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).     

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 lymphokine-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-induced 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+Leu8- 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.     

Natural killer and lymphokine activated killer cell functions in Hodgkin's disease

We report the natural killer (NK) and lymphokine activated killer (LAK) cell activities in peripheral blood lymphocytes (PBL) from untreated patients with Hodgkin's disease (HD) and from healthy donors. The frequency of LAK cell precursors was also studied using limiting dilution analysis (LDA). About 75% of the HD patients had normal NK activity. There was a higher percentage of low NK responders (mean percent NK activity of healthy donors--2 SD) in patients with lymphocyte depletion histologic grade of the disease and those who were in clinical stage IV, suggesting a correlation of decrease in NK activity with poor prognosis. We found efficient LAK activity against the NK-sensitive K562 cells and NK-resistant VIP (melanoma) and T-24 (bladder carcinoma) tumour targets in both low and normal NK responder HD patients, irrespective of the histopathological grade and clinical stage of the disease. In concordance with their good LAK cell activity, HD patients showed a frequency distribution of LAK cell progenitors in the PBL comparable to that of healthy donors.     

人肿瘤浸润淋巴细胞（TIL）的抗肿瘤活性及与外周血淋巴细胞...

In order to search for more efficient effector cells than the classical LAK cells for tumor immunotherapy, antitumor activity of TIL was studied. Although fresh TIL were unable to kill both NK-sensitive K562 and NK-resistant Anip 973 targets, rIL-2 activated TIL from 8 lung carcinoma and 4 gastric cancer patients did express significant cytotoxicity against allogeneic solid tumor targets Antitumor activity of activated TIL was more efficient than that of activated autologous PBL By limiting dilution assay, TIL were shown to have a higher frequency of LAK precursors than PBL.     

Cytolysis of mammary tumor targets by resting, interleukin-2 stimulated and in vitro cultured peripheral blood lymphocytes from breast cancer patients

The cytotoxic capacity of resting, interleukin-2 (IL-2)-stimulated and in vitro cultured (3-5 days in 10 U/ml IL-2 containing media) peripheral blood lymphocytes (PBLs) from breast cancer patients to a panel of established mammary tumor cell lines was ascertained. Significant cytolysis (ranging from 7.8 to 12.4%, at an effector: target ratio of 20:1) of all mammary tumor targets (MCF-7, 734B, ZR-75-1, ZR-75-30, BT-20 and Hs578T) by PBLs was demonstrable in 18 h chromium release assays. Natural killer (NK) cytotoxicity was distinct from IL-2 stimulated (5 U/ml) and in vitro cultured PBL cytotoxicity in that resting PBLs were not cytolytic to RAJI cells, normal breast epithelia (Hs578Bst) and fibroblasts. Basal NK activity against mammary tumor targets was significantly reduced in patients receiving chemotherapy when compared to both untreated patients and normal controls. In criss-cross cold target inhibition studies, ZR-75-1 and K562 targets were not mutually competitive in NK cell assays (using resting PBLs) but were mutually competitive in lymphokine-activated killer (LAK) assays (using in vitro cultured PBLs). In eleven independent experiments, basal NK activity of ZR-75-1 cells was increased by a cold target excess of K562 (8.2 +/- 2.4% vs 30.5 +/- 5.2%, mean +/- SE, p greater than 0.01, cold:hot target ratio = 10:1). Interestingly, no such parallel increase of cytolysis of 734B targets by K562 cells was observed. Basal cytotoxicity against ZR-75-1 and K562 targets was serologically depleted using antibodies to natural killer cells HNK-1 and Leu 11b. Thus mammary tumor cell lines parallel autologous tumor cells, yet show features that are distinct from NK-resistant and sensitive lymphoid cell lines in their susceptibility to natural resistant cytolytic mechanisms.     

Effect of anti-CD3 antibody on the generation of interleukin-2-activated lymphocytes from tumor tissues of gastrointestinal cancer.

