Surface phenotype of Japanese adult T-cell leukemia cells characterized by monoclonal antibodies

We studies the surface phenotype and the functional activities of leukemic cells from three patients with Japanese adult T-cell leukemic (ATL) using the panel of OK and anti-Tac monoclonal antibodies, which react with differentiation antigens and define functionally distinct T- cell subsets or activated and terminally differentiated T cells. The phenotype of ATL cells were determined to be OKT1+T3+T4+T10+T5-T8-Okla1- , although cells from two patients suppressed pokeweed mitogen (PWM) induced normal B-cell differentiation, and cells from all patients lacked helper activity in this system. In addition, after cultivation with PWM, ATL cells from all patients were reactive with anti-Tac monoclonal antibody, and cells from one patient were reactive with OKlal. These findings suggest that ATL cells arise from peripheral mature T-cell subsets and also suggest that the transition of surface phenotype of ATL cells to functionally mature and activated T cells occurs in culture.     

Leukemic cells from some adult T-cell leukemia patients proliferate in response to interleukin-4

The proliferative response of fresh peripheral blood leukemic cells from eight adult T cell leukemia (ATL) patients to interleukin-4 (IL-4) was studied to determine the possibility that the IL-4-mediated T-cell growth pathway is involved in the cell growth of leukemic cells in ATL. Resting lymphocytes from ten normal individuals did not proliferate in response to IL-4. Leukemic cells from two ATL patients did not respond to interleukin-2 (IL-2) or IL-4. Leukemic cells from two patients did respond to IL-2, but not to IL-4. In contrast, a strong proliferative response was observed in the IL-4 culture, but not in the IL-2 culture in the remaining four patients. Chromosome analysis of mitotic cells, performed in one of four patients, confirmed that the cells dividing in response to IL-4 were leukemic cells, but not activated normal lymphocytes. These results indicate the activation of IL-4/IL-4 receptor system in leukemic cells from some ATL patients and suggest the possible involvement of the system in the proliferation of leukemic cells and the leukemogenesis in ATL.     

Defective terminal maturation along monocyte-macrophage lineage in chronic myelomonocytic leukemia

The maturation process of leukemic myelomonocytoid cells derived from peripheral blood of two typical cases of chronic myelomonocytic leukemia was investigated in vitro. In both cases, liquid culture of the leukemic cells in the presence or absence of 12-o-tetradecanoyl phorbol-13-acetate yielded either monocyte-macrophages or mature granulocytes, respectively, when examined by May-Giemsa staining. However, using cytochemical criteria, both of these mature cell fractions were found to be heterogenous and still contained substantial numbers of cells positive for both nonspecific and chloroacetate esterases. Phagocytic activity was markedly defective in the monocyte-macrophages recovered from culture as well as in freshly-isolated peripheral monocytes in both patients. In addition, active oxygen production from isolated monocytes stimulated by zymosan was also decreased in one of the cases. The results indicate that at least in some cases of chronic myelomonocytic leukemia, leukemic cells have the potential to differentiate in vitro to monocyte-macrophages which, however, show only limited development of functions, as well as freshly-isolated monocytes from peripheral blood.     

The effect of a prolonged in vitro exposure to 1-beta-D arabinofuranosylcytosine and deoxycytidine on the survival of normal (CFU-GM) and leukemic (L-CFU) human myeloid progenitor cells in suspension culture

We examined the effect of a 96-h exposure to 1-beta-D arabinofuranosylcytosine (Ara-C) and deoxycytidine (dCyd) in medium lacking an exogenous source of colony-stimulating activity (phytohemagglutinin-stimulated leukocyte-conditioned medium, PHA-LCM) on the survival of normal human committed myeloid progenitor cells (day-7 and day-14 granulocyte-macrophage colony-forming units [CFU-GM] as well as leukemic progenitors (leukemic colony-forming units, L-CFU) derived from myeloblasts obtained from 13 patients with acute nonlymphocytic leukemia (ANLL). Coadministration of Ara-C (20-50 microM) in conjunction with dCyd (50-100 microM) permitted the survival of an average of 9%-57% of day-7 CFU-GM and 32%-65% day-14 CFU-GM, depending upon the relative concentrations of dCyd and Ara-C. In contrast, exposure of leukemic myeloblasts to identical regimens resulted in considerably greater reductions in L-CFU survival, which in general exceeded 90% and in some cases was 100%. In addition, exposure of leukemic myeloblasts to Ara-C and dCyd for 96 h in culture medium lacking PHA-LCM eliminated the secondary plating efficiency (PE2) of leukemic colonies in 11 of 13 samples assayed and reduced values dramatically in the remaining 2. Substantial preservation of CFU-GM formation was also noted when normal bone marrow samples depleted of T cells and marrows obtained from two patients with ANLL in remission were assayed. These studies suggest that in contrast to certain normal committed myeloid progenitor cells, leukemic progenitors, particularly those with self-renewal capacity, are highly vulnerable to a prolonged exposure to Ara-C and dCyd in the absence of an exogenous source of colony-stimulating activity. They also raise the possibility that a combined regimen utilizing chemotherapeutic agents in conjunction with modifications of long-term culture techniques may represent a novel approach to the ex vivo purging of leukemic cells from bone marrow in conjunction with autologous bone marrow transplantation.     

