Generation Of Lymphokine-activated Killer Activity Research Articles

Expansion of Functional NK Cells in Multiple Tissue Compartments of Mice Treated with Flt3-Ligand: Implications for Anti-Cancer and Anti-Viral Therapy

The generation and activity of NK cells appear to be regulated by a particular set of cytokines. We examined the in vivo effects of recombinant human Flt3 ligand (Flt3-L), a recently cloned potent hemopoietic cytokine, on NK cell development in mice. Daily i.p. administration of Flt3-L consistently induced striking increases in both the absolute number and the total cytotoxic activity of mature nonactivated NK cells within various tissues. Dose- and time-dependent increases were observed in the bone marrow (approximately 2- and approximately 11-fold, respectively), thymus (approximately 2.8- and approximately 2.0-fold), blood (approximately 11- and approximately 15-fold), spleen (approximately 10- and approximately 9-fold), and liver (approximately 15- and approximately 39-fold). In addition, IL-2 induced a rapid increase in NK activity, NK cell proliferative responses, generation of lymphokine-activated killer activity, and development of activated adherent NK cells, which were all significantly increased by Flt3-L treatment. Thus, in addition to its recently reported capacity to stimulate dendritic cell production, Flt3-L has a prominent biologic role in NK cell generation in vivo. This is probably a result of selectively induced expansion of NK cell progenitors (pro-NK cells), because Flt3-L stimulates in vitro proliferation of pro-NK cells without affecting the cytotoxicity of mature NK cells. The results also indicate that either alone or in combination with a potent activator of NK cells, such as IL-2, Flt3-L could be used to markedly augment the number and activity of NK cells, especially in the liver. Flt3-L appears to have considerable potential for therapy of both cancer and viral infection.

Interleukin-12 induced cytolytic activity in lymphocytes from recipients of autologous and allogeneic stem cell transplants.

Interleukin-12 (IL-12) has been reported to enhance the cytolytic activity of NK and activated T cells and to induce low levels of lymphokine-activated killer (LAK) activity in normal human lymphocytes. Therapy with IL-12 has induced tumor eradication in murine models. These observations suggest that IL-12 might have a role as treatment for minimal residual disease following transplantation. To determine whether PBL from recipients of autologous (autoSCT) and allogeneic (alloSCT) bone marrow or peripheral blood stem cell transplants respond to IL-12 with generation of LAK activity, PBL were incubated with IL-12 for 5 days, then tested in a 51Cr release assay for lysis of Daudi. PBL from 17 normal 'control' individuals were similarly tested and lysis was observed in only 3/17 (mean 16.9% of the three). By contrast, PBL from 10/12 patients obtained a median of 30 days after autoSCT, exhibited significant IL-12-induced LAK activity (mean lysis, 35.3%, P < 0.005 vs controls). PBL from 18 of 20 patients tested a median of 44 days after alloSCT also exhibited significant LAK activity (mean lysis, 30.0%, P < 0.005 vs controls). In autoSCT recipients, IL-12 and IL-2 at high concentrations (1000 U/ml each) were additive for induction of LAK activity, whereas low, suboptimal concentration of IL-12 (250 U/ml) and IL-2 (1 U/ml) were synergistic in 3/5 experiments. The percentage of PBL expressing IL-12 receptor beta 1 chain (IL-12r beta 1) was higher in stem cell recipients than in normal individuals, P < 0.05. Moreover, a higher percentage of IL-12r beta 1-positive PBL was associated with greater IL-12-induced LAK activity in transplant recipients. These studies demonstrate that PBL obtained early after stem cell transplantation have a higher percentage of cells expressing IL-12r beta 1 and respond to IL-12 with significantly greater LAK cytotoxicity than PBL from normal controls. These results suggest that IL-12 is a potentially attractive candidate for study as consolidative immunotherapy after stem cell transplantation.

Enhanced human lymphokine-activated killer cell function after brief exposure to granulocyte-macrophage-colony stimulating factor.

