Inhibition of Tumor Necrosis Factor-α and β Secretion by Lymphokine Activated Killer Cells by Transforming Growth Factor-β

Transforming growth factor- β (TGF- β ) has a variety of immunosuppressive properties. We investigated the effect of TGF- β secreted by glioblastoma (T98G) cells on the secretion of tumor necrosis factor- α and - β (TNFs) by lymphokine activated killer (LAK) cells stimulated with tumor cells. The supernatant from T98G cells was preincubated with anti-TGF- β l and - β 2 neutralizing antibodies or untreated, and added to a coculture of LAK and Daudi cells. The neutralizing antibodies were added to LAK/Daudi and LAK culture, and natural human TGF- β and recombinant human TGF- β were also added to the LAK/Daudi culture. LAK cells were also cultured with T98G cells, of which the supernatant contained both active and latent forms of TGF-/ β 1 and TGF- β 2, and the neutralizing antibodies were added to the coculture. TNFs activity in the supernatants from LAK/ Daudi cultures was examined by a specific bioassay. Addition of the supernatant from T98G cells to LAK/Daudi culture resulted in the inhibition of TNFs secretion by LAK cells. The inhibition was abrogated by the pretreatment of the supernatants with the anti-TGF- β antibodies. Addition of TGF- β and TGF- β to LAK/Daudi culture inhibited TNFs secretion by LAK cells in a dose-dependent manner. Addition of anti-TGF β - antibodies to LAK culture resulted in an increase of TNFs secretion. These results suggest that, if tumor cells have the capacity to convert TGF- β from a latent to an active form, the active TGF- β suppresses TNFs secretion by LAK cells stimulated with the tumor cells, and that TGF- β secreted and activated by glioblastoma cells suppresses the propagation of immune reaction by inhibiting TNFs secretion by activated lymphocytes adjacent to tumor cells.     

Transfection-induced Tumor Necrosis Factor-α Increases the Susceptibility of Human Glioma Cells to Lysis by Lymphokine-activated Killer Cells: Continuous Expression of Intercellular Adhesion Molecule-1 on the Glioma Cells

To develop more effective adoptive immunotherapy, we transfected the human tumor necrosis factor-α (TNF-α) gene into human glioma cells (U251-SP), which were used as target cells. TNF-α is known to increase both the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of glioma cells and the susceptibility of glioma cells to lymphokine-activated killer (LAK) cell cytolysis. We compared the expression of ICAM-1 induced by TNF-α generated by the TNF-α gene-transfected cells with that induced by exogenously added TNF-α. When the TNF-α gene was transfected into U251-SP cells, the expression of ICAM-1 was detected on the cell surface from 3 days after the transfection and continued until at least 9 days. In contrast, it was expressed only transiently in the case of exogenously added TNF-α. Also, the cytolytic activity of LAK cells induced by transfection-induced TNF-α was significantly stronger than that induced by exogenously added TNF-α. The increased susceptibility was quenched by anti-ICAM-1 monoclonal antibody. These data indicated that continuous expression of ICAM-1 induced by TNF-α gene transfection of glioma cells resulted in higher cytolytic activity of LAK cells.     

Tumor Necrosis Factor α-Gene Therapy for an Established Murine Melanoma Using RGB (Arg-Gly-Asp) Fiber-mutant Adenovirus Vectors

Although adenovirus vectors (Ad) provide high-level transduction efficacy to many cell types, extremely high doses of Ad are required for sufficient gene transduction into several tumors, including melanoma. Here, we demonstrated that the expression of coxsackie-adenovirus receptor, a primitive Ad-receptor, was very low in murine and human melanoma cells. We also found that fiber-mutant Ad containing the Arg-Gly-Asp (RGD) sequence in the fiber knob remarkably augmented gene transduction efficacy in melanoma cells by targeting α_v-integrins. In addition, intratumoral injection of RGD fiber-mutant Ad containing the tumor necrosis factor α gene (AdRGD-TNFα) revealed dramatic anti-tumor efficacy through hemolytic necrosis in an established murine B16 BL6 melanoma model. Ad-RGD-TNFα required one-tenth the dosage of Ad-TNFα to induce an equal therapeutic effect. These results suggest that α_v-integrin-targeted Ad will be a very powerful tool for the advancement of melanoma gene therapy.     

