receptors in CD34+ hematopoietic stem cells - Nature

Leukemia (2002) 16, 1135–1142  2002 Nature Publishing Group All rights reserved 0887-6924/02 $25.00 www.nature.com/leu

Direct signal transduction via functional interferon-␣␤ receptors in CD34+ hematopoietic stem cells J Giron-Michel3, D Weill1, G Bailly1, S Legras2, PC Nardeux1, B Azzarone3, MG Tovey1 and P Eid1 1

Laboratoire d’Oncologie Virale, UPR 9045, CNRS, Villejuif, France; 2Laboratoire d’He´matologie, Hoˆpital Paul Brousse, Villejuif, France; Inserm U506, Hoˆpital Paul Brousse, Villejuif, France

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Affinity purified, freshly isolated CD34+ progenitors were shown to express low levels of type I interferon (IFN) receptors (740 ± 60 binding sites/cell, Kd 0.7 ± 0.04 nM) determined by Scatchard’s analysis using a radiolabelled, neutralizing, monoclonal antibody directed against the IFNAR1 chain of the human type I IFN receptor. Treatment of freshly isolated (day 0), highly purified (⬎95% pure) CD34+ cells with recombinant IFN-␣ resulted in rapid tyrosine phosphorylation and activation of STAT1, Tyk2 and JAK1 as shown by Western immunoblotting. Similarly, IFN treatment was shown by confocal microscopy to result in rapid nuclear localization of the transcription factors IRF1 and STAT2, demonstrating the presence of functional IFN receptors on freshly isolated (day 0) CD34+ cells. The number of specific type I IFN receptor binding sites expressed on hematopoietic progenitor cells increased to some 1440 ± 40 per cell after 11 days of cultivation of CD34+ cells in vitro suggesting that receptor expression increases with cell differentiation. IFN-mediated signal transduction and the inhibitory effect of IFN-␣ on 7 or 14 days CFU-GM and BFUE colony formation was abrogated in the presence of the anti-IFNAR1 mAb, indicating that IFN-␣ acts directly on the proliferation of human hematopoietic progenitor cells via receptor activated signal transduction without excluding the induction of other cytokines or growth factors by residual accessory cells. Leukemia (2002) 16, 1135–1142. DOI: 10.1038/sj/leu/2402492 Keywords: interferon receptors; CD34+ cells; anti-IFNAR1

Introduction Lymphohematopoietic CD34+ stem cells and progenitors are able to proliferate and differentiate into multiple lymphoid lineages, and understanding the mechanisms which control this process remains one of the central questions of modern day biology. Biochemical studies on primitive primary hemopoietic cells have been hindered by the difficulty in obtaining sufficient quantities of highly purified CD34+ cells. Furthermore, analysis of mRNA expression levels using techniques such as the polymerase chain reaction (PCR) does not provide information on protein functionality. Thus, we have adopted an alternative approach based on the use of direct binding studies (Scatchard analysis), which provides information on the level of translation of a receptor product and the level of its expression at the cell surface, coupled with an analysis of receptor signaling leading to the transcriptional activation of effector genes. The proliferation and differentiation of hematopoietic stem cells is regulated by a complex network of cytokines and growth factors,1–3 including the type I interferons (IFN-␣, IFN␤, IFN-␻, and IFN-␶) which are multifunctional cytokines that play an important role in the innate immune response and in

Correspondence: P Eid, Laboratoire d’Oncologie Virale, CNRS-UPR 9045, 7, rue Guy Moquet-94801 Villejuif, France; Fax: 33-1 49 58 34 44 Received 10 September 2001; accepted 29 January 2002

