The effect of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) on granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor cells

AIMS: To determine whether granulocyte macrophage-colony stimulating factor (GM-CSF) production by neuronal precursor (NT2) cells can be regulated by IL-1beta and TNF-alpha. BACKGROUND: We have previously demonstrated GM-CSF expression by neurons of the developing human brain, as well as by NT2 cells. IL-1beta and TNF-alpha upregulate GM-CSF production in glial cells, but GM-CSF regulation in neurons is as yet undefined. We hypothesized that IL-1beta and TNF-alpha would increase GM-CSF mRNA and protein production in NT2 cells. METHODS: The effect of IL-1beta and TNF-alpha on GM-CSF production was assessed by dose response (0 to 2,000 U/ml), and time course (0 to 48 hours incubation) experiments. GM-CSF mRNA and protein production were assessed by quantitative RT-PCR and by ELISA. The effect of these cytokines on cell turnover was determined by BrdU incorporation. RESULTS: IL-1beta increased GM-CSF mRNA and protein expression by NT2 cells. This effect was time and dose dependent, and the effective dose ranging from (20-200 U/ml). TNF-alpha increased GM-CSF mRNA expression to a lesser extent than did IL-1beta (maximal stimulation at 200 U/ml), and a minimal increase in net protein accumulation was noted. Neither cytokine increased NT2 cell turnover. CONCLUSIONS: IL-1beta and TNF-alpha both increase GM-CSF mRNA expression by NT2 cells, but only IL-1beta increases net GM-CSF protein accumulation.     

CD80 (B7-1) expression on human acute myeloid leukaemic cells cultured with GM-CSF, IL-3 and IL-6

Acute myeloid leukaemia (AML) blasts rarely express the B7 family of co-stimulatory molecules and do not elicit a clinically significant autologous T-lymphocyte anti-tumour response. The aim of this study was the in vitro modification of AML blasts to an antigen-presenting cell phenotype characterised by upregulated expression of the co-stimulatory molecule CD80 (B7-1). Circulating AML cells were induced to undergo partial differentiation in culture with the cytokines IL-3, IL-6 and GM-CSF; they exhibited variable upregulation of CD80 and continued to express MHC class I and II. These cells remained viable to day 20, in contrast with normal peripheral blood mononuclear cells (PBMNC), which did not survive under the culture conditions. In contrast to unmanipulated blasts, cultured leukaemic cells expressed B7-1. Where initial cytogenetic abnormalities were present, they were also seen in flow-sorted CD80-expressing cells after culture in cytokines, indicating their malignant origin. The immunogenic potential of these cultured cells was highlighted by allogeneic and autologous mixed lymphocyte reactions, in which both differentiated, but not unmanipulated, blasts produced expansion of T-lymphocyte numbers. Autologous cytotoxic T-lymphocyte (CTL) assays indicated specific killing of B7-1+ leukaemic cells, which was greatly enhanced after priming of the T-lymphocytes by B7-1+ blasts prior to the CTL assay, then enabling the CTL to lyse both unmanipulated and differentiated leukaemic cells.     

Enhancement by transforming growth factor-beta 1 (TGF-beta 1) of the proliferation of leukemic blast progenitors stimulated with IL-3.

