Lymphokine-activated killer cells can kill target cells not only by early killing but also by late killing,and the late killing level against autotumor cell line

Generally, lymphokine-activated killer (LAK) cells kill target cells early after the LAK cells adhere to them. In this study, we describe that LAK cells can also kill at a later time, such as 24-96 h. LAK cells were generated from a cancer patient and healthy volunteers. As target cells, the patient's autotumor cell line H41 was used. When LAK cells were added to the target cells in a culture well, the LAK cells killed the target cells by cell-cell adhesion within 1-4 h (early killing), but not all cells were killed. The LAK cells were then removed. However, the remaining cells ultimately died 24-96 h later (late killing). The late killing was different from the early killing because numerous granules and vacuoles appeared in the cytoplasm. The late killing was not induced by adding supernatant of the LAK cell culture, suggesting that LAK-target cell contact may be necessary for the killing. The cell injury was inhibited by 3-methyladenine (lysosome inhibitor). It suggests that the vacuoles may be caused by activated lysosome. The patient's LAK cells induced late killing at high levels. There was a high percentage of CD8(+)CD16(+) cells in the peripheral blood lymphocytes (PBL). This subset induced late killing more effectively than the CD8(-)CD16(+) subset. Killing was more conspicuous against H41 than against allogeneic cell line T98G. This type of killing is noteworthy for understanding of killing mechanism of LAK cells.     

铂类抗癌药与LILAK细胞联合杀伤人肺腺癌细胞的初步研究

应用ＳＰＣ－Ａ１人肺腺癌细胞系研究了顺铂和上次后的细胞毒效应及其与人外周血ＬＩＬＡＫ细胞的联合效应及机制结果表明：经一定浓度的顺铂或卡铂作用一定时间后，ＳＰＣ－Ａ１细胞可受不同程度的杀伤。经１μｇ／ｍｌ的顺铂处理２４小时，洗去药物后加入ＬＩＬＡＫ细胞，ＳＰＣ－Ａ１细胞被明显杀伤，与未经顺铂处理的对照组相比Ｐ〈０．００１；而卡铂在同样条件下却无此效应。     

Adhesion molecules on murine lymphokine-activated killer cells responsible for target cell killing: a role of CD2.

Lymphokine-activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA-1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA-1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.     

Adhesion Molecules on Murine Lymphokine-activated Killer Cells Responsible for Target Cell Killing: A Role of CD2

Lymphokine-activated killer (LAK) cells were induced from C57BL/6 mouse spleen cells and the effects of culture time on the expression of cell surface phenotypes and cytotoxic activity of LAK cells were determined. The expression of CD2 remarkably decreased after culture of LAK cells for 30 days, while LFA-1, a principal adhesion molecule in LAK cells, and CD3 were not changed by the culture. LAK cells cultured for 90 days completely lost CD2. In accordance with the decrease of CD2, the cytotoxic activity of LAK cells declined but a certain leven was retained even after the complete loss of CD2. The established LAK cell clones were also strongly positive for the expression of LFA-1 but negative for CD2. When the LAK cell clones were transfected with the CD2 cDNA, they started to express CD2 on their cell surface and to show greater binding ability and stronger cytotoxicity to target tumor cells. These results indicated that CD2 plays a role as an adhesion molecule responsible for target cell killing in murine LAK cells.     

Antitumor efficacy of lymphokine-activated killer cells and recombinant interleukin-2 in vivo: survival benefit and mechanisms of tumor escape in mice undergoing immunotherapy

