Lymphokine-activated Killer Effector Research Articles

Increased LAK activity against HIV-infected cell lines in HIV-1+ individuals.

The role of natural killer (NK) cells and their inducible counterparts, lymphokine-activated killer (LAK) cells in AIDS with regard to HIV-1 viral immunosurveillance and the control of secondary opportunistic disease has yet to be established. In this study, we have demonstrated that LAK cells derived from all HIV-1+ groups showed striking increases in their capacity to lyse HIV-1-infected U-937 cells relative to their uninfected U-937 counterparts. Surprisingly, similarly derived LAK cells from healthy seronegative controls showed no differences in their lysis of HIV-1-infected versus uninfected U-937 cells. The differential ability of LAK effectors from seropositive donors to lyse HIV-1-infected targets was demonstrable using a number of U-937 subclones and their HIV-1-infected counterparts. Again, no differences in LAK cell-mediated lysis of HIV-1-infected and uninfected U-937 subclones were observed in seronegative individuals. Our findings that HIV-1+ individuals show selective expansion of non-MHC restricted, HIV-1-directed cytotoxic LAK cells indicate that natural immunity may indeed play a role in HIV-1 viral immunosurveillance.

Evaluating the cytotoxicity of innate immune effector cells using the GrB ELISPOT assay

BackgroundThis study assessed the Granzyme B (GrB) ELISPOT as a viable alternative to the 51Cr-release assay for measuring cytotoxic activity of innate immune effector cells. We strategically selected the GrB ELISPOT assay because GrB is a hallmark effector molecule of cell-mediated destruction of target cells.MethodsWe optimized the GrB ELISPOT assay using the human-derived TALL-104 cytotoxic cell line as effectors against K562 target cells. Titration studies were performed to assess whether the ELISPOT assay could accurately enumerate the number of GrB-secreting effector cells. TALL-104 were treated with various secretion inhibitors and utilized in the GrB ELISPOT to determine if GrB measured in the ELISPOT was due to degranulation of effector cells. Additionally, CD107a expression on effector cells after effector-target interaction was utilized to further confirm the mechanism of GrB release by TALL-104 and lymphokine-activated killer (LAK) cells. Direct comparisons between the GrB ELISPOT, the IFN-γ ELISPOT and the standard 51Cr-release assays were made using human LAK cells.ResultsTitration studies demonstrated a strong correlation between the number of TALL-104 and LAK effector cells and the number of GrB spots per well. GrB secretion was detectable within 10 min of effector-target contact with optimal secretion observed at 3–4 h; in contrast, optimal IFN-γ secretion was not observed until 24 h. The protein secretion inhibitor, brefeldin A, did not inhibit the release of GrB but did abrogate IFN-γ production by TALL-104 cells. GrB secretion was abrogated by BAPTA-AM (1,2-bis-(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid tetra(acetoxymethyl) ester), which sequesters intracellular Ca2+, thereby preventing degranulation. The number of effector cells expressing the degranulation associated glycoprotein CD107a increased after interaction with target cells and correlated with the stimulated release of GrB measured in the ELISPOT assay.ConclusionsBecause of its high sensitivity and ability to estimate cytotoxic effector cell frequency, the GrB ELISPOT assay is a viable alternative to the 51Cr-release assay to measure MHC non-restricted cytotoxic activity of innate immune cells. Compared to the IFN-γ ELISPOT assay, the GrB ELISPOT may be a more direct measure of cytotoxic cell activity. Because GrB is one of the primary effector molecules in natural killer (NK) cell-mediated killing, detection and enumeration of GrB secreting effector cells can provide valuable insight with regards to innate immunological responses.

