Human tumor cell line resistance to chemotherapeutic agents does not predict resistance to natural killer or lymphokine-activated killer cell-mediated cytolysis

Cancer cells selected for resistance to natural product chemotherapeutic agents typically display cross-resistance to a variety of structurally and mechanistically diverse agents, a phenomenon known as multidrug resistance. Preliminary studies involving cells selected for multidrug resistance in vitro have suggested that the development of resistance to these agents might simultaneously confer resistance to some forms of immunotherapy. Using human tumor cell line models, we have investigated the relationship between either intrinsic or selected multidrug resistance and sensitivity to natural killer (NK) or lymphokine-activated killer (LAK) cell-mediated cytolysis. We compared the NK and LAK cell susceptibility of three human tumor cell lines displaying distinct mechanisms of selected drug resistance with that of the parental drug-sensitive lines. We also evaluated the NK and LAK susceptibility of five established renal cell carcinoma lines, all of which were found to be intrinsically resistant to doxorubicin and vinblastine. The drug-resistant cell lines were variably sensitive to NK-mediated lysis. In contrast, all drug-resistant cell lines tested were LAK cell sensitive. The NK and LAK cell-mediated cytolytic sensitivities of the drug-resistant cell lines correlated well with those of the drug-sensitive parental lines, suggesting that susceptibility to lysis was related intrinsically to each tumor type, and not to the resistance phenotype. We attempted to correlated the NK sensitivity of these cells with the cell surface expression of Class I or II histocompatibility antigens, or the presence or absence of the membrane inhibitor of complement-mediated reactive lysis. None of these phenotypic markers were found to predict NK resistance. We therefore conclude that these cells, which are either spontaneously resistant to commonly utilized antitumor agents or are multidrug resistant as a result of drug exposure in vitro, remain sensitive to LAK cell-mediated cytolysis. Our studies suggest that interleukin 2-induced LAK cells may be useful in the therapy of some chemotherapy-resistant cancers.     

Heterogeneity and phenotypic instability of chemotherapeutic and immunologic sensitivity in murine and human melanoma cell clones.

The aim of the present study was to examine the phenotypic heterogeneity of murine and human melanoma cell lines with particular reference to anticancer drug sensitivity, growth pattern and susceptibility to lysis by lymphokine (rIL2) activated killer (LAK cells). Clones selected for a different drug sensitivity were tested to evaluate the stability of such properties after different in vitro passages. A possible relationship between drug sensitivity and LAK susceptibility was also analyzed. The results indicated a high heterogeneity in murine and in human melanoma clones for all the parameters. However, drug sensitivity, which was stable although for only a few passages in an untreated human melanoma, was highly unstable in murine naturally or drug-induced resistant cells. Finally, whereas human drug-resistant clones were sensitive to lysis by LAK cells and an inverse correlation was found with the level of drug resistance, murine clones appeared to be LAK sensitive, and no correlation was found between the level of drug resistance and LAK sensitivity. Our data indicate a different stability in drug response of human and murine cells and a different behaviour of human and murine drug-resistant cells in response to LAK lysis.     

The relationship between multi-drug resistance and resistance to natural-killer-cell and lymphokine-activated killer-cell lysis in human leukemic cell lines.

Results concerning a possible link between susceptibility to natural-cell-mediated immune cytolysis and the multi-drug resistance (MDR) phenotype are conflicting. We evaluated in human acute lymphocytic leukemia the relationship between acquired drug resistance and susceptibility to cytolysis mediated by endogenous, interferon-activated, and interleukin-2-activated natural cytotoxic cells. Eight human leukemia drug-resistant/sensitive cell line pairs were evaluated; drug-resistant sub-lines included those selected for primary resistance to adriamycin, etoposide, teniposide, vincristine, and vinblastine. A majority of P-glycoprotein-positive MDR sub-lines displayed slight but statistically significant resistance to endogenous and/or interferon-activated natural-killer(NK)-cell-mediated lysis, as compared with the drug-sensitive parental type. P-glycoprotein-negative sub-lines displayed variable NK susceptibility; within this group, the variants selected for primary etoposide resistance were more susceptible to NK cytolysis than parental cells. Results of cold-target-inhibition experiments suggest that altered NK susceptibility does not arise solely from modulation of NK target recognition and adherence structures. IL2-activated killer (LAK) cells lysed both drug-sensitive and drug-resistant lines. Two MDR lines selected for primary etoposide resistance displayed enhanced LAK susceptibility. In contrast, the 2 variants selected for resistance to adriamycin exhibited partial resistance to LAK-mediated killing, which could be overcome at high effector-to-target ratios. Our results support the development of interleukin-2/LAK immunotherapy for the treatment of leukemias with acquired drug resistance.     