host defense against virus infection and neoplastic disease.4–6 Recombinant IFN-␣ has found wide application in clinical medicine for the treatment of chronic viral hepatitis, solid tumors, and a variety of hematological malignancies including hairy cell leukemia and chronic myelogenous leukemia (CML).7–9 The mechanism(s) of the anti-tumor effects of IFN␣ remain poorly understood, although it is well established that IFN-␣ exerts both direct anti-proliferative and pro-apoptotic effects, and indirect immune-mediated effects on tumor cells 10.10,11 In the case of hematological malignancies such as CML, IFN-␣ has been reported to inhibit the proliferation of both normal and leukemic progenitor cells.12–18 It remains unclear, however, whether IFN-␣ exerts a direct effect on the proliferation of hematopoietic stem cells, and their immediate progeny committed progenitor cells, or an indirect effect on stromal cells in the bone marrow, and the synthesis of growth factors and other cytokines required for stem cell proliferation.19–22 Interferons exert their characteristic biological effects by binding to high-affinity receptors on the cell surface resulting in the rapid tyrosine phosphorylation and activation of signal transducers and activators of transcription (STATs) which regulate the transcription of interferon-responsive genes. Thus, binding of IFN-␣ to its cell surface receptor activates the receptor-associated Janus tyrosine kinases Tyk2 and JAK123,24 leading to phosphorylation of the two transmembrane polypeptides, IFNAR1 and IFNAR2, which constitute the type I IFN receptor. Both chains of the receptor are members of the cytokine receptor super family and are both required for highaffinity ligand binding and the establishment of biological activity. Tyk2 and JAK1 then phosphorylate STAT1 and STAT2, leading to the formation of a heterodimer which is translocated to the nucleus where it associates with a p48 DNA binding protein, a member of the IRF family of transcription factors (IRF9),25–27 leading to the activation of the transcription of those genes which contain an IFN-sensitive response element (ISRE) within their promoter region. It is the activation of a specific set of IFN-responsive genes within a given cell type which gives rise to the establishment of the characteristic biological activities of the interferons. We show herein, using Scatchard analysis and a highly specific radiolabeled monoclonal antibody directed against the IFNAR1 chain of the type I IFN receptor, which inhibits binding to both receptor chains and which neutralizes the biological activity of all the type I interferons,28,29 that freshly isolated highly purified CD34+ cells (⬎95%) from human umbilical cord blood, possess low levels of functional IFN receptors, the number of which increase during differentiation of myeloid progenitors in culture. These results suggest that IFN-␣ can exert direct effects on the proliferation of early myeloid progenitors.

Type I interferon receptors in CD34+ cells J Giron-Michel et al

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Materials and methods

Isolation of human CD34+ stem cells by affinity chromatography or by magnetic cell sorting (MiniMacs) Human umbilical cord blood (CB) was collected from fullterm newborns following informed consent of the mother at the Hospital St Vincent de Paul (Paris, France). Cord blood mononuclear cells (CB-MNC) were isolated by density (1.077 g/ml) gradient centrifugation (2000 rpm, 20 min) using Ficoll– Hypaque (Seromed, Berlin, Germany). After centrifugation CB-MNC present at the interface were harvested and washed twice with phosphate-buffered saline (PBS). CD34+ cells were separated using the Ceprate LC CD34+ kit (CellPro, Bothell, WA, USA) to yield CD34+ cells with a purity ranging from 65 to 85%. For magnetic cell sorting, the procedure was performed according to the manufacturer’s instructions. Blocking reagent and the antibody QBEND/10 from the direct CD34+ progenitor cell isolation kit (MACS; Miltenyi Biotec, Paris, France) were added simultaneously to the cell suspension, mixed well and incubated for 30 min at 4°C on a rotating wheel. After incubation, cells were washed with PBE (phosphate-buffered saline (PBS), 0.5% bovine serum albumin (BSA), 2 mM EDTA). The cells were re-suspended in 1 ml PBE and applied to the pre-filled column which was placed in a magnetic field. Cells were centrifuged (1500 r.p.m., 10 min, 4°C) and re-suspended with PB buffer (PBS, 0.5% BSA). Only CD34+ progenitor cells were specifically retained by the column matrix and subsequently eluted by removing the column from the magnetic field and flushing with PB. Using this isolation procedure, the purity of CD34+ cells was routinely greater than 95%. The purity of CD34+ cells was determined by incubating cells for 15 minutes at 4°C with PerCP-labeled anti-CD34+ antibody (HPCA-2, clone 8G12, Becton Dickinson, San Jose, CA, USA). Background staining was determined using an irrelevant control PE-IgG1 (Coultronics, Margency, France), or Per-IgG1 (Becton Dickinson) for MACS. Phenotypic analysis was performed using FACscan analyser (Becton Dickinson).