BACILLUS-CALMETTE-GUERIN (BCG) AND 3D TUMORS: AN IN VITRO MODEL FOR THE STUDY OF ADHESION AND INVASION

Purpose To study adhesion, penetration and internalization of BCG and effector-cells to and into three-dimensional in vitro cell aggregates from benign and malignant urothelial origin mimicking small in vitro tumors. Materials and Methods Multicellular spheroids (MCS) were generated by “liquid-overlay” technique. Adhesion and penetration of viable FITC-labelled BCG into MCS from urothelial cancer cell lines and normal urothelial cells was studied by electron microscopy (TEM) and fluorescence microscopy. Spheroid growth during BCG-co-incubation was determined by light microscopy. Peripheral blood mononuclear cells (PBMC) were stimulated with BCG to generate BCG-activated-killer (BAK) cells. The infiltration of these effectors and of lymphokine-activated killer (LAK) cells into MCS was examined at different intervals by means of immunohistochemistry. The resulting cytotoxicity was judged in a 3 H-l-methionine release assay. Results BCG adhered to MCS from tumor cells but not to benign cell MCS. Intracellular internalization of the bacteria was detectable in superficial tumor cell-layers (1-5) whereas BCG was not found in deeper layers. Proliferation of malignant MCS was reduced in the presence of BCG. Benign MCS showed contact inhibition growth arrest, which was not altered by BCG. BAK and LAK effector cells both infiltrated tumor cell MCS as opposed to unstimulated PBMC. In contrast to LAK cells, BAK cells did not infiltrate into benign cell MCS and were not cytotoxic towards them. Conclusion With regard to the clinical situation the selective adhesion and internalization of BCG to malignant cells might explain why BCG has been rarely found in follow-up biopsies in tumor free patients. More interestingly, the selective adhesion of BCG to and infiltration of BAK effector cells into malignant cell spheroids suggests a selective mode of action of BCG.

Strength of binding between leukemic blasts and cytotoxic lymphocytes

Strength of binding (Sb), a new parameter defined by the ratio of binding capacity and dissociation rate constants, was estimated and used for the characterization of conjugate formation between leukemic blasts and cytotoxic lymphocytes. Lymphokine activated killer (LAK) cells, i.e., interleukin-2 activated peripheral blood lymphocytes (PBL) displayed significantly (P<0.001) higher Sb with leukemic blasts when compared to fresh, nonactivated PBL. Interaction of both fresh PBL and LAK effector cells with acute myeloid leukemia (AML) blasts was characterized by a significantly (P<0.05) lower Sb when compared to the K562 cell line. Interaction of LAK effector cells with leukemic cell-lines of lymphoid origin (Daudi, Raji, and HuT78) was characterized by a significantly (P<0.005) lower Sb than with K562 myeloid cell line. Sb for the interaction of natural killer (NK)-derived CD16+/CK56+ LAK with leukemic blasts was significantly (P<0.001) higher than that of T-cell-derived CD3+ LAK. We conclude that calculation of Sb provides a relatively simple and independent way to evaluate systems of interacting cells at a single time and may be used to compare results between different cell systems and laboratories. © 1996 Wiley-Liss, Inc.

Strength of binding between leukemic blasts and cytotoxic lymphocytes.

Strength of binding (Sb), a new parameter defined by the ratio of binding capacity and dissociation rate constants, was estimated and used for the characterization of conjugate formation between leukemic blasts and cytotoxic lymphocytes. Lymphokine activated killer (LAK) cells, i.e., interleukin-2 activated peripheral blood lymphocytes (PBL) displayed significantly (P < 0.001) higher Sb with leukemic blasts when compared to fresh, nonactivated PBL. Interaction of both fresh PBL and LAK effector cells with acute myeloid leukemia (AML) blasts was characterized by a significantly (P < 0.05) lower Sb when compared to the K562 cell line. Interaction of LAK effector cells with leukemic cell-lines of lymphoid origin (Daudi, Raji, and HuT78) was characterized by a significantly (P < 0.005) lower Sb than with K562 myeloid cell line. Sb for the interaction of natural killer (NK)-derived CD16+/CD56+ LAK with leukemic blasts was significantly (P < 0.001) higher than that of T-cell-derived CD3+LAK. We conclude that calculation of Sb provides a relatively simple and independent way to evaluate systems of interacting cells at a single time and may be used to compare results between different cell systems and laboratories.

