Lymphokine-activated killer cells are generated before classical cytotoxic T lymphocytes after bone marrow transplantation in mice

Lymphokine-activated killer (LAK) cells are demonstrable within 2 wk after syngeneic or allogeneic (H-2-compatible) bone marrow transplantation in mice. Classical cytotoxic T lymphocytes (CTL) are not active until at least 4 wk after transplant. Both LAK cells and CTL bear the Thy-1 marker and do not possess the murine natural killer cell marker asialo GM.     

Resistance of lymphokine-activated T lymphocytes to cell-mediated cytotoxicity

We observed that lymphokine-activated T lymphocytes, obtained in short- and long-term cultures following stimulation with recombinant interleukin-2 (rIL-2), are resistant to cell-mediated cytotoxicity. In particular, lymphokine-activated killer (LAK) cells do not undergo self-lysis or lysis by alloreactive cytotoxic T lymphocytes (CTL), in line with recent reports concerning CTL clones. Similar findings were further confirmed in a lectin-dependent cell cytotoxicity assay. LAK cell lysis resistance was not due to an inability to recognize itself, since inactivated LAK cells used as cold competitors inhibited tumor cell lysis in a dose-dependent manner. In contrast, the addition on Day 0 of irradiated LAK cells or alloreactive CTL, as well as a CTL clone having LAK-like activity to rIL-2-stimulated cultures abrogated or strongly reduced LAK cell generation. Therefore, LAK cell precursors were most likely susceptible to the lytic activity of differentiated cytotoxic cells, as no inhibition was detected when cell to cell contact was prevented by using a diffusible chamber culture system. These findings, on the whole, suggest that the emergence of the lysis-resistant phenotype is most likely the result of a selective process occurring in vitro that leads to the elimination of lysis-susceptible lymphocytes present in culture.     

Transforming growth factor-beta inhibits the in vitro generation of lymphokine-activated killer cells and cytotoxic T cells

Summary The effect(s) of purified transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) on the induction and function of lymphokine-activated killer (LAK) cells and cytotoxic T lymphocytes (CTL) was examined. The addition of TGF-beta, but not PDGF, to cultures containing fresh C57BL/6 mouse splenocytes or human peripheral blood lymphocytes plus recombinant interleukin-2 markedly inhibited the development of mouse and human LAK cell activity (measured after 3 days for cytotoxicity against cultured or fresh tumor targets in 4-h ⁵¹Cr release assays). The addition of TGF-beta, but not PDGF, to a one-way, C57BL/6 anti-DBA/2, mixed lymphocyte reaction effectively blocked the generation of allospecific CTL as well. However, TGF-beta did not inhibit the effector function of LAK cells or of allospecific CTL when added directly to the short-term cytolytic assay. A second form of homodimeric TGF-beta, type 2, was also found to be suppressive on the development of murine LAK cells and allospecific CTL. Collectively, these data demonstrate that the peptide TGF-beta is a potent inhibitor of LAK cell and CTL generation in vitro.     

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).     

Characterization of effector-target conjugates for cloned human natural killer and human lymphokine activated killer cells by flow cytometry.

The present studies demonstrate that the intracellular fluorochromes calcein and hydroethidine can be used for quantification of effector-target conjugates involving cloned human natural killer (NK) or interleukin-2 (IL-2) activated human lymphokine activated killer (LAK) cells by dual color flow cytometry without potential artifacts that might result from extensive modification of effector and/or target cell membranes. Cloned NK cells and LAK cells form conjugates with cultured cell lines regardless of susceptibility to lysis. The strength of the interactions in these conjugates was investigated using a variable speed vortexer. Even relatively gentle vortexing disrupted most conjugates involving fresh human peripheral blood lymphocytes (PBL) but only about one-fourth of conjugates between K-562 cells and human PBL that had been cultured with or without IL-2 by this treatment. The rate of conjugate formation for LAK cells was determined to be about 3 times faster than for cloned NK cells, and both rates are considerably faster than the reported rate of formation of cytotoxic T lymphocyte (CTL) target conjugates. The differences in the rate of conjugate formation are apparently not related to target cell specificity, since LAK cells form conjugates with susceptible and resistant cell lines at comparable rates. When effector-target conjugates are incubated at 37 degrees C in the absence of calcium--thereby precluding lysis--the percentage of conjugated LAK or cloned NK cells decreases logarithmically with time. These results suggest that an initial equilibrium between free and conjugated lymphocytes gradually shifts in favor of unconjugated cells.     