BACKGROUND. The efficiency of anti-CD3 antibody (OKT3) for adoptive immunotherapy using lymphokine-activated killer (LAK) cells generated from tumor-infiltrating lymphocytes (TIL), regional lymph node lymphocytes (RLNL), and peripheral blood lymphocytes (PBL) was investigated. METHODS. TIL, RLNL, and PBL derived from 39 patients with gastrointestinal cancers (16 gastric cancers, 17 colorectal cancers, and 6 esophageal cancers) were cultured for 4 weeks with 200 U/ml of recombinant interleukin-2. To one group, solid-phase 10 micrograms/ml OKT3 was added during the initial culture period (day 2 or 4). Cytotoxicity against K562 cells (NK-like activity) and Daudi cells (LAK activity) and the phenotypes of effector cells generated after culturing for 2-3 weeks were studied. RESULTS. Proliferative responses were significantly increased by OKT3 in each type of effector cell (P less than 0.01); in particular, TIL expanded more by OKT3 than PBL and RLNL (P less than 0.01). The population of CD8+ CD11b- cytotoxic T-cells in OKT3-stimulated groups was significantly larger than that in unstimulated groups (P less than 0.01), whereas no differences were observed with CD4+ cells (helper/inducer T-cells) and CD8+ CD11b+ cells (suppressor T-cells). OKT3 enhanced the NK-like activity of TIL and PBL but did not affect their LAK activity. OKT3 suppressed the NK and LAK activity of RLNL. CONCLUSIONS. OKT3 stimulation did not significantly enhance the LAK activity, but the authors propose that OKT3 could be an effective addition to adoptive immunotherapy using TIL due to an increased proliferation and generation of a large cytotoxic T-cell population.     

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.     

Detection of NK activity and antibody-dependent cellular cytotoxicity of lymphocytes by human tumor clonogenic assay--its correlation with the 51Cr-release assay

NK activity of human peripheral blood lymphocytes (PBL) for cells of the human myeloid line K562, and antibody-dependent cellular cytotoxicity (ADCC) of PBL for cells of human lung adenocarcinoma line PC-9 were determined by the human tumor clonogenic assay (HTCA). Incubation of K562 cells or anti-PC-9 serum treated PC-9 cells with PBL before plating inhibited the formation of colonies of these tumor cells. The percent inhibition of tumor cell colony formation was dependent on the effector/target ratio, the incubation time before plating and, in the case of PC-9 cells, on the dilution of anti-PC-9 serum. PBL activated with human T-cell growth factor (TCGF), lymphokine-activated killer (LAK) cells, significantly augmented the inhibition of colony formation of K562 cells, compared to the control lymphocytes. The increase in colony inhibition was dependent on the concentration of TCGF and the time of incubation of PBL with TCGF. The HTCA determining the colony inhibition of K562 cells incubated with LAK or PBL correlated with the 51Cr-release assay (p less than 0.001). The HTCA determining the colony inhibition of anti-PC-9 serum-treated PC-9 cells incubated with PBL also correlated with the 51Cr-release assay (p less than 0.001). We found that the NK activity and ADCC of lymphocytes on K562 and PC9 tumor lines could be detected with HTCA.     

Natural killer and lymphokine-activated killer cell-mediated cytotoxicity in patients with oral cancer

Peripheral blood lymphocytes (PBL) from untreated and treated oral cancer patients, lymph node lymphocytes (LNL) from metastatic (met) and nonmetastatic (non-met) lymph nodes, and tumor infiltrating lymphocytes (TIL) were tested for natural killer (NK) and lymphokine activated killer (LAK) cell cytotoxicity using appropriate targets in a short-term chromium release assay. The results showed that while both NK and LAK functions of PBL from oral cancer patients were comparable to those of normal healthy donors, the NK activity of metastatic and nonmetastatic LNL and TIL was highly compromised. On the other hand, potent LAK activity could be generated from all three lymphoid populations. Individual patients showing low NK activity displayed good LAK cytotoxicity, indicating that endogenous cells with low NK potential have adequate ability to respond to interleukin 2 (IL-2). LAK activity tested on autologous tumour targets revealed that TIL were the best source of LAK cells. followed by PBL and LNL.     

Natural killer and lymphokine-activated killer activities in stomach cancer patients with special emphasis on the effect of 5-fluorouracil, adriamycin and mitomycin-C chemotherapy

