Induction of intercellular adhesion molecule 1 on small cell lung carcinoma cell lines by gamma-interferon enhances spontaneous and bispecific anti-CD3 x antitumor antibody-directed lymphokine activated killer cell cytotoxicity.

The interaction between LFA-1 and its natural ligand, ICAM-1, plays an important role in leukocyte adhesion and signal transduction. LFA-1-mediated T-cell adhesion is generally activated by CD3-mediated signal in association with T-cell receptor-mediated recognition of the antigen/major histocompatibility complex on antigen-presenting cells. In the present study, we compared spontaneous or bispecific antibody (BsAb)-directed LAK cell cytotoxicity against ICAM-1+ or ICAM-1- small cell lung cancer (SCLC) cell lines. gamma-Interferon (IFN-gamma)-induced ICAM-1 expression on ICAM-1- SCLC cell lines, and susceptibility to LAK cells was increased simultaneously. Increased cytolysis of the IFN-gamma-treated SCLC was inhibited by an anti-ICAM-1 monoclonal antibody (mAb). Furthermore, LAK cell cytotoxicity directed by BsAb, which was composed of OKT3 and anti-SCLC mAb, was also increased by the IFN-gamma treatment of SCLC, and this increase was inhibited by an anti-ICAM-1 mAb but not by anti-Class I or anti-CD2 mAb. These results suggest that a prior administration of IFN-gamma would enhance the efficacy of the following specific targeting therapy utilizing BsAb and LAK cells by up-regulating the ICAM-1 expression on tumor target cells. The combinational use of IFN-gamma and anti-CD3 x anti-tumor BsAb might be a promising way of enhancing LAK cell-mediated adoptive immunotherapy in small cell lung cancer patients.     

Decreased susceptibility of lined human gliosarcoma cells to lymphokine-activated killer cell cytolysis by gamma-interferon treatment

The susceptibility of the established cultured gliosarcoma line GI-1 to lymphokine-activated killer (LAK) cells was analyzed with and without interferon (IFN)-gamma treatment of target GI-1 cells. IFN-gamma treatment decreased the susceptibility of GI-1 cells to LAK cell cytolysis in a dose-dependent manner. Acid treatment of GI-1 cells increased their susceptibility to cytolysis compared with untreated cells. IFN-gamma treatment and acid treatment of GI-1 cells respectively increased and decreased the expression of class I HLA antigens on GI-1 cells. The susceptibility of GI-1 cells to LAK cell cytolysis and their expression of HLA class I molecules were inversely correlated. Subpopulation depletion experiments on the LAK cells with monoclonal antibodies and complement revealed that phenotypically natural killer type (CD16+) cells had a high cytotoxic activity against untreated GI-1 cells but a relatively low activity against IFN-gamma-treated GI-1 cells in both the precursor and effector phases. On the other hand, phenotypically T-type (CD3+) cells did not show these tendencies at all in both the precursor and the effector phases.     

Interferon-induced increase in sensitivity of ovarian cancer targets to lysis by lymphokine-activated killer cells: selective effects on HER2/neu-overexpressing cells.

Overexpression of the HER2/neu oncogene in ovarian tumor cells is associated with relative resistance to lymphokine-activated killer (LAK) cell cytotoxicity. Treatment with gamma-interferon (IFN-gamma) (200-2000 units/ml) for 3 days markedly enhanced the sensitivity of HER2/neu-overexpressing ovarian tumor cells to LAK cells but had no effect on the sensitivity of nonexpressing ovarian targets. Increased sensitivity to lysis was associated with an increase in effector-target conjugate formation, the induction of target cell intercellular adhesion molecule 1 (ICAM-1) expression, and the down-regulation of HER2/neu expression. Anti-ICAM-1 antibody blocked the enhanced lysis, indicating that ICAM-1 is important in the increased sensitivity to LAK cells. However, induction of ICAM-1 expression did not correlate well with enhanced sensitivity to lysis; it was maximal after 24 h of exposure to IFN-gamma and still present 24 h after removing IFN-gamma. In contrast, enhanced lysis required 3 days of exposure to IFN-gamma and was reversed within 24 h after removal of IFN-gamma. These data indicate that, although ICAM-1 is necessary, it is not sufficient for the IFN-gamma-induced enhancement of sensitivity to LAK lysis.     

