In vitro and in vivo susceptibility of human leukemic cells to lymphokine activated killer activity

The susceptibility of human myeloid and lymphoid leukemic blasts to the lytic action of recombinant interleukin-2 (rIL-2)-generated lymphokine activated killer (LAK) cells was analyzed. With the exception of the K562 cell line, all 9 leukemic cell lines tested were resistant to the natural killer activity of freshly isolated peripheral blood lymphocytes (PBL) from healthy donors but were susceptible to the lytic action of PBL cultured for 3 days in the presence of rIL-2. Of the 32 primary myeloid and lymphoid acute leukemia samples investigated, the great majority were natural killer cell-resistant but were variably sensitive to LAK effectors. Variations in LAK activity were observed according to the donor of PBL, while little or no difference was documented in the capacity to elicit LAK activity of PBL cultured with 100 or 1,000 U of rIL-2/ml. Pretreatment of the leukemic target cells with neuraminidase did not increase substantially their sensitivity to LAK activity. LAK cells generated from the PBL of patients at the onset of the disease or in complete clinicohematological remission lysed Raji cells as efficiently as normal LAK effectors. Finally, LAK cells were capable of abrogating the tumor growth in nude mice of a human leukemic T cell line. These findings demonstrate the susceptibility in vitro and in vivo of human leukemic blasts to the lytic effect of LAK cells and point to a possible clinical exploitment of this new form of adoptive immunotherapy in the management of acute leukemia.     

Influence of schedule of interleukin 2 administration on therapy with interleukin 2 and lymphokine activated killer cells

The purpose of this study was to compare the toxicity, immunomodulatory changes, and antitumor efficacy of interleukin 2 (IL-2) and lymphokine activated killer (LAK) cell therapy with two durations of IL-2 infusion. Patients with progressive melanoma, non-Hodgkin's lymphoma, renal carcinoma, or colon carcinoma received IL-2 at 3 X 10(6) units/m2/day on days 1-5 and 13-17, either by bolus injection every 8 h (q8h) or by continuous i.v. (CIV) administration. Peripheral blood mononuclear cells were harvested by leukapheresis on days 8, 9, and 10, were incubated in vitro for 5 days for generation of LAK cells, and were infused on days 13, 14, and 15. The first 11 patients were treated with IL-2 q8h, and the subsequent 13 patients were treated by CIV infusion. Toxicity consisted primarily of fever, chills, emesis, diarrhea, weight gain, and edema but did not require intensive care unit support and did not differ significantly between treatment groups. IL-2-induced lymphocytosis on day 8 was higher with CIV than with q8h administration with a mean lymphocyte count/microliter of 5610 +/- 700 (SE) versus 3300 +/- 500. Immunomodulatory changes observed on days 8 and 20 were also greater with CIV IL-2 and included an increase in peripheral blood mononuclear cell IL-2 receptor expression as well as a marked rise in the number of Leu-11+ and Leu-19+ peripheral blood mononuclear cells. The total leukapheresis yield per patient and total number of LAK cells infused per patient were higher with CIV than q8h administration, with 49.8 +/- 4.9 X 10(9) versus 39.4 +/- 5.4 X 10(9) and 42.6 +/- 5.0 X 10(9) versus 34.0 +/- 5.4 X 10(9), respectively. The cells infused displayed phenotypic evidence of activation and exhibited marked lytic reactivity to Daudi, Raji, and HT-144 targets. One complete and one minimal response were observed in 2 of 8 patients with metastatic renal cell carcinoma who received CIV IL-2 and LAK cells. The results show that IL-2 is more biologically active by CIV than q8h administration, as demonstrated by greater rebound lymphocytosis, LAK cell yield, and in vivo immunostimulation.     

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.     