BCR-ABL Does Not Prevent Apoptotic Death Induced by Human Natural Killer or Lymphokine-Activated Killer Cells

The erythromyeloid cell line, K562, the most sensitive target in human natural killer (NK) cell mediated cytotoxicity, is derived from a chronic myeloid leukemia (CML) patient and expresses the characteristic reciprocal translocation t(9;22). The resulting BCR-ABL fusion protein has been shown to mediate the unusual resistance of K562, and other BCR-ABL expressing lines, to apoptosis induced by a variety of agents (irradiation, UV light, cytotoxic drugs). Here we show that human NK and lymphokine-activated killer (LAK) cells, when tested at low effector to target ratio, can readily induce apoptotic death in K562 cells. This was accompanied with classical DNA oligonucleosomal fragmentation, an unexpected finding given the reported lack of such fragmentation when apoptosis is induced in K562 by chemical agents, after downregulation of BCR-ABL. Apoptosis was assessed by several means: morphological studies, 125I-DNA versus 51Cr release, DNA agarose gel electrophoresis, and results were always concordant, with a delayed kinetics for DNA oligonucleosomal fragmentation. Similar data were obtained with a pluripotent human hematopoietic cell line, UT-7, infected with a defective amphotropic p210 BCR-ABL retrovirus. The BCR-ABL expressing subclone UT-7/9, while being no longer sensitive to cytotoxic drugs or to tumor necrosis factor, a lytic mediator to which UT-7 cells are sensitive, underwent apoptotic death when exposed to LAK effector cells to the same degree as the parental UT-7 line. With these targets, DNA oligonucleosomal fragmentation occurred concomitantly with isotope release. Results obtained with several inhibitors of exocytosis strongly suggest that cytotoxic granules mediate NK and LAK cell-induced apoptotic death. In conclusion, NK and LAK cell-induced apoptotic signals, unlike those activated by chemotherapeutic agents, are unaffected by the antiapoptotic action of BCR-ABL. This unique property may support the observed curative effect of allogeneic bone marrow transplantation in CML.

Prolactin increases the susceptibility of primary leukemia cells to NK and LAK effectors

Our previous studies have shown that prolactin (PRL), a pituitary and lymphocyte hormone and a ligand of the cytokine/hemopoietin receptors (R) superfamily, acts synergistically with interleukin (IL)-2 on the development of lymphokine activated killer (LAK) cells and enhances the effects of GM-CSF and IL-3 on myeloid progenitors' proliferation and differentiation. More recently, we have demonstrated that GM-CSF and IL-3 increase the sensitivity of acute myeloid leukemic (AML) cells to LAK activity. Together, these findings have prompted us to study the role of PRL on the target arm of the LAK response. We show here that CD33+ blasts from AML patients express membrane PRL-R and that the PRL/PRL-R interaction is followed by increased susceptibility to natural killer (NK) ( p < 0.02) and LAK ( p < 0.001) cells. As predicted from the dimerization model of PRL-R and in agreement with previous reports, the response of AML blasts to PRL was bell-shaped with a trend peak at 25 ng/ml. Although enhanced lysis occurred at the target recognition level, it was not accompanied by changes in the MHC class I, cellular adhesion molecules, or myeloid differentiation antigens. Cell cycle recruitment and lysis increased concurrently in three cases studied, suggesting a modulatory action of PRL on the expression of putative cycle-related NK/LAK-target structures. Together, these data strengthen the role of PRL in the LAK response.

Regulatory effect of lymphokine-activated killer cells on epidermal proliferation induced by cholera toxin in mice.

We investigated the effects of lymphokine-activated killer (LAK) cells on epidermal hyperplasia induced by cholera toxin (CT). LAK cells showed cytotoxic activity against both tumor cell lines and proliferating normal cells including skin epidermal cells. When 1 x 10(7) LAK cells were injected intradermally together with 1.0 ng of CT, epidermal hyperplasia was markedly suppressed. The LAK effectors inhibiting epidermal hyperplasia showed surface phenotypes of asialo-GM1+, Thy-1+, Lyt-2- and L3T4-, that were different from those of LAK cells killing tumor cells in vitro. Epidermal hyperplasia induced by CT was not suppressed by topical administration of cytokines such as interleukin-2, interferon and tumor necrosis factor. Therefore, the antiproliferative effect of LAK cells might be attributed to their direct action on the epidermal cells.