Mechanisms of selective killing of neuroblastoma cells by natural killer cells and lymphokine activated killer cells. Potential for residual disease eradication.

Widely disseminated neuroblastoma in children older than infancy remains a very poor prognosis disease. Even the introduction of marrow ablative chemotherapy with autologous rescue has not significantly improved the outlook for these children, presumably because of a failure to eradicate minimal residual disease. One additional approach which may hold promise is the use of immunomodulation with cytokines such as IL2 in the setting of minimal residual disease (MDR), for example after intensive chemotherapy and ABMT. However, considerable variability in the susceptibility of neuroblastoma cells to natural killer (NK) and lymphokine-activated (LAK) killing has been observed, and it is presently unclear how NK and LAK cells recognise neuroblastoma cells. In this paper we examine expression of cell adhesion molecules on neuroblastoma to determine which of these modify interaction with NK and LAK cells. We find that LFA-3 (CD58), the ligand for CD2 is of predominant importance in predicting susceptibility of neuroblastoma to the cytotoxic actions of NK and LAK cells, while expression of ICAM-1 (CD54) may also modify susceptibility. These findings were confirmed by blocking experiments in which co-culture of target cells with ICAM-1 and LFA-3 reduced LAK and NK cytotoxicity. Study of the immunophenotypic features of each patient''s neuroblastoma cells before induction of MRD may be valuable in determining the likely effect of IL2 in predicting disease reactivation.     

Interferon-gamma-induced alterations of monocyte susceptibility to lysis by autologous lymphokine-activated killer (LAK) cells

Interleukin-2 (IL-2)-activated killer (LAK) cells specifically lyse human monocytes, which may account for some of the toxicity seen during LAK/IL-2 immunotherapy of cancer patients. In an effort to protect autologous monocytes, we treated monolayer cultures of monocytes with various doses of recombinant human interferon-gamma (IFN-gamma) and assessed their sensitivity to LAK-mediated lysis. IFN-gamma lessens the sensitivity of monocytes to lysis in a dose-dependent manner. Treatment of FMEX, an NK-resistant melanoma tumor cell line, with IFN-gamma did not affect its susceptibility to LAK lysis. Kinetic studies demonstrated that as little as 2 hr incubation with IFN-gamma was sufficient for protection to occur, and that monocytes which were treated with IFN-gamma for 2 hr, washed, and then cultured in medium alone retained their resistance to lysis for at least 4 days. Cold target inhibition studies showed that IFN-treated and untreated monocytes could effectively compete with each other for binding sites on LAK cells. Finally, binding studies demonstrated that there was no significant difference between the number of conjugates formed using either IFN-treated or untreated monocytes. This indicates that resistance to lysis induced by IFN treatment affects a post-binding event and not an initial recognition signal.     

Blockade of natural killer cell-mediated lysis by NCAM140 expressed on tumor cells

Expression of the neural cell adhesion molecule (NCAM) on malignant cells of neuroendocrine, epithelial and hematopoeitic origin has been reported, but its role for tumor cell recognition by the immune system remained uncertain so far. We have studied the cytotoxicity of the natural killer (NK) cell line NK92 and polyclonal NK cells from different donors, against NCAM-deficient and NCAM-transfected tumors. While the pancreatic carcinoma PANC-1 and the glioblastoma T98G showed no enhanced susceptibility to NK lysis after NCAM transfection, de novo NCAM expression in HeLa cervical carcinoma, SHEP neuroblastoma and the multiple myeloma lines RPMI-8226 and LP-1 was associated with significantly decreased lysis by NK cells. Binding of an NCAM-specific monoclonal antibody to NCAM-positive target cells was able to reverse the reduced lysis susceptibility. Conjugate formation of NCAM-expressing tumor cells with NK cells was blocked and could be restored by anti-NCAM. NK cell-expressed NCAM molecules which might engage in homotypic cis- or trans-interactions had no apparent inhibitory function. The known cis-ligands of NCAM, heparan sulfate proteoglycan and L1-CAM, were also not directly involved in NK inhibition. ICAM-1 mRNA and cell surface expression was downmodulated in NCAM-transfected HeLa cells. ICAM-1 is involved in killer cell immune synapse formation. Its downmodulation may therefore contribute to the reduced lysis of NCAM-expressing target cells. We conclude that aberrant expression of NCAM on tumor cells of different histogenetic origin can lead to inhibition of target cell recognition and lysis by NK cells.     

