Autocrine IL-2-dependent growth of a newly established CD3+, CD16-, CD56+, CD57+, J(H)-, TCRbeta-, TCRgamma- leukemia cell line (NOI-90).

Leukemic cells from a 45-year-old male patient with a CD3+, CD56+, CD57+, CD7+ acute lymphoblastic leukemia were cultured in vitro in the absence of any added growth factor for up to 6 years and a continuous lymphoblastoid cell line (NOI-90) was established. NOI-90 cells have the same phenotype and karyotype as initial leukemic cells. Southern blot of DNA from NOI-90 cells showed that TCRbeta, TCRgamma, and J(H) were in germ line. Two and 25% of NOI-90 cells were positive when stained with the IOT14 and 7G7/B6 moAbs, which recognize the CD25 molecule (IL-2R alpha chain); moreover, 4% and 13% of the cells were positive when stained with the TU-27 and mik beta3 moAbs which recognize the CD122 molecule (IL-2Rbeta chain). Equilibrium binding experiments with radiolabelled IL-2 revealed the presence of a small number of high affinity IL-2R on both fresh and continuously growing cells. Media conditioned by NOI-90 cells could induce proliferation of an IL-2-dependent cell line and this IL-2 activity could be detected by a sensitive immunoenzymatic assay using antibodies recognizing distinct epitopes of IL-2. Moreover, IL-2 activity could be adsorbed by immunoaffinity on anti-IL-2 polyclonal purified IgG and the retained molecule displayed a m.w. of 14.5 kDa in SDS-PAGE. In addition, IL-2 immunoreactive molecules could be revealed in the cytoplasm of the cells. Finally, IL-2 fixed on the cell membrane could be detected by indirect immunofluorescence. Although added IL-2 could not induce cell proliferation, monoclonal antibodies against CD25, CD122 and IL-2 could specifically inhibit spontaneous cell proliferation in a dose-dependent manner. NOI-90 cells failed to demonstrate any cytotoxic activity against the K-562, Raji or Daudi cells. These findings indicate that NOI-90 cells are of non-T, non-B, origin lacking NK activity but proliferate under an autocrine pathway which involves, at least partly, the IL-2/IL-2R system.

Nuclear and cytoplasmic expressions of the carbohydrate-binding protein CBP70 in tumoral or healthy cells of the macrophagic lineage.

The expression of the carbohydrate-binding protein CBP70 was analyzed in undifferentiated HL60 cells, HL60 cells differentiated into monocytes/macrophages or granulocytes, healthy monocytes and in vitro monocyte-derived macrophages (MDM) using an anti-CBP70 serum. This study was performed by immunoblotting analysis of nuclear and cytoplasmic extracts before and after N-acetylglucosamine affinity chromatography and by indirect immunofluorescence microscopy. The results of this study show, for the first time, that CBP70 is expressed in the nucleus and the cytoplasm of healthy or leukemic cells of the macrophagic lineage. However, striking differences were observed depending upon the leukemic or normal state of cells and cell differentiation. Indeed, the level of expression and the intracellular distribution of CBP70 were found to be different in undifferentiated HL60 cells and monocytes/macrophages differentiated from these cells. Major differences were also observed according to whether macrophages differentiated from leukemic HL60 cells or healthy monocytes. Thus, the total cellular expression of CBP70 was markedly lower in MDM than in HL60-derived macrophages and the intracellular distribution of the protein was different. Nevertheless, in both cases, the total cellular expression of CBP70 was enhanced during cell differentiation. Another important result is the finding that CBP70 behaviour was totally different when HL60 cells were induced to differentiate into macrophages or granulocytes. These data could therefore suggest that CBP70 is involved in phagocytic cell differentiation. Moreover, we show that an additional 60 kDa polypeptide (p60), recognized by the anti-CBP70 serum, is expressed in HL60 cells differentiated into macrophages or granulocytes as well as in healthy monocytes or MDM but not expressed in undifferentiated HL60 cells. Although CBP70 and p60 appeared to be closely related polypeptides, their relationship remains to be precised. These findings are discussed with regard to data available on galectin-3.

