Lysis of human solid tumor cells by lymphokine-activated natural killer cells

The ability of NK cells to lyse noncultured solid tumor cells was investigated, and the results were compared with lysis of K562. Purified NK cell fractions separated by either Percoll centrifugation or a cell sorter exhibited higher level of lysis against noncultured melanoma cells than did NK-depleted cell fractions. However, the level of lysis was low (less than 10% lysis). Adding recombinant interleukin 2 (rIL 2) to the 4-hr assay induced significant lysis (more than 10%) of noncultured melanoma cells in 18 of 23 (78%) Percoll-enriched NK cell fractions and seven of 11 (64%) sorted Leu-11a+ cells at an E:T ratio of 80 and 10, respectively. In contrast, only two of 13 (14%) PBMC, five of 17 (29%) Percoll-decreased NK cell fractions, and one of 12 (8%) sorted Leu-11a- cells lysed noncultured melanomas in the presence of rIL 2. rIL 2 induced NK cells to lyse noncultured lung and breast cancer cells, as well as melanoma tumors. Exposure of NK cells to 2000 rad radiation abrogated the rIL 2-induced cytotoxicity against noncultured melanomas. Preculture of PBMC for 18 hr with recombinant interferon-gamma (rIFN-gamma) resulted in a modest level of lysis of non-cultured melanomas by sorted Leu-11a+ cells. Adding rIL 2 to the assay increased the cytotoxic activity in both rIFN-gamma-activated Leu-11a+ and Leu-7+ NK subsets. The level of noncultured tumor lysis correlated well with that of K562 lysis in all of the experiments. Purified NK cell fractions in rIL 2 cultures increased cytotoxic activity against noncultured tumor cells with incubation time for up to 3 days, and the level of NK cell-mediated lysis was dependent on both doses of rIL 2 and length of incubation. In contrast, both NK-depleted and sorted Leu-11a- cells demonstrated very low levels of solid tumor lysis after 3-day cultures with a high dose of rIL 2. Killer cell precursors induced by 3-day cultures of sorted cell fractions with rIL 2 and rIFN-gamma were found in both Leu-11a+ and Leu-7+ NK subsets, but not Leu-4+ or Leu-3a+ T lymphocytes. These results indicate that NK cells become cytotoxic for noncultured solid tumor cells by a brief contact with rIL 2, and increase cytotoxic activity after culture with rIL 2.     

Supplementation with selenium augments the functions of natural killer and lymphokine-activated killer cells

This study examined the effects of dietary (2.0 ppm for 8 wk) and in vitro (1×10⁻⁷ M) supplementation with selenium (Se, as sodium selenite) on the activity of spleen natural killer (NK) cells and plastic-adherent lymphokine-activated killer (A-LAK) cells from C57B1/6J male mice. Dietary supplementation with Se resulted in a significant increase in the lytic activity of activated NK cells, and cells from these highly lytic effector cell populations expressed significantly higher numbers of intermediate affinity interleukin-2 receptors (II-2R)/cell. In the presence of high concentrations of II-2 and 1×10⁻⁷ M Se, resting populations of spleen NK cells developed into A-LAK cells that had a significantly enhanced ability to proliferate, as indicated by the significantly higher amounts of nuclear³H-thymidine incorporation, and a significantly augmented cytolytic activity against both NK-sensitive and NK-resistant target cells. Se appears to enhance the lytic activity of activated NK cells and to augment the proliferation, expansion, and lytic activity of A-LAK cells in the presence of high concentrations of Il-2 through its ability to enhance the expression of intermediate affinity Il-2R on these cells.     

