Augmentation of the generation of lymphokine-activated killer cells after a single dose of mitomycin C in cancer patients

Summary The effect of mitomycin C administration on the generation of cytotoxic cells, induced by in vitro activation of peripheral blood mononuclear cells (PBM) with interleukin-2, was studied in patients with various carcinomas. The ability of PBM to generate lymphokine-activated killer (LAK) cell activity against Raji cell targets was significantly augmented 5 and 7 days after a single intravenous dose of 12 mg/m² mitomycin C, when compared to that of PBM obtained before mitomycin C injection. Further, LAK cell activity against autologous tumor cells was also significantly increased after the drug administration. The distribution of lymphocyte subsets exhibited a significant increase in the percentage of CD3⁺ cells after injection, with the elevation of the CD4/CD8 ratio. Furthermore, the proportion of the CD4⁺ Leu8⁺ subpopulation, which identifies inducers of suppression, was significantly reduced. Thus, the decrease in the proportion of suppressor-inducer subsets of PBM might be at least partially, responsible for the augmented generation of LAK cells after mitomycin C administration.     

Concentration-dependent collateral sensitivity of cisplatin-resistant gastric cancer cell sublines

The cisplatin-resistant gastric cancer cell sublines, SNU-601/Cis2 and /Cis10, were 49 and >530 times more resistant to cisplatin, respectively, compared with the drug-sensitive cells, SNU-601/WT. The SNU-601/Cis2 showed cross-resistance to carboplatin, heptaplatin, doxorubicin, mitomycin C, and 5-fluorouracil compared with the SNU-601/WT whereas the SNU-601/Cis10 displayed collateral sensitivity to these drugs with the exception of cisplatin compared with the SNU-601/Cis2, suggesting that the cross-resistance and collateral sensitivity of cisplatin-resistant gastric cancer cells are dependent upon cisplatin concentrations. Altered expression of the antioxidant and transporter genes (metallothionein, catalase, superoxide dismutases, P-glycoprotein, and the breast cancer resistance protein) was involved in these phenotypes of the cisplatin-resistant gastric cancer cell lines.     

Lymphokine-activated killer (LAK) and monocyte-mediated cytotoxicity on tumor cell lines resistant to antitumor agents

In this study we have examined the susceptibility of tumor cell lines exhibiting different patterns of resistance to chemotherapeutic agents, to the cytotoxic action of lymphokine-activated killer (LAK) cells and activated monocytes. The susceptibility of tumor cells with pleiotropic drug resistance to these cytotoxic mechanisms was not different from that of their parental, chemo-sensitive cell lines. Tumor lines used in this study included three human cell lines (LOVO N and LOVO/Dx, I-407 and I-407/Dx, MCF7 and MCF7a) selected for being resistant to doxorubicin and showing a pleiotropic pattern of resistance, and the murine ovarian reticulum cell sarcoma M5076 and its variants resistant to individual antitumor agents (cisplatin, cyclophosphamide and 5-aza-2'-deoxycytidine). These results demonstrate that drug-resistant tumor cell lines, irrespective of the pattern of resistance, were susceptible to the in vitro cytotoxicity mediated by LAK cells and activated monocytes with levels of lysis similar to those of parental chemosensitive lines. Moreover, freshly isolated tumor cells from ovarian cancer patients unresponsive to different chemotherapeutic treatments (operationally drug-resistant) were significantly killed in vitro by LAK cells. These findings support the concept that activated effector cells have the potential to complement conventional chemotherapy by eliminating drug-resistant tumor variants.     

Combined effect of heptaplatin and ionizing radiation on human squamous carcinoma cell lines

Heptaplatin, cis-malonato [(4R,5R)-4,5-bis (amino-methyl)-2-isopropyl-1,3-dioxolane] platinum(II) (SKI-2053R, Sunpla) is a new platinum derivative with anti-tumor activity comparable to cisplatin on various cancer cell lines. Preclinical studies suggest that it is less nephrotoxic than cisplatin. This study was undertaken to examine the combined effect of heptaplatin and ionizing radiation on two established human squamous carcinoma cell lines (NCI-H520, SQ20B). The cytotoxic activity of heptaplatin was concentration-dependent in both cell lines. When low dose heptaplatin was combined with high dose ionizing radiation, there was an additive cytotoxic effect on NCI-H520 cells (P < 0.05), while a moderate dose of heptaplatin and a low dose of ionizing radiation had an additive cytotoxic effect on the growth of SQ20B cells (P < 0.05). FACS analysis and DAPI staining showed that their additive cytotoxic effects were correlated with the induction of apoptosis. Further studies are warranted using heptaplatin and ionizing radiation in squamous cell carcinoma as a substitute for cisplatin.     