Long-term bone marrow culture induces terminal differentiation of human myeloid leukemic cells

Bone marrow cells from a patient with acute myeloblastic leukemia were simultaneously cultured in vitro under conditions that favored the survival of either (1) leukemic progenitors (leukemic suspension culture), or (2) normal progenitors (long-term bone marrow culture). Whereas cells that were morphologically primitive and cytochemically leukemic persisted in leukemic suspension culture, they were progressively and completely replaced in long-term bone marrow culture by neutrophilic granulocytes and subsequently by macrophages. However, Auer rods were present in the maturing myeloid cells, including polymorphonuclear neutrophils, between the 7th and 30th days of long-term bone marrow culture, indicating that they were derived directly from the original leukemic population. This observation suggests that, at least in some patients, selection of cells with the potential for terminal differentiation may be the underlying mechanism responsible for the purging properties that have been attributed to long-term bone marrow culture.     

In vitro differentiation of leukemic cells to eosinophils in the presence of interleukin-5 in two cases of acute myeloid leukemia with the translocation (8;21)(q22;q22).

We demonstrated the significant eosinophilic growth of leukemic cells in the presence of interleukin-5 (IL-5) in 2 of 15 cases of acute myeloid leukemia. These two cases were M2 (FAB classification) with the translocation (8;21)(q22; q22). Bone marrow examination revealed the rather high percentages (6% and 9%) of atypical eosinophils in the total nucleated bone marrow cells in these two cases. In the remaining 13 cases, eosinophils were less than 2% in the nucleated bone marrow cells. In the methylcellulose culture system, 142 +/- 18 or 54 +/- 2 colonies were formed by 5 x 10(4) mononuclear cells in the presence of IL-5 in these two cases. These colonies mainly comprised mature eosinophils. Eosinophils were confirmed by Biebrich scarlet staining and electron microscopic examination using a specific lectin binding assay. The eosinophilic differentiation and proliferation of leukemic cells were also observed in the liquid culture system. It was shown that eosinophils observed in both systems were derived from leukemic cells using the chromosomal marker of leukemic cells, t(8;21). Leukemic cells also differentiated to neutrophils or both neutrophils and eosinophils in response to granulocyte colony-stimulating factor or interleukin-3, respectively, but did not respond noticeably to granulocyte-macrophage colony-stimulating factor. Although IL-5 acts on normal eosinophil committed precursors as a lineage-specific growth factor, at least some leukemic cells reacted to IL-5 and could proliferate and differentiate along eosinophilic pathway. Our findings suggest that atypical eosinophils observed in the bone marrow were derived from the leukemic clone in two cases of AML.     

Growth and differentiation of normal and leukemic human bone marrow cells cultured in diffusion chambers.

Bone marrow from healthy, normal volunteers and patients with acute myelocytic leukemia was cultured in diffusion chambers implanted into cyclophosphamide pretreated mice. Chambers were removed at regularly scheduled intervals over a period of 28 days. Total and differential cell counts were then done on the contents of each chamber. Normal human bone marrow showed an orderly pattern of growth and differentiation which was not found with leukemic bone marrow. Monocytes and macrophages were the predominant cell types in the diffusion chambers filled with normal marrow after day 10 of culture. Although leukemic specimens showed predominantly leukemic cells, a few mature polymorphonuclear leukocytes could be found throughout the entire culture period. Questions about the nature of the defect in acute myelocytic leukemia and the significance of the in vivo culture system are discussed. The results of these studies are compared and contrasted with studies of a similar type.     