Similarities and distinctions between murine natural killer cells and lymphokine-activated killer cells

Pretreatment of mice with rabbit anti-asialo GM1 removes both natural killer (NK) effector cells and NK cells responsive to interleukin 2 (IL-2). Spleen cells from these mice do possess normal lymphokine-activated killer (LAK) activity. Young mice (less than 3 weeks of age) do not have NK activity and do not possess IL-2-inducible NK effector cells. Similarly to anti-asialo GM1-treated mice, LAK cells can be generated from these mice. While these experiments indicate clear distinctions between a certain level of NK and LAK precursors, the distinctions are not as clear when analyzing mice congenitally deficient in NK cells. Beige mice which lack NK effector cells and IL-2-inducible NK cells also lack the ability to generate LAK cells. The relationships and differences between NK- and LAK-cell precursors and effectors are discussed.     

Study of dipeptidyl peptidase IV as a surface marker of human natural killer cells

The enriched populations of natural killer (NK) cells have been obtained using two different approaches. The first one was based on the separation in a density gradient of Percoll and further formation of rosettes with sheep erythrocytes. This method allowed to isolate the population containing approximately 70% of CD16(+)-cells. The second approach consisted in separating lymphocytes on a flow cytofluorometer FACS II. Using this method the population with 80% CD16(+)-cells was isolated from PBMC. Studies on morphological, phenotypical and functional characteristics of the first population revealed that NK cells constituted 70% of total number of cells; T-lymphocytes, 8-lymphocytes and monocytes constituted the minor population (10%, 8% and 1% respectively). Activity of DPIV was determined on both cell populations obtained. As it was shown, approximately 27% of the cells isolated using percoll density gradient and 22% of the cells after the separation on a flow cytofluorometer carried the enzyme molecules on the cell surface. The results of the present study apparently indicate that part of NK cells (about 10%) is characterised by the presence of DPIV on the cell surface.     

Evidence that lymphokine-activated killer cells and natural killer cells are distinct based on an analysis of congenitally immunodeficient mice

The in vitro incubation of lymphoid cells in RIL 2 results in the generation of LAK cells that are broadly lytic to autologous, syngeneic, and allogeneic fresh tumor cells, but which do not lyse fresh, normal cells. Strains of mice with congenital immunodeficiencies were tested both for the presence of NK cells and for their capacity to generate LAK cells after in vitro incubation with IL 2. Splenocytes obtained from two immunodeficient mouse strains (NIH-Beige-Nude and NIH-Beige-Nude-XID) failed to generate LAK cells, but displayed significant activity. Splenocytes from another immunodeficient mouse strain (NIH-Beige-XID) generated LAK cells but did not display NK cell activity. This dissociation of activation of LAK cells from NK cells among the immunodeficient strains indicates that the LAK and NK cell lytic systems are distinct.     

Functional studies on the precursors of human lymphokine-activated killer cells

Human peripheral blood lymphocytes cultured for 4 days in the interleukin 2 (IL-2)-containing cell-free supernatant of the MLA144 cell line (MLA144CM) are cytolytic to NK-susceptible and NK-resistant tumor target cells. This lymphokine-activated killer (LAK) activity is dependent on IL-2 as development of LAK activity is inhibited in the presence of a monoclonal antibody (MoAb) reacting with the IL-2 receptor (anti-Tac). Addition of cyclosporin A (CyA) to mixed lymphocyte cultures inhibits the development of allospecific cytotoxic activity and inhibits the development of IL-2 responsiveness. However, development of LAK activity is unaffected by the inclusion of CyA in the cultures, showing that the LAK precursor can be functionally distinguished from the allospecific cytotoxic precursor cell. Development of LAK activity does not require mature NK cells as shown by the generation of LAK activity from NK inactive human thymocytes and lymph node cells. In addition, depletion of NK activity from human PBL does not impair the development of LAK activity.     