against purified human leukocyte IFN-␣ has been described previously.31 The preparation used in this study had a neutralizing titer of 2 × 105 against 8 IU of recombinant IFN-␣2a. Normal sheep serum was used as a control.

Colony-forming cell assay CD34+ cells were seeded in StemGem medium (hatzfeld얀vjf.cnrs.fr; CNRS, Villejuif, France). The medium was composed of 0.9% methylcellulose supplemented with 30% fetal calf serum (Dominique Deutsher, Brumath, France), 10 mg/ml transferrin, 1 mg/ml detoxified serum albumin (Sigma Chemical, St Louis, MO, USA), 2 mM glutamine (Life Technologies, Cergy-Pontoise, France), 5 × 10−5 M 2-mercaptoethanol (Merck, Darmstadt, Germany), 1.7 units/ml of recombinant human IL3, 10 units/ml of recombinant human IL-6, 3 ng/ml of recombinant human SCF (all obtained from R&D Systems), and 18.3 units/ml of recombinant human GMCSF (Genetic Institute, Cambridge, MA, USA), 2 units/ml of recombinant human EPO (Boehringer, Mannheim, Germany), Approximately 400 enriched CD34+ cells were re-suspended in 0.4 ml Iscove’s modified Dulbecco’s medium (IMDM) supplemented with 10% FCS and mixed with 2 ml of StemGem methylcellulose and 1 ml of this mixture was plated in 35mm Petri dishes. Cultures were incubated for 21 days in 5% CO2 in air and 100% humidity. Each culture was plated in triplicate. The number of colonies containing more than 50 cells was evaluated on days 7 and 14 with an inverted microscope. CFU-GM and BFU-E colonies were determined after 14 days of incubation.

Liquid suspension culture of day 11 progenitor cells Purified CD34+ cells were seeded at a concentration of 103/ml in StemGem liquid medium and incubated at 37°C with 5% CO2 in air for 11 days. The composition of the StemGem liquid medium was the same as for semi-solid medium except that methylcellulose was omitted. Cells were cultured in sixwell flat-bottomed tissue culture plates (Falcon, Becton Dickinson).

Interferons and antibodies Recombinant human IFN-␣2a with a specific activity of approximately 2 × 108 IU/mg was obtained from Hoffman LaRoche (Meylan, France). Recombinant human IFN-␥ with a specific activity of approximately 107 IU/mg was obtained from R&D Systems (Mineapolis, MN, USA). The 64G12 mAb was prepared by immunizing mice with a recombinant protein corresponding to the extra-cellular domain of the IFNAR1 chain of the human IFN-␣/␤ receptor expressed in mammalian cells as described previously.30 The 64G12 mAb is a mouse IgG1 which inhibits both the binding and biological activity of all the human type I interferons tested.28 An irrelevant mouse IgG1 was used as a negative control. Anti-phospho-STAT1 and anti-phospho-Tyk2, were obtained from New England Biolabs (Beverly, MA, USA). AntiSTAT1 (clone IF9A7), anti-STAT2 (S21220), were obtained from Pharmingen-BD (San Diego, CA, USA), and BD Transduction Laboratories (San Jose, CA, USA), respectively. Antiphospho-JAK1 (44–422) and anti-IRF1 (C-20) were obtained from Biosource International (Camarillo, CA, USA), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. A sheep polyclonal anti-human IFN-␣ antibody raised Leukemia