Reinforced Cytotoxicity of Lymphokine-activated Killer Cells toward Glioma Cells by Transfection of the Killer Cells with the γ-Interferon Gene

Lymphokine-activated killer (LAK) cells generated from peripheral blood lymphocytes incubated with recombinant interleukin-2 were transfected with the human γ-interferon (HuIFN-γ) gene by means of liposomes having a positive charge on their surface. The cells secreted significant amounts of HuIFN-γ (reaching more than 5 U/ml) into the culture medium. The HuIFN-γ produced by the cells induced intercellular adhesion molecule-1 (ICAM-1) and enhanced the expression of Fas antigen on the surface of human glioma cells. Also, LAK cells transfected with HuIFN-γ gene exhibited reinforcement of cytotoxicity toward human glioma cell lines (U251-MG and SK-MG-1). Furthermore, the reinforcement was significantly quenched by anti-ICAM-1 and/or anti-TNF-α monoclonal antibody.     

Expression of Membrane-associated Lymphotoxin/Tumor Necrosis Factor-β on Human Lymphokine-activated Killer Cells

A membrane-associated lymphotoxin (LT)-related molecule was detected on human lymphokine-activated killer (LAK) cells by flow cytometric analysis. Kinetic analysis revealed that the LT antigenicity on LAK cells appeared at 9 h after the beginning of culture and was maintained thereafter. By autoradiography, the molecular weight of membrane LT was estimated to be 31 kD and/or 62 kD.     

The Heterogeneity of Target Recognition by Lymphokine-activated Killer Precursor Cells

Lymphokine-activated killer (LAK) cells were generated from peripheral blood lymphocytes (PBL) that were depleted of mature cytotoxic natural killer (NK) cells. PBL NK activity was abolished by pretreatment of effector cells with the toxic lysosomotropic agent l -leucine methyl ester (LME) or by depletion of effector cells by K562 monolayer absorption (MA). Both treatments markedly reduced the proportion of cells expressing NK-associated markers such as CD 16 (Leu 11b, B73.1), Leu 7, and NKH-1 (Leu 19), whereas these treatments had minimal effects on cells expressing T cell markers (CD 3, CD 4, and CD 8). LME and MA also drastically decreased the proportion of K562 target-binding lymphocytes. LAK activity against NK-sensitive and NK-resistant targets can be generated from the NK cell-depleted PBL by incubation with interleukin-2. Peak LAK activity generated from MA-treated PBL was later than the peak of LAK activity generated from either untreated or LME-treated PBL. Although MA of PBL on NK-resistant S4 sarcoma targets had little effect on NK activity, LAK activity against both K562 and S4 targets was reduced. These results suggest that there are at least three LAK precursor subpopulations in PBL: mature NK cells that can bind and kill K562 targets (LME-sensitive and MA-sensitive); "pre-NK"cells that can bind but cannot kill (LME-resistant and MA-sensitive); and non-NK cells that cannot bind and cannot kill K562 targets (MA-resistant).     

Killing of Alveolar Macrophages and of Monocytes that Have Responded to Granulocyte-Macrophage Colony-stimulating Factor by Human Lymphokine-activated Killer Cells

The susceptibilities of human blood monocytes and alveolar macrophages (AM) to cytotoxicity mediated by lymphokine (IL-l)-activated killer (LAK) cells were examined. Monocytes and AM of healthy donors were obtained by counter-flow centrifugal elutriation (CCE) and bronclioalvcolar lavage, respectively. The LAK activity induced by incubation of blood mononuclear cells (MNC) for 4 days with recombinant interleukin 2 (IL-2) was measured by a 4-h ⁵¹Cr release assay. The LAK cells were not cytotoxic to freshly isolated monocytes, but were cytotoxic to autologous fresh AM and monocytes that had been incubated for more than 4 days in medium alone. Blood monocytes that had been incubated for 4- days in medium with granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF) or interleukin 3 (IL-3) were much more susceptible than untreated monocytes to the cytotoxicity of LAK cells. When blood monocytes were separated by CCE into subpopulations of three sizes (small, medium and large), the medium- and large-sized monocytes showed greater responses to GM-CSF in terms of DNA synthesis and colony formation than the small-sized cells. After treatment with GM-CSF for 4 days, these medium and large monocytes were more susceptible than the small monocytes to the cytotoxic action of LAK cells. These results suggest that LAK cells may be important in situ in down-regulating the functions of mature macrophages and blood monocytes that have responded to GM-CSF.     