IL2 triggers a tumor progression process in a melanoma cell line MELP derived from a patient whose metastasis increased in size during IL2/INFalpha biotherapy.

Human melanomas may express both in vivo and in vitro functional IL-Rs and may be expected to directly respond to injected IL2. This may generate biological situations which may be favourable for the patient, but also for tumor progression. Here, we analyse the latter hypothesis. MELP is a melanoma cell line derived from a patient whose metastasis increased in size during IL2/IFN alpha biotherapy [correction of biotheraphy]. These cells have been characterized in vitro for their phenotype and for their sensitivity to IL2. In vitro MELP cells express an IL2-R alpha(+) beta(+) gamma(-) phenotype and IL2 treatment induces the acquisition of new functional characteristics represented (i) by the increased surface expression of two markers of metastatic evolution (ICAM-1 and CD44); (ii) by the stable induction of the IL2-R gamma with the appearance of functional IL2-R beta complex, which are also recognized by GM-CSF; (iii) by the inhibition of transcription of a regulatory cytokine such as IL6; (iv) by a differential effect of IL6 on CD44 surface expression in MELP cells treated or not with IL2 (MILG cells); (v) by the acquisition of faster growth rates and appearance of piling up and multilayer cellular organization; (vi) by the development of rapidly growing tumors in nude mice. IL2 induces in MELP cells a tumor progression process that could mimic the metastatic evolution observed in vivo during biotherapy. Therefore, MELP phenotype may help to define a subset of patients in which IL2 therapy may trigger unfavourable evolution.

Identification of two nuclear N-acetylglucosamine-binding proteins.

Using neoglycoproteins, lectins that recognize different sugars, including N-acetylglucosamine residues, were previously detected in animal cell nuclei. We report herein the isolation of two N-acetylglucosamine-binding proteins from HL60 cell nuclei: i) a 22 kDa polypeptide (CBP22) with an isoelectric point of 4.5 was isolated for the first time and ii) a 70 kDa polypeptide with an isoelectric point of 7.8. This latter protein corresponds to the glucose-binding protein (CBP70) previously isolated, based on the following similarities: i) they have the same molecular mass, ii) they have the same isoelectric point, iii) they are recognized by antibodies raised against CBP70, and iv) both are lectins from the C group of Drickamer's classification. CBP70 appeared to recognize glucose and N-acetylglucosamine; however, its affinity for N-acetylglucosamine was found to be twice that for glucose. The presence in the nucleus of two nuclear N-acetylglucosamine-binding proteins and their potential ligands, such as O-N-acetylglucosamine glycoproteins, strongly argues for possible intranuclear glycoprotein-lectin interactions.

IL-2 production by myofibroblasts from post-radiation fibrosis in breast cancer patients.

The origin of cell activation in post-radiation fibrosis and its chronic extension are still poorly understood. Since local IL-2 cancer treatment sometimes triggers intraperitoneal fibrosis we have analyzed three myofibroblastic cell strains from post-radiation skin fibrosis (FPR7, FPR10 and FPR15) for their interactions with IL-2. In these cells we have observed the surface expression of the two chains of the IL-2R (IL-2R alpha beta), the presence of the 0.9 kb transcript specific for the IL-2 gene and, by flow cytometry with anti-IL-2 mAbs, the presence of IL-2 immunoreactive material inside the cells up to 8 days after subculture. The FPR cell lines secreted IL-2, as determined by ELISA. The secreted IL-2 is biologically active since it sustains the proliferation of the IL-2-dependent murine lymphoid cell line CTLL2 and preincubation with anti-IL-2 blocking mAbs completely abolishes this activity. Overnight incubation of FPR cells with polyclonal anti-IL-2 antibodies leads to a decreased expression of the membrane adhesion molecules ICAM-1 and CD44, suggesting the existence of an autocrine/paracrine loop involved in the surface expression of these antigens. By contrast, in normal adult skin fibroblasts we did not detect IL-2 gene activation. In vivo, IL-2 secretion by post-radiation fibrosis fibroblasts and the subsequent up-regulation of ICAM-1 and CD44 may represent key events during the process that leads to radiation fibrosis.