Subcellular localization of perforin and serine esterase in lymphokine-activated killer cells and cytotoxic T cells by immunogold labeling

CTL, NK cells, and lymphokine-activated killer (LAK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by forming large pores on the plasma membrane of the target cell. Other proteins besides perforin are found in the cytoplasmic granules of effector lymphocytes, and these include a family of serine esterases. Ultrastructural immunogold labeling studies with antibodies against perforin and a serine esterase (MTSP-1, also known as granzyme A and SE-1) show that all the granules of LAK cells and a CTL cell line contain perforin and serine esterase. For both LAK cells and CTL, perforin has been located mostly in the fine granular matrix of the granules, whereas gold particles corresponding to serine esterase have been found in both the matrix and the cap regions of the granules. Results from double immunogold labeling indicate that perforin and serine esterase colocalize to the same granules.     

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.     

Studies on cytotoxicity generated in human mixed lymphocyte culture

Summary High levels of cytotoxic activity against the natural killer (NK) cell-sensitive target K562 and the NK-resistant target UCLA-SO-M14 (M14) can be generated in vitro either by mixed lymphocyte culture (MLC) or by culture of lymphocytes in interleukin 2 (IL2) (lymphokine activated killer (LAK) cells). The purpose of this study was to identify similarities and differences between MLC-LAK and IL2-LAK cells and allospecific cytotoxic T cells. Induction of cytotoxicity against K562 and M14 in both culture systems was inhibited by antibodies specific either for IL2 or the Tac IL2 receptor. Like NK effector cells, the precursors for the MLC-LAK cells were low density large lymphocytes. However these precursors differed from the large granular lymphocytes that mediated NK cytolysis in sensitivity to the toxic lysosomotropic agent L-leucine methyl ester (LME). The resistance of the MLC-LAK precursors to LME indicated that the precursors included large agranular lymphocytes. Although interferon-gamma (IFN-gamma) is produced in MLC and in IL2 containing cultures, it is not required for induction of either type of cytotoxic activity. Neutralization of IFN-gamma in MLC-and IL2-containing cultures with specific antibodies had no effect on the induction of cytotoxic activities. Both allospecific cytotoxic T lymphocyte (CTL) and LAK activities were enhanced by IL2 and IFN-gamma at the effector cell stage. However, the mechanism of cytolysis was different in the two systems. NK- and MLC-induced LAK activities were independent of CD3-T cell receptor complex while CTL activity was blocked by monoclonal antibodies specific for the CD3 antigen. These results suggest that NK and the in vitro induced LAK cytotoxicities are a family of related functions that differ from CTL. Furthermore, MLC-induced and IL2-induced cytotoxicities against K562 and M14 appear to be identical.This work was supported by NIH grant CA34442     

A homogeneous population of lymphokine-activated killer (LAK) cells is incapable of killing virus-, bacteria-, or parasite-infected macrophages

Previous reports have suggested a role for natural killer (NK) cells in directly lysing host cells infected with bacteria and other intracellular microorganisms. Here, we determined the inability of a highly homogeneous population of lymphokine activated killer (LAK) cells to kill macrophages infected with the following intracellular parasites: Mycobacterium avium, Listeria monocytogenes, Legionella pneumophila, Toxoplasma gondii, and Trypanosoma cruzi. In parallel cytotoxicity assays, LAK cells lysed the tumor targets YAC-1 and P815 effectively. Furthermore, we were able to demonstrate that influenza-specific cytotoxic T lymphocytes (CTL), but not LAK cells, were efficient killers of influenza virus-infected macrophages.     