Characterization and modulation of human lymphokine (interleukin 2) activated killer cell induction

Culture of human peripheral blood mononuclear cells with purified interleukin 2 (IL-2) results in the induction of a cytotoxic population of cells capable of lysing autologous tumor cells and natural killer (NK) cell resistant tumor cell lines. The current study was undertaken to characterize biological agents which might modulate the induction of lymphokine (IL-2) activated killer (LAK) cells and to optimize culture conditions for LAK cell induction. Preliminary studies were undertaken to characterize optimal time and IL-2 concentration for induction of LAK cell activity. Subsequently, we demonstrated that: (a) LAK cell induction is inhibited at high (2.5 X 10(6)/ml) cell concentrations and this phenomenon is due to the presence of monocytes; (b) depletion of monocytes allows LAK cell induction at 5-10-fold higher cell concentrations without altering the extent or range of LAK-cell activity; (c) interleukin 1 enhances and alpha- and beta-interferons inhibit IL-2 induced proliferation, without altering LAK cell induction; and (d) gamma-interferon alters neither IL-2 induction of proliferation nor LAK cell activity.     

Cytotoxicity of interleukin 2-induced lymphokine-activated killer (LAK) cells against human leukemia and augmentation of killing by interferons and tumor necrosis factor.

Adoptive immunotherapy with interleukin 2-induced lymphokine-activated killer (LAK) cells or tumor-infiltrating lymphocytes (TIL) and the induction of anti-tumor responses by IL-2 alone having proven to be promising approaches in cancer therapy. The present study investigated the cytotoxicity of LAK cells towards human leukemia cells. LAK cells were generated by culturing peripheral blood mononuclear cells from normal donors for six days in the presence of recombinant interleukin 2 (IL-2). Cytotoxicity was evaluated using a standard 4-h chromium-release assay. A significant lysis of fresh uncultured leukemia cells by IL-2-activated killer cells could be detected in 77 of 150 leukemias examined. The mean Cr-release was 35.7 +/- 12.9% in the LAK cell-sensitive vs 9.9 +/- 5.9% in the resistant leukemias. With a view to the therapeutic utilization of the LAK-cell system, we attempted to improve the efficiency of its cytotoxic mechanisms. Combined application of IL-2 and interferon-alpha, interferon-gamma, or tumor necrosis factor-alpha in cultures for generation of activated killer cells significantly improved the effectiveness of cytotoxic mechanisms. Our results suggest that the performance of adoptive immunotherapy with ex vivo-activated LAK cells and the in vivo induction of cytotoxic immune responses by IL-2 alone or combined with different lymphokines or cytokines may be of value in treating human leukemia, especially when the tumor burden is low, e.g. during maintenance therapy or after bone marrow transplantation to eliminate minimal residual disease or in early relapse.     

Susceptibility of multidrug-resistant human leukemia cell lines to human interleukin 2-activated killer cells.

Considering the possibility to overcome drug resistance by other treatment strategies than chemotherapy we investigated the susceptibility of three independently selected multidrug-resistant sublines of the T-lymphoblastoid leukemic cell line CCRF-CEM to lymphokine-activated killer (LAK) cells. We found that two of the multidrug-resistant sublines were significantly less susceptible targets to LAK cells. A third one, however, was as susceptible as the parental CCRF-CEM cell line. Moreover, a multidrug-resistant subline that reverted to an almost drug-sensitive phenotype was observed to be also revertant for resistance against LAK cells. We found an inverse relationship between the expression of the mdr1 gene (P-glycoprotein) and the susceptibility to LAK cells. Verapamil, a calcium channel blocker, while increasing the drug sensitivity of a multidrug-resistant subline, did not induce a reversal of the suppression of LAK susceptibility. The possibility of enhanced resistance to LAK cells of multidrug-resistant cells should be taken into account when one is looking for therapy strategies to overcome multidrug resistance.     

