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.     

Resistance of chronic lymphocytic leukaemia cells to interferon-alpha generated lymphokine activated killer cells.

Recent studies have shown that, when used in early stage disease, interferon-alpha (IFN-alpha) can produce a fall in the number of malignant cells in the peripheral blood of patients with B-CLL. In this study, we investigated the effect of IFN-alpha on natural killer (NK) cell and lymphokine-activated cell (LAK) activity in patients with B-CLL. In vitro, IFN-alpha (500 U/ml for 18 hours) induced LAK activity in patients with B-CLL (27.7 +/- 9.9%, n = 20), and IL-2 (500 U/ml for 5 days) produced similar activity (35.9 +/- 8.8%, n = 7). Despite the induction of LAK activity by IFN-alpha and IL2 in patients with B-CLL, the malignant cells remained resistant to both allogeneic and autologous LAK effectors. NK activity in patients with B-CLL is also low (23.1 +/- 7.2%, n = 20), and B-CLL cells were resistant to NK cell activity. In cold target competition assays, CLL cells did not compete with labelled K562 or Daudi targets in the NK and LAK assays, suggesting that the malignant cells are not recognised by the effector cells, and this may be related to low level of expression of the adhesion receptors, LFA-1 and ICAM-1. Finally, CLL cells were also resistant to antibody dependent cell mediated cytotoxicity, but were susceptible to antibody dependent complement mediated lysis. These results suggest that it is unlikely that the effects of IFN-alpha in B-CLL are due to the enhancement of NK or LAK activity.     

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.     

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.     

Augmentation of lymphokine-activated killer cell cytotoxicity by monoclonal antibodies against human small cell lung carcinoma

This study examined the lymphokine-activated killer (LAK) cell cytotoxicity on monoclonal antibody (MoAb)-bound tumor cells from the human small cell lung carcinoma cell lines H69 and H128. LAK cells were generated from normal peripheral blood mononuclear cells by incubation with interleukin 2 for 3 or more days. Cells from the LAK culture were cytotoxic to natural killer-sensitive (K562, 84% cytotoxicity) and natural killer-resistant (Daudi, 85%; H69 and H128, 69% and 97%, respectively) cell lines, and to freshly excised human lung (49%) and breast (57%) tumors. LAK cytotoxicity to H69 or H128 cells was significantly augmented by target cell preincubation with the small cell lung carcinoma-reactive MoAbs 1096 (increases of up to 271%) or 5023 (up to 223%). SCLC 5023 or 1096 did not enhance LAK cytotoxicity to Daudi cells of lymphoblastoid origin. Pretreatment of LAK cells with an anti-Fc receptor antibody blocked MoAb augmentation by 1096 or 5023 (but not LAK cytotoxicity), suggesting that LAK-MoAb interaction may be mediated by Fc binding. LAK activity coincided with emergence of a large cell [interleukin 2-stimulated large mononuclear leukocyte (LML)] subset expressing the CD16 and NKH-1 surface determinants. Serial immunophenotyping of the LAK cell culture harvested at Days 3, 5, and 7 indicated that the level of LAK cytotoxicity, with or without MoAb augmentation, correlated with frequency of NKH-1-reactive LMLs. These observations support the hypothesis that LAK cytotoxicity is mediated by a NKH-1-reactive LML subpopulation. Antitumor cytotoxicity may be augmented by tumor-reactive MoAbs through Fc binding to this LML subset.     

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).     

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.     

Natural cytotoxicity of human blood monocytes and natural killer cells and their cytotoxic factors: discriminating effects of actinomycin D

