depleted population, which was devoid of NK cells and LAK precursors (inducible by rIL-2), was capable of generating LAK activity when TCGF was used as the source of lymphokine. LAK cells induced by TCGF from the 211-depleted population lysed a fresh sarcoma and an NK-resistant cultured melanoma tumor target but not the Daudi cell line, which was lysed by rIL-2-induced LAK cells. Lymphoid subpopulations, depleted using NKH1a mAb, behaved similarly, generating high levels of lysis against the two solid tumor targets when cultured with TCGF but not with rIL-2. CD 3-depleted populations showed enrichment for LAK precursors using either rIL-2 or TCGF. These results indicate that while rIL-2-induced LAK precursors cannot be separated from cells with NK activity, TCGF-induced LAK cells can be generated from populations of peripheral blood mononuclear cells without NK activity.     

Human lymphokine-activated killer (LAK) cells: III. Effect ofl-phenylalanine methyl ester on LAK cell activation from human peripheral blood mononuclear cells: Possible protease involvement of monocytes,natural killer cells and LAK cells

Summary We have shown that depletion of monocytes from human peripheral blood mononuclear cells (PBMC) byl-phenylalanine methyl ester (PheOMe) enhanced lymphokine-activated killer cell (LAK) generation by recombinant interleukin-2 (rIL-2) at high cell density. In this study, we have investigated the mechanism of action of PheOMe on LAK activation by using trypsin, chymotrypsin, tosylphenylalaninechloromethanol (TPCK, a chymotrypsin inhibitor), tosyl-l-lysinechloromethane (TLCK, a trypsin inhibitor), phenylalaninol (PheOH), and benzamidine. PBMC were treated with 1–5 mM PheOMe for 40 min at room temperature in combination with the various agents, washed and assessed for their effects on natural killer (NK) activity against K562 cells and monocyte depletion. The treated cells were then cultured with or without rIL-2 for 3 days. LAK cytotoxicity was assayed against⁵¹Cr-labeled K562 and Raji tumor target cells. TPCK at 10 µg/ml partially inhibited depletion of monocytes by PheOMe. TLCK did not prevent depletion of monocytes nor inhibition of NK activity induced by PheOMe. TPCK and TLCK inhibited NK activity by themselves. TPCK but not TLCK inhibited rIL-2 induction of LAK cells. On the other hand, PheOH and benzamidine (analogs of PheOMe) lacked any effect on monocyte depletion but abrogated the inhibitory effect of PheOMe on NK activity. They had no effect on rIL-2 activation of LAK activity enhanced by PheOMe. Trypsin potentiated the inhibitory effect of PheOMe on NK activity and monocyte depletion. Trypsin partially inhibited IL-2 activation of LAK activity enhanced by PheOMe. Chymotrypsin had little effect on NK activity but prevented the inhibitory effect of PheOMe on NK activity. It had little effect on monocyte depletion induced by PheOMe. PheOMe was hydrolysed by monocytes and chymotrypsin to Phe and methanol as determined by HPLC. TPCK inhibited hydrolysis of PheOMe by monocytes. Our data suggest that the effects of PheOMe on monocytes, NK cells and LAK activation involve protease activities of monocytes.     

In vivo effects of recombinant IL-2. I. Isolation of circulating Leu-19+ lymphokine-activated killer effector cells from cancer patients receiving recombinant IL-2

This study was designed to isolate and phenotypically characterize lymphokine-activated killer (LAK) cells generated in vivo during administration of high dose rIL-2 to cancer patients. The development of circulating LAK effector cells in these patients was demonstrated by the ability of fresh PBL to exhibit lytic activity against the NK-resistant Daudi cell line and fresh tumor cells without prior in vitro culture with rIL-2. Kinetic studies demonstrated that circulating LAK effector cells are detectable 4 to 6 wk after the initiation of rIL-2 therapy. Cells isolated by FACS revealed that circulating LAK cells are Leu-19+, Leu-17+ but CD5-. We have previously reported that circulating Leu-19+ cells are heterogeneous with regard to the expression of CD16 and CD8. Since sorting of cells expressing Leu-19 and either low quantities of CD8 or CD16 resulted in cytolytic activity in both the positive and negative fractions, these latter two markers do not identify subpopulations of Leu-19+ cells with or without LAK cytolytic activity. Although all LAK cells generated in vivo were Leu-19+, we generated LAK cells from the Leu-19- subpopulation after in vitro culture with rIL-2, suggesting that at least some of in vitro generated LAK cells are derived from Leu-19- precursor cells. These LAK cells did not, however, express the Leu-19 surface marker. Based on the functional data reported in this paper, we conclude that circulating LAK effector cells are a phenotypically heterogeneous population that express surface Ag in association with NK cells and not T lymphocytes.     