The cytotoxicities of peripheral blood lymphocytes (PBL) and lymphokine-activated killer (LAK) cells were studied to evaluate the effect of chemotherapy on cellular immunity, in 18 patients with unresectable stomach cancer before and after chemotherapy with 5-fluorouracil, adriamycin and mitomycin-C (FAM), and in 21 healthy volunteers. LAK cells were generated in vitro by culturing PBL with 100 U recombinant human interleukin-2 (rH-IL-2)/ml for 72 h. K562 (human myelogenous leukemia), MKN-45 (human stomach adenocarcinoma) and PC-14 (human pulmonary adenocarcinoma) were used as target cells. The cytotoxicity of PBL to K562 and MKN-45 was suppressed in patients with stomach cancer before chemotherapy, compared with that in healthy volunteers (P less than 0.05). The cytotoxicity of LAK cells was significantly higher to all three cell lines tested than that of PBL in both the healthy volunteers and stomach cancer patients (P less than 0.01); however, a lower level of LAK activity was generated in patients with cancer compared to that in the healthy volunteers. FAM therapy did not suppress the cytotoxicities of PBL and LAK cells. The surface markers of PBL and LAK cells were measured, demonstrating that there was no significant change in the percentage of lymphocytes with CD3+, CD4+, CD8+, CD16+ or CD19+ after chemotherapy. The ratios of CD4+ to CD8+ cells in PBL and LAK cells were also not significantly changed after chemotherapy. In the present study, we have demonstrated that the PBL of stomach cancer were defective in generating LAK activity compared to those of controls, but the LAK activity generated from PBL receiving chemotherapy was similar to that from PBL without chemotherapy in stomach cancer patients.     

In vitro generation and antitumor activity of adherent lymphokine-activated killer cells from the blood of patients with brain tumors

A procedure for enrichment in recombinant interleukin-2 (rIL2)-activated natural killer (NK) cells was developed and used for in vitro generation of antitumor effector cells from the peripheral blood of 20 patients with central nervous system (CNS) tumors. In comparison to the patients' unseparated mononuclear cells and nonadherent lymphocytes cultured in the presence of 1000 U/ml of rIL2 for up to 3 weeks, interleukin-2-stimulated lymphoid cells, when purified by adherence to plastic, proliferated better (up to 6,720-fold expansion) and achieved up to five times higher levels of antitumor activity against K562 cell targets and NK-resistant glioblastoma cell targets. Two-color flow cytometry analysis showed that cultures of cells purified by adherence to plastic which had the best proliferation contained 10% or less of CD3+Leu19- T-lymphocytes, while the unseparated lymphokine-activated killer cell cultures which proliferated poorly contained up to 85% of CD3+Leu19- T-cells. Cultures of adherent lymphocytes which reached the highest antitumor cytotoxicity were enriched in CD3+Leu19+ effectors (60-80%); the proportion of CD3-Leu19+ NK-cells was not greater than 25% in these cultures. Thus, using the technique of 24- or 48-h activation in rIL2 and adherence to plastic, and in contrast to the results obtained with cells from normal donors, it was not possible to enrich in activated NK cells from the blood of patients with CNS tumors. Instead of activated NK cells, a population enriched in non-major histocompatibility complex-restricted cytotoxic T-cells (CD3+Leu19+) was obtained in cultures from most but not all patients. Low NK cell activity and elevated numbers of circulating CD3+Leu11+ cells seen in the blood of these patients, previously treated by surgery/radiation/chemotherapy and maintained on steroids, could be responsible for the preferential adherence and subsequent expansion to plastic of IL2-activated non-major histocompatibility complex restricted cytotoxic T-lymphocytes.     

Lymphokine activated killing of fresh human leukaemias

The relative susceptibility of 10 human leukaemias comprising acute phase leucocytes from 5 acute myeloid and 5 lymphoid neoplasms, and 2 immunoblastic lymphomas to killing by peripheral blood mononuclear cells (PBMC), before and after target cell treatment with phytohaemagglutinin (PHA), and by interleukin-2 (IL-2) activated peripheral blood lymphocytes (PBL) was investigated in short term 51Cr release assays using effector cells from 10 allogeneic donors. Optimal lectin-dependent cellular cytotoxicity (LDCC) was verified against K562 and L1210 cells and lymphokine-activated killing (LAK) against K562 and Daudi cells. Under these conditions, the majority of the leukaemias tested revealed only a finite sensitivity to any of the cytotoxic mechanisms, which was dependent on the donor origin of the effectors. The leukaemias were more consistently susceptible to LDCC than LAK and removal of adherent cells to enrich for the latter activity in effector populations, was ineffective. Lymphocytes from a patient in long term (greater than 5 yr) remission exhibited LAK against the autologous target E84, a natural killer (NK)-sensitive acute myelomonocytic leukaemia. These cells failed to cross-compete for lysis of K562 by LAK cells, suggesting the existence of different recognition structure(s) on the two targets.     