Radiolabeling of the monoclonal antibody Affinity purified 64G12 mAb was radiolabeled using a modified chloramine-T method as described previously 28.28,29 Briefly, 30 ␮g of the antibody in 0.2 ml PBS was incubated with 10 ␮l of chloramine-T (Sigma), 0.5 mg/ml diluted in PBS and 1 mCi (10 ␮l) of 125I-Na. After 20 s, the reaction was quenched by addition of 30 ␮l of 1 mg/ml filtered sodium metabisulfite diluted in PBS containing 10 mM tyrosine (Sigma). Unreacted iodide and other low molecular weight reagents were removed by filtration on G-25 Sephadex PD10 columns (Pharmacia Biotech, Saclay, France) equilibrated with PBS, and 0.5% BSA. For equilibrium saturation binding studies, 0.5 or 1.0 × 106 cells were incubated in a final volume of 0.5 ml with various concentrations of 125I-64G12 mAb. After incubation for 2 h at 4°C, cells were washed extensively and bound radioactivity was determined using a gamma counter (LKB 1282). Nonspecific binding was determined by incubation of cells with the 125I-64G12 mAb in the presence of a 200-fold excess of unlabeled 64G12 mAb, and subtracted from the total radioactivity. Results are expressed as the mean


of duplicate determinations. The number of receptors per cell and their dissociation constants were calculated by Scatchard analysis.

Immunoblotting of phosphorylated proteins The 64G12 mAb (20 ␮g/ml) was added or not added to purified CD34+ cells (0.5 million) in 1 ml DMEM culture medium containing 15% FCS, followed by the addition of 1000 IU/ml IFN-␣2 for 15 min at 37°C. The cell pellet was lysed in 2% SDS sample buffer, boiled for 5 min, centrifuged and proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel (7.5%) electrophoresis (SDS-PAGE) under reducing conditions, transferred to polyvinylidene difluoride filters (PVDF, Boehringer Mannheim) and probed with the appropriate primary-specific antibodies for 1 h at 20°C. Protein–antibody complexes were then revealed following incubation for 45 min with a 1/20 000 dilution of a horse-radish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit secondary antibody (Jackson Immunoresearch, Baltimore, MD, USA). PVDF blots were then developed using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Bucks, UK).

Confocal microscopy Purified CD34+ cells were incubated for 30 min with 100 ␮g/ml of the anti-IFNAR1 mAb (64G12) and then activated with 1000 IU/ml IFN-␣2 for 30 min in FCS-free medium. For confocal microscopy of STAT2 and IRF-1, activated cells were fixed and permeabilized with ORTHOpermeafix (Ortho Diagnostic Systems, Raritan, NJ, USA). Cells were then washed once with PBS and incubated for 30 min with 20 ␮g/ml of a rabbit polyclonal anti-STAT2 (Transduction Laboratories, Lexington, KY, USA) or anti-IRF-1 (Santa Cruz Biotechnology). Staining was revealed using 10 ␮g/ml of Alexa FluorTM 488-labeled goat anti-rabbit IgG antibody (Molecular Probes, Montluçon, France). Nuclear coloration was performed with propidium iodide diluted 1/50, 2 ␮g/ml final concentration, (Sigma). Stained cells were washed with PBS, cytocentrifuged in a cytospin 3 (Shandon, Pittsburg, PA, USA) and analyzed by laser scanning confocal microscopy using a Leica TCS Confocal System (Wetzler, Germany). Results

IFN-␣/␤ receptor expression on freshly isolated CD34+ cells and day 11 progenitor cells The results of preliminary experiments showed that the level of IFN-␣/␤ receptor expression on freshly isolated CD34+ cells from human umbilical cord blood was too low to allow quantitative Scatchard analysis to be carried out on highly purified CD34+ cells due to the large number of cells required using radio-labeled IFN-␣2, the low number of CD34+ cells present in cord blood, and the limited volume of blood available. This was overcome by the use of a 125I-labeled monoclonal antibody specific for the human type I IFN receptor, prepared by immunizing animals with a recombinant protein corresponding to the extra-cellular domain of the IFNAR1 chain of the human type I IFN receptor. The 125I-labeled anti-IFNAR1 monoclonal antibody, 64G12, had a significantly higher spe-