Phenotypic and functional heterogeneity of thymic and splenic lymphokine activated killer cells relative to ornithine sensitivity

We have previously reported the selective inhibition of cytotoxic T lymphocytes (CTL) by 10 mM ornithine (ORN) relative to natural killer (NK) cell-derived lymphokine activated killer cells (LAK). To determine if this were due to differences in the progenitor cells or the type of stimulus, we used cortisone-resistant thymocytes (CRT) as a source of mature T cells for induction of LAK and CTL, and compared the results with spleen. Thymic and splenic CTL precursors (CTLp) from C57B1/6 (B6) mice were CD8+, ASGM1-, ORN sensitive. Splenic LAK precursors (LAKp) were CD8-, ASGM1+, ORN resistant when assayed against both YAC-1 and P815 tumor targets. In contrast, CRT-derived LAKp were CD8-, ASGM1+, ORN resistant against YAC-1, whereas LAKp against P815 were CD8+, ASGM1+, ORN sensitive. ORN sensitivity was also observed among CTL and LAK in DBA/2 mice and was associated with CD8+ phenotype. Therefore, our initial observation of differential ORN sensitivity in CTL vs LAK was a function of the progenitor cells; furthermore, CD8+ cytolytic cells are ORN sensitive whether activated by antigen (CTL) or IL-2 (T-LAK).     

Augmentative effect ofNocardia rubra cell-wall skeleton on the induction of human lymphokine-activated killer (LAK) cells by the production of LAK cell helper factor(s)

Summary Nocardia rubra cell wall skeleton (N-CWS) was found to synergistically augment lymphokine-activated killer (LAK) cell generation from human peripheral blood mononuclear cells (PBMC) in the presence of a suboptimal dose of recombinant interleukin-2 (rIL-2). N-CWS increased the number of PBMC expressing IL-2 receptor on their surfaces, and the presence of N-CWS at the early stage of the culture period was essential for the exertion of its augmentative activity on the LAK induction. The predominant phenotype of LAK precursor cells responding to N-CWS and rIL-2 was CD3^– CD16⁺. Culture supernatant from N-CWS-stimulated PBMC was found to act as a substitute for N-CWS in the induction of LAK generation in the presence of rIL-2, suggesting that these cells produced a factor capable of augmenting LAK cell induction (LAK helper factor, LHF). LHF was found to have a molecular mass of 29 kDa by gel filtration, and could also function as a killer helper factor to augment allo-antigen-specific cytotoxic T lymphocyte generation from human peripheral blood T cells as well as murine thymocytes. LHF showed no species specificity, indicating that it is different from IL-4. The enhancing activity of LHF was not neutralized with anti-TNF, anti-IL-1, or anti-IL-1 antibodies. Furthermore, no tumor necrosis factor- (TNF), TNF, IL-1, , IL-2, IL-5, IL-6 or interferon activity was detected in semi-purified LHF during enzyme-linked immunosorbant assay and biological assays. The present findings indicate that LHF produced from N-CWS-stimulated PBMC is a molecule distinct from TNF, TNF, interferon, IL-1, -2, -4, -5, and -6, and suggest that LHF might be a novel lymphokine involved in LAK generation.This work was supported by a Grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan     

Effector and precursor phenotypes of lymphokine-activated killer cells in mice with severe combined immunodeficiency (scid) and athymic (nude) mice

The lineage of lymphokine-activated killer (LAK) cells is poorly understood. To examine the relationship between LAK and natural killer (NK) cells we utilized two congenitally immunodeficient mice, namely severe combined immunodeficient (scid) and athymic (nude) mice that lack T cells but have normal NK cells. LAK activity was evaluated by the ability to lyze NK-resistant P815 cells. When cultured with human recombinant interleukin 2, splenocytes of scid and nude mice could generate LAK activity at levels comparable to or more than those of normal C.B-17 mice. LAK effector cells in these immunodeficient mice as well as normal mice had the phenotype resembling that of NK cells with asialo-GM1 (aGM1) expression. In vivo treatment with anti-aGM1 antiserum completely abolished the induction of LAK activity from splenocytes of normal mice. In contrast, LAK activity in splenocytes of scid and nude mice was still demonstrable even after this treatment, indicating that most LAK precursors in both mice were cells without aGM1 antigen. The aGM1- progenitors for LAK activity, probably in common with NK progenitors, appeared to be more expanded in scid and nude mice than in normal mice. The use of such congenitally immunodeficient mice should be helpful in studying the differentiation step of LAK as well as NK cells from their precursors.     