Expression of adhesion molecules on acute leukemic blast cells and sensitivity to normal LAK activity.

Antigenic expression of CD54 and CD58 adhesion molecules was investigated on leukemic blast samples from ten patients with acute lymphoblastic (ALL) and 14 with acute myeloblastic (AML) leukemia. The mean intensity of fluorescence (MIF) was calculated and correlated with sensitivity of blast cells to the lytic activity of allogeneic lymphokine-activated killer (LAK) cells. CD54 antigen was expressed in all AML cases with an MIF of 11.2 while in ALL, though present in the majority of cases, it was absent in one case and expressed in less than 30% of blasts in two others, with an overall MIF of 3.0. CD58 expression was similar in both groups of patients, with an MIF of 7.6 in ALL and 7.0 in AML. In addition, in six of the ten ALL cases and in two of the 14 AML cases, leukemic blasts proved to be "resistant" to the cytotoxic activity of normal allogeneic LAK effectors. In these "LAK-resistant" patients, the CD54 antigenic expression was lower (p = 0.03) than in "LAK-sensitive" patients with an MIF of 1.7 vs 4.9 in ALL and 1.35 vs 12.9 in AML cases. Finally, blocking of CD54 and/or CD58 receptors on leukemic blasts resulted in a slight reduction of 51Cr release. Findings suggest that CD54 is differently expressed on myeloid and lymphoid blasts and that there is a correlation between CD54 MIF and susceptibility of blasts to the LAK activity.

Inhibition of interleukin-2 dependent immune responses by serum from patients with advanced gastrointestinal cancer

Immunotherapy with high-dose interleukin-2 (IL-2) fails to induce clinical responses in patients with advanced gastrointestinal cancer, but may be effective in patients with malignant melanoma or renal adenocarcinoma. The hypothesis that this failure may be related to immunosuppressive moieties present in patients with advanced gastrointestinal cancer was investigated. Serum samples from 93 patients (32 advanced gastrointestinal cancer, 22 localized gastrointestinal cancer, 13 melanoma/renal adenocarcinoma and 26 age-matched controls) were incubated with peripheral blood lymphocytes from healthy volunteers. The generation of cytolytic lymphokine-activated killer (LAK) cells and the allogeneic mixed lymphocyte response (MLR) were measured in-vitro. LAK effector cytotoxicity (mean % ± SEM) was significantly ( P < 0.05) decreased by serum from advanced gastrointestinal cancer patients (30 ± 3) compared with that from controls (47 ± 3), serum from patients with localized gastrointestinal cancer (49 ± 2) or that from patients with melanoma or renal adenocarcinoma (51 ± 2). MLR responses were also significantly ( P < 0.05) decreased using advanced gastrointestinal cancer patients serum compared to controls. A dose-response phenomenon for suppression of MLR was observed. Serum from patients with melanoma or renal adeno-carcinoma was not significantly different to control samples. The immunosuppressive properties of serum from patients with advanced gastrointestinal carcinoma may abrogate therapeutic attempts using IL-2.

Modulation of target-cell culture conditions alters susceptibility to lymphocyte-mediated lysis.

The culture conditions of the immortalized human breast cell line, 184B5, can be manipulated to evaluate conditions that influence target lysis by activated immune effector cells. Exponentially growing 184B5 cells (EXP) are more susceptible than growth-factor-deprived (removal of epidermal growth factor and bovine pituitary extract) 184B5 cells (GFD) to lysis by lymphokine-activated killer (LAK) cells, activated natural killer (NK) clones, and activated gamma/delta receptor expressing T-cell clones. LAK-cell lysis of contact-inhibited 184B5 cells is similar to lysis of GFD, while 184B5 cells with severe nutrient deprivation are more easily lysed than GFD by LAK cells. In a cold target inhibition assay with LAK effector populations, EXP are better inhibitors than GFD against several targets. Further analysis of the mechanism by which changes of in vitro culture conditions alter target-cell susceptibility to immune-mediated lysis may assist therapeutic strategies that involve combinations of standard therapies with biologic approaches.