Clinical features of childhood Ph1 positive acute leukemia

Clinical, immunologic, cytogenetic and molecular studies were performed on 9 patients with childhood Ph1 positive acute leukemia. FAB-L1 was found in 2 patients, L2 in 5 patients, and M1 and M2 in each patient. Six patients were older than 10 years old, and white blood cell count of 5 patients was more than 10(5)/microliters. All but one patient have died within 18 months. Immunologic analysis revealed that leukemic cells from all patients expressed lymphoid antigens CD10 and CD19, and myeloid antigen, CD13, was expressed on leukemic cells from 3 patients initially, and from 6 patients after short term in vitro culture without stimulation. bcr rearrangements were not observed in 3 patients tested. RNA analysis showed that 5 patients expressed P190bcr-abl pattern and one patient expressed P210bcr-abl pattern using polymerase chain reaction study. We conclude that Ph1 positive acute leukemia had a poor prognosis and differentiate into both myeloid and lymphoid lineages as well as chronic myelogenous leukemia (CML), and that this disease could not be possibly distinguished from CML by use of the molecular studies.     

Effects of low molecular weight B-cell growth factor on proliferation of leukemic cells from children with B-cell precursor-acute lymphoblastic leukemia

Recently, low-molecular-weight B-cell growth factor (LMW-BCGF) has been reported to stimulate growth of leukemic cells from B-cell precursor-acute lymphoblastic leukemia (BCP-ALL). We further investigated the effects of LMW-BCGF on proliferation of leukemic clonogenic (progenitor) and nonclonogenic (progeny) cells from children with BCP-ALL (28 patients) and B-cell ALL (two patients). Patients were either at diagnosis (n = 18) or in relapse (n = 12). Response of leukemic progenitor cells was determined by culturing cells (10(5) cells/mL) in methylcellulose with 0.1 U/mL LMW-BCGF. Colonies (greater than 20 cells) were counted at day 7. The response of the leukemic progeny population was determined by DNA synthesis studies using tritiated-thymidine and by DNA quantitation with propidiumiodide for determination of cell-cycle status. LMW-BCGF supported growth of leukemic progenitor cells from 20 of 28 (71%) BCP-ALL and two of two B-cell ALL patients. Colony numbers ranged from 7 to 2,400 (mean 145, median 45). A dose-response effect in colony growth was noted, with an apparent plateau at approximately 2.0 U/mL LMW-BCGF. Colony cells were primarily of leukemic phenotype (CD19+/CD10+/-). LMW-BCGF also induced significant increases in leukemic progeny cell proliferation as measured by both thymidine incorporation (stimulation indexes of 1.6 to 34) and by cell-cycle assay (percentage S+ G2/M stimulation indexes of 1.6 to 6). LMW-BCGF was more effective in stimulating leukemic proliferation than three recombinant interleukins (rIL-2, rIL-3, rIL-4), although rIL-3 was able to support colony growth in 4 of 11 patients. These results indicate that LMW-BCGF and, to a lesser degree rIL-3, are able to stimulate proliferation of BCP-ALL progenitor and progeny cells, whereas rIL-2 and rIL-4 do not support progenitor cell proliferation and have only marginal effects on leukemic progeny cell proliferation.     

Increased T-cell reactivity to leukemic B cells in chronic lymphocytic leukemia with change of stable disease to its progressive form

Some patients with chronic lymphocytic leukemia (CLL) have a relatively stable clinical course without treatment, but many of these eventually develop progressive disease. We have followed 68 patients over 5½ years by employing conventional surface markers and lymphocyte reactivity, including that of separate enriched T cells to their own leukemic B cells, to phytohemagglutinin (PHA), and to normal allogeneic B cells; we have observed an unusual response in each of the seven patients who developed progressive disease over this period of time. During the stable phase of the disease the T cells from 27 leukemic subjects did not respond to their own leukemic B cells in culture. In the seven patients who developed progressive disease, a significant reactivity of their enriched T cells to their own leukemic B cells occurred. There was no consistent change in T-cell reactivity to PHA or to allogeneic B cells, suggesting a change in the leukemic B-cell populations that was not detected morphologically. The change in reactivity of T cells to leukemic B cells occurred prior to evidence of clinical or laboratory deterioration in one of the seven cases; there was increasing lymphocytosis and lymphadenopathy in six and two instances, respectively; and anemia and thrombocytopenia in the three and one cases, respectively.     