The effect of actinomycin D on target susceptibility to human blood natural killer (NK) cells and monocytes was analysed in direct cell-mediated and their cytotoxic factor-mediated cytotoxicity assays. Treatment of K562 cells with actinomycin D reduced their susceptibility to lysis by non-adherent lymphocytes and Percoll-purified large granular lymphocytes (LGL) in a 4-hr 51Cr-release assay, without affecting their sensitivity to monocytes purified by adherence to autologous serum-coated plastic surfaces. The drug treatment caused no shift in the kinetics of cytotoxicity. In the target binding assay LGL formed fewer conjugates with actinomycin-D-treated K562 cells than with untreated ones, while the binding of monocytes to targets was not reduced by the drug treatment of K562 cells. The cold target competition assay revealed that actinomycin-D-treated cold K562 cells showed less successful inhibition than untreated cold K562 cells. Lymphocytes and monocytes could be induced to release soluble cytotoxic factors, termed natural killer cytotoxic factors (NKCF) and monocyte cytotoxic factors (MCF), respectively, when co-cultured with K562 cells. Both cytotoxic factors lysed NK-sensitive target cells in a 48-hr assay. Actinomycin-D-treated K562 cells reduced or abolished the ability to stimulate the release of NKCF from lymphocytes, whereas they induced MCF secretion from monocytes as effectively as untreated ones. On the other hand, actinomycin D treatment of K562 cells enhanced their susceptibility to NKCF and MCF. This actinomycin-D-induced augmentation of target sensitivity to the cytotoxic factors was restricted to NK-sensitive target cells (K562 and Molt-4). NK-resistant target cells (Raji, YAC-I, EL4 and T blasts) were not lysed by NKCF and MCF even after they were treated with actinomycin D. The capacity of K562 cells to bind NKCF and MCF was not altered by actinomycin D. Treatment of the adherent cell population with OKMI or Leu-MI plus complement abrogated both cell-mediated cytotoxicity and MCF production, while Leu-IIb plus complement was ineffective. These results suggest that the effect of actinomycin-D treatment can be used to distinguish the two distinct types of blood mononuclear cells with natural cytotoxicity, NK cells and monocytes, and that each effector type recognizes different plasma membrane moieties of NK target cells, although the cytotoxic factors released from each effector cell similarly bind to and lyse the target cells.     

Natural killer cell activity and autologous tumor killing activity in cancer patients: overlapping involvement of effector cells as determined in two-target conjugate cytotoxicity assay

The relationship between natural killer (NK) cell activity and autologous tumor killing activity was examined in patients with carcinomatous pleural effusions (PE) by means of a two-target conjugate cytotoxicity assay. Enrichment of large granular lymphocyte(s) (LGL) by discontinuous Percoll gradient centrifugation resulted in an augmentation of cytotoxicity against both K562 cells and tumor cells freshly isolated from PE of the same patients in a 4-hour 51Cr release cytotoxicity assay. At the single-cell level, the LGL-enriched fraction contained an increased number of effector cells that bound to autologous tumor cells and to K562 cells, as well as an increased frequency of cells cytotoxic to these target cells. In the two-target conjugate cytotoxicity assay, a single lymphocyte in the LGL population simultaneously bound to both a fluorescein-labeled K562 cell and a nonfluorescent autologous tumor cell. A significant number of lymphocytes in these mixed two-target conjugates lysed both autologous tumor cells and K562 cells after 6 hours' incubation, although overall lysis of K562 cells was higher than that of autologous tumor cells. These results indicate that a single LGL is involved in the lysis of both autologous tumor cells and K562 cells and thus provide direct evidence of involvement of subsets of NK cells in autologous tumor cell killing.     

Isolation and characterization of cytotoxic granules from human lymphokine (interleukin 2) activated killer cells