Lysis of fresh murine mammary tumor cells by syngeneic natural killer cells and lymphokine-activated killer cells

Summary We have compared the ability of natural killer (NK) cells from two substrains of C3H mice that differ with respect to their susceptibility to the development of mammary adenocarcinomas to lyse fresh syngeneic mammary tumor cells. Single cell suspensions of mammary tumors from retired breeder females were used as targets in 22-h ⁵¹Cr-release cytotoxicity assays with syngeneic NK cells. Tumor cell suspensions were prepared by enzymatic digestion of finely minced tissue followed by centrifugation through a discontinuous Percoll gradient. Effector cells were prepared by passing spleen cells over nylon wool followed by centrifugation through Percoll fraction 7. Syngeneic NK cells had significant levels of lysis against 5/8 tumors studied. NK cells from low risk animals (C3Heb/FeJ) consistently demonstrated greater cytotoxicity against tumor cell preparations than did effectors from the high tumor substrain (C3H/OuJ). Study of cytocentrifuge preparations stained with Wright-Giemsa revealed that the two substrains were identical with respect to the number of azurophilic granules present in the cytoplasm of their NK cells. We have also shown that lymphokine-activated killer (LAK) cells can be generated from splenocytes in C3H mice. While LAK cells from both substrains were capable of lysing fresh syngeneic mammary tumor cells in vitro, LAK cells from the animals at high risk for the formation of mammary adenocarcinomas had greater cytotoxicity against tumor cell suspensions than LAK cells from the low tumor substrain.     

Comparison of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes

Precursors and effectors of murine lymphokine-activated killer cells, natural killer cells, and cytotoxic T lymphocytes are compared. Natural killer cells are resistant to gamma-irradiation (1000 R) whereas precursors of lymphokine-activated killer cells and cytotoxic T lymphocytes are sensitive. Lower doses of gamma-irradiation (500 R) remove precursors for cytotoxic T lymphocytes but not lymphokine-activated killer cells. In addition, lymphokine-activated killer cells are regenerated before classical CTL after sublethal doses of gamma-irradiation. Natural killer cells are resistant to anti-Thy 1 and C' and anti-thymocyte serum, but sensitive to anti-asialo GM1 and complement. Precursors of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Precursors of lymphokine-activated killer cells are partially sensitive to anti-Thy 1 and complement and anti-thymocyte serum, but are resistant to anti-asialo GM1 and complement. Effector cells of cytotoxic T lymphocytes are sensitive to anti-Thy 1 and complement and resistant to anti-asialo GM1 and complement. Lymphokine-activated killer cell effectors are sensitive to anti-asialo GM1 and complement at 24 hr after activation. These effectors are more closely aligned with classical natural killer effectors. Lymphokine-activated killer effectors, 7 days after activation, are resistant to anti-asialo GM1 and complement and sensitive to anti-Thy 1 and complement. Relationships and differences among these cytotoxic subsets are discussed.     

Lysis of human monocytes by lymphokine-activated killer cells

Human peripheral blood leukocytes (PBL), stimulated in vitro with recombinant human interleukin 2 (IL-2) for 2-7 days, were seen to lyse autologous and allogeneic monocytes in a 4-hr 51Cr-release assay. The lymphokine-activated killer (LAK) cells against monocytic cells were selective in that polymorphonuclear leukocytes (PMN) and nonadherent PBLs were not lysed by these cells. Monocytes which had been cultured for 2-7 days served as better targets than uncultured cells. Also, kinetic studies demonstrated parallel activation of cytolytic activity against monocyte targets and FMEX, an natural killer cell-insensitive human melanoma target. Separation of PBLs by discontinuous density centrifugation identified the effector population in the fractions enriched for large granular lymphocytes (LGL). Precursor cells were seen to express CD2, CD11, and some CD16 markers, but not CD3, CD4, CD8, CD15, Leu M3, or Leu 7. The effector population after IL-2 activation retained the phenotype of the precursor cell. These studies indicate that IL-2 can generate LAK cells against monocytic cells, and this cytolytic activity, especially against autologous monocytes, must be taken into account when IL-2 or LAK cells are used for immunomodulation in cancer patients.     