Establishment of Mouse Lymphokine-activated Killer Cell Clones and Their Properties

To assess the properties of lymphokine-activated killer (LAK) cells, we established mouse LAK cell clones from LAK cell lines induced from C57BL/6 mouse spleen cells. Although these clones expressed similar phenotypes to the parent LAK cells, Lyt-2 was expressed in a restricted portion of the clones. All clones were found to express T3 CD₃ and T cell receptor (TcR) αβ on their cell surface. Rearrangement patterns of TcR were the same among the clones derived from the same parent cell line but differed in those from different cell lines as determined by using Cβ₁ and Jβ probes. The molecules responsible for LAK-target cell binding were examined by using a monoclonal antibody (mAb) against lymphocyte function associated antigen 1 (LFA-1). This mAb (termed KBA) showed inhibitory effects on both LAK-target cell binding and cytolytic activity of LAK cell clones, indicating a principal role of LFA-1 in LAK cell clones. The magnitude of perform mRNA expression in LAK cell clones was unrelated to their cytolytic activities.     

Release of Esterase from Murine Lymphokine-activated Killer Cells in Antibody-dependent Cellular Cytotoxic Reaction

Release of granule enzyme(s) (BLT esterase) in the antibody dependent lymphokine-activated killer (LAK) cell-mediated cytotoxic reaction (LAK ADCC) was studied using LAK cells induced from murine splenocytes and thymocytes, various human tumor cells and relevant monoclonal antibodies (mAbs) to the tumor cells. BLT esterase was not significantly released from LAK cells in direct LAK cell-mediated cytotoxic reactions (LAK CMC). However, cultures of LAK cells and IgG-coated target tumor cells resulted in release of the enzyme concomitantly with target cell lysis, although esterase release proceeded faster than target cell lysis. Anti-LFA-1 mAb showed an inhibitory effect on LAK CMC but not on either LAK ADCC or BLT esterase release in the ADCC. These results indicate that exocytosis of granule enzyme from LAK cells is triggered by stimulation of Fc receptor on LAK cells and that LAK CMC and LAK ADCC differ in their lytic mechanism in terms of the release of BLT esterase.     

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.     

Augmentation of Murine Lymphokine-activated Killer Cell Induction by a Factor Produced by Nocardia rubra Cell Wall Skeleton-stimulated T Cells

Four-hour exposure of C3H/HeN mouse spleen cells to Nocardia rubra cell wall skeleton (N-CWS) before 4-day culture with a suboptimal dose of human recombinant interleukin 2 (rIL 2) augmented the induction of lymphokine-activated killer (LAK) cell activity, whereas the treatment with N-CWS alone induced no cytotoxicity. In accordance with this, the IL 2 binding activity of spleen cells was augmented by combined stimulation with N-CWS and rIL 2. The augmented cytotoxicity was mediated by Thy-1.2⁺, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺ cells. Cell cultures in diffusion chambers revealed that N-CWS-treated spleen cells produced a LAK cell induction-helper factor (LAK-helper factor, LHF) when cultured with rIL 2. The LHF production required Thy-1.2⁺, Lyt-1.1⁺, Lyt-2.1⁺ and asialo GM₁⁻ cells, and the coexistence of unstimulated accessory cells was also essential for the LHF production. Cells responding to both LHF and rIL 2 to generate LAK activity were Thy-1.2⁻, Lyt-1.1⁻, Lyt-2.1⁻ and asialo GM₁⁺. The culture fluid of spleen cells stimulated with both N-CWS and rIL 2 contained no tumor necrosis factor (TNF) activity, and the additional stimulation with N-CWS caused no production of either IL 2 or interferon (IFN). Murine recombinant interleukin la (Mu-rIL 1α) could not replace the augmentative effect of N-CWS on LAK cell induction. These results suggest that in the presence of rIL 2, N-CWS stimulates murine T cells to produce LHF that is probably distinct from IL 1, IL 2, TNF and IFN.     