How interleukin-2 can affect human fibroblasts behaviour.

The alpha and beta chains of the Interleukin 2 receptor (IL2R alpha and IL2R beta) were detected at the surface of cultured fibroblastic cells by flow cytometry, using monoclonal antibodies (mAbs) directed against the IL2R alpha and the IL2R beta. These cells bound FITC-IL2 and this binding was inhibited by an excess of cold ligand and by mAbs recognizing the IL2 binding sites of the alpha and beta chains. Internalisation studies show that the fibroblastic IL2R/IL2 complex is internalized at 37 degrees C. By Northern Blot analysis we detected the presence of specific transcripts for the IL2R alpha and IL2R beta genes. Finally, the addition of exogenous IL2 specifically modified the surface expression of different antigens involved in the process of immunosurveillance. Indeed, IL2, at concentrations affecting the high affinity IL2R, caused the down regulation of ICAM-1 protein. IL2 also decreased the surface expression of the class I and class II HLA. By contrast, the use of IL2 concentrations which saturate the intermediate affinity IL2R beta caused the up regulation of the surface expression of the ICAM-1 protein. ICAM-1 is the natural ligand for the LFA-1 integrin expressed at the surface of lymphoid cells. ICAM-1/LFA-1 interactions favour homotypic and heterotypic cell-cell adhesion. Since human fibroblasts express an LFA-1 like molecule, we propose that in these cells IL2 can modify homotypic and heterotypic interactions acting on the surface expression of ICAM-1 protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that lactose binding to CBP35 disrupts its interaction with CBP70 in isolated HL60 cell nuclei.

We have previously reported that two carbohydrate-binding proteins (CBP35 and CBP70) can, under appropriate conditions of affinity chromatography, be isolated from HL60 cell nuclear extracts as a complex. Moreover, we have demonstrated that, during affinity chromatography, the CBP70-CBP35 association can be modified by the binding of lactose to CBP35. To determine whether the CBP70-CBP35 association could be disrupted in the nucleus upon lactose binding to CBP35, the behaviors of CBP70 and CBP35 were analyzed in membrane-depleted nuclei of HL60 cells, incubated with or without lactose. This study was performed by using an antiserum that cross-reacts with CBP35 and CBP70, an antiserum that was specifically raised against CBP70, and a monoclonal antibody (Mac 2) reactive against CBP35. Taken together, the results of indirect immunofluorescent staining, immunoblotting experiments, and quantitative flow-cytofluorometric analysis show that (i) CBP70 and CBP35 are present and colocalized in the nuclei incubated without lactose, (ii) all the CBP70 molecules left the nuclei incubated in the presence of lactose, while CBP35 molecules, probably bound to RNA, remained inside the nuclei, and (iii) glucose failed to have the same effect as lactose. These results strongly suggest that, in membrane-depleted nuclei, CBP35 and CBP70 interactions can be altered by a conformational change of CBP35 induced by the binding of lactose to its carbohydrate-recognition domain.

Human melanoma cells express a functional interleukin-2 receptor.