cific radioactivity than 125I-labeled IFN-␣2, and binding of the 125 I-labeled 64G12 mAb has been shown previously to be a sensitive means of detecting IFN-␣/␤ receptor expression on a number of different human cell lines including Daudi, K562, Ly28 and HL60.28 Thus, in the present study, specific binding of the 125I-64G12 mAb was determined either alone or in the presence of a 200-fold excess of the unlabeled mAb, using freshly isolated CD34+ cells (purity 85% ± 5%) from CB. Experiments were carried out at 4°C until binding equilibrium was reached (routinely 2 h). The results of two independent representative experiments are shown in Figure 1a. CD34+ cells were found to express a single class of high affinity IFN-␣ receptors, with saturation binding occurring at 2 nM. Scatchard analysis revealed that freshly isolated CD34+ cells expressed 740 ± 40 high affinity receptors per cell with a Kd of 0.7 ± 0.04 nM (R2 = 0.989), compared with approximately 4300 receptors per cell and a Kd of 0.8 nM (R2 = 0.980) for Daudi cells determined under the same experimental conditions (data not shown). As CD34+ cells developed into more differentiated cells during incubation at 37°C in vitro, specific binding of 125Ilabeled 64G12 mAb increased markedly and the level of receptor expression rose to some 1440 ± 40 receptors per cell after 11 days in culture, with a Kd of 2.1 ± 0.2 nM (R2 = 0.996) as shown in Figure 1b. These results suggest that specific binding sites for type I IFN increase with the differentiation of progenitor cells. It was estimated from the number of IFN-␣/␤ receptors on CB-MNC that specific binding of 125I-64G12 mAb to accessory cells present in samples of purified CD34+ cells did not exceed 10% of total binding (data not shown).

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Effect of 64G12 mAb on the inhibitory action of IFN␣ on CD34+ cell proliferation In agreement with the results of previous studies,13,14,21 cultivation of human CD34+ cells in methylcellulose in the presence of 1000 IU/ml of IFN-␣ was found to result in 63% ± 5% inhibition of the number of 7 day colonies relative to cultures treated with medium alone (Figure 2). We have shown previously that the 64G12 mAb inhibits both the anti-proliferative and antiviral activities of IFN-␣ in a variety of different cell types.28 It was of interest therefore to determine the ability of the 64G12 mAb to neutralize the effect of IFN-␣ on colony formation by CD34+ cells. Thus, in the presence of 1 ␮g/ml of 64G12 mAb, the inhibition of colony formation observed at 7 days in cultures treated with 1000 IU/ml of IFN-␣ was reduced to only 26% ± 5% (Figure 2a). The suppressive effect of the 64G12 mAb was still apparent when the number of CFU-GM and BFU-E was determined after 14 days in culture (Figure 2b). Thus, in the presence of 1000 IU/ml of IFN-␣ alone the number of CFU-GM and BFUE was inhibited by 60% ± 5% and 62% ± 5%, respectively, 14 days after treatment, relative to untreated control cultures (Figure 1b). In contrast, in the presence of the 64G12 mAb, the inhibitory effect of 1000 IU/ml of IFN-␣ on CFU-GM and BFU-E colony formation was reduced to 20% ± 5% and 28% ± 5% inhibition, respectively. Treatment of CD34+ cells with 1000 IU/ml of IFN-␥ was also found to inhibit colony formation at 7 days relative to control cultures. Treatment of CD34+ cells with IFN-␥ in the presence of 64G12 mAb did not affect 7 day colony formation relative to cultures treated with IFN-␥ alone, indicating that the effect of the 64G12 mAb was specific for type I IFN (Figure 2a). Leukemia


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Figure 2 Effect of IFN-␣ on colony formation by CD34+ cells in the absence or presence of the anti-IFN-␣␤ receptor mAb 64G12. Four hundred purified CD34+ cells (purity 80 ± 5%) were incubated in the presence of 1␮g/ml of the 64G12 mAb for 1 h at 37°C then seeded in the presence of IFN-␣ (1000 IU/ml) or IFN-␥ (1000 IU/ml) and plated in methylcellulose medium. The data presented are representative from a total of three independent experiments. Colonies were counted on day 7 (a) and on day 14 to determine the number of CFUGM and BFU-E (b). The data are presented as the number of colonies formed per 103 cells and expressed as mean ± s.d. of triplicate plates.