Cytolytic and biochemical properties of cytoplasmic granules of murine lymphokine-activated killer cells

The incubation of murine spleen cells in the lymphokine interleukin 2 (IL 2) gives rise to lymphokine-activated killer (LAK) cells capable of lysing fresh tumor cells in short-term lytic assays. During the course of cultures used to generate LAK cells, cytoplasmic granules were prepared and were analyzed for the presence of the cytolysin previously described in large granular lymphocytes (LGL) and cytotoxic T lymphocytes (CTL). Such cytolysin activity is initially undetectable, appears after 2 days of culture, and continues to increase until day 7. The LAK cytolysin has properties similar to those of previously described cytolysins with respect to nonspecific killing of various target cells, rapid kinetics, and absolute dependence on calcium. Antibodies raised against purified LGL tumor granules neutralized the activity of the LAK cytolysin. The precursors of both the LAK cells and the cells bearing the cytolysin are eliminated by treatment with anti-asialo-GM1 and complement, strongly suggesting that the actual LAK effector cells and the cytolysin-bearing cells are identical. Biochemical analysis of the LAK granules indicate that they contain the lysosomal enzyme arylsulfatase. The protein content of granules isolated from various days of culture with r-IL 2 undergoes a dramatic change, with major protein bands around 30,000 daltons becoming prominent, as well as the cytolysin protein band at 70,000 daltons. These data suggest that the mechanism of cell lysis by LAK cells is similar to that of CTL and natural killer-mediated lysis, and each of these forms of lymphocyte-mediated cytolysis is based on a granule exocytosis mechanism.     

Lymphokine-activated killer (LAK) cells. II. Delineation of distinct murine LAK-precursor subpopulations

Lymphokine-activated killer (LAK) cells can lyse a number of tumor target cells regardless of whether the tumors are natural killer (NK) sensitive or resistant. LAK can also lyse autologous lymphoblasts that have been modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS). In this study, we examined the surface markers of murine LAK precursors. It was found that depletion of Thy 1- or Lyt 2-bearing precursor cells abolished the ability of spleen cells to generate LAK against TNBS-self, but had no effect on the generation of LAK against tumor cells. Depletion of asialo-GM1 (AGM1)-bearing precursors abolished the generation of LAK against all target cells tested. Normal spleen cells were fractionated on a Percoll density gradient and two fractions were examined: fraction (Fxn) 3, which is enriched for NK activity but depleted of the ability to generate cytotoxic T lymphocytes (CTL), and Fxn 5, which had no NK activity but was enriched for the ability to generate CTL. Both fractions were capable of generating LAK, although Fxn 5 required a relatively larger amount of interleukin 2 (IL 2). Upon examination of the surface markers of LAK precursors in these fractions it was found that the precursors in Fxn 3 giving rise to LAK against tumors were Thy-1-, Lyt-2-, AGM1+, whereas the precursors in Fxn 5 were Thy-1+, Lyt-2+, AGM1+. The precursors generating LAK against TNBS-self were Thy-1+, Lyt-2+, AGM1+ in both fractions. The time kinetics of LAK generation in both fractions were different, with Fxn 3 showing much earlier kinetics. These data delineate at least two different LAK precursors defined by their buoyant density, by their surface markers, and by their susceptible target cells. These data also may resolve the confusion in the literature regarding the phenotype of LAK precursors.     

Augmentative effect of Nocardia rubra cell-wall skeleton (N-CWS) on lymphokine-activated killer (LAK) cell induction