应用人胎儿脾LAK细胞治疗62例晚期恶性肿瘤病人的疗效观察

采用人胎儿脾ＬＡＫ细胞治疗６２例晚期恶性肿瘤病人。结果表明人胎儿脾ＬＡＫ细胞的临床应用安全可行，多数病人在治疗期间一般状况得到改善。总缓解率为（ＰＲ）２２．５８％，平均缓解时间为１５．９５个月。缓解率与克氏评分和接受４次疗程的ＩＬ－２／ＬＡＫ细胞用量有关。在１８．４个月的治疗期间，死亡率为２０．９７％，平均生存期为１６．５０个月。ＩＬ－２／ＬＡＫ细胞对肾癌、黑色素瘤、结肠癌、肺癌和肝癌的转移灶有较好疗效，对原发灶也有一定作用。     

Selective Enhancement by Tumor Necrosis Factor-α of Vascular Permeability of New Blood Vessels Induced with Agarose Hydrogel-entrapped Meth-A Fibrosarcoma Cells

We have previously developed a simple and quantitative method for assessment of in vivo tumor cell-induced angiogenesis by means of microencapsulation of tumor cells in agarose hydrogel and mouse hemoglobin ELISA (mHb-ELISA). In this article, we report that the new blood vessels induced with agarose-encapsulated tumor cells have the same sensitivity to tumor necrosis factor-α (TNF-α) as the original solid-tumor vessels. Agarose beads (average diameter=200 μm), in which Meth-A fibrosarcoma cells were microencapsulated, were subcutaneously implanted in non-syngeneic ddY mice. Ten days later, extensive angiogenesis was observed on the implanted sites of Meth-A agarose beads, whereas no new blood vessels were induced with cell-free agarose beads. The vascular permeability of the new blood vessels induced with agarose-microencapsulated Meth-A cells was selectively and significantly enhanced by the i.v. injection of TNF-α, and it reached the maximum level at 2 h after the injection of TNF-α. At 4 h after the injection of TNF-α, the vascular permeability was reduced to the basal level. This permeability profile in Meth-A agarose beads in ddY mice is very similar to that in Meth-A solid tumor in syngeneic BALB/c mice. On the other hand, TNF-α-treatment did not affect the vascular permeability of other normal tissues or inflammatory tissue in ddY mice. These results strongly suggest that the new blood vessels induced with agarose-microencapsulated tumor cells have the specific characteristics of tumor vessels. Our in vivo angiogenesis assay system should be useful not only to screen anti-angiogenetic agents, but also to elucidate the mechanism of tumor angiogenesis.     

Lysosome Labilizers Potentiate the Antitumor Effects of Tumor Necrosis Factor-α

Enhancement of in vitro cytotoxic activity of tumor necrosis factor- α (TNF- α ) was observfed in combination with lysosome labilizers, particularly with urokinase-type plasminogen activator (u-PA), tissue-type plasminogen activator (t-PA) and lipoprotein lipase (LPL). The concentration of TNF- α resulting in 50% cytotoxicity to L929 cells was only 20–30% of the value for TNF- α alone, when used in combination with a nontoxic dose of u-PA, t-PA or LPL. Furthermore, combined intravenous (i.v.) administration of TNF- α (3.5 × 10⁵ U/mouse) and u-PA (300 IU/mouse) markedly increased the in vivo antitumor activity of TNF- α to Meth A tumors transplanted into BALB/c mice; the tumor weight in co-administered mice was about 40% of that in mice given TNF- α alone on day 6. The combination therapy of TNF- α (7.0 × 10⁴ U/mouse, i.v.) and u-PA (300 IU/mouse, i.v.) was also effective for L929 tumors in Crj:CD-1(1CR)-nu nude mice compared with the conventional therapy with TNF- α alone. These results suggest that the combination of TNF- α and lysosome labilizers is a promising antitumor therapeutic regimen with clinical potential.     