We studied the effect of transforming growth factor-beta 1 (TGF-beta 1) on colony formation of leukemic blast progenitors from ten acute myeloblastic leukemia (AML) patients stimulated with granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), interleukin-6 (IL-6), or interleukin-1 beta (IL-1 beta). These CSFs and interleukins by themselves stimulated the proliferation of leukemic blast progenitors without adding TGF-beta 1. G-CSF, GM-CSF, and IL-3 stimulated blast colony formation in nine patients, IL-6 stimulated it in five, and IL-1 beta stimulated in four. TGF-beta 1 significantly reduced blast colony formation stimulated by G-CSF, GM-CSF, or IL-6 in all patients. In contrast, TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors from three cases, while in the other seven patients TGF-beta 1 reduced blast colony formation in the presence of IL-3. To study the mechanism by which TGF-beta 1 enhanced the stimulatory effect of IL-3 on blast progenitors, we carried out the following experiments in the three patients in which it occurred. First, the media conditioned by leukemic cells in the presence of TGF-beta 1 stimulated the growth of leukemic blast progenitors, but such effect was completely abolished by anti-IL-1 beta antibody. Second, the addition of IL-1 beta in the culture significantly enhanced the growth of blast progenitors stimulated with IL-3. Third, leukemic cells of the two patients studied were revealed to secrete IL-1 beta and tumor necrosis factor-alpha (TNF-alpha) constitutively; the production by leukemic cells of IL-1 beta and TNF-alpha was significantly promoted by TGF-beta 1. Furthermore, the growth enhancing effect of TGF-beta 1 in the presence of IL-3 was fully neutralized by anti-IL-1 beta antibody. These findings suggest that TGF-beta 1 stimulated the growth of blast progenitors through the production and secretion of IL-1 beta by leukemic cells.     

Regulation of granulocyte-macrophage colony-stimulating factor in human retinal pigment epithelial cells by IL-1beta and IFN-gamma.

GM-CSF production by RPE cells, which form part of the blood-retina barrier, is upregulated by IL-1beta and this increase can be reversed by IFN-gamma. IL-1beta up-regulation is not dependent on PKC but the PKC activator PMA induces low levels of GM-CSF production and acts synergistically with IL-1beta to further increase GM-CSF. Although A23187 and ionomycin stimulated low levels of GM-CSF production, the IL-1beta pathway was cyclosporin A insensitive and did not interact with the calcium pathway. IL-1beta-stimulated GM-CSF mRNA expression and production was strongly dependent on NF-kappaB. IFN-gamma inhibition of the GM-CSF response to IL-1beta acted via NF-kappaB, reducing the translocation of NF-kappaB to the nuclei of RPE cells treated with IL-1beta and IFN-gamma. The results show that IFN-gamma down-regulation acts either directly on NF-kappaB or its activation or by blockade of a pathway upstream of NF-kappaB. However, any such blockade does not involve PKC or intracellular calcium.     

Three-color versus four-color multiparameter cell cycle analyses of primary acute myeloid leukemia samples

Checkpoint alterations that impact cell cycle and apoptosis responses to therapeutic treatments may produce drug resistance in acute myeloid leukemia (AML). To study these, we have developed flow cytometry assays of checkpoint function that also allow quantitation of key molecular regulators of apoptosis and cell cycle. We have used three-color (3C) assays, with FITC-labeled anti-BCL-2 and PE-labeled anti-proliferating cell nuclear antigen (PCNA) antibodies, and the DNA dye 7-aminoactinomycin, to characterize primary leukemia cells identified in DNA x side light scatter (SSC) histograms. We showed that 3C assays are accurate and reproducible in analyses of leukemia cell lines and of primary AML and normal bone marrow samples (Banker et al.: Blood 89: 243-255, 1997; Banker et al.: Leukemia Res 22: 221-239, 1998; Banker et al.: Clin Cancer Res 4: 3051-3062, 1998). To further confirm the validity of our SSC leukemia cell gating and to address whether immunophenotypic AML subsets might have different biologic properties, we have now designed four-color (4C) flow assays to characterize checkpoint status in leukemic blasts specifically identified by surface immunostaining. In modeling this assay strategy, PE/Cy5-labeled anti-CD34 antibody was used to detect blasts, with FITC-labeled anti-BCL-2, PE-labeled anti-PCNA antibodies, and Hoechst 33342 (H33342) DNA dye. Four-color CD34-gated data was concordant with 3C, SSC-gated data for leukemia cell lines and for most primary AML samples with high and intermediate blast counts. BCL-2 and PCNA immunopositivity and sub-G1 apoptosis determinations were different in the CD34-gated versus SSC-gated blasts in particular samples with smaller CD34(+) subsets, suggesting that leukemia samples can contain blast subsets with different biologic properties. On the other hand, PCNA-gated cell-cycle distributions in untreated cells and G1 versus S phase cell-cycle arrests after cytosine arabinoside treatments were completely concordant in 4C and 3C assays. We conclude that both 3C and 4C assays can be used to characterize protein expression and cell-cycle drug response patterns in leukemia blasts, but that 4C assays may additionally allow discrimination of these properties in immunophenotypic leukemia subsets.     