斑马鱼胚胎和肿瘤细胞评价细胞毒药物活性比较研究*

目的:比较斑马鱼胚胎和肿瘤细胞作为药物筛选模型的优缺点.方法:采用MTT法检测顺铂、紫杉醇、阿霉素、5-氟尿嘧啶四种药物对HL-60和Hela细胞的增殖影响;同时,观察药物对斑马鱼胚胎发育的影响.结果:阿霉素、顺铂及紫杉醇作用于HL-60及Hela细胞的IC50均显著高于作用于斑马鱼胚胎的LD50;而5-FU作用于肿瘤细胞和斑马鱼胚胎的结果与其它药物相反;四种抗肿瘤药物对斑马鱼胚胎的生长发育均有致畸作用.结论:斑马鱼胚胎作为细胞毒类药物筛选模型,对于抗微管类药物较为敏感,但对于抗代谢药敏感性较肿瘤细胞差.     

In vitro antitumor effect of LAK cells and alpha interferon in combination on tumor cell lines.

In vitro antitumor effects of LAK cells and alpha-2b-Interferon (IFN) either alone or in combination were evaluated on NK resistant (K562) and NK sensitive (Namalwa, Raji) cell lines. Tumor cells were incubated with LAK cells for 4, 8 and 24 hours at a LAK: tumor cell ratio of 1:1, 10:1, 100:1, or with IFN for 48 and 96 h at the concentrations of 100, 1000, 10,000, 100,000 IU/ml. A clonogenic assay was utilised to enumerate residual cells after in vitro treatment. A positive correlation was found between tumor cell killing and effector: target ratio, IFN of 100:1 incubated for 4 h, and 100 IU/ml of IFN incubated for 48 h were further chosen. A synergistic effect was found when IFN was incubated before LAK cells or contemporarily, but not when IFN was incubated after LAK cells. These findings demonstrate that an additive or a synergistic effect in vitro can be obtained by adding the two agents in different sequences and suggest that a potential utility of LAK cells and IFN in vivo should be tested in clinical trials.     

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.     

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

The human lymphokine-activated killer cell system. V. Purified recombinant interleukin 2 activates cytotoxic lymphocytes which lyse both natural killer-resistant autologous and allogeneic tumors and trinitrophenyl-modified autologous peripheral blood lymphocytes

Culture of human peripheral blood lymphocytes (PBL) in purified natural or recombinant interleukin 2 in the absence of exogenous antigen or mitogen causes the differentiation of nonlytic precursor cells into lymphokine-activated killers (LAK). A titration of purified Jurkat IL-2 (BRMP, FCRC, NIH) IL-2 showed that the relatively low concentration of 5 U/ml was optimal for LAK activation. When the responding PBL were pretreated with either mitomycin C or gamma irradiation, LAK activation did not occur, indicating that proliferation, in addition to differentiation, is required. The spectrum of target cells susceptible to LAK lysis in a 4-hr chromium-51-release assay includes fresh NK-resistant tumor cells and trinitrophenyl (TNP)-modified autologous PBL. Unmodified PBL are not lysed. Cold target inhibition studies indicated that LAK lysis of autologous TNP-PBL is totally inhibited by fresh tumors cells, and that tumor lysis is inhibited by TNP-PBL. Additionally, allogeneic tumors totally inhibit lysis of autologous tumor cells in other cold target studies. These results demonstrate that the lytic activity expressed by LAK is not HLA restricted, is not limited to tumor cells, and is "polyspecific" as indicated by the cross-reactive recognition of multiple target cell types in these cold target inhibition studies.     