Differentiation-related expression of adhesion molecules and receptors on human neuroblastoma tissues, cell lines and variants.

The expression of cellular adhesion molecules (CAM) involved in cell adhesion and immune recognition was measured on neuroblastoma tissue samples, on a neuroblastoma (NB) cell line, SK-N-SH, and on 3 phenotypically different variants, SH-SY5Y, SH-EP, SH-IN, representing neuronal, Schwannian/glial or intermediate NB-cell types. Immunohistochemical analysis of CAM expression by NB and related tumors at different stages of differentiation revealed a co-expression of several CAM (ICAM-1/CD54, LFA-3, VLA-2 and HLA-ABC) associated with low stages and more highly differentiated NB tumors and peripheral neuroepitheliomas (PN). In contrast, N-CAM was uniformly expressed on all NB tumors. Flow cytometric analysis of CAM surface expression by SK-N-SH and variant cells revealed highly variable phenotypes. Expression of ICAM-1, LFA-3, VLA-2 and HLA-ABC molecules was associated with the epithelial cell type represented by the SH-EP variant. In contrast, low expression of these molecules and high expression of N-CAM was associated with the neuronal SH-SY5Y cells. Exposure of the NB cells to differentiation inducers (retinoic acid, 5'-bromodeoxyuridine and phorbol esters) and cytokines (tau-interferon, alpha-tumor necrosis factor) resulted in a variable up-regulation of the expression of all CAMs, except N-CAM, regardless of the type of differentiation induced. In an attempt to establish a link between the pattern of expression of CAM on NB cells and their susceptibility to natural killer (NK) or lymphokine-activated killer (LAK) cell lysis, the analysis revealed that NB cells expressing CAM and a differentiated phenotype were less susceptible to NK lysis, but no difference in the sensitivity of the NB cell types to LAK effectors was observed. Treatment of NB target cells with cytokines or PMA decreased their susceptibility to NK and LAK lysis, while induction of differentiation with RA or BUdR resulted in no changes in the sensitivity to NK and LAK lysis. In conclusion, expression of HLA-ABC and several co-regulated CAMs was shown to be associated with a differentiated phenotype in NB, with an overall decreased sensitivity to NK/LAK effector cells.     

Effect of IFN-gamma treatment and in vivo passage of murine tumor cell lines on their sensitivity to lymphokine-activated killer (LAK) cell lysis in vitro; association with H-2 expression on the target cells

Interferon-gamma (IFN-gamma) treatment or in vivo passage of the murine YAC-1 lymphoma resulted in reduced sensitivity to in vitro lysis by syngeneic murine spleen cells cultured in rIL-2 (LAK-cells). IFN-gamma treatment also rendered the murine B16 melanoma less sensitive to lysis by syngeneic LAK cells, whereas in vivo passage did not alter LAK sensitivity. The reduction in sensitivity to lysis correlated with enhanced expression of cell surface H-2 on the target cells. The possible role of H-2 was studied with a beta 2-microglobulin-deficient, and thus H-2-deficient, variant of the YAC-1 lymphoma. This variant line remained H-2 negative even after IFN-gamma treatment or in vivo passage, and was highly sensitive to LAK-cell-mediated lysis, even after IFN-gamma treatment or in vivo passage. The present results are discussed in relation to IFN-gamma and in vivo induced modulation of MHC class-1 molecules on target cells and the possible consequences for interaction with activated as well as "natural" effector cells.     