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

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

Effect of macrophages on interleukin-2 (IL-2)- and IL-4-induced murine lymphokine-activated killer activity

The enhancing effect of macrophages on interleukin-2 (IL-2)- and IL-4-induced murine lymphokine-activated killer (LAK) activity was investigated in this study. Peritoneal macrophages significantly enhanced LAK activity generated from accessory cell-depleted splenic lymphocytes in both IL-2 and IL-4 cultures. This effect was dependent on the number of macrophages and was not replaced by a factor derived from macrophages or lymphocytes. Macrophages enhanced IL-2- and IL-4-induced LAK activity against both natural killer (NK)-sensitive (YAC-I, P388D1) and NK-resistant (P815) tumor cells. Negative selection of cells with antibodies and complement showed no differences in surface markers between IL-2 LAK effectors and IL-4 LAK effectors generated in the presence of macrophages. These results suggest that the same LAK effector subsets can be enhanced by macrophages in either IL-2 or IL-4 cultures.     

Reversible inhibition of lymphokine-activated killer cell activity by lipoxygenase-pathway inhibitors

Natural killer (NK) cells and lymphokine-activated killer (LAK) cells are anomalous cytotoxic cells which are potentially important in host defense against cancer. Several studies have demonstrated that natural killer (NK) cell activity can be suppressed by chemical inhibitors of the lipoxygenase pathway through inhibition of the production of leukotriene B4 (LTB4). The present study investigated the effects of the lipoxygenase inhibitors BW755C and nordihydroguaiaretic acid (NDGA) on NK and LAK cell activity. NK cell function of fresh peripheral blood mononuclear cells (PBMC) was determined via a standard chromium release assay employing K562 as the tumor target. The LAK cell activity of PBMC which had been stimulated with 10 IU of interleukin-2 for 72 hr was determined against the NK-resistant cell line Daudi. Both BW755C and NDGA inhibited NK and LAK cell function at a variety of concentrations. Indomethacin, a prostaglandin synthesis inhibitor, did not bring about an appreciable diminution in NK or LAK cell activity. Inhibition of NK and LAK cell activities by BW755C and NDGA could be reversed by washing the effector cell suspensions prior to the cytotoxic assay or by adding LTB4 (10(-11)-10(-8) M) directly to the effector:target suspensions. These data indicate that certain arachidonic acid oxidation products of the lipoxygenase pathway are essential for the function of LAK cells.     

Identification of a novel CD56- lymphokine-activated killer cell precursor in cancer patients receiving recombinant interleukin 2.

Circulating lymphokine-activated killer (LAK) cell activity in cancer patients receiving recombinant interleukin 2 (rIL-2) therapy is confined to cells expressing the CD56- surface marker. However, CD56- cells from these patients but not normal individuals have been reported to exhibit LAK cytotoxicity only following in vitro activation with rIL-2. Studies were performed to document the existence of CD56- LAK precursor cells and to phenotypically characterize this population in patients receiving rIL-2 therapy using fluorescence-activated cell sorter-purified CD56- cell subsets. Initial studies confirmed that CD56- cells exhibit NK activity [20 +/- 7 (SE) LU/10(6) cells] but not LAK activity (0 +/- 0 LU/10(6) cells) when evaluated directly from peripheral blood of patients receiving rIL-2. CD56- cells from patients but not normal individuals developed significant LAK cytolytic activity against NK-resistant COLO 205 targets (16 +/- 3 LU/10(6) cells) when cultured for 3 days with 1500 units/ml rIL-2. The CD56- LAK precursor activity was confined to cells expressing a CD56-CD16+ phenotype and a large granular lymphocyte morphology; little or no NK or LAK precursor activity was detectable in CD56-CD5+ T-cells from patients. Phenotypic characterization of CD16+CD56- cells revealed that this population is uniformly CD11a+,CD18+, and CD38+ and is heterogeneous in its expression of CD11b, CD11c, and CD16/Leu 11c. These results indicate that rIL-2 administration induces enhanced LAK precursor activity in a novel population of CD5-CD16+CD56- cells.     