Anti-IFNAR1 mAb (64G12) inhibits tyrosine phosphorylation of the STAT1, Tyk2 and JAK1 proteins involved in signal transduction following IFN␣ activation Figure 1 Analysis of IFN-␣␤ receptor expression on freshly isolated CD34+ cells and 11 day progenitor cells. Interferon receptor expression was analyzed in freshly isolated CD34+ cells from human cord blood (a), or on progenitor cells after 11 days cultivation of CD34+ cells in liquid medium (b), using the 125I-labeled anti-IFN-␣␤ receptor monoclonal antibody, 64G12, as described in the Materials and methods. The data presented are representative of two independent experiments for both CD34+ and progenitor cells. The purity of CD34+ cells was 85% (±5%). As shown in the insert of panel a, CD34+cells were estimated to possess 740 ± 60 binding sites per cell with a Kd = 0.7 ± 0.04 nM (R2 = 0.989) by extrapolation of the linear portion of the Scatchard plot, and 1440 ± 40 binding sites per cell, Kd = 2.1 ± 0.2 nM (R2 = 0.996) for 11 day progenitor cells as shown in the insert of panel (b). Leukemia

Binding of IFN to its cell surface receptor results in rapid tyrosine phosphorylation and activation of the various components of the signal transduction pathway leading from the cell surface to the nucleus. We have shown previously that the anti-IFNAR1 monoclonal antibody, 64G12, neutralizes the biological activities mediated by IFN-␣, ␤ and ␻28–30 as well as the formation of the ISGF3 complex (IFN-stimulated gene factor 3) in nuclear extracts from human cell lines.32 In order to elucidate the events which follow the binding of IFN-␣ to the cell surface and which culminate in inhibition of the proliferation of CD34+ cells, we analyzed the tyrosine phosphorylation and activation of the various components of the IFN signal transduction pathway following IFN treatment in


the presence or absence of the anti-IFNAR1 monoclonal antibody 64G12. It has been shown previously that IFNAR1, IFNAR2, Tyk2 and JAK1 are all maximally tyrosine phosphorylated within 10 min, and STAT1 and STAT2 within 60 min, of contact of cells with IFN-␣.23–27 The tyrosine phosphorylation of STAT1, Tyk2 and JAK1, was therefore analyzed 15 min after treatment of freshly isolated highly purified CD34+ cells (⬎95% purity) with IFN-␣ in the presence or not of the 64G12 mAb. As shown in Figure 3a, tyrosine phosphorylation is clearly stimulated in extracts of purified CD34+ cells treated with IFN-␣ compared to extracts from excipient treated control cells (Figure 3a). Furthermore, tyrosine phosphorylation of STAT1, Tyk2 and JAK1, is effectively abrogated in extracts of purified CD34+ cells treated with IFN-␣ in the presence of the 64G12 mAb (Figure 3a). As shown in Figure 3b the quantities of protein applied were comparable for all the cell extracts. Similar results were also obtained using extracts of highly purified CD34+ cells isolated from bone marrow (data not shown).