Lymphokine-activated killer (LAK) cell function can be generated in peripheral blood mononuclear cells (PBMC) after brief exposure of high dose interleukin-2 (IL-2) over the course of 1 or 2 days' culture in plain culture medium (IL-2-pulsed PBMC). The aim of the present study was to investigate the ability of granulocyte-macrophage-colony stimulating factor (GM-CSF) to augment LAK induction in low dose IL-2-pulsed PBMC derived from patients with cancer undergoing immunotherapy with IL-2. Peripheral blood mononuclear cells were collected from patients with cancer receiving a 5-day cycle of local (intraperitoneal or intrapleural) infusions with IL-2. The cells were incubated with IL-2 in the presence or absence of GM-CSF for 1 hour and then tested as effectors against allogeneic tumor cells and LAK-sensitive cell lines. Granulocyte-macrophage-colony stimulating factor at doses between 10 and 100 ng/ml was synergized with low dose IL-2 (100 IU/ml) in the generation of LAK activity in PBMC. Lymphokine-activated killer cell-mediated cytotoxicity derived from PBMC cultures incubated with IL-2 and GM-CSF was significantly higher (up to three-fold) compared with that generated with IL-2 alone. The GM-CSF-induced enhanced LAK activity was maintained when tested at day 5. GM-CSF increased the percentages of IL-2 receptor (R) positive (+) and CD8+ cells in the IL-2-pulsed PBMC. In contrast to CD56+ cells, highly purified CD8+ cells isolated from PBMC pulsed with IL-2 and GM-CSF responded with increased LAK activity, thus representing the cell-type that mediates the augmenting effect of GM-CSF. Major histocompatibility complex (MHC) molecules or the CD3 surface antigens were not involved in the GM-CSF-mediated enhancement of LAK induction because anti-MHC class I and class II monoclonal antibodies (MoAb) or MoAb against the CD3 molecules remained without any effect in this system. The GM-CSF-mediated LAK-enhancement was IL-2-dependent because MoAb against IL-2 receptor completely inhibited the generation of LAK activity. The use of GM-CSF for the enhancement of IL-2-induced LAK activity in 1 hour cultures may improve clinical results in cancer immunotherapy. In addition, implementation of this procedure could eliminate the high cost of cell culture which usually accompanies IL-2/LAK cell therapy as well as eliminate the known toxic side effects associated with this kind of therapy.

Generation of Lymphokine-Activated Killer Activity in Rodents but Not in Humans Is Nitric Oxide Dependent

The role of nitric oxide (NO) in the generation of lymphokine-activated killer (LAK) cells was investigated. Here we report that L-arginine analog NG -monomethyl-L-arginine (NMMA), a specific inhibitor of nitric oxide synthase, prevents LAK cell generation from cultured rat splenic cells. Accumulated NO endproduct nitrite (NO-2), as measured in the supernatants of rat splenic cells, correlated well with the generation of LAK cells. In contrast, cell proliferation induced by rIL-2 or by Con A was not affected by NMMA. Similarly, phenotypic expression of CD25 in rIL-2-stimulated cultures was unaffected. Furthermore, we could not observe differences in percentages of CD5-CD8+ cells (NK and LAK cell phenotype markers in rats) between rIL-2-stimulated cultures performed in the presence or absence of NMMA. LAK cell generation could no longer be blocked if NMMA was added to the rat cell cultures 24 hr after rIL-2 stimulation. To further confirm the role of NO in LAK cell generation, rat splenic cells were cultured in medium without L-arginine. Under such conditions rIL-2 could not induce LAK cell generation. Hemoglobin, which is a scavenger of NO, also inhibited LAK cell generation. Finally, addition of sodium nitroprusside (SNP) which releases NO in cultures was able to overcome blocking effects of NMMA. To attempt the identification of NO-producing cells, lysosomotropic agent, L-leucine methyl ester (LME), was used. Generation of LAK cell activity was virtually abolished in cell cultures treated with LME. Addition of SNP to cultures, however, sufficed to restore LAK cell generation. These results suggest that LAK cell precursors depend on a exogenous NO supply from other cell types in order to display their full cytotoxic potential. Similar results were also obtained by using mouse splenocytes as responder cells. In contrast, NMMA did not affect generation of LAK cells from human peripheral blood or spleen mononuclear cells.

