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.     

Sensitivity of fresh and cultured ovarian tumor cells to tumor necrosis factor,interferon-alpha 2, and OK-432

Summary The in vitro sensitivity to rTNF, rIFN-2, and OK-432 of 11 freshly derived human ovarian tumors and 2 established tumor cell lines was examined in a cytotoxic assay using the ⁵¹Cr release test. Nine fresh lines were sensitive to rTNF, 8 to OK-432, and only 2 were sensitive to rIFN-2. Cytotoxicity by rIFN-2 was of lesser magnitude than the cytotoxicity mediated by rTNF or OK-432. The time of exposure and the concentration of BRM required for maximal cytotoxicity varied from line to line. Two fresh tumor cell lines and 1 established cell line (PA-1) were sensitive to all 3 BRMs, while 2 other FOCs and 1 cell line (SKOV-3) were resistant to all BRMs. The remaining FOC showed an intermediate degree of sensitivity. These results demonstrate the existence of heterogeneity of ovarian carcinoma tumor cell lines to lysis by BRMs. Among the FOCs, the 2 endometrioid carcinomas tested were highly sensitive to rTNF, whereas the serous carcinomas were more sensitive to OK-432. Low grade tumors were more sensitive to BRM than high grade tumors, and tumor extension did not correlate with sensitivity to the BRM. When tumor targets were exposed to more than 1 BRM added either simultaneously or sequentially, the net cytotoxic effect achieved was usually inferior to the sum cytotoxicity obtained by each BRM alone. Furthermore, rTNF and OK-432 were cytostatic to most ovarian tumor cell lines examined. The results of this study demonstrate that certain BRMs exert a direct effect on fresh ovarian tumor cells independently of host factors. These findings suggest that in vitro screening of a patient's tumor cells for sensitivity to a particular BRM prior to therapy could be beneficial for the proper identification of patients most likely to benefit from the treatment.Supported in part by a grant from the Concern Foundation, by a gift from Chugai Pharmaceuticals, Japan, and in part by grants CA 35791 and CA 12800 from the National Cancer Institute, DHHS Abbreviations used: BCG, bacille calmette-Guérin; BRM, biological response modifier; CP, Corynebacterium parvum; FOC, fresh ovarian cancer; HBSS, Hanks' balanced salt solution; rIFN-2, recombinant interferon-alpha 2; rTNF, recombinant tumor necrosis factor; NK, natural killer     

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     

Tumor targets stimulate IL-2 activated killer cells to produce interferon-gamma and tumor necrosis factor

Lymphokine-activated killer (LAK) cells are cytotoxic for a variety of autologous and allogeneic tumor cells as well as modified autologous cells. It is assumed that LAK cells lyse their targets solely by direct cell to cell contact, possibly involving the degranulation and exocytosis of pore-forming elements, similar to that observed with cytotoxic T lymphocytes and NK cells. Reported here are studies demonstrating that LAK cells release factor(s) that are cytotoxic for a human breast carcinoma cell line, MCF-7, when stimulated with tumor cells. The factor(s) are slow acting and maximum cytotoxicity is observed only in a 72-h cytotoxic assay. The ability of LAK cells to secrete cytotoxic factor(s) is dependent on both the ratio of LAK cells to stimulating tumor cells as well as the length of their coincubation. A number of similarly slow acting cytokines that are cytostatic and/or cytotoxic for tumor cells have been described. We tested the ability of specific polyclonal antibodies directed against TNF, IFN-alpha, IFN-beta, and IFN-gamma to neutralize the cytotoxic supernatant activity. Only antibodies specific for IFN-gamma and TNF were neutralizing. We measured the amounts of IFN-gamma and TNF in the cytotoxic supernatants and determined that increased amounts of IFN-gamma and TNF were released after LAK cell-tumor cell interactions compared to supernatants of LAK cells alone or tumor cell alone. Comparable concentrations of human rIFN-gamma and rTNF resulted in similar levels (50 to 90%) of MCF-7 cell cytotoxicity as those observed with the stimulated LAK cell supernatants. We thus concluded that the majority of the cytotoxic activity released by LAK cells when stimulated with tumor cells was attributed to the synergistic activities of IFN-gamma and TNF. The significance of these observations in relation to the possible mechanisms by which LAK cells mediate cytolysis is discussed.     

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.     