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.     

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug-resistant colon carcinoma cells (LoVo) than on their wild-type counterparts

The occurrence of resistance to cytotoxic agents in tumor cells, associated with several phenotypic alterations, is one of the major obstacles to successful anticancer chemotherapy. A new strategy to overcome MDR of human cancer cells was studied, using BSAO, which generates cytotoxic products from spermine, H(2)O(2) and aldehyde(s). The involvement of these products in causing cytotoxicity was investigated in both drug-sensitive (LoVo WT) and drug-resistant (LoVo DX) colon adenocarcinoma cells. Evaluation of clonogenic cell survival showed that LoVo DX cells are more sensitive than LoVo WT cells. Fluorometric assay and treatments performed in the presence of catalase demonstrated that the cytotoxicity was due mainly to the presence of H(2)O(2). Cytotoxicity was eliminated in the presence of both catalase and ALDH. Transmission electron microscopic observations showed more pronounced mitochondrial modifications in drug-resistant than in drug-sensitive cells. Mitochondrial functionality studies performed by flow cytometry after JC-1 labeling revealed basal hyperpolarization of the mitochondrial membrane in LoVo DX cells. After treatment with BSAO and spermine, earlier and higher mitochondrial membrane depolarization was found in LoVo DX cells than in drug-sensitive cells. In addition, higher basal ROS production in LoVo DX cells than in drug-sensitive cells was detected by flow-cytometric analysis, suggesting increased mitochondrial activity in drug-resistant cells. Our results support the hypothesis that mitochondrial functionality affects the sensitivity of cells to the cytotoxic enzymatic oxidation products of spermine, which might be promising anticancer agents, mainly against drug-resistant tumor cells.     

Differential lysis of melanoma clones by autologous recombinant interleukin 2-activated lymphocytes. Relationship with spontaneous resistance to doxorubicin (Dx)

To investigate whether human melanoma cells intrinsically resistant to autologous LAKs do exist, and whether a relationship between the level of lysis of LAKs and spontaneous drug resistance can be identified at the clonal level, we studied 44 clones obtained from a metastatic melanoma lesion. The antigenic phenotype of clones revealed a marked heterogeneity in the expression of HLA antigens of classes I and II. The clones were subsequently tested for sensitivity to autologous LAK and for spontaneous resistance to Dx. No clone resistant to autologous LAK was found, although a considerable range of lysis was noted with a normal frequency distribution. Growth in agar of the 2 clones in which lysis was least pronounced (6 and 26) was completely inhibited after co-culture with LAKs, indicating a lack of absolute resistance to these effectors. Spontaneous resistance to Dx, evaluated as ID50, revealed instead that the majority of clones had a low ID50. The frequency distribution of clones showed a left-skewed curve. The percentage of specific 51Cr-release and the ID50 for Dx could be correlated in 25 clones by linear regression. Sensitivity to LAK did not correlate with HLA classes I or II or melanoma-associated antigen expression. These results support the contention that increased LAK sensitivity of tumor cells is associated with drug resistance.     

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).     

人LAK细胞的诱导及其生物特性的研究

LAK cell activity of peripheral blood from normal donors was induced by interleukin 2 (IL-2). Optimal induction of LAK cells was obtained by 3-4 day incubation of PBMC in the presence of 30-40 units/ml of IL-2. Several cell lines Raji, CEM, HeLa, 109 (esophageal carcinoma) and OC (ovarian carcinoma) which were NK cell resistant tumor cells could be lysed by LAK cells. PHA was unable to markedly induce the LAK cell activity. Although both LAK and NK cell activity was decreased by mitomycin C treatment, their degree of susceptibility to such treatment was quite different. NK cells were more sensitive to mitomycin C than LAK cells. In contrast to the significant inhibition of the NK cell-mediated lysis by the addition of anti-CD 2 monoclonal antibody during cytotoxicity assay LAK cell-mediated lysis was not affected. These results indicate that LAK cells are capable of lysing a variety of tumor cells that are resistant to NK cells and their biological characteristics are also different.     

Efficacy of an anti-CD138 immunotoxin and doxorubicin on drug-resistant and drug-sensitive myeloma cells.

