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.     

Lysosome rich cells contain the lytic activity of lymphokine-activated killer cell populations

Summary Little is known regarding the effectors of lymphokine-activated killer activity. Lysosomotropic agents such as quinacrine can be used to positively sort for lysosome rich cells in natural killer (NK) cell populations. We therefore decided to use this agent to sort lymphokine-activated killer (LAK) cells to characterize their lysosomal content. We found that the positively sorted population contained all the LAK activity, i.e., lysis of NK-resistant tumor cells (B16 melanoma cell line), with the negatively sorted cells having no killing activity. Therefore separation of interleukin-2-incubated cells for LAK activity could be accomplished using sorting after quinacrine staining. The treatment of positively sorted LAK cell populations with L-leucine methyl ester, a lysosomotropic dye which inhibits killing by lysosome rich cells, caused abrogation of killing of the B16 tumor by the treated populations. Single cell conjugate assays were also done on these sorted cells, with positively sorted cells forming the highest and negatively sorted cells the lowest percent of conjugates. Our data therefore indicates the important role of lysosome rich cells in the LAK cell population in the murine system.This work was supported by NIH grants R01 CA42962 and K04 CA0122, and by intramural funds from the Norris Cancer Center     

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.     

Leukoregulin up-regulation of tumor cell sensitivity to natural killer and lymphokine-activated killer cell cytotoxicity

The present investigation demonstrates that leukoregulin, a cytokine secreted by natural killer (NK) lymphocytes up-regulates the sensitivity of tumor cells to lymphokine-activated killer (LAK) cell cytotoxicity. It has been previously established that leukoregulin increases the sensitivity of sarcoma, carcinoma and leukemia cells to natural killer (NK) cell cytotoxicity. Tumor cells were treated with leukoregulin for 1 h at 37 degrees C and tested for sensitivity to NK and LAK cytotoxicity in a 4-h chromium-release assay. NK-resistant Daudi, QGU and C4-1 human cervical carcinoma cells became sensitive to NK cytotoxicity after leukoregulin treatment, and their sensitivity to LAK was increased two- to sixfold. Y-79 retinoblastoma cells, which are moderately sensitive to NK and very sensitive to LAK, became increasingly sensitive (two- to four-fold) to both NK and LAK cell cytotoxicity. Recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), recombinant interleukin-1 (alpha and beta), recombinant interferon gamma, recombinant tumor necrosis factor or combinations of the latter two failed to up-regulate tumor cell sensitivity to NK and LAK cell cytotoxicity. However, treatment with recombinant interferon gamma for 16-18 h, GM-CSF and interleukin-1 beta for 1 h induced a state of target cell resistance to both NK and LAK cell cytotoxicity. Leukoregulin may have an important physiological function in modulating NK and LAK cell cytotoxicity by increasing the sensitivity of target cells to these natural cellular immunocytotoxicity mechanisms.     

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).     

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.     

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.     

Natural killer and lymphokine-activated killer cell functions in chronic myeloid leukemia

Summary The natural killer (NK) and lymphokine-activated killer (LAK) cell activities of peripheral blood lymphocytes from chronic myeloid leukemia (CML) patients in remission and from healthy donors have been studied. Regression analysis to compare both cytotoxic responses in individual donors and the frequency of LAK cell precursors was also carried out. About 42% of CML patients in remission showed low NK activity (less than the mean percentage NK activity of healthy donors — 2 SD) and were categorised as low NK responders. The stage of remission or the drugs used to bring about remission did not influence the NK status. The LAK activity of low NK as well as normal NK responder CML patients was significantly low against the NK-sensitive K562 cell line and the NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumor targets, as assessed by linear regression analysis. Allogeneic leukemic cells were more resistant to killing, especially by patients' LAK cells. The frequency analysis of LAK cell precursors revealed a significant reduction in the LAK cell progenitor frequency in CML patients in remission.     