Celecoxib increases lung cancer cell lysis by lymphokine-activated killer cells via upregulation of ICAM-1

The antitumorigenic mechanism of the selective cyclooxygenase-2 (COX-2) inhibitor celecoxib is still a matter of debate. Using lung cancer cell lines (A549, H460) and metastatic cells derived from a lung cancer patient, the present study investigates the impact of celecoxib on the expression of intercellular adhesion molecule 1 (ICAM-1) and cancer cell lysis by lymphokine-activated killer (LAK) cells. Celecoxib, but not other structurally related selective COX-2 inhibitors (i.e., etoricoxib, rofecoxib, valdecoxib), was found to cause a substantial upregulation of ICAM-1 protein levels. Likewise, ICAM-1 mRNA expression was increased by celecoxib. Celecoxib enhanced the susceptibility of cancer cells to be lysed by LAK cells with the respective effect being reversed by a neutralizing ICAM-1 antibody. In addition, enhanced killing of celecoxib-treated cancer cells was reversed by preincubation of LAK cells with an antibody to lymphocyte function associated antigen 1 (LFA-1), suggesting intercellular ICAM-1/LFA-1 crosslink as crucial event within this process. Finally, celecoxib elicited no significant increase of LAK cell-mediated lysis of non-tumor bronchial epithelial cells, BEAS-2B, associated with a far less ICAM-1 induction as compared to cancer cells. Altogether, our data demonstrate celecoxib-induced upregulation of ICAM-1 on lung cancer cells to be responsible for intercellular ICAM-1/LFA-1 crosslink that confers increased cancer cell lysis by LAK cells. These findings provide proof for a novel antitumorigenic mechanism of celecoxib.     

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.     

Recombinant gamma interferon provokes resistance of human breast cancer cells to spontaneous and IL-2 activated non-MHC restricted cytotoxicity

Natural and lymphokine activated killer cells (NK and LAK) are believed to play an important role in the control of tumour progression and metastasis. Their specific receptors on tumours cells are still unknown. Several studies suggest that these cells recognise and eliminate abnormal cells with deleted or reduced expression of MHC class I molecules. Previous reports suggest that interferons (IFN), by increasing MHC class I expression on target cells, induce resistance to killing by NK cells. We investigated the role of MHC molecule expression by two human breast cancer cell lines T47D and ZR75-1 in their susceptibility to NK and LAK cells. These two cell lines spontaneously express low levels of HLA class I antigens but no HLA class II molecules. After IFN-gamma treatment they both overexpressed MHC class I and de novo expressed class II molecules as detected by flow cytometry, quantified by a radioimmunoassay and analysed by two-dimensional gel electrophoresis. Opposed to untreated cells these IFN-gamma treated cells were resistant to NK and LAK lysis. Furthermore, preincubation of IFN-gamma treated breast cancer cells with F(ab')2 fragments of monoclonal antibodies to HLA class I and HLA class II molecules was unable to restore lysis. In contrast, several complete monoclonal antibodies including anti-HLA class I and HLA class II induced the lysis of target cells whether or not they had been treated by IFN-gamma. The therapeutic use of monoclonal antibodies directed against antigens expressed on tumour cells (ADCC) in conjunction with interferon therapy should be discussed in lymphokine-based strategies for treatment of cancer patients.     

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.     

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.     

Induction of human monocyte susceptibility to lymphokine-activated killer cell lysis by granulocyte-macrophage colony-stimulating factor

We have recently reported that cultured human monocytes are susceptible to lysis by autologous lymphokine-activated killer (LAK) cells. In an attempt to modulate the sensitivity of monocytes to LAK-mediated lysis, monocytes were cultured in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF was found to enhance the susceptibility of monocytes to lysis by LAK cells by 2- to 5-fold over that of untreated cells in a dose-dependent manner. As little as 10 units of GM-CSF per milliliter was sufficient to induce increased sensitivity. In a kinetics study, susceptibility of monocytes increased after 2 days of incubation with GM-CSF, with peak sensitivity occurring from 4 to 6 days of culture. The effect of GM-CSF appeared to be specific for monocytes within the circulating peripheral blood cells because nonadherent cells (NAC) and granulocytes, which are normally resistant to LAK-mediated lysis, did not become susceptible after treatment with GM-CSF. In cold-target inhibition experiments, unlabeled GM-CSF-treated monocytes, but not untreated monocytes, could block the lysis of FMEX, a human melanoma tumor cell line, as well as freshly isolated tumor cells. Finally, LAK cells specifically bound to GM-CSF-treated monocytes in significantly higher percentages than to control monocytes. In summary, our results indicate that GM-CSF was capable of enhancing the susceptibility of monocytes to LAK lysis possibly via increased binding or expression of target structure(s).     