Elevated low density lipoprotein receptor activity in leukemic cells with monocytic differentiation

The receptor-mediated degradation of 125I-low density lipoprotein (LDL) was compared in normal white blood cells and leukemic cells. The cells were isolated from the peripheral blood and bone marrow of healthy subjects and patients with newly diagnosed leukemia. The cells from most of the 40 consecutive patients with acute myelogenous leukemia showed markedly higher degradation rates as compared to mononuclear cells and granulocytes from peripheral blood and nucleated cells from the bone marrow of healthy individuals. Leukemic cells from patients with monocytic (FAB-M5) or myelomonocytic leukemia (FAB-M4) exhibited the highest degradation rates. The rate of receptor-mediated degradation of 125I-LDL was also high in leukemic cells from all three patients with chronic myelogenous leukemia in blast crisis, as well as in two of three patients with acute undifferentiated leukemia. In contrast, leukemic cells isolated from two patients with acute lymphoblastic leukemia showed low rates. In most cases, there was little difference in LDL receptor activity between leukemic cells isolated from peripheral blood and those from bone marrow. Hypocholesterolemia was a frequent finding in the leukemic patients. There was an inverse correlation between the plasma cholesterol level and the rate of receptor-mediated degradation of 125I-LDL by the leukemic cells. Studies are now in progress to investigate the possibilities of using LDL as a carrier of cytotoxic drugs in the treatment of leukemia.     

CD34(+) acute myeloid and lymphoid leukemic blasts can be induced to differentiate into dendritic cells.

CD34(+) hematopoietic stem cells from normal individuals and from patients with chronic myelogenous leukemia can be induced to differentiate into dendritic cells (DC). The aim of the current study was to determine whether acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells could be induced to differentiate into DC. CD34(+) AML-M2 cells with chromosome 7 monosomy were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNFalpha), and interleukin-4 (IL-4). After 3 weeks of culture, 35% of the AML-M2 cells showed DC morphology and phenotype. The DC phenotype was defined as upmodulation of the costimulatory molecules CD80 and CD86 and the expression of CD1a or CD83. The leukemic nature of the DC was validated by detection of chromosome 7 monosomy in sorted DC populations by fluorescence in situ hybridization (FISH). CD34(+) leukemic cells from 2 B-ALL patients with the Philadelphia chromosome were similarly cultured, but in the presence of CD40-ligand and IL-4. After 4 days of culture, more than 58% of the ALL cells showed DC morphology and phenotype. The leukemic nature of the DC was validated by detection of the bcr-abl fusion gene in sorted DC populations by FISH. In functional studies, the leukemic DC were highly superior to the parental leukemic blasts for inducing allogeneic T-cell responses. Thus, CD34(+) AML and ALL cells can be induced to differentiate into leukemic DC with morphologic, phenotypic, and functional similarities to normal DC.     

Flow cytometric analysis of expression of interleukin-2 receptor beta chain (p70-75) on various leukemic cells

We analyzed the expression of the interleukin-2 receptor (IL-2R) beta chain (p70-75) on various leukemic cells from 44 patients by flow cytometric analysis using the IL-2R beta chain-specific monoclonal antibody (MoAb), designated Mik-beta 1, which has been recently developed. Flow cytometric analysis demonstrated the expression of the IL-2R beta chain on granular lymphocytes (GLs) from all eight patients with granular lymphocyte proliferative disorders (GLPDs), on adult T-cell leukemia (ATL) cells from all three patients with ATL, and on T-cell acute lymphoblastic leukemia (T-ALL) cells from one of three patients with T-ALL. Although GLs from all the GLPD patients expressed the IL-2R beta chain alone and not the IL-2R alpha chain (Tac-antigen: p55), ATL and T-ALL cells expressing the beta chain coexpressed the alpha chain. In two of seven patients with common ALL (cALL) and in both patients with B-cell chronic lymphocytic leukemia, the leukemic cells expressed the alpha chain alone. Neither the alpha chain nor the beta chain was expressed on leukemic cells from the remaining 28 patients, including all 18 patients with acute nonlymphocytic leukemia, five of seven patients with cALL, all three patients with multiple myeloma, and two of three patients with T-ALL. These results indicate that three different forms of IL-2R chain expression exist on leukemic cells: the alpha chain alone; the beta chain alone; and both the alpha and beta chains. To examine whether the results obtained by flow cytometric analysis actually reflect functional aspects of the expressed IL-2Rs, we studied the specific binding of 125I-labeled IL-2 (125I-IL-2) to leukemic cells in 18 of the 44 patients. In addition, we performed 125I-IL-2 crosslinking studies in seven patients. The results of IL-2R expression of both 125I-IL-2 binding assay and crosslinking studies were in agreement with those obtained by flow cytometric analysis. These results indicate that flow cytometric analysis using MoAbs, anti-Tac, and Mik-beta 1 is useful for detecting the expression of the IL-2R chains.     