Reversal of Tumor Necrosis Factor Resistance in Tumor Cells by Adriamycin via Suppression of Intracellular Resistance Factors

Tumor necrosis factor (TNF) and various chemotherapeutic drugs show synergistic antitumor effects in vitro and in vivo , though the mechanism is not clear. Based on our previous finding that endogenous TNF (enTNF) acts as an intracellular resistance factor against exogenous TNF by scavenging oxygen free radicals (OFR) with induced manganous superoxide dismutase (MnSOD), we examined the suppression of these resistance factors by chemotherapeutic drugs and the resulting increase in TNF cytotoxicity. Pretreatment of HeLa cells, which produce an appreciable amount of enTNF and show apparent TNF resistance, with TNF followed by adriamycin (ADM) resulted in an additive effect, whereas pretreatment with ADM followed by TNF resulted in a synergistic effect. After treatment of HeLa cells with ADM, the expression of enTNF was remarkably suppressed and MnSOD activity was decreased by one-half. These results indicate that suppression of the intracellular resistance factors, i.e., enTNF and MnSOD, by ADM plays an important role in the mechanism of the synergistic antitumor effect of TNF in combination with ADM.     

Expansion of Lung Vα14 NKT Cells by Administration of α-Galactosylceramide-pulsed Dendritic Cells

NKT cells, a novel murine lymphoid lineage bearing an invariant T cell receptor encoded by Vα14 and Jα281 gene segments, recognize a specific ligand glycolipid, α-galactosylceramide (α-GalCer) in a CDld-dependent manner. Recent research has revealed that activated Vα14 NKT cells have dramatic antitumor effects against a wide variety of tumor cell lines in vivo and in vitro. Here, we demonstrate strong in vivo antitumor effects brought about by treatment with α-GalCer-pulsed dendritic cells in comparison with in vitro -activated Vα14 NKT cells. Furthermore, we show a significant expansion of endogenous Vα14 NKT cells in the lung following the administration of α-GalCer-pulsed dendritic cells. The feasibility of immunotherapy with α-GalCer-pulsed dendritic cells is discussed.     

Augmentation and Suppression of Release of Tumor Necrosis Factor from Macrophages by Negatively Charged Phospholipids

We recently reported that some lipid species of cell membranes and lipoproteins induced the growth of peripheral macrophages. In this study, the effects of phospholipids on tumor necrosis factor (TNF)-releasing activity of macrophages were examined. Ten to 20 μg/ml of cardiolipin, which is a suboptimal concentration for macrophage growth-stimulation, augmented macrophage TNF release triggered by lipopolysaccharide (LPS) in vitro. This priming effect appeared with 1 day of preincubation and was still potent on day 3, whereas the priming effect of interferon-γ (IFN-γ) peaked at 3 h and then gradually decreased. In contrast, a high concentration of cardiolipin (40 μg/ml) which is optimal for the induction of macrophage growth, completely suppressed LPS-triggered TNF release from not only untreated macrophages but also IFN-γ-primed macrophages. The suppressive effect was potent even with 3 h preincubation, was still potent on day 3, and was not abolished by indomethacin. Cardiolipin had scarcely any effect on the triggering activity of LPS. Similar augmentative and suppressive activities were observed in peroxidized phosphatidylserine, which is also highly active in inducing macrophage growth, but was not found in native phosphatidylserine, which is less active, nor in phosphatidylcholine, which is an inactive species toward macrophage growth. These results suggest that lipids may be important endogenous factors in regulating both activation and growth states of peripheral macrophages.     