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     

Antiviral effect of lymphokine-activated killer cells: characterization of effector cells mediating prophylaxis

Lymphokine-activated killer (LAK) cells generated by cultivation of C57BL/6 mouse spleen cells in the presence of recombinant interleukin-2 were transferred into natural killer (NK) cell-deficient suckling mouse recipients. These mice were then challenged with either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis (LCMV) and sacrificed 3 days later. No interleukin 2 infusions were given. Mice receiving as few as 5 x 10(5) LAK cells had several 100-fold decreases in spleen MCMV titers as compared with untreated mice. This treatment had no effect on spleen LCMV titers. The LAK cell cultures contained 10 to 17% NK 1.1+, 50 to 55% Lyt-2+, and 33 to 50% immunoglobulin D+ cells. Double fluorescence labeling and in vitro cytotoxicity assays with fluorescence-activated cell sorting revealed at least two mutually exclusive killer cell populations. NK 1.1+ LAK cells resembled freshly isolated activated NK cells with regard to target cell range (YAC-1 cell killing greater than L-929, P815, and EL-4 cell killing), large granular lymphocyte (LGL) morphology, and decreased ability to lyse interferon (IFN)-treated target cells. Lyt-2+ LAK cells lysed the targets mentioned above but at lower levels and without the differences in susceptibility mentioned above. These Lyt-2+ LAK cells also had a decreased ability to lyse IFN-treated targets, in contrast to classic cytotoxic T lymphocytes, which lyse IFN-treated targets far more efficiently than untreated targets. Purified populations of LAK cells obtained by fluorescence-activated cell sorting were used in the antiviral protection model. The results showed that protection against MCMV could be mediated by NK 1.1+, NK 1.1-, Lyt-2+, Lyt-2-, and IgD- populations but not by IgD+ cells. The five protective populations all had in common the LGL phenotype and cytotoxic activity in vitro. The IgD+ population did not contain LGLs, lyse target cells in vitro, or mediate an antiviral effect in vivo. These results suggest that LAK cells may be therapeutically useful against certain virus infections (MCMV) but not others (LCMV) and that despite their heterogeneity in antigenic phenotype and cytotoxic activity, their pattern of antiviral activity in vivo resembles that of NK cells, which protect against MCMV but not LCMV.     

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     

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.     

Induction of LAK-like cells in the peritoneal cavity of mice by inactivated Candida albicans

We have investigated the effect of multiple administrations of inactivated Candida albicans (CA) cells on induction of non-MHC-restricted antitumor cytotoxic responses both in normal and congenitally athymic (nude) mice. Intraperitoneal inoculation of CD2F1 mice with five doses of 2 x 10(7) CA cells over a 2-week interval was associated with the induction of peritoneal exudate cells (PEC) that mediated natural killer cell activity. These cells, in contrast to those elicited by a single dose of CA, killed both NK-sensitive and NK-resistant tumor target cells in vitro. This broad-spectrum, antitumor cytotoxicity peaked 1 day after the last injection of CA, and decreased to control values within 6 (NK-resistant) or 14 (NK-sensitive target cells) days. Cytotoxicity could be recalled to a high level by a boosting injection of CA or a major mannoprotein-soluble antigen (MP) from the Candida cell wall, given 30 days after multiple CA treatment. Upon a 24-hr in vitro incubation, CA-induced peritoneal immunoeffectors lost their killing activity unless human recombinant interleukin-2 (rIL-2) was added to cultures. The non-MHC-restricted cytotoxic PEC activity induced by CA was mainly associated with nonadherent, nonphagocytic large granular lymphocytes (LGL) which exhibited the following phenotypes: (i) asialo GM1+, Lyt 2.2-, and partially Thy 1.2+ (effectors active against NK-sensitive targets) and (ii) asialo GM1+, Lyt 2.2-, and Thy 1.2+ (effectors active against NK-resistant targets). Nude mice also responded to multiple CA inoculations by displaying high cytotoxic activity against NK-sensitive targets and significant cytotoxicity against NK-resistant targets. This cytotoxicity could be recalled on Day +30, and the cytotoxic effectors involved were highly sensitive to anti-asialo GM1 plus complement treatment. Overall, the results add further experimental evidence to the wide range of immunomodulatory properties possessed by C. albicans, and demonstrate that the majority of antitumor cytotoxic activity induced by fungal cells was due to lymphokine-activated killer (LAK)-like effectors.     