Lymphoblastoid cell lines from patients with chronic lymphocytic leukemia: identification of tumor origin by idiotypic analysis.

Multiple lymphoblastoid cell lines have been derived from two patients with chronic lymphocytic leukemia with an associated monoclonal immunoglobulin (Ig) band. Idiotypic antisera raised against the monoclonal serum Ig bands were shown to be specific for the membrane Ig of the patients' leukemic cells. The idiotypic determinants in these patients thereby constitute tumor-specific antigens. Surface and intracellular immunofluorescence studies utilizing these idiotypic antisera were used to identify the cell lines of leukemic origin. These studies showed that certain cell lines from each patient were derived from the leukemic cells while other cell lines were derived from residual normal B lymphocytes. The leukemic cell lines were variable and contained different percentages of lymphoid cells with the idiotype-specific membrane Ig and, in addition, different percentages of plasma cells with intracellular Ig of the same specificity. Specific Ig synthesis was also demonstrated by hemagglutination-inhibition analysis of cell line supernatants. Aside from Ig specificity, no differences have been found between the leukemic cell lines and those derived from normal cells. One of the leukemic cell lines was cloned in soft agarose. All the clones were shown to be of leukemic origin.     

Adenosine deaminase (ADA) in leukemia: clinical value of plasma ADA activity and characterization of leukemic cell ADA

Adenosine deaminase (ADA) activity was measured in plasma, erythrocytes, and mononuclear cells from 18 patients with acute and chronic leukemia. High levels of ADA activities were found in plasma, erythrocytes, and mononuclear cells from patients with acute leukemia, especially acute lymphoblastic leukemia, and blastic crisis of chronic myeloid leukemia. Serial determination of plasma ADA activities was done in 9 patients with acute leukemia. All patients untreated or in relapse had an elevation of plasma ADA activity, which decreased to normal or subnormal levels during complete remission. On starch gel electrophoresis, plasma ADA in leukemic patients separated into two bands. The major band showed a mobility identical to that of normal red cells and mononuclear cells, and the minor band corresponded to that of normal plasma ADA. Enzymatic and immunological studies were performed on ADA from leukemic cells of acute myeloid and lymphoblastic leukemia. There were no differences in Michaelis constant for adenosine, thermostability, electrophoretic mobility, immunological reactivity, and specific activity between ADA of leukemic cells and normal mononuclear cells. These results strongly suggest that the increased ADA activity in leukemic cells is caused by an increased synthesis of a structurally normal enzyme and that increased plasma ADA activity in leukemic patients reflects an increment of leukemic cells in bone marrow. Therefore, serial determination of plasma ADA activities seems to provide a good indicator of the total mass of leukemic cells in bone marrow.     

Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid

The recent finding that retinoic acid induces terminal granulocytic differentiation of the human promyelocytic leukemia cell line, HL-60, prompted an investigation of the sensitivity to this inducer of human myelocytic leukemia cells in primary suspension culture. Of the 21 leukemic specimens, only cells from the two patients with acute promyelocytic leukemia differentiated in response to retinoic acid. After an incubation period of 5--7 days in 1 microM retinoic acid, the cells from these two patients showed extensive morphological and functional maturation. Thus, because it appears that retinoic acid specifically induces granulocytic differentiation of leukemic promyelocytes, this compound may have therapeutic utility in the treatment of acute promyelocytic leukemia.     

Correlation of in vitro culture pattern and Q-banded karyotype in acute nonlymphocytic leukemia