The studies described in this paper showed that the combination of i.v.-transferred lymphokine-activated killer (LAK) cells and i.p. injections of recombinant interleukin-2 (RIL-2) was highly effective in vivo in reducing established pulmonary metastases of natural killer cell-resistant, MCA-105 sarcoma and B16 melanoma in mice. A 3-day in vitro incubation of normal C57BL/6 splenocytes in medium containing pure RIL-2 generated LAK cells that, when combined with RIL-2, reduced the mean number of established pulmonary micrometastases of the B16 melanoma and of the MCA-105 sarcoma from 179 and 140, respectively (in groups treated with Hanks' balanced salt solution alone), to 12 (P = 0.01) and 6 (P = 0.01), respectively. This combined immunotherapy also consistently resulted in significant prolongation of survival in mice with established, 3-day or 10-day pulmonary metastases of the MCA-105 sarcoma. Mice autopsied at time of death revealed a massive involvement of tumor in the lungs and liver in the group receiving Hanks' balanced salt solution alone compared to a small number of residual large lung or liver metastases in the group receiving LAK cells plus RIL-2. Experiments were designed to test whether variants existed in the original tumor cell inoculum that were resistant to killing by LAK cells and thus could account for the metastases that "escaped" the combined immunotherapy of LAK cells plus RIL-2 in vivo. Metastases of the MCA-105 sarcoma that escaped the combined therapy of LAK cells plus RIL-2 were dissected from the organs of mice upon autopsy and directly tested for susceptibility in vitro to lysis by LAK cells in 4-h and 18-h 51Cr release assays. Target cells derived from the metastases were lysed to an equivalent extent as those prepared from a fresh MCA-105 sarcoma that was growing s.c. In addition, successful reduction of pulmonary metastases established by the i.v. infusion of MCA-105 sarcoma cells obtained from metastases that escaped a prior round of therapy with LAK cells and RIL-2 could be achieved in vivo by the combined immunotherapy as well as by high doses of RIL-2 alone. Culture adapted, natural killer cell-resistant B16 melanoma cells surviving two successive treatments with LAK cells in vitro remained as susceptible to LAK cell lysis as untreated B16 melanoma cells in 18-h 51Cr release assays.(ABSTRACT TRUNCATED AT 400 WORDS)     

The cytotoxic action of lymphokine activated killer cells upon the human glioma cell line U251 is stimulated by bispecific monoclonal antibody (MoAb) constructs

The ability of IL-2 stimulated mononuclear cells to kill the human glioblastoma cell line U251 has been investigated. Highest cytotoxic activity was generated in low cell density cultures incubated for 15 days with 250–1000 U/ml IL-2. Sub-optimal killing was noted, with cells only exposed to IL-2 for three days. Under the latter conditions, bispecific monoclonal antibodies (MoAbs) of either anti-CD3 or anti-CD16 and an anti-NCAM MoAb stimulated LAK cell activity markedly. Anti-CD16 conjugates were found more effective than anti-CD3 and (Fab)₂ constructs more efficacious than those made with whole Ig molecules. Maximal stimulation of LAK cell activity was noted with bispecific MoAbs. Little effect was observed with either single or mixtures of monomeric MoAbs. Furthermore, no effect of bispecific MoAbs was observed when target cells lacked expression of NCAM. These results could be of clinical importance as it is not always feasible to screen LAK cells for optimal activity before administration to patients. Whilst bispecific MoAbs have no effect on optimally stimulated LAK cells, they are not inhibitory and can stimulate killing under sub-optimal IL-2 stimulation.     

Alpha-interferon and lymphokine-activated killer cells in hairy cell leukemia

Lymphokine-activated killer (LAK) cell activity generated from peripheral blood was tested in 6 patients with typical hairy cell leukemia, 3 not on treatment with alpha-interferon (alpha-IFN) and 3 receiving therapy. In all cases, substantial killing of the LAK-sensitive target Daudi was observed, but hairy cells, whether or not they had been pretreated with alpha-IFN, were uniformly resistant to LAK lysis. The hairy cells were also resistant to LAK cell killing generated from normal peripheral blood mononuclear cells. alpha-IFN added at various times during LAK generation had little or no effect on LAK activity. It is concluded that LAK cells are not important in mediating the beneficial effects of alpha-IFN in hairy cell leukemia.     

The roles of Fas and perforin in LAK T-cell/bispecific antibody-mediated killing of the murine B-lymphoma cells,BCL1

The aim of this study was to construct bispecific F(ab′)₂ [anti-CD3 × anti-BCL₁ idiotype (Id)] Abs (BsAbs) which would enable lymphokine-activated killer (LAK) T cells to kill Id⁺ mouse BCL₁ lymphoma cells, and to determine the mechanism(s) underlying cell death. Using 4-day activated LAK T cells from either perforin-knockout mice or FasL-deficient gld mice, we show that the Fas pathway, but not perforin, is required for BsAb-mediated LAK T-cell-induced killing of BCL₁ cells. © 1996 Wiley-Liss, Inc.     

Relation of natural killer cell line NK-92-mediated cytolysis (NK-92-lysis) with the surface markers of major histocompatibility complex class I antigens, adhesion molecules, and Fas of target cells.