Gene expression and secretion of cytokines and cytokine receptors from highly purified CD56+ natural killer cells stimulated with interleukin‐2, interleukin‐7 and interleukin‐12

Interleukin (IL)-2 IL-7 and IL-12 stimulate the generation of lymphokine-activated killer activity and proliferation in natural killer (NK) cells by different mechanisms. In this study, we have compared the ability of IL-2, IL-7 and IL-12 to induce expression of cytokines and cytokine receptors both at the gene and protein level. IL-2 and IL-12 stimulated the CD56+ NK cells to release significant amounts of soluble p55 and p75 tumor necrosis factor receptor (TNFR), whereas less amounts of soluble TNFR were detected in IL-7-stimulated cultures. The p55 and p75 TNFR mRNA were expressed in resting NK cells, and no further induction was observed after cytokine-stimulation. Compared to the effects of IL-2, IL-7 induced lower, but substantial levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA, and IL-7 was a more potent GM-CSF-inducing stimulus than IL-12. IL-12 induced higher levels of interferon-gamma (IFN-gamma) mRNA than did IL-2, and IL-7 only weakly influenced the IFN-gamma expression. In accordance with the mRNA studies, IL-7 induced the secretion of high amounts of GM-CSF and no or low levels of IFN-gamma, whereas high amounts of IFN-gamma and low levels of GM-CSF were detected in supernatants from IL-12-stimulated NK cells. In conclusion, IL-2, IL-7 and IL-12 differentially regulate expression of cytokines and cytokine receptors both at the gene and protein level.

On the Relative Roles of Interleukin-2 and Interleukin-10 in the Generation of Lymphokine-Activated Killer Cell Activity

Induction of cytokine gene transcription by recombinant human IL-2 (rhIL-2) in peripheral blood mononuclear cells (PBMC) from healthy donors was studied by qualitative polymerase chain reaction. In all donors tested, optimal doses of rhIL-2-induced transcription of genes encoding for IL-5, GM-CSF, IFN-γ and TNF-α whereas transcription of IL-1-α, IL-3, IL-4, and IL-6 genes could only be detected in about half of the donors. Moreover, we observed that different doses of rhIL-2 were needed to induce transcription of different cytokine genes. In contrast, transcription of IL-2 and IL-10 genes was only observed in a minority of donors, irrespective of the concentration of rhIL-2 used. Since IL-10 displays a well-characterized inhibitory activity on the synthesis of cytokines possibly involved in the generation of lymphokine-activated killer (LAK) cells, we asked whether the absence of IL- 10 gene transcription plays a role in the induction of LAK cells. Thus, we tested the effects of different doses of rhIL-10 on the rhIL-2-driven generation of LAK activity. Interestingly, rhIL-10 dose-dependently inhibited the production of IFN-γ and TNF-α induced by IL-2, but had no effects on PBMC proliferation and generation of LAK activity. Similarly, purified CD3-/CD16+ lymphocytes, the precursors of LAK effector cells, could be optimally induced by low doses of rhIL-2 to proliferate and generate MHC-unrestricted cytotoxic activity against NK-resistant targets in the presence of rhIL-10. Altogether, our results indicate that rhIL-2 induces transcription of a preferential pattern of cytokine genes, with the IL-10 gene being infrequently transcribed. On the other hand, rhIL-10 shows diverse effects on rhIL-2-triggered PBMC activation, in that it inhibits IFN-γ and TNF-α production but does not affect PBMC proliferation or generation of LAK activity.

Up-regulation of IL-2 induced lymphokine activated killer cell activity by cisplatin and FK-565: involvement of calcium ion.

As reported earlier, the IL-2 induced lymphokine activated killer (LAK) activity is significantly up-regulated in the presence of cisplatin/FK-565. Based on these observations, we have investigated whether calcium is involved in the generation of LAK activity by IL-2 alone or along with CP/FK-565. We have shown that treatment of PBMC with IL-2 for four days caused an increase in intracellular free calcium and in ATP levels, which were further significantly enhanced when LAK cells were generated in the presence of CP/FK-565. Depletion of calcium resulted in decreased cytotoxic activity. Addition of tumor cells to LAK cells, generated in the presence of IL-2 alone or along with CP/FK-565 caused an instant rise in intracellular free calcium which was significantly decreased when an increase in intracellular free calcium was observed in calcium-free, EGTA-containing buffer. These data suggest that calcium is required for the activation and manifestation of lytic activity by LAK cells. Further, we observed that the increase in intracellular free calcium is not associated with the blastogenic response of peripheral blood mononuclear cells in response to treatment of IL-2 alone or together with CP/FK-565.