Interferon is able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells

It is known that IL-2 induces lymphocytes to produce interferon-gamma (IFN-gamma) and this IFN type is particularly efficient in inducing tumor cell resistance to natural killer (NK) cell-mediated lysis. We have investigated the effect of IFN on tumor cell sensitivity to LAK cell-mediated cytotoxicity. Pretreatment of the human K562 leukemia and HHMS melanoma with IFN-gamma and the Daudi lymphoma with IFN-alpha caused a significant reduction in sensitivity to lysis by human LAK cells generated in vitro in the presence of human recombinant IL-2 (100 U/ml). The LAK activity was mediated by cells expressing NK cell markers (CD16,NKH1) as well as by cells with T cell markers (CD3, CD5). IFN-treated K562 cells were protected from lysis mediated by all these populations. Supernatants from LAK cultures containing IFN-gamma were able to induce NK and LAK resistance when used to pretreat K562 overnight. Antibodies to IFN-gamma but not to IFN-alpha were able to neutralize this activity. Taken together, these results indicate that the production of IFN-gamma by LAK cells may be of importance in induction of tumor cell resistance to LAK cell-mediated lysis.     

Drug resistance in rat colon cancer cell lines is associated with minor changes in susceptibility to cytotoxic cells

The development of resistance to anticancer drugs urges the search for different treatment modalities. Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility. We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS). In a 4-h⁵¹Cr-release assay we found no difference in susceptibility to NK cell lysis. No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay. In a prolonged 20-h⁵¹Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS). None of the cell lines was completely resistant to lysis by aLAK cells. Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.     

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

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

Sensitivity of ovarian tumor cells to effector cells generated by various biological response modifiers

The sensitivity of freshly derived human ovarian tumors (FOT) to various allogeneic cytotoxic effector cells stimulated by recombinant interleukin 2 (rIL-2), recombinant interferon alpha 2 (rIFN-alpha 2), OK-432, and concanavalin A was examined using the 51Cr release assay. Peripheral blood lymphocytes (PBL) of normal female donors were used as source of effector cells. Incubation of PBL with these biological response modifiers for 24 h generated effector cells with high natural killer activity, and only 20% (1/5) of the FOT examined were susceptible to lysis. By contrast, 83% (5/6) of the FOT were sensitive to lymphokine-activated killer (LAK) cells generated by rIL-2. OK-432 and concanavalin A activation of PBL also generated cytotoxic cells, though the cytotoxic activity against FOT was much less than that obtained by LAK cells. The addition of OK-432 to LAK culture medium containing rIL-2 generated effector cells with higher cytotoxicity against FOT than cultures with IL-2 alone. However, the addition of rIFN-alpha 2 in LAK culture medium resulted in the generation of effector cells with lower cytotoxicity. The addition of rIL-2, rIFN-alpha 2, or OK-432 to LAK cells during the in vitro cytotoxicity assay had no significant effect. When FOT target cells were pretreated with OK-432 they became more sensitive to LAK than nontreated tumor cells. However, pretreatment with rIL-2 or rIFN-alpha 2 did not influence cytolysis. These results suggest that the generation of LAK cells in vitro using rIL-2 plus OK-432 may be a more effective way to prepare these cells for adoptive immunotherapy in the treatment of ovarian cancer.     

Long-term killing of natural killer-resistant target cells by interferon-alpha-, interferon-gamma-, and interleukin-2-activated natural killer cells

The sensitivity of target cells to natural killer (NK) cell-mediated cytotoxicity was investigated. Five target cell lines were examined for susceptibility to killing by activated NK cells in a 4-hour cytotoxicity assay: one of them (K562) was highly sensitive, while the other four were resistant. However, the four NK-resistant target cell lines were fully susceptible to lysis when the assay was extended to 24 h. The cytotoxic cells that killed the NK-resistant target cells in a 24-hour assay were plastic- and nylon wool-nonadherent human peripheral blood mononuclear cells (PBMC) and their cytotoxicity was increased by interferon-alpha, interferon-gamma, and interleukin-2. Further, the cytotoxic activity of PBMC in the long-term assay was associated with large granular lymphocytes purified on a Percoll gradient, that killed the NK-sensitive cell line K562 in a 4-hour assay. All of the above are general criteria to qualify the cytotoxic cells as NK cells. Thus, the NK-resistant phenotype may not reflect absolute immunity to NK-mediated lysis, but it may reflect the different rates at which various target cell lines can be killed.     

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     

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.     