Flow cytometric analysis reveals that 5 human melanoma cell lines (M14, IGR3, ME1477, JUSO, GLL19) express both alpha and beta chain of the interleukin 2 receptor (IL-2R alpha and IL-2R beta). These chains are able to specifically bind IL-2 and to form high-affinity heterodimers (IL-2R alpha beta). Analysis of poly A+ RNAs by Northern blot reveals the presence of typical transcripts for both the IL-2R alpha gene (3.6 kb) and the IL-2R beta gene (4 kb). Reverse transcription/polymerase chain reaction analysis allowed transcripts for the IL2R gamma (p64) gene to be detected in 3 of these melanoma cell lines (M14, IGR3, ME 1477). Incubation with human recombinant IL-2 modifies in IL-2R alpha+beta+gamma+ (M14) the expression of several surface molecules: down-regulation of ICAM-1, HLA class I and HLA-DR and up-regulation of CD44. IL-2 is also active on IL-2 alpha+beta+gamma- cell lines since it decreases ICAM-1 and HLA class-II expression at the surface of JUSO cells. Down-regulation of ICAM-1, whose expression in melanoma cells is a marker of tumor progression, is detectable within 3 hr in M14 cells and is maximal after 48 hr incubation, at IL-2 concentrations corresponding to the high-affinity heterodimers. This feature is specific since it is partially inhibited by MAbs directed against the IL-2 binding site of the IL-2R alpha (MAR93, 10T14) and IL-2R beta (MiK beta 1, TU27) chains. Our data support the notion of a direct effect of IL-2 on human melanoma cells. Modulation of the expression of surface molecules which is important for the interaction with immunocompetent cells or for tumor progression, could have a role to play during in vivo IL-2 treatment of human melanomas.

Interleukin 2 production and interleukin 2 receptor expression by human immature leukemic T cells.

In order to determine the role of interleukin 2 (IL2) on the proliferation of leukemic cells from patients with T-cell acute lymphoblastic leukemia (T-ALL) we studied the production of IL2, the function of IL2 receptors (IL2R) expressed on T-ALL cells and their IL-2-dependent in vitro proliferation. Leukemic cells from six out of 17 T-ALL/T-cell non-Hodgkin's lymphoma patients with a prothymocyte (stage I) or a mature thymocyte (stage III), but not with a common thymocyte (stage II) phenotype, could proliferate, in a dose-dependent manner, in response to recombinant IL2 (rIL2) and anti-Tac and TU27 moAbs as well as polyclonal anti-IL2 purified immunoglobulin G could inhibit this IL2-induced cell proliferation. Both crude or/and Amicon-concentrated media conditioned by T-ALL cells from 10 out of 13 tested patients contained IL2 activity as assessed by colorimetric biological and immunoenzymatic assays; this biologic activity was due to a 14.5 kDa molecule adsorbed by anti-IL2 antibodies in an immunoaffinity assay. Although less than 10% of fresh leukemic cells expressed IL2R alpha (Tac) chain, a 24 h cell incubation in the absence of any mitogenic stimulation induced IL2R alpha chain expression in five out of 13 patients (11-83% Tac+ cells). Morever, Tac mRNA transcripts could be detected in fresh cells from all 10 patients tested. Staining of fresh leukemic cells with an IL-2R beta-chain-specific monoclonal antibody and flow cytometry analysis revealed that 4-13% of leukemic cells were positive. Binding experiments with 125I-rIL2 showed a small number of high affinity IL2R on fresh cells from three T-ALL patients (114-200 sites/cell, dissociation constant = 101-181 pm). Finally, antibodies against IL2R alpha, IL2R beta and IL2 could inhibit both IL2 driven and spontaneous cell proliferation of most patients' T-ALL cells, although in some cases an heterogenous pattern of inhibition was observed. Taken together, these findings strongly suggest that an IL2/IL2R-dependent mechanism could be involved in the proliferation of some T-ALL cells.

Expression of the interleukin-2 receptor on human fibroblasts and its biological significance.