Multidrug resistance is an increasing problem in the treatment of cancer. We evaluated in vitro the effect of an anti-CD138 plasma-cell-specific immunotoxin (IT, B-B4-SO6) in combination with the chemotherapeutic drug doxorubicin on drug-sensitive and drug-resistant variants of the multiple-myeloma (MM)-derived cell line RPMI8226 and freshly isolated malignant-myeloma cells. Drug-resistant RPMI8226 cells were still sensitive to the IT, although to a lesser extent than drug-sensitive cells. In the clonogenic assay, using 10 nM B-B4-SO6, at least 5 logs kill was found for drug-sensitive RPMI8226 cells, vs. 2.5 logs kill for the drug-resistant RPMI8226 cells. When a sub-optimal dose of 1 nM IT was combined with 3 ng/ml doxorubicin, which was toxic for drug-sensitive but not for drug-resistant cells, an additive effect was found for drug-sensitive RPMI8226 cells. The IT did not influence the sensitivity of resistant cells for doxorubicin. We therefore speculate that this type of IT, may be of more value in combination with primary chemotherapy. The effect of B-B4-SO6 on malignant-myeloma cells of patients was investigated in a viability assay. Both drug-sensitive and drug-resistant cells from MM patients were sensitive to B-B4-SO6. After 2 days, a 50% kill of malignant cells was found when 10 nM IT were used. Doxorubicin was effective only on sensitive cells, and there was a tendency for an additive effect in the combination of these cells.     

Increased susceptibility of IFN-gamma-treated neuroblastoma cells to lysis by lymphokine-activated killer cells: participation of ICAM-1 induction on target cells.

We have investigated the effect of interferon-gamma (IFN-gamma) treatment of neuroblastoma cells on the susceptibility to lysis by lymphokine-activated killer (LAK) cells and examined the participation of cell-adhesion molecules on the target cells in LAK cell lysis. Untreated neuroblastoma cells expressed lymphocyte-function-associated antigen 3 (LFA-3) and neural-cell-adhesion molecule (NCAM), but did not express MHC-class-I, MHC-class-II, or intercellular-adhesion molecule I (ICAM-I). IFN-gamma treatment of neuroblastoma cells induced the expression of MHC-class-I and ICAM-I antigens, but did not affect the expression of MHC-class-II, LFA-3, and NCAM. This was accompanied by an increased susceptibility to lysis by LAK cells. Anti-ICAM-I antibody inhibited partially the increased sensitivity of IFN-gamma-treated neuroblastoma cells to LAK cell lysis, and blocked completely the increase in binding of LAK cells observed after IFN-gamma treatment of the target cells. These results suggest that the increased LAK sensitivity of IFN-gamma-treated neuroblastoma cells is partially attributable to the induction of ICAM-I on neuroblastoma cells and indicate that post-binding events also play a role in the increased sensitivity to LAK cell lysis observed after IFN-gamma treatment.     

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.     

Lysis of autologous melanoma cells by tumor-infiltrating lymphocytes: association with clinical response

Tumor-infiltrating lymphocytes (TILs) can be grown in vitro in medium containing interleukin-2 (IL-2). In clinical trials at the Surgery Branch of the National Cancer Institute, patients with metastatic malignant melanomas were treated with IL-2 plus the adoptive transfer of autologous TILs. At the time of treatment, TILs were assayed for in vitro lysis of fresh autologous and allogeneic melanoma cells and Daudi cells. Patients were evaluated for clinical response 4-8 weeks later. Lysis of autologous tumor cells by TILs was significantly higher for responding than for nonresponding patients. Tumor cells from responding and nonresponding patients were equally sensitive to lysis by allogeneic lymphokine-activated killer (LAK) cells. There was no difference between TILs from responding and nonresponding patients for lysis of LAK-sensitive Daudi cells, which was low in most cases and demonstrated that TIL lysis of autologous tumor cells was not due to LAK cells. The observed association of autologous tumor cell lysis by TILs with clinical response suggests that the development of culture methods to optimize lysis of autologous tumors may lead to increased response rates using this TIL treatment regimen.     

Antitumor effects of a bispecific antibody targeting CA19-9 antigen and CD16.