Cytotoxic granules were isolated from human lymphokine-activated killer (LAK) cells and analyzed for their biochemical properties. Isolated granules of approximately 85-95% purity were obtained by differential centrifugation followed by discontinuous Percoll gradient centrifugation. The murine lymphocyte granule marker N-alpha-carbamazepine-L-lysine thiobenzyl ester-esterase as well as cytotoxic activity toward the human tumor cell lines K562, Raji, Daudi, and CEM were associated with LAK granule fractions. Granule-associated N-alpha-carbamazepine-L-lysine thiobenzyl ester-esterase activity increased in recombinant interleukin 2 expanded human LAK cells in parallel with cytotoxic activity for Raji tumor cell targets. Cytotoxic LAK cell granules mediated calcium-dependent killing of the tumor cell lines K562, Raji, Daudi, and CEM. However, no calcium-dependent hemolytic activity was found. Preincubation of human granules with calcium, a treatment which totally inactivates the hemolytic and cytotoxic activity of murine lymphocyte granules [perforin 1 (P1)] had no effect on human LAK granule cytotoxicity for nucleated cells. Human LAK granules appear to contain P1 detected as cross-reactive antigen detected by mouse anti-P1 and human anti-C9 in Western blot analysis. In addition, Northern blot analysis of polyadenylated RNA isolated from human LAK cells using a murine P1 complementary DNA probe showed a cross-hybridizing 2.8- to 3.0-kilobase mRNA species identical in size to murine P1 mRNA. These results demonstrate that despite similar biochemical composition, functional differences exist between human and murine cytotoxic granules. Human LAK granules were synthesized in response to recombinant interleukin 2 activation and appeared in parallel with cytotoxicity for tumor targets, suggesting an important role for LAK granules in tumor cell cytotoxicity by human LAK cells.     

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.     

以SMMC—7721为靶细胞MTT法检测LAK细胞活性方法的建立

目的建立以贴壁生长的癌细胞为靶细胞、MTT法检测LAK细胞活性的方法。方法用MTT比色法测定LAK细胞对体外培养的K562、Raji、SMMC－7721细胞的杀伤活性。结果LAK细胞对已知的NK敏感细胞K562及NK耐受细胞Raji均有明显杀伤作用,贴壁生长的SMMC-7721肝癌细胞对NK细胞不敏感,而对LAK细胞敏感,在效靶比为40：1时,LAK细胞对肝癌细胞的杀伤率达90％以上,其杀伤效应随效靶比增高而增高。结论SMMC-7721细胞可作为检测LAK细胞活性的靶细胞。以贴壁细胞作为靶细胞的MTT法检测LAK细胞活性较传统的悬浮细胞如Raji作为靶细胞具有灵敏、准确、简单、可靠的优点,更适用于国内一般实验室开展。     

Inhibition of colony formation of drug-resistant human tumor cell lines by combinations of interleukin-2-activated killer cells and antitumor drugs

The cytotoxicity of interleukin-2-activated killer (LAK) cells with or without anticancer drugs against cell lines with acquired drug resistance was evaluated in vitro by colony assay. Human non-small cell lung cancer cell lines, PC-9 and PC-14, human leukemia cell line, K-562, and their sublines resistant to cisplatin (CDDP), PC-9/CDDP and PC-14/CDDP, and to adriamycin (ADM), K-562/ADM, were used as target cells. PC-9/CDDP demonstrated a marked increase in susceptibility to killing by both peripheral blood lymphocytes (PBL) and LAK cells, as compared to the parental cell line, PC-9. The cytotoxicity of PBL and LAK cells against PC-14/CDDP and K-562/ADM was similar to that against their parental cell lines. Moreover, the combination of LAK and CDDP had a synergistic effect on PC-14 and PC-14/CDDP.     

Lymphokine-activated killer activity of tumor-associated and peripheral blood lymphocytes isolated from patients with ascites ovarian tumors

Peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) were isolated from 36 patients with advanced ovarian adenocarcinoma and peritoneal effusions for study of lymphokine-activated killer activity. PBLs and TALs cultured in vitro for 3-5 days in the presence of interleukin-2 (IL-2, supernatant of the MLA 144 gibbon cell line, or human recombinant IL-2) expressed higher levels of cytotoxicity as compared to cells cultured in medium alone, against natural killer (NK)-susceptible (K562) or NK-resistant targets (Daudi and the human ovarian carcinoma cell line SW626). When ovarian tumor cells, freshly isolated from carcinomatous ascites or surgical specimens, were used as target cells in the cytotoxicity assay, 8 of 14 PBLs and 5 of 7 TAL preparations lysed the autologous tumor after treatment with IL-2, while no spontaneous reactivity was observed in any of the 14 patients tested. Although levels of lysis were usually relatively low, these data demonstrate that PBLs and TALs from ovarian cancer patients (TALs usually exhibiting low NK activity) when stimulated in vitro by IL-2 acquire some cytotoxic potential against the autologous tumor.     