The combined effect of lymphokine activated killer cell and radiation therapy on rat brain tumorin vitro

Thein vitro effect of a combined treatment with lymphokine activated killer (LAK) cell and radiation therapy on rat brain tumor was examined using⁵¹Cr release assay. The tumor cell-line used in this experiment was 9L rat brain tumor derived from a Fischer 344 rat. LAK cells were obtained by culturing rat lymphocytes with recombinant human interleukin 2 for at least 3 days. The cytotoxic activity of the LAK cells was examined by⁵¹Cr release assay. Irradiation was done by exposing the microtiter plate in which the¹⁵Cr labeled 9L cells and LAK cells were cultured to a¹³⁷Cs gamma cell unit. Without irradiation, there was 18% cytotoxicity in the 1:100 tumor-to-LAK cell ratio specimen after 24 hrs cocultivation. However, if 5 Gy of irradiation was given, followed by 12 hrs incubation, the cytotoxicity was enhanced significantly at the same cell ratio (30%). This enhancement effect was the most prominent when the cell ratio was 1:100 and the irradiation dose was 5 Gy. To generate the enhancement effect, an incubation time of over 8 hrs both before and after irradiation was required. The supernatant of the LAK cells showed 19.8% and 11.4% cytotoxicity with and without irradiation, respectively. This result indicates the participation of a cytotoxic factor released from LAK cells.This work is supported in part by grant from Univeristy of Tsukuba Project Research.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

Cytotoxic cell function and phenotypic analysis of peripheral blood mononuclear cells in cancer patients treated with low-dose interleukin-2 and mitomycin C

We previously found that the ability of peripheral blood mononuclear cells (PBM) of cancer patients to generate lymphokine-activated killer (LAK) cells became remarkably augmented after mitomycin C administration. On the basis of the clinical finding, we designed a treatment regimen comprised of 12 mg/m² mitomycin C i. v. on day 1 and 700 U/m² recombinant interleukin-2 (IL-2) i.v. every 12 h from day 4 through day 8. Of 25 patients with advanced carcinoma, 9 had a partial response and 3 had a minor response. Cytotoxic cell function, including natural killer activity, lymphokine-activated killer (LAK) activity, and the ability to generate LAK cells, and lymphocyte subsets in PBM was measured 1 day before and after either the first or second course of this therapy. The relationship between these parameters and the clinical antitumor response to this treatment was examined. Although the cytotoxic activities were significantly augmented after either the first or second treatment course, no positive correlation was observed between the changes in these cytotoxic activities and the clinical response to this therapy, when patients who either showed a partial response or whose disease remission was partial or minor were defined as responders. Further, phenotypic analysis showed a significant increase in CD2⁺, CD3⁺ CD4⁺ and CD4⁺Leu8^– cells after the firs course, and CD25⁺ cells after either the first or second course of this treatment. The precentages of CD2⁺ and CD25⁺ cells were significantly elevated only in responders but not in nonresponders, suggesting the increase in these subsets was related to clinical response.     

Differential effects of BCNU on T cell,macrophage, natural killer and lymphokine-activated killer cell activities in mice bearing a syngeneic tumor

Summary Chloroethylnitrosoureas have been used widely to treat human and experimental animal tumors. We have earlier observed that >90% of the mice transplanted with syngeneic tumors survive following treatment with nitrosoureas such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and furthermore, they resist subsequent challenge with the same tumor. The present investigation was initiated to determine the mechanism by which BCNU brings about this effect. Treatment of tumor cell targets in vivo or in vitro with BCNU, increased their susceptibility to macrophage (MØ)-mediated cytotoxicity as measured in a direct cytotoxicity assay or in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. In contrast, the antitumor cytotoxicity caused by cytotoxic T lymphocytes (CTL), natural killer (NK) cells, or lymphokine-activated killer (LAK) cells, was not altered following BCNU treatment of tumor targets. Studies were also conducted to investigate the direct effect of BCNU in vivo on various cytotoxic effector cells. For this purpose, MØ, NK, LAK, and CTL activities from BCNU-treated-tumor-bearing mice were screened for cytotoxicity against untreated tumor targets in vitro. It was observed that tumor-specific CTL and LAK cell activity increased in BCNU-treated tumor-bearing mice when compared to untreated controls while the cytotoxic potential of NK cells and MØs was not altered. The present study suggests that antitumor drugs such as BCNU are not only tumoricidal but also selectively act in a variety of ways at both the effector and target cell level, leading to overall enhanced antitumor immunity and high rate of cures from the syngeneic tumor challenge.The work at Virginia Polytechnic Institute and State University was supported by NIH grants CA45009 and CA45010 and by a Biomedical Research Support Grant. The work at University of Kentucky was supported by NIH grants CA34052 and CA33629 and by a grant from the Tobacco and Health Institute     

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.     