Lymphokine-activated killer cells in rats: generation of natural killer cells and lymphokine-activated killer cells from bone marrow progenitor cells

The coculture of rat bone marrow cells with recombinant interleukin-2 induced the generation of cells mediating natural killer (NK) activity and subsequent lymphokine-activated killer (LAK) activity depending upon the dose of IL-2 and time of culture. NK activity was detected as early as 4 to 5 days after the addition of IL-2 and could be evoked with as little as 5 to 50 U/ml. The induced NK cells had large granular lymphocyte (LGL) morphology and expressed 0X8 and asialo GM1 surface markers but did not express 0X19 or W3/25 markers. LAK activity was detected only after 5 days of culture, and required above 100 U/ml IL-2. Cells mediating LAK activity also expressed 0X8 and asialo GM1 but not 0X19. The generation of detectable NK and subsequent LAK activity was due to induction of early progenitor cells and not contaminating mature LGL/NK cells within the bone marrow population since of removal of such mature NK cells with L-leucine methyl ester (L-LME) did not affect the subsequent generation of either activity. Moreover, the removal of actively dividing cells as well as mature NK cells from the bone marrow by treatment with 5-fluorouracil (5-FU) in vivo enriched the remaining bone marrow population for both NK and LAK progenitor cells. The phenotype of the L-LME- and 5-FU-resistant NK and LAK progenitor cells within populations of bone marrow was determined by antibody plus complement depletion analysis. Although treatment of normal bone marrow with anti-asialo GM1 + C reduced the induction of NK and LAK activity in 5-day cultures, treatment of 5-FU marrow with anti-asialo GM1 + C did not affect either activity. Treatment with a pan-T cell antibody + C did not affect the development of NK or LAK activity under any conditions. Thus, the 5-FU-resistant NK/LAK progenitors were asialo GM1 negative but became asialo GM1+ after induction by IL-2. Finally, evidence that bone marrow-derived LAK cells were generated directly from the IL-2-induced NK cells was obtained by treating the IL-2-induced LGL/NK cells with L-LME.(ABSTRACT TRUNCATED AT 400 WORDS)     

Interleukin-6 enhances the induction of human lymphokine-activated killer cells

Summary Human peripheral blood mononuclear cells develop a powerful lytic capacity when cultured in vitro with interleukin-2 (IL-2), becoming lymphokine-activated killer cells (LAK cells). As part of an investigation into means of influencing this process, the effect of other cytokines has been examined. In this study we describe the ability of interleukin-6 (IL-6) to regulate the induction and function of human LAK cells. The results show that substitution of IL-6 for IL-2 did not lead to the development of functional LAK cells, nor was IL-6 able to alter the lytic capacity of established LAK cells. However, when IL-6 was included with IL-2 during the induction phase of the LAK cells, the resulting cells displayed considerably greater lytic activity than those prepared with IL-2 alone. This effect was IL-6 dose-related. These results indicate that LAK cell development may be positively regulated in vitro; the implications of this observation for the clinical usage of LAK cells are discussed.     

Studies of serum-free medium for the generation of lymphokine-activated killer cells

We examined a serum-free medium (designated as TYI 101) for the generation of lymphokine-activated killer (LAK) cells from human lymphocytes, regional lymph node lymphocytes (RLNL) and peripheral blood lymphocytes (PBL). TYI 101 medium consisted of, in addition to nutrient mixture, transferrin, insulin, fetuin, sodium selenite, 2-mercaptoethanol, o-phosphorylethanolamine, chick egg yolk and porcine kidney extract. These hormones were effective for supporting RLNL proliferation as assessed by (³H)-thymidine uptake. When human lymphocytes from two different sources were cultivated with recombinant interleukin 2 (rIL-2) in TYI 101 medium, LAK activity was generated. In cultures of PBL from a healthy donor, LAK cells were generated in TYI 101 medium as efficiently as in RPMI 1640 medium supplemented with 10% human AB-type serum (RPMI-AB). In cultures of RLNL from lung cancer patients, LAK activity obtained in TYI 101 medium was about sixty-five percent of that in RPMI-AB. However, the addition of a small amount of AB-type serum improved the generation of LAK activity, LAK cell expansion, and cell viability in TYI 101 medium. We conclude that TYI 101 medium can be used for the generation of LAK cells from human lymph node lymphocytes with supplementation of none or only a reduced amount of human serum.Abbreviations IMDM Iscove's Modification of Dulbecco's Medium - rIL-2 recombinant Interleukin - LAK Lymphokine-Activated Killer - RLNL Regional Lymph Node Lymphocytes - PBL Pheripheral Blood Lymphocytes - PBS Phosphate-Buffered Saline - RBC Red Blood Cells - RPMI-AB RPMI 1640 medium supplemented with 10% human AB-type serum Address for offprints: Tsukuba Research Laboratory, Japan Synthetic Rubber Co., Ltd., 25 Miyukigaoka, Tsukuba-shi, Ibaraki, 305 Japan     