Inhibition by fibrin coagulation of lung cancer cell destruction by human interleukin-2-activated killer cells.

We examined the effect of fibrin coagulation on tumor cytotoxicity mediated by human lymphokine (IL-2)-activated killer (LAK) cells. LAK cells were induced from peripheral blood mononuclear cells (MNC) by culture with recombinant IL-2 for 4 or 5 days, and LAK cell-mediated cytotoxicity against tumor cells was assessed by 51Cr release assay in the presence or absence of plasma from normal subjects and lung cancer patients. Plasma did not affect the phase of induction of LAK activity by IL-2, but dose-dependently inhibited the effector phase of LAK cell-mediated cytotoxicity against Daudi cells. Similar inhibition of LAK cell-mediated cytotoxicity was observed on pretreatment of Daudi cells and human lung cancer cell lines with human fibrinogen plus thrombin. A parallel relationship was found between the amount of fibrinogen in plasma of lung cancer patients and inhibition of LAK cytotoxicity. This inhibition was reduced by addition of anticoagulants (heparin or argatroban). These findings suggest that fibrin coagulation on tumor cells protects them from LAK cell-mediated tumor cytotoxicity in malignant lesions and that a combination of an anticoagulant drug and IL-2/LAK therapy may be effective for treatment of lung cancer patients.     

Natural cytotoxicity of human blood lymphocytes and monocytes and their cytotoxic factors: effect of interferon on target cell susceptibility

The effect of interferon (IFN) on target cell susceptibility to human natural killer (NK) cells and monocytes was analyzed in direct cell-mediated and their cytotoxic factor-mediated cytotoxicity assays. Treatment of K562 cells with IFN resulted in a decrease in their sensitivity to lysis by nonadherent lymphocytes and Percoll-purified large granular lymphocytes (LGL) when tested in a 4-hour 51Cr release assay. In contrast, the treatment did not affect the target susceptibility to monocytes purified by adherence to autologous serum-coated plastic surfaces. In the target-binding assay with LGL or monocytes the number of conjugates was not altered after IFN treatment of K562. Lymphocytes and monocytes were induced to release soluble cytotoxic factors, termed "natural killer cytotoxic factors (NKCF) and monocyte cytotoxic factors (MCF)," respectively, when co-cultured with K562. Both NKCF and MCF lysed K562 in a 48-hour microcytotoxicity assay or in an 18-hour 51Cr release assay in the presence of dactinomycin. IFN-treated K562 reduced or completely lost their ability to stimulate the release of NKCF, whereas they triggered MCF secretion as effectively as did the untreated K562. When lymphocytes or monocytes were pretreated with IFN, they released NKCF or MCF with augmented lytic activity. In contrast to the sensitivity to NK cell-mediated lysis, IFN pretreatment of K562 induced no change in their susceptibility to NKCF and MCF. When IFN was added to NKCF and MCF assays, the cytotoxicity was enhanced. The addition of IFN to K562 that had been pretreated with NKCF or MCF and washed resulted in no increase in lysis. The capacity of K562 to absorb the lytic activity of NKCF and MCF was not altered by IFN. These results indicate that IFN treatment of target cells can be used to distinguish the two distinct types of blood mononuclear cells with natural cytotoxicity, NK cells and monocytes, and that each effector cell type is stimulated to release cytotoxic factors by the different target determinant after the initial effector-target cell binding.     