GHRH antagonist when combined with cytotoxic agents induces S-phase arrest and additive growth inhibition of human colon cancer

Treatment of colon cancer with an antagonist of growth hormone-releasing hormone (GHRH), JMR-132, results in a cell cycle arrest in S-phase of the tumor cells. Thus, we investigated the effect of JMR-132 in combination with S-phase-specific cytotoxic agents, 5-FU, irinotecan and cisplatin on the in vitro and in vivo growth of HT-29, HCT-116 and HCT-15 human colon cancer cell lines. In vitro, every compound inhibited proliferation of HCT-116 cells in a dose-dependent manner. Treatment with JMR-132 (5 μM) combined with 5-FU (1.25 μM), irinotecan (1.25 μM) or cisplatin (1.25 μM) resulted in an additive growth inhibition of HCT-116 cells in vitro as shown by MTS assay. Cell cycle analyses revealed that treatment of HCT-116 cells with JMR-132 was accompanied by a cell cycle arrest in S-phase. Combination treatment using JMR-132 plus a cytotoxic drug led to a significant increase of the sub-G₁ fraction, suggesting apoptosis. In vivo, daily treatment with GHRH antagonist JMR-132 decreased the tumor volume by 40–55% (p < 0.001) of HT-29, HCT-116 and HCT-15 tumors xenografted into athymic nude mice. Combined treatment with JMR-132 plus chemotherapeutic agents 5-FU, irinotecan or cisplatin resulted in an additive tumor growth suppression of HT-29, HCT-116 and HCT-15 xenografts to 56–85%. Our observations indicate that JMR-132 enhances the antiproliferative effect of S-phase-specific cytotoxic drugs by causing accumulation of tumor cells in S-phase.     

Structurally similar diazenes exhibit significantly different biological activity

We have previously synthesized various diazenecarboxamides (subsequently referred to as diazenes) that were cytotoxic to several tumor cell lines. To increase their biological activity, the structure has been modified appropriately. In the present study we examined the effects of N¹-phenyl-N²-(2-pyridinylmethyl)diazenedicarboxamide (RL-337) obtained from the previously examined cytotoxic compound N¹-phenyl-N²-(2-pyridinyl)diazenecarboxamide (JK-279), and compared them with those of diazene JK-279. Using a modified colorimetric MTT assay, the cytotoxicity of RL-337 was determined on human cervical carcinoma HeLa cells, glioblastoma A1235 cells, and prostate adenocarcinoma PC-3 cells. The possible synergistic effect of diazene RL-337 with cisplatin, doxorubicin, and vincristine, and its influence on intracellular GSH content was examined on HeLa cells. Diazene RL-337 was cytotoxic against all three human tumor cell lines, being more cytotoxic to HeLa cells than diazene JK-279. The higher efficacy of RL-337 than of JK-279 can be connected with higher basicity of the 2-picoline moiety present in the former diazene comparing with the pyridine fragment that is a part of the latter. The diazene RL-337 acted synergistically with cisplatin, doxorubicin, and vincristine (diazene JK-279 exhibited synergistic effect only with cisplatin). Glutathione (determined by Tietze's method) was not a target molecule of diazene RL-337 (but was for JK-279, as shown earlier). After just 1 h treatment with diazene RL-337, the cells started to lose membrane integrity. There was no cleavage of caspase-3 in RL-337-treated samples, and the majority of cells died 6 h after the treatment through necrosis (previously, apoptosis-like cell death was detected for diazene JK-279). Thus, although diazenes JK-279 and RL-337 are very similar in their structure, they exhibit widely different biological activity.     

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)     

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.