Relations between IL-3-induced proliferation and in vitro cytokine secretion of bone marrow cells from AML patients.

The influence of IL-3 on the bone marrow cells of 53 patients with acute myeloid leukaemia (AML) was investigated after 72 h suspension in cultures by analysing the proliferation of blasts and the secretion of cytokines. The titres of IL-1beta IL-6, TNF-alpha and IL-3 were measured in the supernatants of these cultures with ELISA tests. Comparing the percentage of cells in S-phases of control cultures and cultures with IL-3, the leukaemias were divided into two growth pattern groups: IL-3-insensitive (n=19) and IL-3-sensitive (n=34) leukaemias. The IL-3-insensitive AML cells show a greater ability for autonomous growth, first by the increase of S-phase in the control culture compared with the S-phase in vivo (P=0.0486) and second, by the higher constitutive secretion (control culture) of IL-1beta P =0.0004), IL-6 ( P =0.0395) and TNF-alpha P=0.0005). The IL-3-induced secondary cytokine secretion is also different in the two growth pattern groups. Whereas in the IL-3-insensitive AML cells a moderate increase of IL-1beta (1.48-fold increase) was present, in the IL-3-sensitive AML cells a 4.72-fold increase of IL-1beta 2.71-fold increase of IL-6 and 11.81-fold increase of the TNF-alpha titre could be detected. Overall, the data show an inverse correlation between the ability of AML cells to respond to IL-3 with increase of an S-phase and the constitutive secretion of IL-1beta, II-6 and TNF-alpha. A further effect of IL-3 is the induction of secondary cytokine secretion in the bone marrow of IL-3-sensitive growing AML cells.     

Production of granulocyte colony-stimulating factor and granulocyte/macrophage-colony-stimulating factor after interleukin-1 stimulation of marrow stromal cell cultures from normal or aplastic anemia donors.

We have studied stromal cell function in naive or interleukin-1 (IL-1)-stimulated (100 pg/ml) long-term marrow cultures (LTC) from 12 normal donors and 21 patients with severe aplastic anemia (AA). Conditioned media (CM) from normal LTC contained levels of erythroid burst-promoting activity (BPA) and granulocyte/macrophage (GM) colony-stimulating activity (CSA) comparable to those previously described (Migliaccio et al., [1990] Blood, 75:305-312). The addition of IL-1 to these cultures increased the level of CSA and, specifically, of granulocyte colony-stimulating factor (G-CSF) released. Anti-GM-CSF antibody neutralized BPA and CSA in normal naive LTC CM but only the CSA in the CM from IL-1-stimulated LTC. Since the concentrations of GM-CSF, as detected with a specific immunoassay, did not increase after IL-1 treatment, these data suggest that IL-1-stimulated cultures contain an unidentified growth factor having BPA. CM from AA stromal cells contained levels of CSA comparable to those observed in normal stromal cell CM but had significantly lower levels of BPA. Neither anti-GM-CSF nor anti-IL-3 antibodies neutralized the BPA in AA stromal cell CM. This activity may be related to that found in the CM of IL-1-treated normal stromal cells. In nearly 50% of stromal cell cultures of AA patients, addition of IL-1 failed to increase the BPA, CSA, or G-CSF. The presence of an inhibitor in naive or IL-1-treated AA stromal cell CM was excluded by adding the CM to IL-3-stimulated cultures. These findings suggest that G-CSF and GM-CSF genes are differentially regulated in the marrow microenvironment. Furthermore, a marrow microenvironment, deficient in BPA production and, in some cases, unresponsive to IL-1 could contribute to marrow failure in some patients with AA.     