Role of CD18 in lymphokine activated killer (LAK) cell-mediated lysis of human monocytes: comparison with other LAK targets

The role of CD18, identified as the beta chain of the CD11 family of adhesion glycoproteins, in the lysis of normal autologous monocytes by interleukin-2-activated killer (LAK) cells was explored. The addition of several preparations of anti-CD18 monoclonal antibodies (MAbs) to the incubation mixture of LAK cells and various target cells indicated that lysis of autologous monocytes, K562 erythroleukemia tumor cells, FMEX melanoma tumor cells, and fresh ovarian tumor cells were readily inhibited by all anti-CD18 antibodies tested. Kinetic experiments demonstrated that significant inhibition of lysis occurred if RH1-38 antibody was added up to 2 hr after LAK cells were added to target cells. By the use of selective coating of targets and effector cells with RH1-38, it was determined that anti-CD18 antibody inhibited lysis at the effector cell level but not at the target cell level, although CD18 was detectable on the surface of monocyte targets by FACS analysis and immunoprecipitation. Furthermore, specific binding of LAK cells to all targets tested was not affected by the presence of anti-CD18, indicating that lysis of target cells was blocked at a post-binding event. Finally, of the 3 alpha chains associated with CD18, only antibodies to LFA-1 (CD11a) partially blocked binding of LAK cells to monocytes and tumor cells. It is possible, then, that both CD11a and CD18 may work in concert to effect the lysis of target cells by LAK cells.     

The high lysability by LAK cells of colon-carcinoma cells resistant to doxorubicin is associated with a high expression of ICAM-1, LFA-3, NCA and a less-differentiated phenotype.

A human colon-carcinoma cell subline resistant to doxorubicin (LoVo/Dx), previously shown to be more lysed than the chemosensitive subline LoVo/H by different immune effectors, is reported here to be similarly susceptible to direct, anti-proliferative effect of soluble cytokines (TNF-alpha and/or IFN-gamma). More adhesion molecules ICAM-1, LFA-3 and NCA were expressed on LoVo/Dx than on LoVo/H, while no significant amounts of CEA were detectable on the cell surface or in culture supernatant of either tumor subline. Anti-ICAM-1, anti-LFA-3 and anti-NCA monoclonal antibodies (MAbs) caused a marked reduction of lysis by interleukin-2 (IL-2) activated lymphocytes (LAK) of LoVo/Dx, whereas a lower effect was evident on LoVo/H. A pool of these antibodies was able to further increase the inhibition of the LAK lysis of both sublines. LoVo/Dx displayed a less differentiated phenotype as assessed by morphology, in vitro growth and altered or increased expression of markers such as desmoplakin and vimentin respectively, and disappearance of mucin. Treatment of LoVo sublines with differentiating agents (dimethylformamide and retinoic acid) led to a decreased expression of all adhesion molecules studied, accompanied by increased resistance to LAK-mediated lysis. These data indicate that sensitivity of chemoresistant tumor cells to cytotoxic effectors depends on the level of expression of adhesion molecules, including NCA, and is related to differentiation stage.     

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.     

Therapeutic efficacy of interleukin-2 activated killer cells against adriamycin resistant mouse B16-BL6 melanoma.