Bispecific murine monoclonal antibodies that target tumor and Fc gamma RIII (CD16) can promote relevant tumor lysis by large granular lymphocytes. For these antibodies to be clinically useful, their properties should be maintained in vivo, where competing human immunoglobulin, shed target antigen, and shed CD16 may be encountered. At a minimum, bispecific antibody antitumor effects should be preserved in whole blood. Furthermore, potentiation of tumor lysis should be reflected by demonstrating the ability of bispecific antibody-retargeted effector cells to infiltrate and mediate lysis of organized tumor. If these characteristics are demonstrated, and there is evidence of in vivo efficacy of bispecific antibody-based therapy in a relevant animal model, further clinical development of such antibodies would be warranted. In this report the ability of CL158 bispecific antibody supernatants to mediate lysis of SW948 tumor growing in monolayer is shown to be preserved in the presence of interleukin 2-activated whole blood. When SW948 cells were grown in vitro as multicellular human tumor spheroids, incubation with interleukin 2-activated lymphocytes (LAK cells) and CL158 led to structural and widespread necrosis. This was dependent on CL158 and resistant to competition by pooled human immunoglobulin or interleukin 2-exposed whole blood. These effects were not promoted by the monospecific antibodies produced by the parent clones of CL158 and were not observed when the IgG2a variant of CA19-9 antibody, which mediates conventional antibody-dependent cellular cytotoxicity, was used instead of its bispecific derivative. To examine the efficacy of bispecific antibody-based treatments on in vivo tumor, scid mice bearing early s.c. SW948 xenografts were treated with interleukin 2 for 5 consecutive days, supplemented by three i.v. injections of 10(7) human LAK cells and various antibodies. Treatment of mice bearing SW948 tumors with LAK cells did not retard tumor growth, but when CL158 was added, significant delays in tumor growth were observed. Tumor growth delay required treatment with both LAK cells and the bispecific antibody. Treatment with the IgG2a variant of CA19-9 antibody, alone or with LAK cells, had no effects on tumor growth. Although the mechanisms of these antitumor effects require further study, it is clear that human LAK cell treatment of animals bearing early, established s.c. tumors is enhanced by the addition of bispecific antibodies with relevant binding characteristics. When compared with the IgG2a isotype variant of CA19-9 monoclonal antibody, this bispecific antibody offers the advantages of preservation of activity in physiological conditions, infiltration and disruption of organized tumor in vitro, and antitumor effects in a relevant xenograft model.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Generation and partial characterization of melanoma sublines resistant to lymphokine activated killer (LAK) cells. Relevance to doxorubicin resistance

To see whether a tumor cell population may contain cells resistant to lymphokine-activated killer (LAK) lymphocytes, cells from a LAK-sensitive melanoma line (Me 665/2) were co-cultured with LAKs. Three sublines were obtained after 1, 2 or 3 immunoselection cycles. Immunoselected (IS) sublines show reduced proliferation, decreased reactivity to the monoclonal antibody (MAb) R24 and appeared morphologically more differentiated in comparison with the parental Me 665/2 line. A progressively reduced sensitivity to LAKs was observed in IS sublines with a more than 8-fold reduction in LAK susceptibility. A reduced complement (C)-mediated lysis was also observed in IS sublines. Since we have previously shown that LAK sensitivity of melanoma cells may be associated with Doxorubicin (Dx) resistance, the sensitivity to Dx was tested in these lines. An augmented sensitivity to Dx was noted in IS sublines as compared with Me 665/2. The differences in LAK susceptibility between the IS sublines and the parental Me 665/2 line remained stable for 2 weeks but declined and disappeared thereafter. These results indicate that (1) a LAK-sensitive tumor line may contain a subpopulation of cells which are significantly less lysed by LAKs; (2) a correlation between LAK sensitivity and susceptibility to C-mediated lysis is also present; and (3) increased sensitivity to Dx is evident in the IS sublines.     

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.     

Lymphokine-activated killer cells: lysis of fresh syngeneic natural killer-resistant murine tumor cells by lymphocytes cultured in interleukin 2