Heterogeneity of the lymphokine-activated killer cell phenotype.

Lymphokine-activated killer cells (LAK) are functionally defined by their ability to mediate the MHC-unrestricted lysis of a range of tumor targets, while sparing normal cells. They can also lyse TNP-modified normal syngeneic lymphoblasts. We show here that lysis of TNP-modified targets is mediated by CD8+ LAK in a self-MHC-restricted manner, whereas lysis of tumor targets is largely by CD8- LAK and is MHC-unrestricted. LAK generated from the immune-deficient strains Balb/c nude and C.B-17 scid lyse tumor targets as effectively as LAK from normal mice but do not lyse TNP-modified normal targets. Further, lysis of TNP-modified targets, but not tumors, can be inhibited by antibody to the T cell receptor complex. These experiments strongly suggest that recognition of TNP-modified targets is not accomplished by the same mechanism as that of tumors. Rather, they are consistent with recognition of TNP-modified targets by CD8+ LAK cells being mediated via recognition through the T cell receptor.     

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.     

The nonclassical major histocompatibility complex molecule Qa-2 protects tumor cells from NK cell- and lymphokine-activated killer cell-mediated cytolysis

The cytotoxic activity of NK cells is regulated by class I MHC proteins. Although much has been learned about NK recognition of class I autologous targets, the mechanisms of NK self-tolerance are poorly understood. To examine the role of a nonpolymorphic, ubiquitously expressed class Ib Ag, Q9, we expressed it on class I-deficient and NK-sensitive B78H1 melanoma. Presence of this Qa-2 family member on tumor cells partially protected targets from lysis by bulk lymphokine-activated killer (LAK) cells. H-2K(b)-expressing B78H1 targets also reduced LAK cell activity, while H-2D(b) offered no protection. Importantly, blocking with F(ab')(2) specific for Q9 or removal of this GPI-attached molecule by phospholipase C cleavage restored killing to the level of vector-transfected cells. Experiments with LAK cells derived from H2(b) SCID and B6 mice established that NK1.1(+)TCR(-) NK and NK1.1(+)TCR(+) LAK cells were the prevalent cytolytic populations inhibitable by Q9. Treatment of mice with poly(I:C) also resulted in generation of Q9-regulated splenic cytotoxicity. LAK cells from different mouse strains responded to Q9, suggesting that the protective effect of this molecule is not detectably influenced by Ly49 polymorphisms or the presence/absence of Q9 in NK-harboring hosts. We propose that Q9 expressed on melanoma cells serves as a ligand for yet unidentified NK inhibitory receptor(s) expressed on NK1.1(+) NK/T cells.     

High expression of NKR-P1 is not an absolute requirement for natural killer activity in BDIX rats