Development of multidrug-resistance (MDR) remains a major cause of failure in the treatment of cancer with chemotherapeutic agents. In our efforts to explore alternative treatment regimens for multidrug-resistant tumors we have examined the sensitivity of MDR tumor cell lines to lymphokine activated killer (LAK) cells. Adriamycin (ADM) resistant B16-BL6 melanoma, L1210 and P388 leukemic cell lines were tested for sensitivity to lysis by LAK cells in vitro. While ADM-resistant B16-BL6 and L1210 sublines were found to exhibit at least 2-fold greater susceptibility to lysis by LAK cells, sensitivity of ADM-resistant P388 cell was similar to that of parental cells. Since ADM-resistant B16-BL6 cells were efficiently lysed by LAK cells in vitro, the efficacy of therapy with LAK cells against the ADM-resistant B16-BL6 subline in vivo was evaluated. Compared to mice bearing parental B16-BL6 tumor cells, the adoptive transfer of LAK cells and rIL2 significantly reduced formation of experimental metastases (P less than 0.009) and extended median survival time (P less than 0.001) of mice bearing ADM-resistant B16-BL6 tumor cells. Results suggest that immunotherapy with LAK cells and rIL2 may be a useful modality in the treatment of cancers with the MDR phenotype.     

Susceptibility of adult acute lymphoblastic leukemia blasts to lysis by lymphokine-activated killer cells

To help understand host-tumor relationships in adult acute lymphoblastic leukemia (ALL) and to better define potential indications for interleukin-2 (IL-2) treatment in this disease, the relationship between the susceptibility of leukemia cells of 22 patients with ALL to lysis by allogeneic lymphokine-activated killer (LAK) cells and characteristics of the leukemia was studied. Lymphocytes were activated in the presence of 1000 U/ml recombinant IL-2 for 5 days. The lysis of ALL cells was studied by the release of 51Cr. The average lysis of ALL cells by control, unactivated lymphocytes was 1.2 +/- 2.4% and by LAK cells 8.9 +/- 8.6%. The susceptibility of leukemic cells to lysis did not correlate with the expression of lymphoid or myeloid differentiation markers or expression of the adhesion molecules CD54 (ICAM-1) and CD58 (LFA-3). Leukemic cells of the FAB I2 subtype were significantly more resistant to lysis than those of the other subtypes (average lysis 1.4 +/- 3.0% versus 12.3 +/- 8.2%, p = 0.003). The susceptibility to lysis did not correlate with the other initial characteristics of the leukemia. The 11 patients in whom 8% or more of leukemic cells were lysed by allogeneic LAK cells survived significantly longer than the 11 patients whose blast cells were less susceptible to lysis (p = 0.04). It is concluded that IL-2 treatment might be of benefit in adult ALL, particularly in non-L2 FAB subtypes and during complete remission to possibly delay relapse and prolong survival.     

Altered expression of P-glycoprotein and cellular adhesion molecules on human multi-drug-resistant tumor cells does not affect their susceptibility to NK- and LAK-mediated cytotoxicity.

Drug resistance has been associated with resistance to NK- and LAK-cell-mediated cytotoxicity. We evaluated this issue in human cell lines, using multiple myeloma cells (8226) and 2 multi-drug-resistant (MDR) sublines selected using doxorubicin (8226/Dox40) and mitoxantrone (8226/MR40). In parallel, we studied the human breast carcinoma cell line series MCF7, MCF7/D40 and MCF7/Mitox. Unlike the sensitive parental cell lines, all 4 sublines display MDR-patterns of resistance, with the P-glycoprotein pump (P-170) detected only in the doxorubicin-selected sublines. Flow cytometric and immunocytochemical analyses showed expression of cellular adhesion molecules ICAM-I and LFA-3, and MHC-Class-I (MCF7/D40 only), to be decreased in the doxorubicin-selected MDR-sublines, whereas expression of CD56 (Leu 19) was strongly up-regulated in 8226/Dox40. Lysis of P-170-positive MDR tumor cells by NK or LAK cells was, however, unaffected by these alterations, suggesting redundancy in effector:target-cell adhesion pathways. Mitoxantrone-selected tumor cells did not display P-170, nor did they show altered expression of cellular adhesion molecules. Their susceptibility to NK or LAK cytolysis was also unimpaired as compared to the parental cell lines. Clinically, these results imply that immunotherapeutic modalities aiming at increased natural killer functions deserve full consideration even in patients who have become refractory to further cytostatic drug treatment.