Summary The present study elucidated that N-CWS augments the cytolytic activity against 3LL tumor cells of LAK cells from N-CWS-immunized mice administered i.p. with rIL-2. This augmentative effect of N-CWS was not seen when the LAK cells were prepared from normal mice. The cytolytic activity was predominantly expressed in the NAPC prepared from the site of injection of rIL-2, and repeated administrations of rIL-2 were required to induce and maintain this potent cytolytic activity in vivo. Serological analysis revealed that the LAK cells were positive for Thy 1.2 and asialo GM1 antigens and that they were not classical CTL or NK cells. The administration of rIL-2 statistically prolonged the MST of mice bearing LAK-sensitive 3LL cells but not the MST of mice bearing LAK-resistant EL-4 leukemia. Furthermore, combination therapy with N-CWS and rIL-2 prolonged the MST of the mice more than the therapy with rIL-2 alone. These results suggest that LAK cells potentiated with N-CWS would be useful for immunotherapy of malignant neoplasms. Abbreviations used: N-CWS, Nocardia rubra cell-wall skeleton; rIL-2, recombinant interleukin 2; LAK, lymphokine-activated killer; RPMI 1640, Roswell Park Memorial Institute 1640; FCS, fetal calf serum; TCM, tumor culture medium; PC, peritoneal cells; NAPC, nonadherent PC; APC adherent PC; MST, mean survival time; NK, natural killer; E:T ratios, effector to target ratios; Poly I:C, polyinosinic-polycytidylic acid; CTL, cytotoxic T lymphocytes; RLNC, regional lymphnode cells     

Asialo GM1 as an accessory molecule determining the function and reactivity of cytotoxic T lymphocytes

The expression and function of asialo-GM1 (AsGM1) in alloreactive cytotoxic T lymphocytes (CTL) was studied. We have shown previously that the cytotoxic reactions mediated by AsGM1+-cloned CTL were blocked by anti-AsGM1 or by purified AsGM1. To further determine the role of AsGM1 in CTL-mediated cytotoxicity, we examined the correlation between this blocking effect and the expression of AsGM1 on effector and target cells. Now we found that the blocking by anti-AsGM1 was largely dependent on the expression of AsGM1 on the effector cells in a dose-dependent fashion. The expression of AsGM1 on target cells had only little effect on the blocking of cytotoxic reactions by anti-AsGM1 or AsGM1. A threefold difference was seen in the blocking of AsGM1+ and AsGM1- targets. The observation was in sharp contrast to the effectors as no blocking was ever seen with AsGM1- CTL. Similar to CTL effectors, we found that the expression of AsGM1 and L3T4 were mutually excluded on mitogen-activated T cells, despite the fact that they could coexpress in resting T cells. The expression of AsGM1 on CTL effectors was associated with the antigen-nonspecific natural killer (NK)-like or lymphokine-activated killer (LAK)-like activity exerted by the alloreactive CTL. All AsGM1+ CTL possessed LAK activity against antigen-unrelated tumor targets, and the AsGM1- CTL only displayed antigen-specific alloreactivity. The LAK activity was associated with the expression of AsGM1 on effectors, and was not related to the AsGM1 expression on target cells. These findings indicate that the AsGM1 expressed on alloreactive CTL may function as an accessory molecule for T-cell receptors in the antigen-specific alloreactive cytotoxicity mediated by AsGM1+ CTL. The expression of AsGM1 may also be related to the activation of an NK-like apparatus in these CTL. Therefore, AsGM1 not only may be involved in cytotoxic reactions mediated by AsGM1+ CTL, it may also modulate the specificity of the CTL cytotoxicity.     

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.     

Lymphokine-activated cell-associated antigen involved in broad-reactive killer cell-mediated cytotoxicity

Spleen cells from rats which had been hyperimmunized with mouse lymphokine-activated killer (LAK) cells, were fused with the mouse myeloma cell line, P3 X 63 Ag8.653. Antibodies secreted by 1500 cultures were selected by their blocking effect on LAK cell-mediated cytotoxicity in the absence of complement. Two monoclonal antibodies (KBA4 and KBA6) greatly inhibited the cytotoxic activity of LAK cells, which were induced from mouse spleen cells by culture with recombinant human interleukin 2 (r-IL-2). These antibodies also blocked the cytotoxic activity of natural killer (NK) cells, but activated macrophages (A-M phi) were only slightly sensitive to them. However, no effect of the antibodies on the cytotoxic activity of cytotoxic T lymphocytes (CTL) was detected. These data suggest that the specific antigen, lymphokine-activated cell-associated (LAA) antigen, defined by these monoclonal antibodies may be associated with the recognition mechanisms of broad-reactive killer (BRK) cell-mediated cytotoxicity. The observation that low levels of LAA antigen are distributed in all lymphoid cells and that it was significantly enhanced by treatment of the cells with r-IL-2 suggests that the antigen may be involved in lymphocyte-activation mechanisms. We also found that the LAA antigen consists of two distinct polypeptides with Mr of 180,000 and 95,000 Da, which are similar to that of LFA 1 antigen. However, the biological characteristics of LAA antigen did not coincide with those of LFA 1. Therefore, KBA MAb may recognize a carbohydrate epitope distinct from that of LFA 1.     