 NKR-P1 has been identified as a triggering structure selectively expressed on rat natural killer (NK) cells and adherent lymphokine-activated killer (A-LAK) cells. In vivo treatment with anti-NKR-P1 monoclonal antibody (mAb 3.2.3) was shown to induce complete inhibition of NK cytotoxicity and elimination of LAK cell precursors in Lewis and Fisher rat strains. We investigated the effects of mAb 3.2.3 in a colon tumor model in BDIX rats. Inoculation of animals with mAb 3.2.3 even at very high doses induced a strong but incomplete inhibition of NK cytotoxicity in nylon-wool-non-adherent spleen and peripheral blood cells. Generation of adherent A-LAK cells from their spleen precursors was also strongly but not fully inhibited. We also investigated the effect of treatment with mAb 3.2.3 on the tumorigenicity of the NK-sensitive REGb cell line. When subcutaneously inoculated in syngeneic animals, REGb cells induce tumors that first grow for 2 weeks, then spontaneously regress and disappear. In contrast with previous results using anti-asialoGM1, no significant difference in tumor growth was observed between rats treated with mAb 3.2.3 and control animals, even with a long-term treatment. In vitro, mAb 3.2.3 exhibited the same incomplete efficiency. Nylon-wool-non-adherent spleen cells treated with mAb 3.2.3 plus complement were completely free of 3.2.3^bright cells, but retained a substantial NK activity and generated LAK cells after culture with IL-2. After an overnight incubation in standard medium of 3.2.3-depleted spleen cells, 3.2.3^bright cells were partially recovered and the NK cytotoxic activity, as well as the generation of LAK cells, was significantly enhanced. These results suggest that a strong expression of NKR-P1 is not required for BDIX mononuclear cells to exhibit NK function and generate LAK cells under IL-2 activation. Received: 11 July 1995 / Accepted: 16 November 1995     

Mouse tumors are heterogeneous in their susceptibility to syngeneic lymphokine-activated killer cells and delineate functional subsets in such effectors

We have analyzed whether lymphokine-activated killer (LAK) cells, generated from C57BL/6J (B6) spleen cells at different times after recombinant interleukin-2 (rIL-2) culture, could be heterogeneous in their ability to lyse a variety of tumor targets. When tested 3 days after exposure to 250 U/ml rIL-2 (day-3 LAK cells) a significant lysis was detected with the natural-killer(NK)-sensitive YAC lymphoma, the NK-resistant P815 mastocytoma, three different syngeneic melanomas and a syngeneic fibrosarcoma (group 1 targets), whereas no lysis was observed with a reticulum cell sarcoma, two different lymphomas or concanavalin A blasts, all of B6 origin (group 2 targets). LAK cells cultured for 5 days, however, lysed group 2 targets and showed a parallel increase of cytotoxic activity against group 1 targets. At day 7, LAK activity declined on all targets examined. In cold-target inhibition studies, the lysis of group 1 tumor targets by day-3 or day-5 LAK cells could be inhibited only by group 1 and not by group 2 unlabelled tumor cells. All group 1 tumors could effectively compete each other. Conversely, the lysis of group 2 tumor targets by day-5 LAK cells was inhibited by both group 1 and group 2 targets. These data indicate the presence of separate LAK effectors that appear to arise with different time kinetics and have different recognition structures. In vitro antibody depletion at the effector level showed that day-3 LAK cells with cytotoxic activity against group 1 tumors were ASGM1+. Day-5 LAK cells included both ASGM1+ and Lyt2+ effectors and both populations, although to a different extent, contributed to the lysis of all targets. Our results indicate that LAK cells are functionally heterogeneous. This heterogeneity is defined by their susceptible target cells and cannot be ascribed to different (Lyt2+ versus ASGM1+) lineages.     

Analysis of the murine lymphokine-activated killer (LAK) cell phenomenon: dissection of effectors and progenitors into NK- and T-like cells