Evidence for Shared Receptor Proteins for Human Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor in the Human M-07 Cell Line

Abstract

The biologic response of the human leukemia cell line M-07 to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 4 (IL-4) is mediated by a low number of high affinity receptors. Cross-competition studies revealed that IL-3 and GM-CSF partially inhibited the specific binding of the heterologous radiolabeled ligand, whereas IL-4 binding was not affected by these cytokines. The molecular mechanism of cross-competition was investigated by chemical crosslinking and immuno-precipitation. Trimolecular receptor complexes consisting of a major 73kDa and two minor 120 and 128kDa membrane proteins for IL-3, and a major 84kDa and two minor 120 and 130 kDa proteins for GM-CSF were found on M-07 cells. The 73 and 84kDa proteins represent distinct and non-linked membrane proteins and are identical with the cloned, low affinity IL-3 and GM-CSF receptor proteins (Gearing et al, 1989, Hayashida et al, 1990). The higher molecular weight proteins share common binding sites as evidenced by immunoprecipitation of double-crosslinked membranes. The 120/128kDa proteins are most likely identical with the recently cloned and shared β-subunit of the IL-3 and GM-CSF receptor (Kitamura et al, 1991) containing a single or two IL-3 and/or GM-CSF molecules.     

Dexamethasone regulation of the expression of cytokine mRNAs induced by interleukin-1 in the astrocytoma cell line U373MG

BSF-2/IL-6, GM-CSF and IL-1 beta mRNAs were induced by recombinant IL-1 in human astrocytoma cell line U373MG. The induction of BSF-2/IL-6 and IL-1 beta mRNAs did not require de novo protein synthesis while that of GM-CSF mRNA required a newly synthesized protein. Dexamethasone inhibited the induction of these cytokine mRNAs by IL-1. This process seems to require continued protein synthesis. These results suggest that the production of these cytokines are positively and negatively controlled by IL-1 and glucocorticoids, respectively, in astrocytes.     

The effects of recombinant CSF-1 on the blast cells of acute myeloblastic leukemia in suspension culture

Recombinant hemopoietic colony-stimulating factors (CSFs), including GM-CSF, G-CSF and IL-3, have been shown to be effective stimulators of both self-renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF-1 (or M-CSF), on the growth of leukemic blasts in culture. CSF-1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF-1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF-1 may be useful in the treatment of selected cases of AML.     

Induction of interleukin-3 by interleukin-1 in the absence of other exogenous stimuli

We have previously reported that lipopolysaccharide (LPS) could induce the production of interleukin-3 (IL-3) by mouse spleen cells. In the present study, we show that recombinant human interleukin-1, in the absence of other stimuli, is able to induce the production of IL-3. IL-3 was detected in the supernatants of adult, although neither in young nor in nude mouse splenocytes and was assessed by its capacity to support the growth of the IL-3-dependent FDC-P2 cell line. The presence of IL-3 was antigenically confirmed with a monoclonal anti-IL-3 antibody. Both recombinant IL-1 alpha and IL-1 beta had similar potential for inducing IL-3 production. IL-3 activity was detected in the supernatants of cells cultured in the presence of 100 pg/ml IL-1; maximal IL-3 levels were obtained with 10-30 ng/ml IL-1. Kinetic studies of IL-1-induced IL-3 production indicated that 4-6 days of culture were required for optimal production, whereas 1-2 days were sufficient in cultures stimulated with concanavalin A. Recombinant IL-6 failed to induce significant amounts of IL-3, and TNF alpha induced only weak IL-3 production. GM-CSF but not M-CSF could lead to the appearance of IL-3 in spleen cell culture supernatants. Removal of macrophages decreased the production of IL-3 induced by LPS and GMF-CSF though did not affect the IL-3 production induced by IL-1. This observation suggests that IL-1 production might be an intermediate event in IL-3 production induced by LPS and GM-CSF through the activation of macrophages. IL-3 was detected in culture supernatants of B-cell-depleted splenocytes indicating that T-cells were the source of IL-3. Surprisingly T-cell-depleted populations could also produce IL-3 upon IL-1 stimulation. Preliminary experiments with an autoreactive CD4- CD8- V beta 8+ clone suggested that these cells might also be involved in the described IL-3 production.     