In vivo antitumor activity of anti-CD3-induced activated killer cells

This study investigates the potential of the alpha CD3-induced killer cells for use in adoptive immunotherapy of tumor growth. The alpha CD3-induced, activated, killer cells (CD3-AK) were generated in DBA/2 (H-2d) splenocytes by preactivation with alpha CD3 and were then cultured in the presence (CD3-AK [alpha CD3+]) or absence (CD3-AK [alpha CD3-]) of alpha CD3. The conventional lymphokine-activated killer (LAK) cells were induced by culturing DBA/2 splenocytes with purified human recombinant interleukin 2. Testing their in vitro cytotoxicity against syngeneic mastocytoma P815, CD3-AK (alpha CD3+) cells gave the highest levels of cytotoxicity and were 20-fold higher than LAK cells and 200-fold higher than CD3-AK (alpha CD3-) cells. However, the cytotoxic activity of LAK or CD3-AK (alpha CD3-) cells was augmented by preincubating them with alpha CD3 for 3 h; then, the difference in cytotoxic activity was reduced from 20- to 4-fold and from 200- to 2-fold, respectively. The in vivo antitumor activity of these killer cells paralleled the in vitro activity. In tests using tumor neutralization experiments, 80-100% of the mice that were challenged with 1 x 10(2) P815 cells remained tumor free after receiving 5 x 10(6) CD3-AK (alpha CD3+) cells. When the challenge dose increased to 1 x 10(3) and to 1 x 10(4) cells, giving CD3-AK (alpha CD3+) cells slowed down the rate of tumor growth but only 20% of the mice remained tumor free. The untreated LAK cells or CD3-AK (alpha CD3-) cells did not induce any protection. After preincubation with alpha CD3 for 3 h, the CD3-AK (alpha CD3-) cells provided protection in 30% of the challenged mice. The phenotype of effectors for mediating the in vitro and in vivo antitumor activities was found to be Thy1+, CD4-, and CD8+ cells. Flow microfluorometry analysis showed that the higher levels of cytotoxic activity obtained with CD3-AK (alpha CD3+) cells could not be simply explained by the increase of CD8+ cells, and the cytotoxic activity of individual CD3-AK (alpha CD3+) cells appeared to be much higher than that of the LAK cells. After tumor growth was established for 1-2 days, giving CD3-AK (alpha CD3+) cells slowed down the rate of tumor growth, and 20% of the mice remained tumor free. These results indicate that CD3-AK cells may be used in the immunotherapy of tumor growth.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct killing of interleukin-2-transfected tumor cells by human neutrophils

We have previously established that human polymorphonuclear cells (PMN) express IL-2Rβ- and γ-chains and that addition of IL-2 maintains the viability of PMN by preventing these cells from undergoing programmed cell death. The purpose of this study was to examine whether IL-2-releasing tumor cells are capable of stimulating PMN tumoricidal activity. We therefore investigated the ability of PMN to kill IL-2-transfected tumor cells using normal human PMN directed against the murine mammary adenocarcinoma TS/A engineered to release high amounts of murine IL-2 (3,600 U, B6) compared with TS/A parental cells and TS/A tumor cells transfected with the neomycin-resistance (NEO) gene only. The potency of PMN as IL-2-induced killer cells was indicated by the low number of cells required for killing (effector cell:target cell ratio 10:1) and the degree of tumor cell lysis (68 ± 10%). Evidence for the role of IL-2 as a mediator of tumor cytotoxicity by PMN was substantiated by inhibition of tumor killing with anti-IL-2 and anti-IL-2Rβ monoclonal antibodies (MAbs). Furthermore, in vivo depletion of mature granulocytes using MAb RB6-8C5 resulted in B6 adenocarcinoma growth, thereby confirming a direct role for IL-2-activated PMN in tumor cytolysis. Lastly, we suggest that one possible mechanism involved in IL-2-induced PMN cytotoxicity against the B6 clone occurs via the nitric oxide pathway, which could be inhibited upon addition of the arginine analog, N^G-monomethyl-L -arginine. © 1996 Wiley-Liss, Inc.     