Differential effects of chemotherapy-induced and HIV-1-induced immunocompromise on NK and LAK activities using breast cancer and HIV-1 seropositive patient populations

This study contrasts the effect of chemotherapy-induced and viral-induced (HIV-1) immunocompromise on natural killer (NK) and lymphokine-activated killer (LAK) cell function. The ability of NK and LAK cells isolated from the peripheral blood of healthy controls, breast cancer patients receiving or not receiving adjuvant chemotherapy, and HIV-1 seropositive individuals to lyse K562 and U937 targets was determined. Exponential regression analysis of the cytolytic data was used to derive the cytolytic variables A (indicative of the maximal cytolytic kill of a target) and k (indicative of the lytic efficiency of individual effector cells). Overall LU20 values were ascertained and adjusted to incorporate absolute lymphocyte numbers. Such analysis indicates that the cytolytic NK and progenitor LAK cell pools are diminished in breast cancer patients receiving chemotherapy. However, the ability of individual NK and LAK cells from treated patients to lyse targets remain unchanged. In contrast, the diminution of NK and LAK cell function in HIV-1 seropositive individuals is associated with reductions in both NK and LAK cell pool sizes as well as their cytolytic functions.     

Conjugate Formation by Leukemic Blasts from Acute Myeloid Leukemia with Cytotoxic Lymphocytes

Conjugate formation by AML blasts with fresh peripheral blood lymphocytes (PBL) and lym-phokine 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.     

Interleukin-2-activated lymphocytes from brain tumor patients. A comparison of two preparations generated in vitro

Two preparations of human recombinant interleukin-2 (rIL-2)-activated lymphocytes from patients harboring malignant brain tumors were characterized as autologous-stimulated lymphocytes (ASL) and lymphokine-activated killer (LAK) cells. ASL were generated from Ficoll-Paque-isolated, nonadherent, defibrinated peripheral blood lymphocytes (PBL) that were stimulated overnight with phytohemagglutinin (PHA) and cultured with rIL-2 (100 U/ml) for 10 days. LAK cells were produced by culturing all PBL in rIL-2 (500 U/ml) for 4 days. In 4-hour chromium release assays, LAK cells showed greater cytotoxicity than ASL against natural killer (NK)-sensitive and NK-resistant tumor cell lines; by 18 hours, the effectiveness of ASL equaled that of LAK cells. By electron microscopic study, PBL, LAK cells, and ASL showed differences. The helper/inducer to suppressor/cytotoxic ratio (T4+/T8+) of PBL, LAK cells, and ASL was 1.1:1, 1.0:1, and 0.4:1, respectively. ASL, when compared with PBL or LAK cells, have a significantly higher percentage of MO1+/DR+ and T8+/9.3+ subpopulations. ASL and LAK cells, used for the therapy of gliomas, are distinct.     

Ewing's sarcoma: ex vivo sensitivity towards natural and lymphokine-activated killing

We studied the ex vivo cell-mediated cytotoxicity of natural killer (NK) and lymphokine-activated killer (LAK) cells against continuously cultured Ewing's sarcoma cells from 3 different patients. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, the mean (+/- 1 SD) cytolysis by fresh purified large granular lymphocytes (NK cells) was 20 +/- 8, 25 +/- 2, and 21 +/- 3% in Ewing's sarcoma cell lines 6647, 5838, and A4573, respectively. Under identical conditions, NK cells lysed 56 +/- 7% of K562 (a standard NK target), and 3 +/- 3% of Daudi (a standard NK-resistant LAK target). When compared to fresh unseparated peripheral blood mononuclear cells (PBMC), purified NK cells did not exhibit an enhanced cytotoxic reactivity against either Ewing's sarcoma target. In contrast, LAK cells (i.e., PBMC that were preincubated for 4 days in the presence of rIL-2) were highly cytotoxic against all three Ewing's sarcoma targets. LAK activity was dependent on the concentration of rIL-2 used in PBMC cultures. Optimum cell-mediated toxicity against the standard LAK target Daudi (99 +/- 10% cytolysis at 50:1 E:T ratio) was achieved at rIL-2 concentrations of 1,000 u/ml. LAK cells grown under these conditions were also effective against Ewing's sarcoma cells. At an E:T ratio of 50:1, 86 +/- 16, 85 +/- 16, and 67 +/- 13% inhibition was observed in 6647, 5838, and A4573 cells, respectively, as compared to 17 +/- 10, 19 +/- 15, and 29 +/- 11% cytolysis by fresh uninduced PBMC. In summary, our results suggest that rIL-2-induced LAK-type immune effector cells may be of some therapeutic value in the treatment of poor prognosis Ewing's sarcoma.