Comparative analysis of proinflammatory cytokines in plasma of mice exposed to radiation or in combined radiation injury

Male mice (CBA x C57BL/6)F1 were used for the experiments throughout this study. Blood serum levels of IL-1 beta, IL-6, TNF-alpha, IL-3, and GM-CSF were evaluated by means of enzyme-linked immunosorbent assay at 3, 6, 24, 48 and 72 hours after 7 Gy gamma-irradiation alone or combined injury (irradiation + thermal burn). Radiation as well as combined injury did not cause any important alterations of IL-1 beta, TNF-alpha, IL-3, and GM-CSF concentrations in the system circulation. Combined injury revealed more enhanced serum levels of IL-6 versus only irradiated mice. A possible significance of this phenomenon at the combined radiation and thermal burns' pathogenesis is discussed.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Multiple amino acid substitutions suggest a structural basis for the separation of biological activity and receptor binding in a mutant interleukin-1 beta protein.

Receptor binding and biological activity properties of human interleukin-1 beta can be dissociated by mutating a single amino acid, arginine 127, to glycine (IL-1 beta R----G) [Gehrke et al. (1990) J. Biol. Chem. 265, 5922-5925]. The mechanism underlying the reduced biological activity has been examined by replacing arginine 127 with several other amino acids, followed by determination of biological activity using a T-helper cell proliferation assay. Mutant IL-1 beta proteins containing lysine, glutamic acid, tryptophan, or alanine in place of arginine 127 maintain biological activity. These data strongly suggest that IL-1 beta biological activity is not directly dependent upon the specific properties of charge, hydrophobicity/hydrophilicity, or side-chain group presented by the residue at position 127. Molecular modeling analyses indicate that the structural integrity of the antiparallel beta-strand 1/12 pair is disturbed in the glycine 127 mutant protein. Collapse of beta-strand 1 into a hydrated space between strands 1, 2, and 4 could structurally alter a cleft in IL-1 beta that contains a cluster of highly conserved amino acids, including a key aspartic acid residue [Ju et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2658-2662]. Mutagenesis data and the differential activities of the IL-1 beta R----G and IL-1 receptor antagonist proteins in stimulating early and late gene expression [Conca et al. (1991) J. Biol. Chem. 266, 16265-16268] suggest that multiple receptor-ligand contacts, exclusive of those required for receptor binding, are required for the stimulation of full IL-1 biological activity.     

Cytokine production by a megakaryocytic cell line

Summary The regulation of megakaryopoeisis by cytokines is not yet well understood. It is possible that autocrine loops are established during megakaryocyte growth and differentiation, aiding in the maturation of these cells. The CHRF-288-11 human megakaryoblastic cell line has been examined for cytokine production in growing cells and cells stimulated to differentiate by the addition of phorbol esters. It has been demonstrated that these cells produce RNA corresponding to the interleukins IL-1α, 1β, 3, 7, 8, and 11, granulocyte-macrophage colony stimulating factor (GM-CSF), stem cell factor (SCF), transforming growth factor-β (TGF-β), tumor necrosis factor-α (TNF-α), interferon-α (INF-α), and basic fibroblast growth factor (bFGF). Additionaly, RNA corresponding to the receptors for IL-6, GM-CSF, SCF, INF-α,β, bFGF, and monocyte colony stimulating factor (M-CSF) were also expressed by the cells. The receptor for TNF-α was detected immunologically. Analysis at the protein level demonstrated that significant amounts of INF-α, TNF-α, GM-CSF, SCF, IL-1α, and a soluble form of the IL-6 receptor were produced by the cells. Addition of phorbol esters to CHRF-288-11 cells enhances their megakaryocytic phenotype; such treatment also results in increased secretion of INF-α, TNF-α, and GM-CSF. These results suggest that potential autocrine loops are established during the differentiation of CHRF-288-11 cells, which may alter the capability of the cell to differentiate. These findings are similar to those recently obtained for marrow-derived megakaryocytes (Jiang et al.) suggesting that CHRF-288-11 cells provide a useful model system for the study of cytokine release during megakaryocyte differentiation.     