Both cytogenetic abnormalities and in vitro growth patterns of leukemic cells have prognostic significance in acute nonlymphocytic leukemia (ANLL). The present study was undertaken to analyze the interrelationship between these two factors and response to therapy. Blast cells from 43 patients with de novo ANLL, four patients with secondary ANLL, and two patients with ANLL following a preleukemic phase were studied using both in vitro culture in methylcellulose and quinacrine chromosome banding techniques. In 19 patients with de novo ANLL, minimal growth in culture (≤ 5 colonies without prominence of small clusters) was noted (Pattern I). Blast cells from the remaining 24 patients formed numerous abnormal clusters and colonies in a continuum distribution (Pattern II). Sixty-three percent of Pattern I patients had completely normal karyotypes (NN). Only 29% of Pattern II patients were NN. No Pattern I patient had only abnormal karyotypes (AA), while 25% of Pattern II patients were AA (p<0.05). All six patients with secondary leukemia or leukemia following a preleukemic phase demonstrated both Pattern II growth and cytogenetic abnormalities. Fifty-six percent of Pattern I patients with de novo ANLL responded to chemotherapy, 35% of Pattern II patients responded, and 0% of patients with secondary or post-preleukemic ANLL responded. The correlation between the presence of cytogenetic abnormalities in leukemic cells and a pattern of excessive, abnormal growth in vitro coupled with the trend toward poor prognosis in these patients suggests that cytogenetically abnormal cells may have a proliferative advantage both in vitro and in vivo.     

Divergent patterns of marrow cell suspension culture growth in the myeloid leukemias: correlation of in vitro findings with clinical features.

Cellular recovery, maturation, and colony-forming cell (CFC) generation patterns of bone marrow cells from 23 patients with acute, subacute, and chronic myeloid leukemia (AML, SML, and CML) were studied using liquid and agar culture techniques. Increased recovery of proliferative myeloid cells from liquid culture was noted in 6 of 8 AML patients at diagnosis or relapse and 5 of 7 untreated SML patients. Patients with either AML or SML with rapid clinical progression exhibited greater recovery of cells in vitro with less maturation than patients with more stable disease. Studies from 3 patients with CML showed normal to increased cellular recovery with normal maturation. Three of 4 studies of AML patients followed sequentially in apparent remission, but with impending relapse, exhibited increased numbers of myeloblasts and promyelocytes, whereas 28 of 32 studies performed during stable remission were normal. The normally observed increase in CFC during liquid culture was absent in most leukemic marrow samples studied (3 of 4 AML, 4 of 6 SML, and 2 of 3 CML). Persistent low recovery of CFC during AML remission was associated in 3 patients with short remission duration. These studies indicated the potential utility of these techniques for the clinical evaluation of patients with myeloid leukemia and for studying factors involved in the progression of these diseases.     

Analysis of the growth enhancing effect of L-ascorbic acid on human leukemic cells in culture

The effect of L-ascorbic acid (LAA) on the in vitro colony growth of leukemic cells from the marrows of patients with acute nonlymphocytic leukemia was studied using a modified agar culture method featuring daily feeding. In 10 of 31 patients (32%) the growth was enhanced with supplementation of LAA in culture. The average number of colonies was reduced to 26% if LAA was deleted from culture. This growth enhancing effect appears to be specific to LAA: neither glutathione which has redox potential similar to LAA nor the acidification of culture medium with HCI to the pH of medium containing LAA is effective. This effect is also selective for leukemic marrows, and normal marrow colonies are not affected over wide range of LAA concentrations (0 to 300 microM) which are achievable in human in vivo.     

Induction of NK and LAK activities in human lymphocyte culture by a cytosol fraction from leukemic myeloblasts and by monoclonal antibody CD 3

The stimulating effect of cytosol fraction (F3) isolated from human myeloblasts (m.w. ranging from 30 to 100 kDa) and monoclonal antibody CD 3 (MEM 57) was tested on NK and LAK cell activities in peripheral mononuclear cells (PMNC) of normal donors and leukemic patients. The F3 fraction added in 10% of the total volume of RPMI 1640 medium induced significantly increased lytic activity of normal lymphocytes or IL-2 activated lymphocytes against K 562 cells in 3-day culture. Similarly, the proliferation of both cell cultures was enhanced by F3. The effect of F3 on NK and LAK cell activities in culture of PMNC from leukemic patients was less pronounced and synergic action of F3 did not occur, whereas the proliferation of leukemic cells was significantly enhanced. MEM 57 increased the cytotoxicity and cell proliferation in 3-day culture of normal lymphocytes at concentrations ranging from 10 to 250 ng/ml. MEM 57 stimulated also NK cytotoxicity estimated in freshly isolated normal lymphocytes. The deficient NK cell activity observed in PMNC of leukemic patients was induced by MEM 57 or its combination with IL-2 in 3-day culture. These observations indicate that combination of more immunomodulating agents can lead to shortening of the incubation time necessary for correction of the NK cell defective activity in PMNC of leukemic patients.