Altered conjugate formation and altered apoptosis of multidrug-resistant human leukemia cell line affects susceptibility to killing by activated natural killer (NK) cells

Most leukemias that exhibit P-glycoprotein (P-gp)-associated multidrug resistance (MDR) exhibit reduced susceptibility to immune cytotoxicity mediated by natural killer (NK) cells. To explore this phenomenon we investigated N6/ADR, a doxorubicin-selected, P-gp-positive variant of the human acute lymphoblastic leukemia (ALL) cell line NALM6. Each stage of the NK cytolytic pathway, (binding, activation and killing) was evaluated to identify the alterations responsible for the reduced cytotoxicity of the variant relative to its drug-sensitive parental line. The major cause of the decreased susceptibility to NK cytolysis was found to be reduced conjugate formation by the MDR variant. Activation of NK effectors by parental and MDR cells with concomitant release of tumor necrosis factor-alpha (TNF-alpha) correlated with conjugate formation. N6/ADR was also more resistant than NALM6 to antibody-dependent cellular cytotoxicity and to cytotoxic factors released from NK cells as measured both by 51Cr-release and by DNA fragmentation. This is the first report of a P-gp-positive leukemic line that exhibits reduced conjugate formation as well as increased resistance to NK-mediated killing mechanisms. Our results suggest caution in the use of NK-based immunotherapy as an alternative treatment for multidrug-resistant leukemias.     

CD5+ B cells from bovine leukemia virus infected cows are activated cycling cells responsive to interleukin 2.

Most of the B cells from bovine leukemia virus (BLV) infected cows in persistent lymphocytosis (PL) were known to express the CD5 T-cell marker but it was not known whether this peculiar membrane phenotype relates to an activation state. It was demonstrated that these B cells were also flagged by two other membrane markers normally borne by cells belonging to the myeloid lineage (namely CD11b and CD11c). Moreover, cell cycle analysis illustrated that a significant percentage of these B cells (greater than 15%) left their resting (G0/G1) status and progressed through the cell cycle. In addition, T-cell-depleted peripheral blood mononuclear cells from animals in PL were shown to proliferate in response to a IL-2-containing supernatant (MLA 144). These results indicate that the CD5+ B cells from BLV-infected cows in PL are activated cells.     

Mechanisms of selective killing of neuroblastoma cells by natural killer cells and lymphokine activated killer cells. Potential for residual disease eradication.

Widely disseminated neuroblastoma in children older than infancy remains a very poor prognosis disease. Even the introduction of marrow ablative chemotherapy with autologous rescue has not significantly improved the outlook for these children, presumably because of a failure to eradicate minimal residual disease. One additional approach which may hold promise is the use of immunomodulation with cytokines such as IL2 in the setting of minimal residual disease (MDR), for example after intensive chemotherapy and ABMT. However, considerable variability in the susceptibility of neuroblastoma cells to natural killer (NK) and lymphokine-activated (LAK) killing has been observed, and it is presently unclear how NK and LAK cells recognise neuroblastoma cells. In this paper we examine expression of cell adhesion molecules on neuroblastoma to determine which of these modify interaction with NK and LAK cells. We find that LFA-3 (CD58), the ligand for CD2 is of predominant importance in predicting susceptibility of neuroblastoma to the cytotoxic actions of NK and LAK cells, while expression of ICAM-1 (CD54) may also modify susceptibility. These findings were confirmed by blocking experiments in which co-culture of target cells with ICAM-1 and LFA-3 reduced LAK and NK cytotoxicity. Study of the immunophenotypic features of each patient''s neuroblastoma cells before induction of MRD may be valuable in determining the likely effect of IL2 in predicting disease reactivation.     

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.     

Phorbol myristate acetate-induced expression of high-affinity interleukin 2 receptors and production of interleukin 2 by human acute lymphoblastic leukemia T cells.