Lytic activity against primary AML cells is stimulated in vitro by an autologous whole cell vaccine expressing IL-2 and CD80

Despite being of the myeloid lineage, acute myeloid leukaemia (AML) blasts are of low immunogenicity, probably because they lack the costimulatory molecule CD80 and secrete immunosuppressive factors. We have previously shown that in vitro stimulation of autologous peripheral blood mononuclear cells (PBMCs) with primary AML cells modified to express CD80 and IL-2 promotes proliferation, secretion of Th1 cytokines and expansion of activated CD8⁺ T cells. In this study, we show that allogeneic effector cells (from a healthy donor or AML patients) when stimulated with IL-2/CD80 modified AML blasts were able to induce the lysis of unmodified AML blasts. Effector cells stimulated with IL-2/CD80AML blasts had higher lytic activity than cells stimulated with AML cells expressing CD80 or IL-2 alone. Similarly, AML patient PBMCs primed with autologous IL-2/CD80 AML cells had a higher frequency of IFN-γ secreting cells and show cytotoxicity against autologous, unmodified blasts. Crucially, the response appears to be leukaemia specific, since stimulated patient PBMCs show higher frequencies of IFN-γ secreting effector cells in response to AML blasts than to remission bone marrow cells from the same patients. Although studied in a small number of heterogeneous patient samples, the data are encouraging and support the continuing development of vaccination for poor prognosis AML patients with autologous cells genetically modified to express IL-2/CD80.     

Cellular immune responses in acute leukaemia patients with severe chemotherapy-induced leucopenia; characterization of the cytokine repertoire of clonogenic T cells

 T lymphocytes are important both for the host defence against infections and probably also as antileukaemic effector cells in patients with acute leukaemia. To investigate the T lymphocyte cytokine repertoire of clonogenic T lymphocytes, CD4⁺ and CD8⁺ T lymphocyte clones were prepared from acute leukaemia patients with chemotherapy-induced cytopenia (leucocytes <0.5×10⁹/l). A majority of both CD4⁺ and CD8⁺ clones secreted detectable interleukin-2 (IL-2), IL-10, IL-13, granulocyte/macrophage-colony-stimulating factor and interferon γ (IFNγ) in response to phytohaemagglutinin + accessory cells (Epstein-Barr-virus-transformed B cell line, 80-Gy-irradiated). The CD4⁺ clones showed significantly higher levels of IL-10 secretion than the CD8⁺ clones. Decreased levels of IL-2, IL-13 and IFNγ were observed when acute myelogenous leukaemia (AML) blasts were used instead of cells from the B cell line as accessory cells during phytohaemagglutinin activation, but the differences in IL-13 and IFNγ levels were reversed by addition of exogenous IL-2. On the basis of these results we conclude: (i) the remaining clonogenic T lymphocytes derived from acute leukaemia patients with therapy-induced leucopenia can respond to activation with a broad cytokine response, and T-cell-derived cytokines may then contribute to cytokine responses during complicating infections in these patients; (ii) although T cells can modulate AML blast functions and mediate antileukaemic effects, the leukaemia blasts will also modulate T cell functions and alter the cytokine profile of activated T lymphocytes. Received: 6 November 1997 / Accepted: 5 March 1998