Induction of human lymphokine-activated killer cells by IFN-alpha and IFN-gamma

By traditional definitions, NK cells can be activated by cytokines to exhibit two functionally distinct levels of cytotoxicity. Whereas IL-2-mediated activation of NK cells leads to the development of lymphokine-activated killer (LAK) cytotoxicity, characterized by the acquisition of cytolytic activity against NK-resistant targets, IFN-treated NK cells become activated without the acquisition of novel cytolytic specificities. In this study we show that NK cells activated by 18 to 24 h of stimulation with either IFN-alpha or IFN-gamma do acquire LAK cytolytic activity, demonstrated by the ability of IFN-treated PBMC to lyse NK-resistant COLO 205 cells as well as fresh tumor targets. The level of IFN-alpha-induced LAK activity was significantly greater than that induced by IFN-gamma, although IL-2-induced LAK activity was considerably greater than IFN-alpha-induced LAK cytotoxicity. Maximal IFN-induced LAK cytotoxicity occurred after 24 h of culture, and occurred with the use of IFN-alpha at 500 U/ml and IFN-gamma at 1000 U/ml. Whereas neutralizing antibody experiments demonstrated that IFN-alpha-induced LAK activation did not involve the participation of endogenously produced IL-2, the partial inhibition (63%) of IFN-gamma-induced LAK cytotoxicity by anti-IL-2 and of IL-2-induced LAK by anti-IFN-gamma (33.3%) indicates that the induction of LAK cytotoxicity by either of these individual cytokines involves the endogenous production and participation of the other cytokine. Similar to IL-2-induced LAK cells, phenotypic analysis revealed that IFN-alpha/gamma LAK cells were Leu-19+, although the Leu 19"dim"+ subset exhibited greater IFN-induced LAK activity than the Leu-19"bright"+ subset. The results of this study clearly demonstrate that IFN-alpha and IFN-gamma induce classic LAK activity and IFN-gamma plays a participatory role in the optimal induction of LAK cells by IL-2.     

Role of lymphokine-activated killer cells as mediators of veto and natural suppression

Fresh bone marrow cells have veto activity but little if any NK activity. By contrast, lymphokine-activated bone marrow cells have potent natural suppressor as well as veto activity, and also have cytolytic activity characteristic of lymphokine-activated killer cells. Veto activity of fresh bone marrow cells is eliminated by 9 Gy irradiation and by depletion of cells expressing Qa-2, but is unaffected by removal of cells expressing Thy-1, Qa-5, Ly-5, or asialo GM1. By contrast, the veto and NS activities of lymphokine-activated bone marrow cells are both abrogated by C lysis depletion of cells expressing Qa-2, Qa-5, Thy-1, asialo GM1, NK1, and Ly-11, but are unaffected by depletion of cells expressing Ly-2. Bone marrow cells depleted of Qa2+ cells fail to generate veto or natural suppressor activity when cultured in Con A-conditioned medium, unlike bone marrow cells depleted of mature NK1.1+ NK cells. Cloned NK cell line F8 is able to mediate both natural suppression and veto. These findings indicate that bone marrow veto and natural suppression are not mediated by T or NK cells present de novo in the bone marrow, but are dependent on proliferating cells that phenotypically resemble pre-NK cells. The progeny of these cells have the phenotype and functional activity of lymphokine-activated killer cells, and are capable of directly mediating both veto and natural suppression.     