The effect of phorbol myristate acetate (PMA) on the expression of interleukin 2 receptors (IL-2R), production of IL-2 and IL-2-dependent proliferation of acute lymphoblastic leukemia T cells (T-ALL cells) from 10 patients was studied. First, the effect of PMA on the expression of cell surface markers was assessed: a decrease of CD3 and CD4, and an enhanced expression of CD8 molecule were observed on T-ALL cells. Moreover, PMA exhibited an heterogenous effect on various activation-associated molecules such as a decreased expression of transferrin receptor and T10 molecule and an induced expression of 4F2 and CD9 molecules. It is known that functional high-affinity IL-2R are composed of at least two IL2 binding molecules, the alpha (p55) and beta (p70) chains. We found that PMA induced the expression of both IL-2R alpha and IL-2R beta chains, as well as IL-2 production by T-ALL cells. These effects were time- and dose-dependent. Cross-linking experiments with 125I-labelled recombinant IL-2 (125I-rIL-2) revealed both p55 (IL-R alpha) and p70 (IL-2R beta) IL-2-binding polypeptides, whereas binding equilibrium assays on PMA-treated cells demonstrated the presence of a low number (31-413) of high-affinity binding sites/cell in five out of six cases analysed, as well as intermediate affinity IL-2R (1234-3919 sites/cell) in four out of six cases, according to the time of incubation with PMA. In two cases tested high-affinity IL-2R on PMA-treated T-ALL cells could internalize 125I-rIL-2 at 37 degrees C. PMA alone enhanced the spontaneous proliferation of T-ALL cells in three cases, whereas a clear synergy between IL-2 and PMA could be detected in three patients' cells. Moreover, exogenous rIL-2 enhanced cell proliferation of PMA-preincubated T-ALL cells in four cases studied. Taken together, these observations indicate that a short-term incubation of T-ALL cells with PMA can activate the IL-2/IL-2R system on these cells without inducing strong modifications of their differentiation status. These results thus suggest that this system may be involved in the proliferation process of some activated immature T cells.     

Reduced 1alpha-hydroxylase activity in human prostate cancer cells correlates with decreased susceptibility to 25-hydroxyvitamin D3-induced growth inhibition

Evidence from epidemiological, molecular, and genetic studies suggests a role for vitamin D in the development and/or progression of prostate cancer. In experimental models and clinical trials, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] was shown to exert antiproliferative, prodifferentiating, and antimetastatic/invasive effects on prostatic epithelial cells. Because the direct clinical application of 1,25(OH)2D3 is limited by the major side effect of hypercalcemia, we investigated the potential therapeutic utility of its less calcemic precursor, 25-hydroxyvitamin D3 [25(OH)D3], which is converted locally within the prostate to 1,25(OH)2D3 by 1alpha-hydroxylase. Quantification of 1alpha-hydroxylase activity in human prostatic epithelial cells by enzyme-substrate reaction analyses revealed a significantly decreased activity in cells derived from adenocarcinomas compared with cells derived from normal tissues or benign prostatic hyperplasia (BPH). In growth assays, we found that 25(OH)D3 inhibited growth of normal or BPH cells similarly to 1,25(OH)2D3. In contrast, in primary cultures of cancer cells and established cell lines, the antiproliferative action of 25(OH)D3 was significantly less pronounced than that of 1,25(OH)2D3. Our results indicate that growth inhibition by 25(OH)D3 depends on endogenous 1alpha-hydroxylase activity, and that this activity is deficient in prostate cancer cells. This finding has ramifications for both the prevention and therapy of prostate cancer with vitamin D compounds.     

Efficacy of chemoimmunotherapy with cyclophosphamide, interleukin-2 and lymphokine activated killer cells in an intraperitoneal murine tumour model

We have previously reported on the efficacy of intraperitoneal (i.p.) immunotherapy with interleukin-2 (IL-2) and adoptively transferred lymphokine activated killer (LAK) cells in an i.p. murine tumour model. Because of a dose-limiting toxicity associated with IL-2, cures are seldom observed. The development of treatment strategies that combine components that augment or synergise with the antitumour activity of IL-2 is crucial for the successful use of IL-2 in a clinical setting. Because of the known toxicity of high-dose IL-2 or high dose cyclophosphamide (CY) treatment, the goal of our experiments was to investigate the efficacy of chemoimmunotherapy with low or moderate doses of cyclophosphamide (CY) in combination with low or moderate doses of IL-2 with or without adoptively transferred LAK cells. Assessment of i.p. tumour growth 14 days after tumour inoculation, using the peritoneal cancer index (PCI) scoring system, demonstrated that combination treatment of established (day 3) i.p. tumour was clearly superior to single modality treatment. The effect was further enhanced by a second dose of CY at the end of a course of IL-2. Combination treatment led to a significant survival benefit. About 25% of the mice were cured, even when the dose of tumour cells at inoculation was increased. These experiments demonstrate the efficacy of combined treatment with IL-2, LAK cells and CY. Further research should be directed at the design of treatment schedules based on repetitive courses of chemoimmunotherapy associated with little toxicity.     