Pregnancy Is Associated With a Reduction in the Pattern of the Cytotoxic Activity Displayed by Lymphokine‐Activated Killer Cells

Several modifications in the homeostasis of the maternal immune system have been implicated in the survival of the fetoplacental graft. We have investigated the adaptive response of the cytotoxic nonmajor histocompatibility complex (MHC)-restricted effector lymphocytes in pregnancy, and have found that the spontaneous lytic activity against both natural killer (NK)-sensitive and NK-resistant target cells is either decreased or lacking in peripheral blood mononuclear cells (PBMNC) from pregnant women. Recombinant interleukin-2 (rIL-2) normalizes the cytotoxic activity of PBMNC from pregnant women against NK-sensitive target cells in a dose- and time-dependent manner, without modification in the normal amounts of HNK-1+, CD16+ (Leu 11, or CD11b+ (OKM-1) present in these effector populations. However, the pattern of lytic activity against NK-resistant target cells found in PBMNC from pregnant women after short- and long-term incubation with rIL-2 was reduced in comparison with that observed in PBMNC from nongravid women in similar conditions. Moreover, rIL-2 incubation of PBMNC from pregnant subjects was not associated with an enhancement of the lytic binding against NK-resistant target cells. These findings demonstrate that pregnancy is not only associated with a reduction in the NK lytic activity of PBMNC, but also with a reduction in the generation of lymphokine-activated killer activity, in terms of the pattern of lytic activity developed.

Generation of lymphokine-activated killer activity in T cells. Possible regulatory circuits.

CD4+ and CD8+ T cells do not develop significant lymphokine-activated killer (LAK) activity when PBL are cultured with IL-2 or even when they are activated with a T cell stimulus such as OKT3 mAb. The possibility that a T cell regulatory mechanism prevents the development of LAK activity by CD4+ or CD8+ cells in OKT3 mAb and IL-2 cultures was tested by depleting CD8+ or CD4+ cells from PBL before stimulation with OKT3 and IL-2. Under these conditions, the remaining CD4+ and CD8+ cells were able to generate non-MHC-restricted lysis of NK-resistant tumor targets. Our data suggested that a regulatory signal was present in the culture to prevent the development of lytic function by T cells. T cells removed from the PBL cultures were, upon culture with IL-2, able to generate high LAK activity, suggesting that inhibition of the CD4+ or CD8+ T cell-mediated LAK activity was an active ongoing process, which blocked the lysis at the level of the activated cell and not the precursor cell. Mixing experiments demonstrated that the CD4+ or the CD8+ cells isolated from the PBL cultures were able to inhibit the development of lytic function in the CD4-depleted and CD8-depleted cultures. Transforming growth factor-beta (TGF-beta) has been shown to block LAK activity of NK cells in IL-2-stimulated cultures. When TGF-beta was added to CD4(+)- or CD8(+)-depleted cultures, it also inhibited LAK activity of T cells in a dose-dependent fashion, without interfering with T cell growth. Lytic activity returned to activated levels when TGF-beta was removed from the culture medium, thereby demonstrating the reversibility of TGF-beta inhibition.

IL-7 induces human lymphokine-activated killer cell activity and is regulated by IL-4.

Induction of lymphokine-activated killer (LAK) activity by IL-2 has been described and characterized as broadly cytolytic activity against both fresh and cultured tumors. rIL-7 in the absence of IL-2 also induces LAK activity in human cells. This activity is unique for IL-7, because it is not shared by other cytokines including IL-1, IL-4, IL-6, and TNF-alpha. IL-7 also induces either de novo or increased expression of the surface markers CD25 (Tac, IL-2R alpha-chain), CD54 (ICAM-1), Mic beta 1 (IL-2R beta-chain) and CD69 (early T cell activation Ag). IL-7-induced LAK activity is independent of IL-2 secretion, because it is not abrogated by IL-2 antisera. The LAK precursor responding to IL-7 stimulation is enriched in the null cell fraction as has been demonstrated for IL-2-induced LAK cells. TGF-beta and IL-4 interfere with generation of LAK activity by IL-7. Anti-IL-4 antiserum enhances IL-7-induced LAK activity and augments induction of surface marker expression by IL-7. This may be indirect evidence that IL-7 stimulation leads to induction of IL-4 activity. Our results describe the activation of mature lymphoid cells by IL-7. This and the previously described role of IL-7 in lymphohemopoiesis makes it a cytokine of potential therapeutic value for treatment of immunodeficiency states and possibly the immunotherapy of cancer.

The inhibitory effect of human interferon alpha on the generation of lymphokine-activated killer activity.