Combined effect of epirubicin and lymphokine-activated killer cells on the resistant human breast cancer cells

Accumulating evidence suggests the concept that epirubicin and lymphokine-activated killer (LAK) cells cytotoxicity may be mediated by free radicals generation and P-glycoprotein-positive (Pg-p⁺) cancer cells are more sensitive for LAK cells than their drug-sensitive parental lines. We tested this hypothesis further by exposing drug-sensitive (WT) and epirubicin-resistant MCF-7 human breast tumor cells to epirubicin and LAK cells. Subsequently, we monitored cell proliferation as a measure of cytotoxicity. The cytotoxicity of epirubicin, LAK, and LAK + epirubicin (1/10 of IC₅₀) was evaluated in 400-fold epirubicin resistant MCF-7 EPI^R (P-glycoprotein overexpressing) and drug-sensitive MCF-7 WT cells. IC₅₀ values were measured using the MTT cytotoxicity test. The MCF-7 EPI^R cells exhibited an increased susceptibility to LAK cells than did the MCF-7 WT cells. P-gp⁺ MCF-7 EPI^R cells were lysed by human LAK cells to a greater extend than were their drug-sensitive counterparts. LAK + epirubicin combined treatment increased susceptibility of MCF-7 WT and MCF-7 EPI^R cells to LAK cells cytotoxicity. For both cell lines, cytotoxicity was dependent upon the concentration of the epirubicin and effector cell/target cell (E/T) ratio. The resistance of MCF-7 EPI^R cells to epirubicin appears to be associated with a developed tolerance to superoxide, most likely because of a tree-fold increase in superoxide dismutase (SOD) activity and 13-fold augmented selenium dependent glutathione peroxidase (GSH-Px) activity. Acting in concert, these two enzymes would decrease the formation of hydroxyl radical from reduced molecular oxygen intermediates. The addition of SOD decreased cytotoxicity of epirubicin and LAK cells. Taken together, these observations support the role of oxygen radicals in the cytotoxicity mechanism of epirubicin and suggest further that the development of resistance to this drug by the MCF-7 EPI^R tumor cells may have a component linked to oxygen free radicals. It is proposed that production of reactive oxygen species by the treatment of epirubicin and LAK cells can cause cytotoxicity of MCF-7 WT and MCF-7 EPI^R cells. SOD, catalase, GSH-Px, GST (glutathione S-transferase), and GSH (reduced glutathione) must be considered as part of the intracellular antioxidant defense mechanism of MCF-7 WT and MCF-7 EPI^R cells against reactive oxygen species.     

Differential expression of lymphokine-activated killer cells and natural killer cells in adoptive transfer experiments utilizing fractionated bone marrow

Precursors of murine natural killer (NK) cells and lymphokine-activated killer (LAK) cells can be distinguished by utilizing an adoptive transfer system in which donor bone marrow is fractionated on Percoll discontinuous gradients. Although precursors of LAK cells are present in all fractions, one fraction (greater than 65% Percoll) contains LAK precursors and is depleted of NK precursors. Both in vitro NK activity and in vivo hybrid resistance is abrogated in recipients of bone marrow from the greater than 65% Percoll fraction, whereas LAK activity can be readily demonstrated.     