Generation and characterization of lymphokine-activated killer cells against fresh human leukemia cells

Lymphokine-activated killer (LAK) cells generated from 15 acute leukemia patients in remission showed significant levels of cytotoxicity against Daudi 1A4, a natural killer-resistant cell line. This indicates that lymphocytes of leukemia patients in remission could respond to interleukin-2 to generate conventional LAK cells. However, LAK cells caused lysis of autologous leukemia cells at considerably lower levels in seven out of the 15 patients, with the exception of one case (48.6% cytolysis). None of the remaining eight patients exhibited LAK activity against autologous leukemia cells. On the other hand, patients' LAK could lyse allogeneic leukemia cells including those resistant to autologous LAK. Thus, patients' LAK seem not to be defective in lysis of leukemia cells. In the cold target competition analysis, the binding of patients' LAK to leukemia cells could be inhibited by autologous and allogeneic leukemia cell competitors, implying that almost all leukemia cells could be recognized by patients' LAK. Most LAK cells from normal donors showed significant lysis of allogeneic leukemia cells, but some leukemia cells were found to be resistant to lysis. LAK cells against both leukemia cells and Daudi 1A4 were phenotypically heterogenous, and were predominantly observed in the T3- fraction in the precursor phase. In the effector phase, whereas LAK activity against leukemia cells was also predominantly shown in the T cell-depleted fraction, similar levels of LAK activity against Daudi 1A4 were found in both the T cell-depleted and -enriched fractions.     

Preparation of NK-enriched LAK cells--their potential cytotoxic and ADCC activities

We examined several culture methods to induce proliferation of natural killer (NK) cells from peripheral blood mononuclear cells (PBMC). In the presence of IL-2, a remarkable proliferation of NK cells was observed when PBMC were co-cultured with MMC-treated K562, which is known as a highly sensitive in vitro target cell for the NK assay. Addition of OK-432 or TNF-alpha and IL-1 beta also induced marked NK proliferation in a dose dependent manner. These NK-enriched lymphokine activated killer (LAK) cells showed highly cytotoxic activities against various MHC class I positive or negative tumor cells. They also showed potent ADCC activities against Herceptin-coated SK-BR-3, a HER2/neu positive breast cancer cell line. These results indicated that NK-enriched LAK cells are potent effector cells, and suggested novel therapeutic strategies for nonspecific immunotherapy as well as target immunotherapy in combination with anticancer antibodies, such as Herceptin.     

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.     

In vivo induction of lymphokine-activated killer cells by interleukin-2 splenic artery perfusion in advanced malignancy

In an effort to stimulate in vivo LAK cell activity at relatively nontoxic doses, 20 patients with advanced metastatic malignancy (13 renal cell carcinoma, 6 melanoma, 1 lymphoma) were treated with recombinant human interleukin-2 (IL-2) by continuous 5-day splenic artery perfusion using the femoral approach. Two treatment cycles were administered 3 weeks apart; IL-2 doses ranged from 1.5-4 x 10(4) Cetus units/kg/day. Peripheral blood lymphocyte cytotoxicity in a 4-h 51Cr release assay was measured using as tumor cell targets K562 for natural killer (NK) activity, Daudi for LAK, and Daudi plus in vitro IL-2 for inducible LAK (I-LAK). For the 20 patients, an increase in mean peak percent cytotoxicity from pretreatment levels was seen for NK (36% to 53%), LAK (8% to 37%) and I-LAK (20% to 53%) activity, all significant at P = 0.001. On day 43, 16 days after completing the second cycle of treatment, NK activity remained elevated at 47% and I-LAK at 40% (P = 0.008 and 0.01, respectively). Lymphocyte phenotype analysis by flow cytometry demonstrated increases from pretreatment levels in Leu 11+ (13 to 23%), Leu 19+ (10 to 21%), Leu 11+ 19+ (7 to 17%), IL-2r+ (4 to 17%), and HLA-DR+ (12 to 25%) subsets, all significant at P less than or equal to 0.01. Dose effect was studied at 3 dose levels: 1.5, 3, and 4 x 10(4) Cetus units/kg/day. At the higher doses mean peak NK (57%) and I-LAK (57%) activity were greater than at the low dose (42 and 31%, respectively), both significant at P less than 0.05. A trend to positive dose effect was seen in LAK activity (P = 0.08). Splenic artery perfusion with IL-2 can result in significant in vivo peripheral LAK cell generation as well as enhancement of I-LAK and NK activity that persists at least 16 days after the cessation of treatment. Such sustained activity would not be expected with conventional high dose i.v. therapy.