Effect of recombinant human interleukin 4 on human monocyte activity

Recombinant human interleukin 4 (rhuIL-4), a lymphokine that reportedly stimulates tumoricidal activity in mouse macrophages, is currently undergoing clinical studies to determine its efficacy in the treatment of cancer. IL-4 is known to participate with other cytokines to regulate growth and differentiation of various hematopoietic cells as well as modulate the immune response. Little is known about the effect of rhuIL-4 on human monocyte tumoricidal activity. The purpose of these studies was to examine the effect of rhuIL-4 on human peripheral blood monocytes. Peripheral blood monocytes isolated from normal donors failed to demonstrate tumoricidal activity or interleukin 1 secretion after treatment with rhuIL-4 in vitro. Furthermore, monocyte-mediated cytotoxicity induced by recombinant human gamma-interferon plus muramyl dipeptide was suppressed in a dose-dependent manner by rhuIL-4. This reduction in cytotoxicity corresponded to a reduction in IL-1 production and secretion. Further investigation of rhuIL-4 and its role in the cytokine network is necessary for the development of effective immunotherapy in cancer patients.     

Immunotherapy of murine sarcomas using lymphokine activated killer cells: optimization of the schedule and route of administration of recombinant interleukin-2

Interleukin-2 (IL-2) at high doses or at low doses in concert with lymphokine-activated killer (LAK) cells can produce regression of established pulmonary and hepatic metastases from a variety of tumors in mice. IL-2 appears to mediate its antitumor effect through the generation of LAK cells in vivo from endogenous lymphocytes and by the stimulation of host and transferred LAK cell proliferation in tissues. In this paper we have investigated different strategies for IL-2 administration to determine which regimen produced maximal in vivo proliferation and optimal immunotherapeutic efficacy of LAK cells. Tissue expansion of lymphoid cells was assessed using an assay of in vivo labeling of dividing cells by the thymidine analogue, 5-[125I]iododeoxyuridine. The therapeutic effect of the different IL-2 administration protocols was determined by evaluating their efficacy in the treatment of established, 3-day pulmonary metastases from sarcomas in mice. The selection of IL-2 injection regimens for evaluation was based upon pharmacokinetic studies of IL-2 in mice. A single i.v. or i.p. dose yielded high peak IL-2 levels that could be measured for only a few hours after injection, while IL-2 given i.p. thrice daily produced titers that were detectable throughout the study periods (greater than or equal to 6 units/ml of serum after 100,000 units of IL-2 i.p. thrice daily). Using the proliferation and therapy models, we tested the same cumulative daily doses of IL-2 administered by i.v. or i.p. once daily, or i.p. thrice daily regimens. The i.p. thrice daily protocol stimulated greater lymphoid cell proliferation in the lungs, for example, than did the other regimens. Similarly, 300,000 units of IL-2 divided i.p. thrice daily were more successful in reducing metastases (n = 16) than was the entire dose given i.v. once daily (n = 190; P less than 0.05) or i.p. once daily (n = 71; P less than 0.05). When compared to the i.p. or i.v. once daily protocols, the i.p. thrice daily regimen for IL-2 also produced greater proliferation of exogenous LAK cells, as well as a more effective therapeutic outcome when IL-2 was combined with transferred LAK cells. Thus, sustained, lower levels of IL-2 were more effective than brief, high peak titers for stimulation of proliferation and antitumor activity. We then evaluated the effect of duration of IL-2 treatment as well as the number of LAK cell injections in the two models.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro activity of vinorelbine on human leukemia cells.