Resistance ofHER2/neu-overexpressing tumor targets to lymphokine-activated-killer-cell-mediated lysis: evidence for deficiency of binding and post-binding events

HER2/neu-overexpressing tumor cell lines are relatively resistant to lymphokine-activated killer (LAK) cell cytotoxicity when compared toHER2/neu-nonexpressing lines.HER2/neu ⁺ targets were also resistant to binding by LAK large granular lymphocytes (LGL) as shown by visualization at the single-cell level, a target monolayer binding assay and in cold target inhibition experiments.HER2/neu ⁺ LAK-resistant ovarian cell lines demonstrated an absence of ICAM-1 expression while expression of LFA-3, N-CAM, laminin and ₁ integrins was comparable to that ofHER2/neu ^– targets. In contrast, theHER2/neu ⁺ breast cell line, SKBR-3, which was also resistant to lysis and binding by LAK LGL, demonstrated normal expression of ICAM-1. Anti-ICAM-1 antibodies blocked binding and lysis ofHER2/neu ^– carcinoma targets by LAK cells, further supporting the notion that lack of ICAM-1 expression onHER2/neu ⁺ cells contributes to their resistance. The modest binding and lysis ofHER2/neu ⁺ targets by LAK cells was significantly inhibited by anti-LFA-1 antibodies, suggesting the existence of another counter-receptor for LFA-1 onHER2/neu ⁺ targets. The following also supported deficiencies in post-binding events whenHER2/neu ⁺ cells resisted the lytic activity of LAK cells: (a) when the relative resistance to effector cell binding was overcome by exogenous lectin,HER2/neu ⁺ cell lines were still resistant to LAK cytolysis, and (b)HER2/neu ⁺ targets were resistant to perforin-containing granule extracts obtained from the CTLL-R8 cytotoxic lymphocyte cell line. These results indicate that deficiency in effector binding as well as post-binding events contributes to the resistance ofHER2/neu-overexpressing tumor targets to LAK-cell-mediated lysis.Supported by research funds of the Veteran's Administration, the California Institute for Cancer Research and Jonsson Cancer Center core grant CA 16042 funded by NIH     

Gs protein-coupled adenosine receptor signaling and lytic function of activated NK cells

The effect of adenosine and its analogues on the cytotoxic activity of IL-2-activated NK cells was investigated. Adenosine is an endogenous ligand for four different adenosine receptor (AdoR) subtypes (AdoRA1, AdoRA2A, AdoRA2B, and AdoRA3). Increased concentrations of adenosine were found in ascites of MethA sarcoma or in culture medium of 3LL Lewis lung carcinoma growing under hypoxic conditions. We hypothesize that intratumor adenosine impairs the ability of lymphokine-activated killer (LAK) cells to kill tumor cells. The effect of AdoR engagement on LAK cells cytotoxic activity was analyzed using AdoR agonists and antagonists as well as LAK cells generated from AdoR knockout mice. Adenosine and its analogues efficiently inhibited the cytotoxic activity of LAK cells. CGS21680 (AdoRA2A agonist) and 5-N-ethylcarboxamide adenosine (NECA) (AdoRA2A/ADoRA2B agonist) inhibited LAK cell cytotoxicity in parallel with their ability to increase cAMP production. The inhibitory effects of stable adenosine analog 2-chloroadenosine (CADO) and AdoRA2 agonists were blocked by AdoRA2 antagonist ZM 241385. Adenosine and its analogues impair LAK cell function by interfering with both perforin-mediated and Fas ligand-mediated killing pathways. Studies with LAK cells generated from AdoRA1-/- and AdoRA3-/- mice ruled out any involvement of these AdoRs in the inhibitory effects of adenosine. LAK cells with genetically disrupted AdoRA2A were resistant to the inhibitory effects of adenosine, CADO and NECA. However, with extremely high concentrations of CADO or NECA, mild inhibition of LAK cytotoxicity was observed that was probably mediated via AdoRA2B signaling. Thus, by using pharmacological and genetic blockage of AdoRs, our results clearly indicate the prime importance of cAMP elevating AdoR2A in the inhibitory effect of adenosine on LAK cell cytotoxicity. The elevated intratumor levels of adenosine might inhibit the antitumor effects of activated NK cells.     

Effects of beta-2 microglobulin anti-sense oligonucleotides on sensitivity of HER2/neu oncogene-expressing and nonexpressing target cells to lymphocyte-mediated lysis.