The Effects of Pentoxifylline on the Generation of Human Lymphokine-Activated Killer Cell Cytotoxicity

Pentoxifylline (PTX) is a methylated xanthine that has been shown to reduce the toxicity of Interleukin-2 (IL-2) therapy in animal models, possibly by inhibiting secretion of tumor necrosis factor-alpha. However, the use of PTX to reduce IL-2 toxicity in cancer patients would be advantageous only if PTX did not abrogate antitumor effector mechanisms. We therefore tested the effects of PTX on the induction of lymphokine-activated killer (LAK) cell reactivity in human peripheral blood mononuclear cells (PBMCs). LAK precursor and effector activity were defined as lysis of Daudi cells by PBMCs after incubation with IL-2 at 1,000 U/ml (LAKp) or without incubation (LAKe). PBMCs from four healthy subjects were cocultured for 5 days with IL-2 and PTX (0-1.0 mM). LAKp was inhibited in a PTX dose-dependent manner, as the mean % lysis at an E:T ratio of 50:1 was 62, 43, and 8% in the presence of 0, 0.1, and 1.0 mM PTX, respectively. To determine the effect of in vivo PTX on LAKp activity, four healthy subjects were tested after treatment with PTX at 2 g/day for 3 days. LAKp activity was detected in all four and was not inhibited by autologous serum containing PTX and biologically active metabolites. Because lymphocytes "preactivated" by IL-2 in vivo may respond to PTX differently than resting cells, two patients were tested after a 5 day infusion of IL-2 at 6 x 10(6) U/m2/day. PBMCs from both displayed LAKp activity, with far less inhibition by PTX than in resting cells (4, 17, and 37% inhibition at 0.05, 0.1, and 1.0 mM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

On the Relative Roles of Interleukin-2 and Interleukin-10 in the Generation of Lymphokine-Activated Killer Cell Activity

Induction of cytokine gene transcription by recombinant human IL-2 (rhIL-2) in peripheral blood mononuclear cells (PBMC) from healthy donors was studied by qualitative polymerase chain reaction. In all donors tested, optimal doses of rhIL-2-induced transcription of genes encoding for IL-5, GM-CSF, IFN-γ and TNF-α whereas transcription of IL-1-α, IL-3, IL-4, and IL-6 genes could only be detected in about half of the donors. Moreover, we observed that different doses of rhIL-2 were needed to induce transcription of different cytokine genes. In contrast, transcription of IL-2 and IL-10 genes was only observed in a minority of donors, irrespective of the concentration of rhIL-2 used. Since IL-10 displays a well-characterized inhibitory activity on the synthesis of cytokines possibly involved in the generation of lymphokine-activated killer (LAK) cells, we asked whether the absence of IL- 10 gene transcription plays a role in the induction of LAK cells. Thus, we tested the effects of different doses of rhIL-10 on the rhIL-2-driven generation of LAK activity. Interestingly, rhIL-10 dose-dependently inhibited the production of IFN-γ and TNF-α induced by IL-2, but had no effects on PBMC proliferation and generation of LAK activity. Similarly, purified CD3-/CD16+ lymphocytes, the precursors of LAK effector cells, could be optimally induced by low doses of rhIL-2 to proliferate and generate MHC-unrestricted cytotoxic activity against NK-resistant targets in the presence of rhIL-10. Altogether, our results indicate that rhIL-2 induces transcription of a preferential pattern of cytokine genes, with the IL-10 gene being infrequently transcribed. On the other hand, rhIL-10 shows diverse effects on rhIL-2-triggered PBMC activation, in that it inhibits IFN-γ and TNF-α production but does not affect PBMC proliferation or generation of LAK activity.

Conjugate formation by leukemic blasts from acute myeloid leukemia with cytotoxic lymphocytes.

Conjugate formation by AML blasts with fresh peripheral blood lymphocytes (PBL) and lymphokine activated killer (LAK) effectors was studied by flow cytometry. Leukemic blasts formed very low numbers of conjugates with fresh PBL and were resistant to natural killer (NK) cytotoxicity. When LAK effectors were used a significant increase in conjugate formation was observed, which in the majority of cases was followed by an increased killing. There was a positive correlation between the percentages of conjugates formed by AML blasts with LAK effectors and the susceptibility to lysis. No significant difference in binding activity between the CD3+ and CD56+ LAK subpopulations was found. There was no correlation between the expression of ICAM-1, LFA-3 and Transferrin receptor (CD71) and the conjugate formation. The blocking of CD71 on the control K562 cell line reduced the conjugate formation with LAK effectors but no such effect could be observed with CD71+ AML blasts.