Natural killer (NK) cell line NK-92 has recently been established by Klingemann et al. In this study, we compared the NK-92-mediated cytolysis (NK-92-lysis) with the killing of healthy volunteers' NK cells and lymphokine-activated killer (LAK) cells. The NK-92-lysis was partially different from the NK- and LAK-lysis. 1) The NK-92 could kill most of major histocompatibility complex (MHC) class I antigen-positive tumor cells. 2) The NK cells killed a myeloid leukemia cell line K562, but the NK-92 showed low killer activity against it. 3) The LAK cells could not kill a CD58-deficient cell line OKM-2T, whereas the NK-92 could kill it sufficiently. 4) The NK-92 could not kill CD54-, CD102-deficient cell lines T98G and U373MG; however, the LAK cells could kill them. Blocking tests using specific antibodies revealed the reason for these differences. The K562 expressed relatively low levels of CD54 and CD102. When the K562 was pretreated with anti-CD54 and anti-CD102, the NK-92 could not kill it at all, whereas the NK cells could still kill it, although the killing level decreased. The NK-92 could not kill the anti-CD54- and anti-CD102-treated OKM-2T. The LAK cells could not kill anti-CD58-treated U373MG and T98G. These findings suggest that NK-92-lysis may require the CD54 and CD102 but that NK-lysis does not require them as much, whereas the LAK-lysis may be rather in relation with the CD58. The NK-92 has high killer activity, and may be applicable for clinical use. However, it should be considered that the NK-92 cannnot kill CD54-, CD102-deficient tumor cells.     

人LAK细胞对肺腺癌靶细胞的杀伤活性及形态学观察

LAK activity could be induced by IL-2 from human PBL. After co-culture of LAK cells with Anip973 human lung adenocarcinoma cells, LAK-tumor conjugates were microscopically observed. Formation of the conjugates paralleled well with the expression of LAK activity (P less than 0.001), suggesting that LAK cells' killing of Anip973 cells depended on their close contact. Under electron microscope, LAK cells had various shapes and certain degrees of motility. Ten minutes after co-culture, close binding between microvilli of LAK cells and tumor cells could be seen. Four hours after co-culture, Anip973 cells bound with LAK cells developed numerous membrane blebs with cytoplasm and nucleus very condensed, which was followed by destruction of cell structure and appearance of cell debris. These features are consistent with apoptosis rather than a cytolytic mechanism of cell death. These findings suggest that LAK cells share certain common features with CTL and NK cells on killing tumor cells.     

Decreased sensitivity of drug-resistant cells towards T cell cytotoxicity.

Killing of target cells by cytotoxic T cells is mediated by induction of apoptosis requiring functional death pathways. Kill is mediated either by the CD95 or the perforin/granzyme pathway. We found that SH-EP neuroblastoma cells are preferentially killed via CD95, while in the T leukemia cell line CEM CD95 and perforin/granzyme are involved. In both types of cell lines, cells resistant to CD95- and drug-induced apoptosis are crossresistant to cytotoxic T cell kill. Resistant cells show decreased apoptosis and deficient activation of caspases indicated by decreased cleavage of the prototype caspase substrate PARP. Preincubation with the caspase inhibitor zVAD-fmk strongly decreased LAK cell kill in sensitive cells. Although parental CEM cells could be sensitized for LAK kill by preincubation with doxorubicin, resistance could not be reverted in doxorubicin or CD95 resistant CEM cells. These data demonstrate the crossresistance in induction of apoptosis by different cytotoxic regimens in tumor cells and may have implications for the immunotherapy of tumors in which apoptosis resistance was induced by previous chemotherapy.     

Target lysis by human LAK cells is critically dependent upon target binding properties, but LFA-1, LFA-3 and ICAM-1 are not the major adhesion ligands on targets.