Normal splenocytes that are cultured in the lymphokine, interleukin 2 (IL-2), for as short as 2 days develop lytic activity for fresh syngeneic natural killer-resistant tumor cells as well as natural killer-sensitive YAC cells in a 4-hr 51Cr release assay. Lymphokine-activated killer (LAK) cells do not lyse syngeneic fresh lymphocytes but do lyse syngeneic concanavalin A-induced lymphocyte blasts. Lysis is not due to the presence of lectin or xenogeneic serum and appears to be an intrinsic property of lymphocytes activated in IL-2. The activation appears universal in that lymphocytes from all strains of mice activated in this manner exhibited similar patterns of lysis for fresh tumor target cells. To characterize the cells responsible for this lysis, we analyzed the phenotypic expression of surface markers on these cells with depletion techniques using monoclonal antibody and complement. These studies indicate that the precursor of the LAK cell is Thy-1+ and nonadherent to plastic or nylon wool. Lysis of syngeneic tumor was inhibited when LAK cells were treated with an anti-Thy-1.2, or anti-Lyt-2.2 monoclonal antibody and complement but not with anti-Lyt-1.2 monoclonal antibody and complement, indicating that the observed lytic activity was due to a Thy-1+ Lyt-1-2+ cell. Furthermore, LAK cell-mediated lysis could be inhibited by the addition of anti-Lyt-2 or LFA-1 monoclonal antibody to cytotoxicity assays. Cold target inhibition analysis revealed that the syngeneic tumor cells were lysed by recognition of a determinant not present on normal lymphocytes or lymphocyte blasts. This lysis of fresh solid tumor cells by lymphoid cells grown in IL-2 may be of value in the study of tumor-host immunological interactions. The biological significance of tumor lysis by IL-2-activated cells requires further study.     

Interaction between tumor and immune system: the role of tumor cell biology

In present work the role of tumor cell biology upon different conditions of their interaction with the cells of immune system is discussed. The presented data show that it tumor cell biology that in many cases determines tumor antigen recognition and realization of cytotoxic action of killer cells. Here also we discuss own data obtained with the use of experimental tumor models (transplantable MC-rhabdomyosarcoma, B16 melanoma) and human tumors (soft tissue sarcoma, melanoma, breast cancer, ovarian cancer, cervical cancer). It was demonstrated that various tumors have different sensitivity to antitumor action of activated (LAK) and non-activated lymphocytes. Recent studies on the role of tumor cells in expansion and activity of different suppressor cells (MDSC, Treg, Th17, M-2 macrophages, etc.) are overviewed. The role of organ-specificity and interaction of the components of microenvironment (cells of immune system and stroma, tumor cells, endothelial cells, extracellular matrix) are discussed in details. Along with the presentation of modern views on some patterns that characterize the development of drug resistance of different tumors and capabilities of immune system cells to participate in lysis of resistant tumor cells, the authors present their own data illustrating that resistant tumor cells reveal increased sensitivity to LAK. An analysis of the involvement of a number of molecules, lymphocytes, and tumor cells in the phenomenon of elevated sensitivity to LAK action allowed to conclude that tumor cells reveal high sensitivity at the background of decreased E-cadherin expression and increased CD40 expression.     

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.     

Generation of lymphokine-activated killer cells in strain 2 guinea pigs and their use in the therapy of L2C, an acute B-cell leukemia

Lymphokine-activated killer (LAK) cells were induced by incubating strain 2 guinea pig splenocytes or lymph node-derived cells in recombinant human interleukin-2 (IL-2) for 3-5 days. These effector cells had the morphology of lymphoblasts and were able to lyse murine P815 tumor cell targets. Fresh, unstimulated, guinea pig effectors were not capable of lysing these targets. The therapy of the L2C leukemia, an acute B-lymphoblastic leukemia of strain 2 guinea pigs, using LAK cells and recombinant IL-2 was examined. Antitumor effects were demonstrated by premixing LAK and tumor cells prior to intradermal injection in Winn type assays and then measuring the growth of local tumor and survival of the animals. In further experiments i.p. administration of LAK cells, 4 h following tumor cell inoculation by the i.p. route, prolonged the survival of treated animals. The best results in this i.p. therapy model were obtained with a 10-fold excess of LAK cells over tumor cells plus additional treatment with 1000 units of IL-2 for 20 days. This resulted in a 10-day increase in median survival of treated animals. Despite these in vivo antitumor effects, lytic activity of LAK effector populations against L2C targets could not be demonstrated in vitro. The potential synergy between LAK cells, IL-2, and a monoclonal antibody directed against the idiotype of the neoplastic cell surface immunoglobulin was also investigated. In these experiments enhanced survival of the combined treatment group, beyond that of either singly treated group, was not found. This study shows that LAK cells are useful agents in the therapy of a widely disseminated, aggressive, B-cell lymphoblastic leukemia. The use of such effectors, even in cases where in vitro lysis of the target tumor cell cannot be demonstrated, is encouraged by these results.