Kinetic analysis of cytotoxic lymphocyte—target cell interaction as quantified by dual parameter flow cytometry

The kinetics of conjugate formation between leukemic cell lines (K562 and Daudi) and lymphokine-activated killer (LAK) cells was studied. A flow cytofluorometry method using double immunofluorescence staining was applied. During the first 15 min of incubation of LAK effectors with leukemic targets, a rapid binding occurred, followed by a plateau phase lasting until 30 min of observation. A considerable, yet not statistically significant, between-donor variability was noticed. A mathematical model of conjugate formation kinetics, based on the analogy to enzyme kinetics, was formulated and validated. Parameters of the model were related to the binding capacity of effector and target cells, and to the lifetime of conjugates and free cells. The concordance of theoretical curves with experimental data proved that the described model can be considered as a useful tool for the evaluation of kinetic and dynamic characterization of conjugate formation between leukemic targets and LAK effectors.

Human lymphokine-activated killer cells are cytotoxic against cells infected with Toxoplasma gondii.

Experiments were conducted to determine whether human lymphokine-activated killer (LAK) cells are cytotoxic against cells infected with Toxoplasma gondii. Nylon wool nonadherent (NWNA) peripheral blood lymphocytes, as well as purified natural killer cell (NK) (CD3- CD16+ CD56+) and T (CD3+ CD16- CD56-) cells obtained from five healthy T. gondii seronegative volunteers exhibited minimal cytotoxic activity against T. gondii-infected cells. When standard LAK (S-LAK) cell preparations were induced by incubation of NWNA cells with recombinant interleukin 2, induction of remarkable cytotoxic activity against T. gondii-infected cells. When standard in LAK cell preparations from each of the volunteers. The phenotype of the LAK precursor and effector cells varied depending on the target cell used. Whereas the precursor and the effector cells of most of the LAK activity against K562 and Daudi cells were cells with NK phenotype, when T. gondii-infected cells were used as targets, both cells with NK and T cell phenotypes were precursors and effectors of the lysis. When cytotoxic activity of S-LAK cells was compared with the activity of adherent LAK (A-LAK) cells, A-LAK cells displayed higher cytotoxic activity against T. gondii-infected cells, as well as against K562 and Daudi cells. Cold target inhibition experiments suggested that there is a subset of LAK effector cells capable of lysing both T. gondii-infected cells and Daudi cells, whereas other subsets preferentially or exclusively lyse one of these target cells.

Interleukin-4 differentially regulates interleukin-2-mediated and CD2-mediated induction of human lymphokine-activated killer effectors.

Natural killer (NK) cells can be differentiated into lymphokine-activated killer (LAK) effectors following stimulation with interleukin (IL)-2. This induction can be negatively regulated by IL-4. In this study, we demonstrate that the stimulation of NK cells through the CD2 pathway with (9-1 + 9.6) monoclonal antibodies can also induce these cells to secrete tumor necrosis factor-alpha (TNF-alpha) and to differentiate into LAK effectors. More importantly, our data indicate that, in contrast to the IL-2-induced LAK generation, the anti-CD2-triggered LAK activity was not regulated by IL-4. IL-4 was found to enhance the LAK activity as well as NK cell proliferation following activation with anti-CD2 by a mechanism involving, at least in part, an increased TNF-alpha production. Using immobilized monoclonal antibodies against the Fc receptor (Fc gamma RIII or CD16) for NK stimulation, we also observed that the anti-CD16-induced LAK activity was not inhibited by IL-4. These data further point to a pivotal role of TNF-alpha as a regulatory cytokine in anti-CD2-induced LAK generation, and suggest that IL-4 could serve as a discriminatory factor between two distinct pathways involved in the activation of non-MHC-restricted cytotoxicity.

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.