Induction of antitumor activities in spleen cells from mice and rats activated with lipopolysaccharide immobilized on beads

Summary Lipopolysaccharides (LPS) were coupled to polystyrene beads in order to apply the LPS without toxicity. The antitumor activity of the LPS-immobilizing beads was studied in experiments in vitro and in vivo. In vitro studies showed that spleen cells from C3H/HeN mice stimulated by beads immobilizing LPS fromEscherichia coli produced cytolytic activity as strong as that of lymphokine-activated killer (LAK) cells. Spleen cells from Sprague-Dawley rats stimulated by beads immobilizing LPS fromSalmonella minnesota produced cytolytic activity stronger than that of LAK cells. However, spleen cells stimulated by beads immobilizing each component of the LPS separately could not induce cytolysis. Contact stimulation, even for a brief period, sufficed for cytolytic activity, and was enhanced by culture for 48–72 h. Through in vivo studies, the suppression of tumor growth and a prolongation of the survival time were observed in tumorbearing mice injected with spleen cells activated by beads immobilizing LPS fromE.coli, and in mice injected with LAK cells. The effect of the activated spleen cells was stronger than that of the LAK cells. In rats bearing metastatic tumors, spleen cells activated by beads immobilizing LPS fromS.minnesota suppressed lung metastases more strongly than did LAK cells. These findings indicate that LPS immobilized by beads induced killer cells more strongly than interleukin-2. Ex vivo immunomodulation with LPS-immobilizing beads can be applied usefully as an anticancer treatment.     

Inhibition of murine lymphokine-activated killer (LAK) cell activity by adherent cells

The effects of adherent cell depletion, indomethacin, and prostaglandin E2 (PGE2) on murine LAK cell activity were investigated. Removal of plastic adherent cells from splenocyte suspensions either prior to 5-day culture with 1000 U/ml of recombinant human IL-2 (rIL-2) or prior to assay resulted in an enhanced LAK cell cytotoxicity compared to that of whole spleen cell suspensions. Indomethacin enhanced LAK cell cytotoxicity of whole splenocyte suspensions if present during the culture period, but had no effect on whole splenocyte or adherent cell-depleted cell suspensions if added just prior to assay. PGE2 suppressed LAK cell activity of nonadherent splenocyte but not whole splenocyte suspensions when present during the culture period. In vivo treatment of mice with indomethacin enhanced cytotoxicity directed toward both LAK sensitive, natural killer (NK) resistant (P-815) and LAK, NK sensitive (YAC-1) tumor cell targets. Splenocytes from indomethacin-treated mice cultured with additional indomethacin and rIL-2 exhibited highest LAK cell activity. The results from this study indicate that LAK cells are regulated by adherent cells which suppress LAK cell activity. This suppression can be reversed both in vitro and in vivo by indomethacin. This study has important implications for the possible clinical use of indomethacin in the potentiation of in vivo and in vitro LAK cell activity for immunotherapeutic protocols.     

Correlation of immunomodulatory and therapeutic activities of interferon and interferon inducers in metastatic disease

The mechanism of therapeutic activity of recombinant murine interferon-gamma (rMu IFN-gamma) and the IFN inducer polyinosinic-polycytidylic acid solubilized with poly-L-lysine in carboxy methyl cellulose (pICLC) in treating metastatic disease was investigated by comparing effector cell augmentation with therapeutic activity in mice bearing experimental lung metastases (B16-BL6 melanoma). Effector cell functions in spleen, peripheral blood, and lung (the organ with tumor) were tested after 1 and 3 weeks of rMu IFN-gamma or pICLC administration (intravenous, three times a week). In these studies, natural killer (NK), lymphokine-activated killer (LAK), cytolytic T lymphocytes (CTL) (against specific and nonspecific targets), and macrophage tumoricidal and tumoristatic activities were measured. rM IFN-gamma and pICLC had therapeutic activity and immunomodulatory activity in most assays of immune function examined. Specific CTL activity of pulmonary parenchymal mononuclear cells (PPMC), but not in splenocytes or peripheral blood lymphocytes (PBL), during week 3 and not during week 1, correlated with the therapeutic activity of rMu IFN-gamma and of pICLC. Macrophage tumoricidal activity in PPMC, but not in alveolar macrophages, also correlated with the therapeutic activity of rMu IFN-gamma, but the opposite was true for the therapeutic activity of pICLC. NK activity of PPMC, but not of splenocytes or PBL, during week 1 correlated with the therapeutic activity of pICLC; in contrast, NK activity at any site did not correlate with the therapeutic activity of rMu IFN-gamma. LAK activity at any site did not correlate with the therapeutic activity of either agent.     