In this study, we have investigated the expression of the alpha and beta chains of the IL-2 receptor (IL-2R alpha, IL-2R beta) both at the membrane and at transcriptional levels during the lifespan of human embryonic fibroblasts. Here we show that the mAbs IOT14 and MIK beta 1 directed against the IL-2 binding sites of the IL-2R alpha and IL-2R beta respectively, stain human embryonic fibroblasts early in their life span. Data from [125I]rIL2 cross-linking experiments show the simultaneous expression of two IL-2 binding peptides of 70 and 55 kDa respectively on embryonic young fibroblasts as on lymphoid activated cells. The p55 and the p70 IL-2 binding peptides are shown to be specific for the IL-2R alpha and to the IL-2R beta by the finding that these bands are abolished by excess amounts of cold IL-2 and mAbs directed against the IL-2 binding sites of the alpha and beta chains. Scatchard analysis after [125I]IL-2 labelling shows the presence of both high affinity (150 sites with a Kd of 147 pM) and low affinity (1100 sites with a Kd of 4 nM) IL-2 binding sites. Northern blot and dot blot analysis show the presence of specific transcripts for the IL-2R alpha and IL-2R beta genes in early passaged fibroblasts. By contrast, in senescent cultures, only the IL-2R beta transcript were detected. Finally, IL-2 at low concentrations (36 pM) down modulates the level of the intercellular adhesion molecule ICAM-1 in young but not in senescent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Human immunodeficiency virus-infected adherent cell-derived inhibitory factor (p29) inhibits normal T cell proliferation through decreased expression of high affinity interleukin-2 receptors and production of interleukin-2.

Adherent cells from HIV-infected subjects as well as in vitro HIV-infected normal adherent cells produce spontaneously a 29-kD (p29) factor that inhibits mitogen-induced proliferation of normal T cells. p29 mediates a partial dose-dependent inhibition of total protein synthesis in both nonstimulated and PHA-activated cells that is associated with impaired PHA-induced expression of IL-2 receptor (IL-2R)alpha chain, HLA-class II molecules, and production of IL-2 by these cells; conversely, p29 does not modify the expression of IL-2R beta chain, 4F2, CD9, or transferrin receptor, or the production of IL-1 and TNF alpha by the cells. 1 h preincubation of the cells with p29 is sufficient to detect its biologic activity and added rIL-2 abrogates p29-induced inhibition of IL-2R alpha chain expression; however, p29 does not display any biologic effect on already expressed IL-2R alpha chains. The impaired expression of IL-2R alpha chain mediated by p29 is not due to a decreased accumulation of the corresponding mRNA transcripts, but is associated with a two-fold increase of intracellular cAMP. Binding experiments with 125I-rIL-2 reveals that p29 induces a 50% decrease in the number of both high and low affinity IL-2R per cell. p29 also inhibits alloantigen-induced proliferation of PBMC, whereas it does not modify IL-2-dependent proliferation of 48-h PHA-blasts that already express high affinity IL-2R. These findings indicate that p29 mediates its biologic activity during early stages of T cell activation affecting the expression of high affinity IL-2R and production of IL-2, through a nontranscriptional mechanism involving an increase of intracellular cAMP.

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.

Abnormal expression of IL-2R beta (p70)-binding polypeptide on HIV-infected patients' cells.

The constitutively expressed IL-2R beta (p70) chain participates in the formation of high-affinity (h.a.) IL-2R and transduces IL-2-mediated signals to normal cells. Its expression on HIV-infected patients' PBMC was evaluated and was found to be decreased in both nonstimulated CD4+ and CD8+ cells. Mitogenic cell stimulation induced IL-2R beta chain expression on both CD4+ and CD8+ cells from asymptomatic and persistent generalized lymphadenopathy patients but not on those from AIDS patients. Comparison of mean fluorescence intensity of IL-2R beta positively stained cells from normals and patients did not reveal significant differences. Cross-linking of 125I-rIL-2 on patients' PHA-blasts revealed decreased signals corresponding to both IL-2-binding chains and, in some cases, neither IL-2R alpha nor IL-2R beta chains could be detected. Decreased expression of IL-2R beta polypeptide was associated with impaired accumulation of the corresponding mRNA transcripts. Binding experiments with 125I-rIL-2 under h.a. conditions showed a decreased number of IL-2-binding sites/cell which was more pronounced in patients with AIDS than in patients with less advanced clinical stages. In vitro HIV infection of normal PHA-blasts also resulted in a decreased number of h.a. IL-2R/cell. High concentrations of rIL-2 in the absence of other mitogenic stimuli induced a decreased cell proliferation and expression of the IL-2R alpha chain and did not enhance the constitutive NK and the generation of LAK activity in several patients, suggesting an impaired IL-2R beta chain-mediated cell activation.