Murine as well as human lymphokine-activated killer (LAK) cells have been reported to have several characteristics of T lymphocytes and to be clearly distinct from natural killer (NK) cells. The present study of murine LAK cells showed that cytotoxic cells generated in the presence of interleukin 2 IL 2 were heterogeneous with respect to cell surface markers of progenitor as well as effector cells. Negative selection of cells with antibodies and complement or positive selection by fluorescence-activated cell sorting unequivocally showed that LAK effector cells consisted of at least two clearly distinct populations, the relative contribution of which was dependent on donor organ and target cells studied. Approximately 40% of the cytotoxic activity of spleen-derived effector cells active against the NK-resistant targets EL-4 or MCA-5 was eliminated by treatment with antibodies to the NK-markers asialo-GM1 and NK 1 (NK-LAK). Approximately 60% of cytotoxic activity was associated with cells expressing the T cell marker Lyt-2, lacked NK 1, and was lacking or expressed only small amounts asialo-GM1 (T-LAK). The NK-LAK cells were of greater importance for the cytotoxic activity against the standard NK target YAC-1, although T-LAK cells also excerted significant cytotoxicity against this cell line. Limiting dilution analysis estimated that the minimal frequency of precursors developing into cells with cytotoxic activity against EL-4 was 1/6700 in spleen and 1/4200 in peripheral blood. The frequency of cells developing into cytotoxic effectors against YAC-1 cells was 1/3700 and 1/1450 in spleen and peripheral blood, respectively. Depletion of progenitor cells from spleen or peripheral blood expressing NK 1 or Lyt-2 by treating the cells with antibodies to these structures and complement indicated that NK-1-expressing cells were the dominating progenitor of the LAK cells irrespective of target cells used. Culture of murine lymphoid cells from spleen or peripheral blood with high concentrations of IL 2 results in the emergence of two different killer cell populations with phenotypic similarities to NK and T cells, respectively, both being able to kill targets resistant to resting NK cells. In contrast to numerous earlier reports, we concluded that LAK cells are heterogeneous with respect to surface markers, with a major population of LAK cells apparently representing IL 2-activated cells expressing cell surface markers associated with NK cells.     

Phenotypic and functional analysis of lymphokine-activated killer (LAK) cell clones

Summary Lymphokine-activated killer (LAK) cells are generated by the culture of peripheral blood lymphocytes with interleukin-2 (IL-2). A variety of cells, including T-lymphocytes and natural killer (NK) cells, can be activated by IL-2 to exhibit the ability to kill multiple tumor and modified-self targets. Recent reports indicate that culture conditions can determine the phenotype of cells expressing LAK activity. Using limiting dilution techniques, we first generated cloned LAK cells with three culture conditions: autologous human serum (AHS)+IL-2; AHS+IL-2+0.1 g/ml phytohemagglutinin and fetal bovine serum and IL-2. We determined that all but one of the 47 LAK cell clones generated with the three culture conditions were CD3⁺ and T-cell like; one NK-like clone was observed. Clones that were cytotoxic for one target could generally kill multiple targets, and the absence of phytohemagglutinin did not significantly affect the ability of the LAK cell clones to kill multiple targets. The presence of phytohemagglutinin was, however, necessary for the long-term maintenance of proliferation and cytotoxic activity of the LAK cell clones. The mechanism by which LAK cells kill tumor targets is not known. We here demonstrate that LAK cells and LAK cell clones can produce interferon- and tumor necrosis factor (TNF) when stimulated with an erythroleukemia cell, K562. Five of the six CD3⁺, LAK cell clones tested could be stimulated by K562 cells to produce both interferon- and TNF. However, the ability of the cloned LAK cells to kill K562 cells, as measured in a 4-h ⁵¹Cr-release assay, did not correlate with their ability to produce these cytokines. Furthermore, specific antibodies that neutralize the cytotoxic activity of interferon- and TNF did not inhibit killing of K562 cells by LAK cells as measured with a 4-h cytotoxic assay. The cytostatic and cytotoxic activities of interferon- and TNF for tumor cells are well documented, but these cytolytic activities are slower acting and exhibit their maximum effect after 48–96 h. We here propose that LAK cells kill tumor targets by a combination of cell-to-cell-mediated killing and by the release of slower acting cytostatic/cytotoxic cytokines that can inhibit the growth of tumors some distance from the effector cells.This work is supported in part by grants from the Arizona Disease Research Commission (3364-000000-1-1-AP-6621) and the National Institutes of Health (Grants GM 34121, CA-17094 and CA-23074)