Vinorelbine (VNR) is a semi-synthetic Vinca rosea alkaloid that has been employed both as a single agent and in combination, and has shown significant antitumor activity. As little is known about VNR activity on human leukemia, we studied its in vitro cytotoxic effect on human leukemia cell lines (FLG 29.1, HL60, K562, Balm 4, CEM and Daudi) and on fresh leukemia cells from 28 patients: 2 acute myeloid leukemia (AML); 3 chronic myeloid leukemia in blastic phase (CML-BP); 5 acute lymphoblastic leukemia (ALL); 18 B-chronic lymphatic leukemia (B-CLL), employing the colorimetric INT assay and determining the IC50. We observed that VNR exerts its cytotoxic activity on leukemic cell lines in a dose-dependent fashion. The lymphoid cell lines appear more sensitive than the myeloid ones to the VNR-dependent growth inhibition. A similar pattern was noticed for leukemia cells in primary cultures. VNR is not effective on CML-BP cells, shows variable activity on the AML and ALL cells and is very effective against B-CLL cells. VNR inhibited the growth of fresh B-CLL cells from 15 of 18 patients, the IC50 doses ranging from 4 ng/ml to 83 microg/ml (doses coinciding with the plasma levels obtained in clinics). These observations strongly suggest that VNR could be useful in clinics for the treatment of B-CLL.     

Cytotoxic effects on viable human leukemic cells by combinations of lymphokine activated killer cells and monoclonal antibodies

Earlier studies with individually phenotyped monoclonal antibody combinations and complement or lymphokine activated killer (LAK) cells showed that many acute myeloid leukemic cells were resistant to these cytotoxic agents when used singly. Therefore, a combination of both agents was studied. When the leukemic target cells were submitted to killer cells activated with 100 or 800 IU of recombinant interleukin-2 (rIL-2), only averages of 6.0 and 16.7% of the targets were killed respectively. When the remaining, refractory cells were confronted with a cocktail of individually phenotyped monoclonal antibodies and complement, an additional significant cell kill was obtained, but it amounted to only between 7.4 and 5.5% (for LAK-100 and LAK-800, respectively). In contrast, of the target cells initially refractory to the same cocktail of monoclonal antibodies, all were cross-resistant both to LAK-cells activated with 100 and to those activated with 800 IU of rIL-2. This cross-resistance was caused neither by sub-optimal LAK-cell activation, nor by antibody blocking of hypothetical LAK-cell receptors, since pre-incubation with monoclonal antibodies without complement did not inhibit LAK-cell cytotoxicity. Although only partial cross-resistance was found in the present study, it still remains that only a minority of the tumor cells could be killed. A higher in-vitro cell kill should be attempted prior to clinical trials in order to avoid clinical effects resembling those of a partial surgical tumor resection.     

Local regional promotion of tumor growth after abdominal surgery is dominant over immunotherapy with interleukin-2 and lymphokine activated killer cells

Various investigators have shown that tumor growth can be facilitated by surgery, that an operation can suppress immune functions, and that these phenomena may be related. With a murine intraperitoneal (i.p.) tumor model, we investigated whether an operation could promote tumor growth and, if so, whether this effect could be successfully overcome by immunotherapy with interleukin-2 (IL-2) and lympokine activated killer (LAK) cells. Treatment with IL-2 and LAK cells was highly effective in unoperated mice. When a laparotomy was done 4 days before i.p. tumor inoculation, tumor growth was enhanced in all experiments, and the effect of immunotherapy was completely abrogated. This effect was local regional; an incision on the back of the mice did not affect tumor growth or outcome of treatment. Tumor growth in the scar area was usually excessive. Furthermore, these effects were found to be temporary. Promotion of tumor growth and abrogation of IL-2 + LAK effects were seen only from 4 days to up to 2 weeks after laparotomy but were lost 5 weeks after surgery. These results suggest that growth factors present in healing wounds may cause promotion of tumor growth and are dominant over the effects of IL-2 and LAK cell therapy.