Production of a tac inhibitory activity by adherent cells of HIV-infected subjects at different clinical stages.

Studying the mechanisms of the impaired T-cell colony growth from HIV-infected subjects, we have demonstrated that depletion of adherent cells from some patients' peripheral blood mononuclear cells enhanced the plating efficiency of T colony-forming cells. We report here that media conditioned by patients' cells but not normal adherent cells could inhibit the expression of the interleukin-2 receptor alpha (IL-2R alpha) chain but not the IL-2R beta chain in a dose-dependent manner. This inhibitory activity was produced by macrophage-monocyte cells since they displayed the My9+ My7+ OKM1+ phenotype and since adherent cell depletion by complement-mediated cytotoxicity with the My9 monoclonal antibody completely abrogated production of the inhibitory activity. A similar inhibitory activity, which could not be recognized by anti-p24 or anti-gp 120 monoclonal antibody or purified human anti-HIV immun]gobulin G in Western blot assays, could also be detected in culture supernatants of in vitro HIV-infected normal adherent and U937 leukemic cells. Production of IL-2R alpha chain inhibitory activity was associated with a decreased mitogen-induced expression of IL-2R alpha chain on patients' PBMC in 8 of 10 studied cases. Its production could be detected in 82, 58, and 91% of media conditioned by adherent cells from stage II, III, and IV patients, respectively. The amount of IL-2R alpha chain inhibitor released by patients' adherent cells increased during the deterioration of the patients' clinical status, and zidovudine treatment completely abrogated its production in all patients. These findings strongly suggest that production of IL-2R alpha chain inhibitory activity is involved in the pathophysiology of the impaired T-cell responses during HIV infection and could be of clinical relevance during the patients' follow-up.

Intracytoplasmic detection of the Tac (p55) chain of interleukin-2 receptor in pre-B leukemic cells associated with a constitutive expression of Tac mRNA.

The pre-B Reh-6 leukemic cells do not express membrane interleukin-2 (IL-2)-R alpha (Tac or p55) chain; however, their incubation with PMA induces the expression of both high and low affinity IL-2-R. Northern analysis of nonstimulated Reh-6 as well as leukemic cells from patients with acute B cell precursor lymphoblastic leukemia displayed a constitutive expression of p55 mRNA transcripts, which could be enhanced by PMA. Both actinomycin-D and cycloheximide could abrogate PMA-induced p55 membrane expression, suggesting the need for de novo mRNA and protein synthesis. The increased PMA-induced p55 mRNA accumulation was an early event (4 hr) and could be enhanced, specifically, by rIL-2 because anti-Tac moAb inhibited this rIL-2-mediated effect. Immunofluorescence and cross-linking studies using 125I-rIL-2 failed to reveal membrane-associated p55 protein on both Reh-6 and patients' leukemic cells. Conversely, immunogold staining and electron microscopy studies, revealed p55 immunoreactive molecules in the cytoplasm but not in the nucleus of all Reh-6 cells. Using a sensitive EIA, p55 molecules could be detected in cell lysates but not the culture supernatants of Reh-6 cells, suggesting that p55 was not released into the culture medium. These results indicate that constitutively expressed p55 mRNA on pre-B leukemic cells is translated into a relative immunoreactive protein that cannot be expressed on cell surface for unknown, yet, reasons.

Interleukin 2 receptors.