Mechanism of the Combined Antitumor Effect of Natural Human Tumor Necrosis Factor-α and Natural Human Interferon-α on Cell Cycle Progression

We have studied the mechanism of the synergistic effect of the combination of tumor necrosis factor-α (TNF-α) and interferon-a (IFN-α) on cell cycle progression using two-parameter flow cytometry in vitro and an immunohistochemical staining method in vivo. The cells used were human colon cancer cell line RPMI 4788 in vitro and in vivo, and human breast cancer cell line MX-1 and human renal cancer cell line NAMKO-1 in vivo. In the in vitro experiment, the cell cycle progressed normally as time elapsed in the control group. However, in the group treated with TNF-α and IFN-α in combination (combination group), it appeared that the transition from the S phase to the G₂/M phase was blocked, and the cells that accumulated in the S phase died. In the in vivo experiment with male nude mice of a CD-1 genetic background, the antitumor effect on all three kinds of cancer cells was significantly greater in the combination group than in the control group. The cell labeling index on staining with bromodeoxyuridine in the combination group became markedly larger and the mitotic index smaller than in the other groups. From these results, it was concluded that in the combination group, both in vitro and in vivo , tumor cells markedly accumulated in the S phase and their progression from the S phase to the G₂/M phase in the cell cycle was inhibited.     

Enhancement of Blood Stasis and Vascular Permeability in Meth-A Tumors by Administration of Hyperthermia in Combination with Tumor Necrosis Factor

Blood stasis and vascular permeability induced by tumor necrosis factor (TNF) in Meth-A tumors transplanted in BALB/c mice were significantly enhanced by hyperthermia at 40°C for 30 min immediately following TNF administration. A dose-dependent, sustained decline in the intratumoral blood flow rate occurred following the administration of TNF alone (i.v.; 5 × 10³, 5 × 10⁴, or 5 × 10⁵ JRU/kg) and was enhanced by the administration of hyperthermia in combination with the TNF, even though no decline occurred with hyperthermia alone. The combination of TNF at 5 × 10⁵ JRU/kg and hyperthermia resulted in a blood flow ratio (ratio of blood flow after administration to that before) of 0.47 at 1 h compared with a ratio of 0.65 at 1 h after TNF alone. The blood flow in normal skin sites did not decrease in any case. The permeability of the intratumoral vasculature similarly increased in a dose-dependent manner after the administration of TNF alone and was further increased by combination with hyperthermia, even though no increase occurred with hyperthermia alone. The mean permeability in mice receiving TNF alone at 5 × 10⁵ JRU/kg was 1.35 times that in untreated mice. In mice receiving TNF at the same dose together with hyperthermia, the ratio was increased to 1.65. The results suggest that TNF selectively suppresses intratumoral blood flow, that this effect is enhanced by mild hyperthermia, and that the mechanism of the suppression by TNF with or without hyperthermia partly involves an increase in blood vessel permeability.     

Differential Activation of Lymphokine-activated Killer Cells with Different Surface Phenotypes by Cultivation with Recombinant Interleukin 2 or T-cell Growth Factor in Gastric Cancer Patients

Fourteen days' culture of human peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL 2) or T cell growth factor (TCGF) results in the generation of lymphoklne-activated killer (LAK) effector cells which have the unique property of lysing natural killer (NK)-resistant human tumor cells, Daudi, as well as NK-sensitive, K562 cells. LAK cells were generated from both normal and gastric cancer patients' PBL. However, LAK cell activities induced by rIL 2 or TCGF decreased with the progress of the tumor growth. In addition, TCGF-induced LAK cell activities were found to be lower than the rIL 2-indnced LAK cell activities. Different mechanisms may be involved in the decreases of the rIL 2-induced and TCGF-induced LAK cell activities. This study further demonstrates that the cell types involved are also heterogeneous, as determined by phenotypic characteristics. The LAK-effector cell type was analyzed by two-color flow cytometry. RIL 2-induced LAK cells showed increased proportions of CD4^±Leu 8^- and Leu 7^±CD16^-, and a decreased proportion of CD8^±CD11^- cells, which are believed to be associated with killer T cell functions. In contrast, TCGF-induced LAK cells revealed significantly increased proportions of CD8^±CD11^-and CD4^±Leu 8^- cells, and a decreased proportion of Leu 7^±CD16^- cells. Thus, LAK cells with different surface phenotypes were induced by the cultivations with rIL 2 and with TCGF.