Nuclear localization of IFN-␣ activated STAT2 and IRF1 IFN signal transduction culminates in nuclear translocation of an activated transcription complex containing a number of transcription factors including IRF1 and STAT2. To assess the effects of IFN-␣ on the nuclear translocation of activated transcription factors in CD34+ cells, cells were treated with 1000 IU/ml of IFN-␣ in the presence or absence of the 64G12 mAb, and analyzed by confocal microscopy as described in Materials and methods. The nuclear localization of IRF1 following treatment of CD34+ cells with IFN-␣ is shown in Figure 4. The upper panel shows that the nuclear localization of IRF1 in IFN-␣-treated CD34+ cells is inhibited in the presence of the 64G12 mAb. Similarly, STAT2, which is specific to the transduction of the type I IFNs, and which forms a complex with STAT1 and IRF9 (p48 DNA binding protein) is clearly translocated to the

nucleus following IFN treatment of CD34+ cells as shown in Figure 4 (lower panel). Furthermore, the nuclear localization of STAT2 is again inhibited by treatment of CD34+ cells with the 64G12 mAb which neutralizes the biological activity of type I IFN (Figure 4, lower panel).

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Discussion Interferon-␣ is used extensively for the treatment of myeloproliferative disorders, hairy cell leukemia (B cell leukemia) and chronic myeloid leukemia.22,33–35 Although numerous studies have shown that IFN-␣ inhibits the proliferation of leukemic progenitor cells,15–19 relatively little is known, however, about the effect of IFN-␣ on the proliferation of hematopoietic progenitor cells from normal individuals,36 and in particular whether the proliferation of such cells is influenced directly by IFN, or indirectly by cytokines and growth factors induced by the action of IFN on bone marrow stromal cells. Interferons exert their characteristic biological effects by binding to high-affinity receptors on the cell surface resulting in rapid tyrosine phosphorylation and activation of STAT proteins which regulate the transcription of IFN responsive genes resulting in the characteristic biological activities of the interferons. Thus, IFN sensitivity is determined by receptor expression, and the biological activity of IFN-␣ is proportional to the number of IFN-␣ receptors expressed on the cell surface.37–39 It was of interest, therefore to determine whether freshly isolated CD34+ cells from human cord blood express detectable levels of IFN receptors, and if so whether such receptors are functional. Thus, we have shown for the first time that freshly isolated CD34+ cells from human cord blood express approximately 740 ± 60 IFN-␣/␤ receptors per cell, compared with 4300 IFN-␣/␤ receptors per cell detected on the human B cell line Daudi using either radiolabeled IFN, or the same 125I-64G12 binding assay used in this study (P Eid, unpublished results). The number of IFN-␣/␤ receptors detected on freshly isolated CD34+ cells is significantly greater than the 36 ± 1 GM-CSF receptors per cell,40 and the absence

Figure 3 Inhibition of tyrosine phosphorylation of the components of the signaling pathway in CD34+ cells following IFN-␣ activation in the presence of the 64G12 mAb. Highly purified (⬍95% purity) freshly isolated (day 0) CD34+ cells (5 × 105 per lane) were treated (+) or not (−) with anti-IFNAR1 mAb 64G12 (100 ␮g/ml) followed by the addition of IFN-␣2 (1000 IU/ml) for 15 min at 37°C. The cells were lysed under reducing conditions and protein extracts were separated by SDS/7.5% PAGE, transferred to PVDF membranes and probed with anti-phosphoSTAT1, anti-phospho-Tyk2 and anti-phospho-JAK1 antibodies (panel a). After stripping, the filters were re-probed individually with the antiSTAT1, anti-Tyk2 and anti-JAK1 antibodies in order to demonstrate that similar amounts of proteins were present in all lanes (panel b). Immunoreactive bands were visualized by ECL Western blotting system (Amersham, UK). Leukemia


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Figure 4 Effect of IFN-␣2 on nuclear translocation of STAT2 and IRF1 in CD34+ cells. Freshly isolated (day 0), and highly purified (⬎95%) CD34+ cells were fixed, permeabilized, and stained with anti-STAT2, or anti-IRF-1 (green spots) and propidium iodide (for nucleal staining, red spots). Yellow spots indicate nuclear localization. (a) Resting CD34+ cells; (b) CD34+ cells 30 min after IFN-␣2 activation; (c) CD34+ cells 30 min after treatment with neutralizing anti-IFNARI antibodies followed by 30 min treatment with IFN-␣2.