Interleukin 2 receptors (IL-2-R) are composed of (at least) two chains: the alpha chain (or p55 or Tac) and the beta chain (or p70 or p75). The association of both chains constitutes high affinity IL2-R complexes (Kd congruent to 10-100 pM), whereas the alpha-chain or the beta chain alone bind IL-2 with low (Kd congruent to 5-50 nM) and intermediate (Kd congruent to 1 nM) affinities, respectively. The beta chain (but not the alpha chain) mediates IL-2 internalization and signal transduction (proliferation or differentiation) to the cell, thus only high and intermediate affinity IL-2-R are functional. High affinity IL-2-R are expressed on activated normal and leukemic T and B lymphoid cells. Low affinity IL-2-R (Tac alone) are seen on stimulated T cells, B cells, and monocytes, as well as on various leukemic cells. The p70 chain alone is constitutively expressed by subsets of normal resting T and B cells, LGL and NK cells and we have recently demonstrated its presence on leukemic cells from various hemopoietic lineages. The finding of IL-2-R on non-lymphoid cells may disclose new functions for IL-2.

Phorbol myristate acetate induces both high affinity and low affinity interleukin 2-receptors on a pre-B leukemic cell line.

The human pre-B leukemic cell line Reh6 does not constitutively express the Tac molecule (p55), which is one of the two polypeptide chains of the interleukin 2-receptor (IL2-R). Cell incubation with Phorbol Myristate Acetate (PMA) induces expression of the Tac molecule in a dose and time-dependent manner. Binding experiments with radiolabelled recombinant IL2 (rIL2) revealed both high and low affinity IL2-R (225 +/- 105 sites/cell with a Kd of 130 +/- 51 pM and 24060 sites/cell with a Kd of 17.3 nM respectively), whereas unstimulated Reh6 cells only expressed intermediate affinity Reh6 cells revealed the presence of two polypeptide chains of mol. wts 55,000 (Tac molecule) and 70,000, as in normal activated T cells, while the 70,000 mol. wt chain alone was observed in unstimulated Reh6 cells. IL2-R-bearing Reh6 cells could absorb rIL2 in a dose-dependent manner and this absorption was inhibited by a monoclonal antibody against the Tac molecule (anti-Tac). Moreover, partial internalization of IL2 bound under high affinity conditions occurred at 37 degrees C. IL2-R expressed on PMA-induced cells were functional since rIL2 specifically enhanced the proliferation in vitro of PMA-treated cells in semi-solid but not liquid cultures. These findings thus demonstrate an IL2-dependent mechanism of proliferation in vitro of pre-B leukemic cells induced by PMA, which can express high affinity, functional IL2-R.

Presence of a p70 IL-2-binding peptide on leukemic cells from various hemopoietic lineages.

Recent studies have shown that IL-2R are composed of at least two polypeptide chains of 55 kDa (Tac or alpha-chain) and 70 to 75 kDa (p70 or beta-chain). The association of both chains forms high affinity IL-2R, whereas each chain alone binds IL-2 with a low (alpha-chain) or intermediate (beta-chain) affinity. So far, the p70 peptide has been found, in the absence of the Tac peptide, on the surface of lymphoid cells of T, B, or NK lineage. In this study, we investigated whether leukemic cells of various hemopoietic lineages expressed the p70 IL-2-binding protein. We found that both fresh leukemic cells obtained from patients, and cells from established leukemic lines of T cells, B cell, and myeloid origin constitutively expressed a p70 IL-2-binding protein on their surface, as detected by affinity cross-linking of radioiodinated IL-2. IL-2 binding and cross-linking to these cells was completely inhibited in the presence of an excess unlabeled rIL-2, but not with an anti-Tac mAb. Binding experiments on pre-B and myeloid cell lines revealed intermediate affinity IL-2R, whereas both high and intermediate affinity IL-2R were detected in T leukemic cells. The intermediate affinity binding of 125I-rIL-2 to the leukemic cell lines MOLT4 and Reh6 was inhibited by the TU27 mAb, which recognized the p75 chain of IL-2R. Moreover, the TU27 mAb could stain the K562, KM3, and MOLT4 (weakly) cell lines by indirect immunofluorescence. A high dose of rIL-2 (400 U/ml) enhanced the proliferation of cells from one out of three patients with acute myeloblastic leukemia, but it did not induce differentiation of the cells in any of three cases. Thus the finding of p70 IL-2-binding molecules on immature lymphoid and nonlymphoid hemopoietic cells should disclose new biologic functions for IL-2.