The generation of lymphokine-activated killer (LAK) activity and the proliferative response to human recombinant interleukin-2 (IL-2) were significantly reduced by the presence of human recombinant leukocyte interferon (IFN alpha) in cultures of human peripheral blood mononuclear cells (PBMC). Mature natural killer (NK) cells can be depleted from PBMC with the toxic lysosomotropic agent L-leucine methyl ester. The generation of cytotoxic cells from lymphocytes depleted in leucine methyl ester was also inhibited by indicating that the IFN-alpha effect is not limited to mature cytotoxic NK cells. Depletion of adherent cells from PBMC did not affect the suppression of LAK induction by IFN-alpha. Surface marker analyses of Tac antigen and transferrin receptor (TfR) showed that the presence of IFN alpha throughout the culture period significantly suppressed the typical increase in IL-2-induced Tac- and TfR-positive cells. In contrast, IFN alpha treatment before and after IL-2 culture enhanced LAK cytotoxic activity. Therefore, combinations of these biological response modifiers for clinical use should take into account the dual effect of IFN alpha on key features of the IL-2 response.

Corticosteroids inhibit the generation of lymphokine-activated killer activity in vitro.

In phase-I clinical trials of adoptive immunotherapy using lymphokine-activated killer (LAK) cells plus recombinant interleukin-2 (rIL-2) (Cetus) for the treatment of malignant glioma, we observed that blood mononuclear cells (MNC) from patients dependent on dexamethasone for management of cerebral edema produced substantially less LAK activity as compared to MNC of normal blood donors or glioma patients not receiving steroid therapy. Therefore, we examined the in vitro effects, brought about by therapeutically attainable concentrations of various corticosteroids, on the proliferative response, production of gamma interferon (IFN-gamma), and induction of LAK activity from blood MNC of normal donors. Incubation in media containing rIL-2 (1000 U/ml) with either dexamethasone, hydrocortisone, methylprednisolone, or prednisolone profoundly affected all of these parameters. First, while 0.01 micrograms/ml of either dexamethasone or hydrocortisone caused a slight enhancement of the mitogenic response of lymphocytes to phytohemagglutinin, a dose-dependent decline occurred as concentrations increased to 10 micrograms/ml. The addition of prednisolone and methylprednisolone elicited a dose-dependent inhibition of lymphocyte proliferation over the entire concentration range tested. At 0.1 microgram/ml or higher, dexamethasone, hydrocortisone, methylprednisolone and prednisolone significantly (P less than 0.02) inhibited the production of IFN-gamma: respectively 18.9%, 4.4%, 2.2%, and 12.3% of the IFN-gamma produced by MNC in the absence of steroids. All four corticosteroids inhibited the induction of LAK activity. Compared to MNC that had been incubated with 1000 U/ml rIL-2 alone, MNC cultured with rIL-2 and 10 micrograms/ml either dexamethasone or prednisolone demonstrated significantly lower cytotoxicity (P less than 0.05) for the natural-killer-cell-resistant cell line, Daudi. Culturing MNC with hydrocortisone had a more dramatic result, causing a significant decline (P less than 0.01) in lytic activity at both 1.0 micrograms/ml and 10 micrograms/ml, while incubation with methylprednisolone produced a significant drop (P less than 0.02) in LAK-mediated cytotoxicity at 0.1 micrograms/ml as well as 1.0 micrograms/ml and 10 micrograms/ml. When cytotoxicity was expressed as lytic units per million effectors, a dose-response decline in lytic activity was once again apparent, with hydrocortisone, methylprednisolone and prednisolone showing significant inhibition (P less than 0.05) at both 1.0 micrograms/ml and 10 micrograms/ml and dexamethasone at 10 micrograms/ml (P less than 0.01). These results indicate that corticosteroids commonly used in the management of cere

Obligatory role of IFN-gamma in induction of lymphokine-activated and T lymphocyte killer activity, but not in boosting of natural cytotoxicity.