The cytotoxicity mediated by the CD2+ CD3- lymphocyte subset, either NK or LAK, is puzzling since no specific antigen recognition structures, equivalent to the CD3-associated heterodimer T-cell receptor, have been recognized on these cells so far. The possibility exists that the CD3- cytotoxic effectors recognize their targets through non-specific adhesion mechanisms. The goal of this study was: (a) to examine the correlation between binding properties and susceptibility to lysis of 6 informative target cell lines; (b) to evaluate the role, as ligands on these targets, of adhesion molecules such as LFA-1, LFA-3 and ICAM-1. The effectors used in this study were IL-2-activated LGL, predominantly CD3-, or highly purified CD3- lymphocytes from normal human donors. The 6 target lines studied included 2 pairs of EBV-transformed B-cell lines (721 LCL vs. 721.134, and MM vs. MM-10F2) in which the parental lines were resistant to lysis while HLA variants were susceptible. A third pair was the Daudi Burkitt cell line, susceptible to LAK lysis, and an HLA-positive transfected Daudi line which was more resistant to lysis. The binding properties of these targets to LAK effectors (conjugate formation) were evaluated using a sensitive double fluorescence flow cytometry method. In each pair examined, the susceptible targets formed more conjugates and were surrounded by more cytotoxic LAK effectors than their resistant counterparts, indicating that the conjugation properties of targets are closely correlated with their susceptibility to LAK lysis. The expression of adhesion molecules on the informative targets was examined by indirect immunofluorescence and their role was evaluated by inhibition of lysis after pre-coating the targets with the relevant antibodies. The differences in the expression of the classical cell-cell adhesion molecules LFA-1, LFA-3 and ICAM-1 on the target surfaces were only marginal, insufficient to explain the striking differences in susceptibility to lysis and in binding properties. Coating the target cells with antibodies directed against these adhesion determinants had no effects on the lysis of susceptible target cells. The same antibodies reacting with the LAK effectors did inhibit lysis. Taken together, these results suggest that, on the targets, presently undefined membrane adhesion structures may have a major role in conjugate formation between target and CD3- effectors and determine the susceptibility of the targets to lysis.     

Effect of human interleukin 3 on the susceptibility of fresh leukemia cells to interleukin-2-induced lymphokine activated killing activity.

Pretreatment of acute myeloblastic leukemia cells with the hemopoietic growth factor interleukin 3 (IL3) increased their susceptibility to lymphokine activated killing (LAK) but did not affect their constitutive resistance to native natural killer activity. In addition, IL3 treatment did not alter the LAK cell-mediated killing of CD34+ hemopoietic progenitors present in normal bone marrow. Increased 3H-thymidine uptake was generally observed after IL3 treatment. However, failure to proliferate in response to IL3, observed in some cases, did not prevent changes in LAK susceptibility. Enhanced lysis of IL3-treated leukemic cells was accompanied by a moderate increase of the effector-target binding. Increased LAK susceptibility was already observed at 18 h, while optimal cytolysis and expression of the cell adhesion molecule (CAM) LFA-3 (CD58) by IL3-treated AML cells were concomitantly observed at later culture times. In contrast, the CAM ICAM-1 (CD54) was not modulated by IL3, nor were significant changes in the expression of either CAMs observed in normal hemopoietic cells. Blocking experiments with the anti-CD58 monoclonal antibody demonstrated a variable neutralizing effect on the IL3-induced increase of LAK activity, depending on the leukemia cell studied. The effect described here, together with the known role of IL3 in normal hemopoiesis makes it a factor of potential therapeutic value for the treatment of leukemic patients.     

A-NK细胞抗肝癌Walker256瘤株的实验研究

目的:观察A-NK细胞的体外生长与增殖,以及杀伤肿瘤细胞的能力,研究A-NK细胞局部注射的体内抗肿瘤作用。方法:用淋巴细胞分离液分离单个核细胞(PBMC),培养LAK细胞和A-NK细胞,分别将LAK细胞、A-NK细胞和Walker-256瘤株细胞接种于培养板中,培养24h后,用四甲基偶氮唑盐(MTT)方法测定吸光度,计算肿瘤杀伤率。复制鼠肝癌模型,分别将LAK、A-NK细胞经肝动脉注入鼠肝癌模型中,观察两组动物的生存时间。结果:A-NK细胞的增殖明显快于LAK细胞(P<0.05),A-NK细胞对肿瘤细胞的杀伤活性比LAK细胞强(P<0.01),A-NK细胞能显著延长肝癌动物模型的生存期,与LAK细胞比较,具有显著性差异(P<0.01)。结论:A-NK细胞具有增殖快,抗瘤活性强,在体内能抑制肿瘤生长,延长荷瘤动物的生存期。     

Cell killing by antibody-drug conjugates

Antibody-drug conjugates (ADCs) are designed to specifically bind to and kill cells expressing their target antigens. In addition to the obvious requirement of the presence of the target antigen on the cell surface, several other factors contribute to the sensitivity of target cells to the action of ADCs. These include (i) the rate of internalization of the ADC, (ii) its proteolytic degradation in late endosomes and lysosomes and the subsequent release of cytotoxic drug, and (iii) the intracellular concentration of the released drug. In addition to killing antigen-expressing cells, some ADCs were found to kill bystander cells irrespective of their antigen expression. This review summarizes the current knowledge of the mechanisms of killing of antigen-expressing and bystander cells by antibody-drug conjugates.     