The mechanism by which HER2/neu overexpressing tumor cells resist NK, LAK, and LDCC cytotoxic lymphocytes was investigated. Resistance was not explained by a delay in kinetics of lysis, concurrent resistance to TNF, or a diminished expression of the transferrin receptor. HLA-class I expression, however, was markedly elevated compared to HER2 nonexpressing targets suggesting a reason for resistance. To test the role of class I, we selectively decreased expression by incubation of targets with beta-2 microglobulin anti-sense oligonucleotides. Anti-sense-treated HER2+ targets, displaying levels of class I comparable to HER2- targets, were still markedly resistant to cytotoxic effectors. Down-regulation of class I expression in HER2- carcinoma cells also had no effect on sensitivity to cytotoxicity by anti-sense treatment of Raji and U937 targets resulted in enhanced sensitivity to NK and LAK effectors but not to T cells mediating LDCC. These data indicate resistance to cytotoxicity in HER2-expressing targets cannot be solely explained by heightened expression of class I. The data also support the concept that class I expression regulates sensitivity to NK and LAK cells (but not LDCC effectors) in selected targets.     

Human oncogene-transfected tumor cells display differential susceptibility to lysis by lymphokine-activated killer cells (LAK) and natural killer cells

NIH 3T3 tertiary transfectants containing the N-ras or c-Ha-ras oncogenes derived from human tumors were tested for susceptibility to lymphokine-activated killer (LAK) cell and natural killer (NK) cell lysis. N-ras tertiary transfectants contained a human acute lymphocytic leukemia-derived N-ras oncogene. C-Ha-ras transfectants contained either the position 61-activated form of the oncogene (45.342, 45.322, and 45.3B2) or the position 12-activated form (144-162). In 4 hr 51Cr release assays, seven of seven in vivo grown human oncogene transfected NIH 3T3 fibroblasts were lysed by murine LAK effectors, whereas six of seven were lysed by human LAK effectors. There was no difference in susceptibility to lysis between cells transfected with the N-ras oncogene, the position 61 activated c-Ha-ras oncogene, or the position 12 activated c-Ha-ras oncogene. Cultured NIH 3T3 fibroblasts, as well as in vitro and in vivo grown NIH 3T3 tertiary transfectants were resistant to lysis by murine NK effectors and were relatively resistant (4/6 were not lysed) to lysis by human NK effectors. We conclude that human oncogene-transfected tumors are susceptible to lysis by both murine and human LAK cells while being relatively resistant to lysis by murine and human NK cells. Different oncogenes or the same oncogene activated by different point mutations do not specifically determine susceptibility to lysis by LAK or NK. Also the presence of an activated oncogene does not appear to be sufficient for inducing susceptibility to these cytotoxic lymphocyte populations.     

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.     

IL-6 enhances the cytotoxic activity of thymocyte-derived CD56+ cells.

Thymocyte-derived lymphokine-activated killer (LAK) cells were used as a model for the study of the cytokine driven development of cytotoxicity. These cells are devoid of initial cytotoxic activity but upon culture in IL-2 they develop into cytotoxic effectors. The parameters of the response of thymocytes to IL-6 are similar to that of PBL in that IL-6, at concentrations as low as 1 mu/ml, increases cytotoxicity of thymocyte-LAK cells when generated in low doses (25-50 mu/ml) of IL-2. IL-6-enhanced thymocyte-LAK cytotoxicity is observed when tested against both NK-resistant and NK-sensitive tumor cell lines. IL-6 alone does not induce any cytotoxicity from thymocytes nor does IL-6 change the time course of thymocyte-LAK cell generation in IL-2 culture. IL-6 does not affect DNA synthesis, total cell number, proportion of CD56+ cells, or the expression of IL-2R (both P55 and P75 glycoproteins) in IL-2-cultured thymocytes. Instead, IL-6 used to treat mature thymocyte-LAK effector cells for as little as 1 hr prior to 51Cr-release assay increases LAK cytotoxicity. This enhancement is abrogated by pretreatment of effector cells with cycloheximide, suggesting that protein synthesis is required for IL-6 to enhance LAK cell activity. The precursor phenotypes of IL-6-responsive thymocyte-LAK cells are CD3-/CD5-. The effector phenotypes of IL-6-enhanced thymocyte-LAK cells are CD5-/CD56+. Thus, IL-6 depends on synthesis of rapid-turnover proteins to act on mature CD56+/CD5- LAK cells to increase their cytotoxic function.     

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.