Resistance of Different Tumor Cells to Lysis by Lymphokine Activated Killer Cells Can Be Mediated by Distinct Mechanisms

Lymphokine activated killer (LAK) cells have been shown to exert a potent cytotoxic effect on many histologically different tumors and virally infected targets. Most normal cells but very few tumors have proven resistant to LAK lysis. The availability of two LAK resistant tumors, P815r, a murine mastocytoma, and SNUC-1, a human colon carcinoma, allowed us to study the phenomenon of LAK lysis. We examined the role of surface molecules on targets, which mediate binding to LAK cells, by cold target competition experiments and lectin dependent cellular cytotoxicity assays.The results showed that in the murine system, P815r cells do not compete for lysis of the LAK sensitive target B16 whereas other LAK sensitive murine targets compete. Alternatively, in the human system, SNUC-1 cells compete for lysis of the LAK sensitive target SNUC-4 as do other LAK sensitive human tumor cells. Furthermore, inducing binding of target and effector cells with lectin reverted the resistance of P815r but not SNUC-1 targets to lysis by LAK cells. These results imply that distinct stages of the lytic pathway might be involved in the resitance of different tumors to killing by LAK cells. The murine cell line is resistant to lysis because it cannot bind LAK cells. The human target, which does bind LAK, was insensitive to the effects of tumor necrosis factor alpha (TNF-α), a lymphokine released by LAK effectors and possibly involved in their lysis. Resistance to TNF-α was not mediated by the presence of endogenous short-lived proteins in the SNUC-1 targets. The elucidation of mechanisms of resistance may provide a tool to improve current protocols of adoptive immunotherapy as well as insights as to how tumor cells are or are not killed by LAK effectors.

A member of the tetra spans transmembrane protein superfamily is recognized by a monoclonal antibody raised against an HLA class I-deficient, lymphokine-activated killer-susceptible, B lymphocyte line. Cloning and preliminary functional studies.

The IA4 mAb was identified among a series of antibodies raised in BALB/c mice after immunization against a HLA class I-deficient, lymphokine-activated killer (LAK)-susceptible EBV-B lymphocyte line. The IA4 antibody was selected because of its high expression, in the range of 10(5) to 25 x 10(5) sites/cell, on several B lymphocyte lines (EBV-transformed or Burkitt) and monocytic lines such as HL60 and U937, and because its expression was correlated with both target susceptibility to LAK lysis and reduced expression of HLA class I surface Ag on two pairs of EBV-B-transformed cell lines (721/721.134 and MM/10F2). Despite the strategy followed to raise the mAb and the correlation mentioned above, no direct role of the IA4 molecules in LAK susceptibility has been established, since the IA4 molecule is poorly expressed on the sensitive targets Daudi and K562; moreover, the IA4 antibody did not affect reproducibly the in vitro killing of positive target cells by LAK effectors. The IA4 antibody was poorly immunoprecipitating and the surface molecule recognized was identified by gene cloning following an expression strategy using a U937 cDNA library transfected in COS cells, and a screening strategy based on membrane expression of IA4 molecule. The IA4 cDNA is virtually identical to "R2," a mRNA species previously identified in activated human T cells by subtractive hybridization. The IA4 cDNA contains an open reading frame coding for a protein 267 amino acids long with four potential transmembrane domains and one large external hydrophilic domain of about 110 amino acids, possibly glycosylated. The encoded protein belongs to a family of surface molecules, the tetra spans transmembrane protein superfamily, all displaying the four transmembrane domains, expressed on various cell types including lymphocytes (CD9, CD37, CD53, TAPA-1), melanoma cells (ME491), and intestinal cells (CO-029). These molecules have been reported to be involved in cell activation and cell death. Surprisingly, the Schistosoma mansoni Ag Sm23 displays significant homologies with this family. The IA4 molecule is a widely distributed surface marker expressed on circulating lymphocytes and monocytes, newborn thymocytes, and the cell lines mentioned above. The IA4 molecule expression is up-regulated upon cell activation. Weakly expressed on resting peripheral T and B lymphocytes and large granular lymphocytes (NK), its expression roughly doubles after activation by PHA, staphylococcus aureus Cowan I, and IL-2, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)