Murine natural killer cells express the Ly24 (Pgp-1) marker on their surface

The Ly24 (Pgp-1) marker is expressed on some, but not all, mature T lymphocytes. It has recently become apparent that the development of Ly24- T lymphocytes is dependent on the presence of an intact thymus and that virgin Ly24- T cells rapidly acquire this marker upon antigenic or mitogenic stimulation. Although natural killer (NK) cells can develop and function in the absence of an intact thymus, some NK cell subsets express certain markers normally associated with T lymphocytes. The experiments in this report were undertaken to determine if NK cells express Ly24 and whether such an expression could be used to delineate distinct NK cell subsets. We found that mature functional NK cells expressed the Ly24 marker as defined by the monoclonal antibody 9F3. Double-color fluorescence analysis using C57BL/6 splenocytes (whose NK cells express the NK1.1 marker) showed all the NK1.1+ cells to be Ly24+ as well. For C3H/HeN (an NK1.1- strain), double-color fluorescence analysis utilizing asialo GM1 and Ly24 revealed a distinct subset positive for both markers and containing most of the functional NK cell activity. Whereas the Ly24 marker did not illuminate an NK cell subset, these findings demonstrate that this determinant can be useful for the further characterization and isolation of NK cells.     

Molecular mechanisms of cytolysis induced by human lymphokine-activated killer cells

It has been shown that lysis of tumor target cells caused by lymphokine-activated killers is possible both upon a direct contact and in the presence of isolated nongranular cytotoxic proteins. The contact of cytolytic lymphocytes with K-562 cells leads to Fas L activation on the lymphocyte membrane and secretion of a broad spectrum of soluble cytotoxic proteins immunologically related to Tag 7 described earlier. These proteins can form inactive complexes, which are reactivated upon heating and addition of ATP. The proteins induced discrete cytolytic processes in tumor cells, differing in the rate of cytolysis and the mechanism of the apoptotic signal transduction. Fast processes (revealed in 3 h) mediated by caspases, and slow ones (in 24 h) with the supposed involvement of mitochondria were detected. A scheme for the lymphokine-activated killer interaction with target tumor cells is proposed.     

Assessment of human natural killer and lymphokine-activated killer cell cytotoxicity against Toxoplasma gondii trophozoites and brain cysts

Because previous work has suggested that NK cells may be important in host resistance against the intracellular parasite Toxoplasma gondii we examined whether human NK cells and lymphokine-activated killer (LAK) cells have activity against trophozoites and cysts of this organism in vitro. A method to radiolabel Toxoplasma trophozoites with 51Cr was developed and direct cytotoxic activity was determined by using modifications of the standard 51Cr release assay. Viability of 51Cr-labeled trophozoites assessed by both methylene blue staining and trypan blue exclusion was greater than 90%. Significantly more 51Cr was released by anti-Toxoplasma antibody and C than by antibody in the absence of C. Incubation of trophozoites with freshly isolated human NK cells or NK cells activated with either rIL-2 or rIFN-alpha did not result in significant release of 51Cr (specific lysis was 0 to 2.3%). In contrast, the average specific lysis of radiolabeled trophozoites by LAK cells was significant (specific lysis was 7.8% +/- 1.1, p less than 0.01). In a series of separate experiments, preincubation of radiolabeled trophozoites with heat-inactivated normal or Toxoplasma antibody-positive human serum increased the cytotoxicity of LAK cells from a mean specific lysis of 15% +/- 4.5 to 39% +/- 8.5, respectively (p less than 0.05), as assessed by 51Cr release. Because previous work has shown that radioisotope release from parasites may be nonspecific, separate experiments were performed to determine the cytotoxicity of LAK cells against antibody-coated trophozoites by using ethidium bromide-acridine orange staining to assess effector cell damage. LAK cells had a mean specific lysis of 51% against antibody-coated trophozoites by ethidium bromide-acridine orange staining. Preincubation with heat-inactivated Toxoplasma-antibody positive human serum did not increase activity of rIL-2-activated NK cells against 51CR-labeled trophozoites. Neither human NK cells (freshly isolated or activated by rIL-2 or rIFN-alpha) nor LAK cells were cytotoxic for purified preparations of cysts of Toxoplasma isolated from the brains of chronically infected mice.