of detectable levels of the CXC chemokine receptor for the IFN-␥-inducible protein IP 10 and IFN-␥-inducible monokine Mig,41 reported previously for freshly isolated CD34+ cells. Primitive progenitor cells can be induced to proliferate and differentiate in vitro by the supply of different combinations of cytokines.42 Thus, progenitor cells derived from CD34+ cells cultivated for 11 days in liquid suspension culture were found to express some 1440 ± 40 IFN-␣/␤ receptors per cell. Although this is only a two-fold increase in receptor expression relative to the levels detected on freshly isolated CD34+ cells. FACS analysis revealed that only 2.2% of the progenitor cells were found to express the CD34+ marker after 11 days in liquid suspension culture (data not shown). The number of IFN-␣/␤ receptors seemed to increase during the differentiation of CD34+ cells. Although the biological significance of these data can hardly be ascertained, these results are in keeping with previous reports that IFN-␣/␤ receptor expression increases during the course of monocyte and megakaryocyte differentiation in vitro.43,44 Similarly, in vitro maturation of CD34+ progenitors is accompanied by an increase in the number of GM-CSF receptors per cell while the Kd remains unchanged.40 The CXC chemokine receptor for the IFN-␥-inducible proteins IP 10 and Mig also becomes detectable after cultivation of CD34+ cells in vitro in the presence of GM-CSF.41 The number of IFN-␣/␤ receptors detected on 11 day CD34+ progenitors is somewhat lower, however, than the 1910 to 2070 IFN-␥ receptors per cell reported previously for 6 day erythroid colony-forming cells.45 In common with numerous other cytokines binding of IFN␣ to its cell surface receptor is followed by rapid tyrosine phosphorylation and activation of specific STAT proteins by Janus kinases.26,46 Activated STAT proteins then dimerize, and translocate to the nucleus leading to activated transcription of a specific set of IFN-responsive genes.6,25 Thus, treatment of freshly isolated and highly purified CD34+ cells with IFN-␣ was shown to result in rapid tyrosine phosphorylation of STAT1, Tyk2 and JAK1 indicating that freshly isolated (day 0) CD34+ cells possess functional type I IFN receptors. The specificity of this activation was demonstrated by its abrogation Leukemia

when CD34+ cells were treated with IFN-␣ in the presence of a neutralizing antibody specific for the human type I IFN receptor. We have also shown using confocal microscopy that phosphorylation of STAT proteins in freshly isolated (day 0) CD34+ cells is followed by nuclear translocation of a complex of transcription factors containing STAT2 and IRF1, preceding transcriptional activation of IFN responsive genes and the establishment of the biological effects of IFN-␣. These results contrast with those obtained for GM-CSF by Wheadon et al,40 who reported that freshly isolated (day 0) CD34+ cells which express very low numbers of GM-CSF receptors, are hyporesponsive to GM-CSF as determined by activation of downstream signaling. Thus, JAK 2 activation was detected in one of four experiments only and the level of STAT 5 activation obtained was relatively low, and there was no apparent activation of MAP kinase by GM-CSF. Indeed, CD34+ did not exhibit maximal downstream signaling from the GM-CSF receptor until some 3 days in culture. The detection of significant numbers of functional IFN-␣/␤ receptors on freshly isolated (day 0) and highly purified (⬎95% purity) CD34+ cells, and the abrogation of the inhibitory effect of IFN-␣ on the formation of 7 or 14 day CFUGM and BFU-E colonies when CD34+ were cultivated in the presence of an anti-IFN-␣/␤ receptor monoclonal antibody, is in favor of a direct action of IFN-␣ on the proliferation of CD34+ cells via receptor-mediated signal transduction without excluding an indirect effect on the induction of other cytokines or growth factors by residual accessory cells. Acknowledgements This work was supported by grants from Corixa Inc, and the Association Nouvelles Recherches Biome´dicales. References 1 Kurata H, Arai T, Yokota T, Arai K. Differential expression of granulocyte–macrophage colony-stimulating factor and IL-3 receptor


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