Membrane-associated Lymphotoxin Expression and Functional Analysis of Lymphokine-activated Killer Cells Derived from Tumor-infiltrating Lymphocytes

The expression of a membrane-associated lymphotoxin molecule (mLT) on lymphokine-activated killer (LAK) cells obtained from 18 patients with malignant tumors and its role in the tumor cell killing mechanisms were investigated. LAK cells from tumor-infiltrating lymphocytes (TIL-LAK cells) were mainly composed of CD3-positive cells, whereas LAK cells from peripheral blood lymphocytes (PBL-LAK cells) were mainly composed of CD16- and CD56-positive cells. However, mLT was found to be expressed on TIL-LAK cells as well as PBL-LAK cells. The degree of mLT expression correlated with the killing activity of LAK cells towards L929 cells (r=0.806, P <0.01, n = 15), but not with that towards Daudi or K562 cells. Although the degree of mLT expression correlated with the amount of secreted lymphotoxin (LT) in the supernatant of LAK cell culture, the secreted LT itself could not account for the tumor cell killing activity of LAK cells. Polyclonal rabbit anti-LT antibody partially inhibited the killing activities of LAK cells towards L929 cells and this inhibition was found in the combination of autologous tumor cells and PBL-LAK cells. These findings suggest the possibility that the mLT-related cytotoxicity is involved in the tumor cell killing mechanisms of TIL-LAK cells as well as PBL-LAK cells.     

Epimedokoreanin C,a prenylated flavonoid isolated from Epimedium koreanum,induces non-apoptotic cell death with the characteristics of methuosis in lung cancer cells

Methuosis is a novel type of non-apoptotic cell death characterized by accumulation of cytoplasmic vacuoles. Identification of molecules that induce methuosis may provide alternative therapeutics for cancers that are refractory to apoptosis. Epimedokoreanin C (EKC) is a prenylated flavonoid isolated from a Chinese herb Epimedium koreanum. In this article, we described that EKC reduced cell viability accompanied by extreme vacuolation in human lung cancer cells. The EKC-induced cell death was clarified as non-apoptosis based on the absence of apoptotic changes. The vacuoles stimulated by EKC were supposed to be derived from macropinocytosis based on the engulfment of extracellular fluid tracer, Lucifer Yellow. The vacuoles acquired some characteristics of late endosomes supported that EKC-induced cell death could be described as methuosis. Rac1 and Arf6 were found to be regulated inversely after EKC treatment. Blocking Rac1 activation with the specific Rac1 inhibitor EHT 1864 prevented the accumulation of vacuoles induced by EKC markedly, suggested that the regulation of Rac1 and Arf6 was at least partial mechanism involved in EKC induced methuosis. EKC synergized the effects of doxorubicin and etoposide, demonstrating the effectiveness of using EKC to synergize conventional chemotherapy. Collectively, EKC was demonstrated as a methuosis-like cell death inducer in lung cancer NCI-H292 and A549 cells. It has the potential to be used as an attractive prototype for developing drugs that could kill apoptosis-resistant cancer cells.     

Lymphokine-activated killer (LAK) cells. Analysis of factors relevant to the immunotherapy of human cancer

Lymphokine-activated killer (LAK) cells can be generated by incubating fresh peripheral blood lymphocytes (PBL) in Interleukin-2 (IL-2). LAK cells kill fresh autologous and allogeneic human tumor cells in vitro. This study analyzes aspects of LAK cells that make them a promising candidate for the adoptive immunotherapy of human cancer. LAK cells can be generated from PBL of normal individuals and tumor-bearing patients. Pure, recombinant IL-2 generates LAK cells capable of killing a wide variety of tumors including sarcomas and cancers of the colon, pancreas, adrenal gland, and esophagus. Thirty-six of 41 (88%) fresh, noncultured, human tumor cell suspensions prepared from surgical specimens were lysed by LAK cells in a standard 4-hour chromium-release assay. Normal PBL were not killed. LAK cells can be expanded in vitro for periods longer than 2 months, potentially more than 10(20)-fold, while maintaining lytic ability. These results and the demonstrated efficacy of LAK cells in the therapy of murine tumors make LAK cells a candidate for clinical use in the adoptive immunotherapy of human cancer.