The biological role of the murine IFN-gamma endogenously secreted during cell activation has been probed by the use of a rat mAb (AN18) that specifically neutralizes its activity. When An18 mAb is added to the cultures of BALB/c and C57BL/6 nylon nonadherent spleen cells (naSpc) stimulated for 18 h with 1000 U of IL-2, the normally released IFN-gamma can no longer be detected in the supernatants and the IL-2-induced proliferative response is markedly reduced as compared with control cultures set up in the presence of an unrelated rat mAb. By contrast, the increased natural cytotoxicity is not affected. The 96-h culture of both BALB/c and C57BL/6 naSpc in the presence of 1000 U of IL-2 resulted in marked lymphocyte proliferation and generation of lymphokine-activated killer activity. The presence of An18 mAb strongly inhibited both functions. Similarly, when naSpc were stimulated with mitomycin C-inactivated allogeneic leukocytes in the presence of An18 mAb, the normal proliferative response and specific cytotoxicity were almost abolished, whereas the secretion of IL-2 was in no way affected. Lymphocytes recovered from 3-day cultures stimulated by IL-2 or allogeneic cells in the presence of An18 mAb displayed a decrease of the expression of the p55 chain of IL-2R, as shown by flow cytofluorimetry. Moreover, binding experiments with 125I-labeled IL-2 showed that lymphocytes from allostimulated cultures set up in the presence of An18 mAb display a decreased number of low affinity and almost no high affinity IL-2R as compared with control cultures. These data show that endogenous IFN-gamma plays an obligatory role for the de novo induction of cytolytic activity in lymphokine-activated killer cells and CTL, most probably by affecting the membrane expression of high affinity IL-2R.

Transforming growth factor-beta 1 (TGF-beta 1) and recombinant human tumor necrosis factor-alpha reciprocally regulate the generation of lymphokine-activated killer cell activity. Comparison between natural porcine platelet-derived TGF-beta 1 and TGF-beta 2, and recombinant human TGF-beta 1.

We have investigated the ability of porcine-platelet-derived transforming growth factor-beta 1 (TGF-beta 1) to inhibit the generation of lymphokine-activated killer (LAK) cells by human rIL-2. The results demonstrate that TGF-beta 1, in a dose-related manner, significantly inhibits rIL-2-induced LAK cell activity against Daudi and COLO target cells and, to a lesser degree, against K-562 cells. Maximal inhibition was obtained by the addition of TGF-beta 1 at the time of culture initiation and, to a lesser degree, on day 1. Only minimal inhibition was obtained when TGF-beta 1 addition was delayed until day 2 of culture or when added directly into the LAK cell assay. Additional studies demonstrated that porcine platelet-derived TGF-beta 2 and human rTGF-beta 1 inhibited LAK cell generation similar to that obtained with TGF-beta 1. The inhibition of LAK cell activity by TGF-beta 1 was reversed by the addition of human rTNF-alpha at the initiation of culture. In addition, rTNF-alpha synergized with suboptimal levels of rIL-2 in the generation of LAK activity. After stimulation with rIL-2, LAK cells produced significant levels of IFN-gamma, TNF-alpha, and TNF-beta. TGF-beta 1 inhibited the production of these cytokines in a dose-related manner. The results extend the previous known activities for human rTNF-alpha and TGF-beta 1 and further demonstrate the reciprocal relationship between these two molecules in the regulation of certain immune functions.

Surface markers of human lymphokine-activated killer cells and their precursors. Analysis at the population and clonal level.

Lymphokine-activated killer (LAK) activity was first analyzed on PBL populations fractionated on the basis of the expression of T11 or T3 antigen. LAK cell precursors were found to be present in both T11+ and T11- populations, but only in the T3- cell fraction. The generation of LAK activity in highly purified T3- populations of PBL was not accompanied by expression of T3 antigen during a 5-day culture period. LAK activity was next analyzed at the level of limiting dilution clonal microcultures. T11+T3- and T11+T3+ cells, cloned under optimal culture conditions, gave a frequency of proliferating cells of approximately 1 cell in 1.25 for T11+T3+ and 1 cell in 10 for T11+T3- cells. Clones were screened for their ability to lyse fresh ovarian carcinoma cells and K562 target cells. The majority of LAK clones were derived from the T11+T3- cells; moreover, most of the clones derived from these cells displayed LAK activity. Clones displaying LAK activity lysed a panel of fresh or cultured tumor target cells, but failed to lyse PHA-activated lymphoblasts. Surface marker analysis indicated that all the clones had maintained the original T11/T3 phenotype. Whereas 2 T3+ selected LAK clones expressed the T8+T4- phenotype, only 1 out of 9 T3- clones was T8+T4-, all the others lacking both T4 and T8 antigens.

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 MLA 144 cell line (MLA 144CM) 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.