Antitumour efficacy of lymphokine-activated killer cells loaded with ricin against experimentally induced lung metastases

Summary Adoptive transfer of tumour-specific T lymphocytes loaded with ricin into tumour-bearing mice exerts a transient therapeutic effect against locally induced tumours [Cerundolo et al. (1987) Br J Cancer 55: 413]. As transferred cells preferentially locate in the lung, we studied the therapeutic effect of ricin-loaded, lymphokine-activated killer (LAK) cells on lung metastases induced by M4 or B16-F1 (F1) tumour cell injection. In vitro studies demonstrated that ricin-treated LAK cells were about 100-fold more efficient than untreated LAK cells in inhibiting growth of the ricin-sensitive M4 tumour cell line. This effect was most likely due to the released ricin, as treated and untreated LAK cells inhibited the relatively toxin-resistant F1 cell line to the same extent. Ricin treatment did not alter the tissue distribution of intravenously (i.v.) injected LAK cells, which selectively localized in the lung early after inoculation, whether or not metastases were present. Adoptive transfer experiments showed that ricintreated LAK cells were significantly more efficient than untreated LAK cells in inhibiting M4- but not F1-induced lung metastases. These results indicate that LAK cells are able to deliver a therapeutic concentration of antineoplastic compounds directly to the lung.Research activities were partially developed in relation to a contract with the National Program of Pharmacological Research (Rif. 078606), by the Italian Consortium for Antitumoral Vectors (C.I.V.A) for the Italia Ministry of University and Scientific and Technological Research     

Lymphokine activated killer cells

Various subpopulations of human leukocytes may be induced by lymphokines to exert cytotoxic activity. In man major histocompatibility complex non-restricted tumor cell lysis by interleukin-2 (IL-2) induced peripheral blood lymphocytes is attributed mainly to natural killer cells. These T cell receptor negative large granular lymphocytes are called lymphokine activated killer (LAK) cells. In order to explore the potential of LAK cells in tumor therapy, several clinical studies have been conducted, using IL-2 alone or in combination with ex vivo IL-2-activated peripheral blood lymphocytes. Objective responses have reproducibly been achieved only in renal cell carcinoma and malignant melanoma and were associated with considerable toxicity. In view of restricted efficacy and increasing doubts as to whether LAK cells indeed account for the in vivo observed responses, more recent strategies focus on tumor antigen specific cytotoxic T cells or tumor infiltrating lymphocytes. Successful translation of this approach into clinical practice, however, may be dependent on some basic problems of tumor immunology to be solved which were thought to be by-passed by the LAK cell approach.     

Additive effects of antitumor drugs and lymphokine-activated killer cell cytotoxic activity in tumor cell killing determined by lactate-dehydrogenase-release assay

Summary The effect of pretreatment with antitumor drugs on lymphokine-activated killer (LAK) cell cytotoxic activity, determined by lactate-dehydrogenase(LDH)-release assay, was investigated. LAK cells were induced by incubating peripheral blood lymphocytes of healthy donors in medium containing interleukin-2 (IL-2) and monoclonal anti-CD3 antibody for 6–7 days. A human lung squamous carcinoma cell line, SQ-5, was used as an adherent target. After 24 h exposure of the target cells to cisplatin, doxorubicin, or mitomycin C, the drugs were washed off and LAK cells were added at an E/T ratio of 5. During further incubation for 48 h, LDH release from cisplatin- or doxorubicin-pretreated target cells was markedly higher than that from non-pretreated target cells. The combination of cisplatin and LAK cells has an additive cytotoxic effect and that of mitomycin C and LAK cells does not; there may also be an additive effect late in the toxicity mechanism between doxorubicin and LAK cells.