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.     

Lymphokine-activated killer cells in rats. IV. Developmental relationships among large agranular lymphocytes, large granular lymphocytes, and lymphokine-activated killer cells

The developmental relationships among large agranular lymphocytes (LAL) large granular lymphocytes (LGL) and the activation of these cells into lymphokine-activated killer (LAK) cells by rIL-2 was investigated. Highly enriched populations of LAL were isolated from Fischer 344 spleen cells by a combination of nylon-wool filtration (to remove B cells and macrophages), treatment with a pan T cell antibody plus complement (to remove T cells) and incubation in L-leucine methyl ester (to remove LGL). The resultant cells were highly enriched in morphologically identifiable LAL which expressed asialo GM1 and partially expressed the OX8 surface marker. The enriched LAL did not contain detectable NK cytotoxic activity, did not express pan T cell (OX19), Ia, Ig, or laminin surface markers and contained less than 0.2% LGL. Incubation of LAL in a low dose of rIL-2 (100 U/ml) induced the generation of LGL having NK activity within 24 h of culture. Longer culture periods (48 h) resulted in a continued increase in the percentage of LGL and higher levels of NK activity. However, with this low dose of rIL-2, little or no LAK activity (i.e., reactivity against NK-resistant target cells) was generated. With a high dose of rIL-2 (500 U/ml), LAL responded by first generating LGL with NK activity (within 24 h), with subsequent generation of LAK activity by 48 h. Evidence that the development of granular lymphocytes from LAL was responsible first for NK activity and then LAK activity was demonstrated by depletion of the generated granular NK or LAK effector cells by second treatments with L-leucine methyl ester. Concomitant with the induction of LGL with NK or LAK activity, rIL-2 also caused LGL to proliferate and expand four- to five-fold in 48 h. This occurred in the presence of high or low dose rIL-2. These results indicate that LAL are the precursors of LGL/NK cells, that LAL, LGL/NK cells and LAK cells appear to represent sequential developmental or activation stages and that LAL may comprise major source of LAK progenitors in lymphoid populations having few LGL or mature active NK cells.     

IL-4 inhibits IL-2-mediated induction of human lymphokine-activated killer cells, but not the generation of antigen-specific cytotoxic T lymphocytes in mixed leukocyte cultures

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.     

IL-4 regulation of murine lymphokine-activated killer activity in vitro. Effects on the IL-2-induced expansion, cytotoxicity, and phenotype of lymphokine-activated killer effectors

The in vitro incubation of B6 splenocytes with purified, mouse rIL-4 for 4 to 5 days was sufficient to generate lymphokine-activated killer (LAK) activity. In addition, rIL-4 augmented LAK cytotoxic activity when combined with rIL-2, as measured in a 4 h 51Cr-release assay against fresh, syngeneic MCA-sarcoma (MCA-102 and MCA-105) cells. Interestingly, this augmentation was not observed against the cultured YAC-1 target. LAK generation and augmentation of cytotoxicity by rIL-4 was species-specific, because human rIL-4 (up to 20,000 U/ml) failed to elicit these effects in the mouse splenocyte cultures. When 5-day B6 LAK cells (splenocytes incubated in rIL-2 at 1000 U/ml for 5 days) were split and recultured in the combination of rIL-2 plus rIL-4 for 4 additional days at least a twofold greater expansion in cell number resulted compared to similar cells cultured in either rIL-2 or rIL-4 alone. Moreover, LAK cells expanded in rIL-2 plus rIL-4 exhibited substantial increases in in vitro cytolytic activity (on a per cell basis) against MCA-102 and MCA-105 sarcoma cells, but not against YAC-1 targets. FACS analysis or negative selection using Lyt-2 or NK-1.1 mAb plus C revealed no differences in effector phenotype(s) of LAK cells expanded in rIL-2 alone compared to rIL-2 plus rIL-4 to account for the differences observed in both expansion and cytolytic activity by rIL-4. The majority of cells was Thy-1+, Lyt-2+, T3+, and ASGM-1+. However, a marked increase in the granule-associated serine esterase, BLT-E, was found only in LAK cells expanded in the combination of both lymphokines. Collectively, these studies show that rIL-4 has potent regulatory activities on splenic LAK generation, expansion, and cytotoxic function in the mouse.