Effects of Interferon Gamma on Native Human Acute Myelogenous

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O R IGINA L A R T IC L E

Elisabeth Ersvaer Jrn Skavland Elling UlvestadBjrn Tore Gjertsen ystein Bruserud

Effects of interferon gamma on native human acute myelogenousleukaemia cells

Received: 21 December 2005 / Accepted: 14 March 2006 / Published online: 13 April 2006 Springer-Verlag 2006

Abstract T cell targeting immunotherapy is now con-sidered a possible strategy in acute myelogenous leu-kaemia (AML), and IFNc release may then contributeto the antileukaemic effects. We investigated the effects

of IFNc on native human AML cells. Normal T cellscould be activated to release IFNc in the presence ofAML cells. Furthermore, high levels of CD119 (IFNcreceptor a chain) expression were observed for all 39patients examined. Receptor expression was decreasedafter exposure to exogenous IFNc, and receptor ligationcaused Stat1 phosphorylation but no phosphorylationof the alternative messengers Erk1/2. The effect ofexogenous IFNc on AML blast proliferation wasdependent on the local cytokine network and IFNc (1)inhibited proliferation in the presence of exogenousIL1b, GM-CSF, G-CSF and SCF; (2) had divergenteffects in the presence of IL3 and Flt3 (65 patients

examined); (3) inhibited proliferation in the presence ofendothelial cells but had divergent effects in the presenceof fibroblasts, osteoblasts and normal stromal cells (65patients examined). IFNc increased stress-induced(spontaneous) in vitro apoptosis as well as cytarabine-induced apoptosis only for a subset of patients. Fur-thermore, IFNc decreased the release of proangiogenicCXCL8 and increased the release of antiangiogenicCXCL911. We conclude that IFNc can be released inthe presence of native human AML cells and affectAML cell proliferation, regulation of apoptosis and thebalance between pro- and antiangiogenic chemokinerelease.

Keywords Acute myeloid leukaemia Interferongamma Cytokines Apoptosis Proliferation

Abbreviations AML: Acute myeloid leukaemia

Introduction

Interferon gamma (IFNc) (reviewed in [1, 2]) is a type IIinterferon synthesized by T lymphocytes and naturalkiller cells. It has pleiotropic effects on a wide range oftarget cells. Among the important effects of IFNc are (1)upregulation of MHC class I and induction of MHCclass II protein expression by a variety of immuno-competent as well as nonimmune cells; (2) regulation ofhumoral immune responses; and (3) modulation of therelease of a variety of other immunomodulatory cyto-

kines such as IL12 and tumour necrosis factor (TNF) a.IFNc-induced gene expression is important for severalof these effects, but the molecular mechanisms have notbeen characterized in detail.

Acute myelogenous leukaemia (AML) is an aggres-sive malignancy characterized by accumulation ofimmature myeloid cells [3]. The overall disease-freesurvival is less than 50% even for younger patients whocan receive the most intensive therapy [3]. However, theexperience from allotransplanted patients shows that Tcells can mediate posttransplant anti-AML effects andthereby contribute to improved prognosis. Targeting ofautologous T cells is therefore considered a possible

therapeutic approach [4, 5]. T cell release of antiprolif-erative, proapoptotic or antiangiogenic cytokines is thena possible antileukaemic effector mechanism, and IFNcis of particular interest because it is released at highlevels by human T cells. These high levels have beenobserved both for normals, AML patients receivingintensive chemotherapy and allogeneic stem cell recipi-ents [6, 7]. IFNc has also been tried in the treatment ofAML, and the results from these initial small clinicalstudies suggest that IFNc can mediate antileukaemiceffects in vivo [8, 9].

E. Ersvaer (&) J. Skavland B. T. Gjertsen . BruserudInstitute of Medicine, Section for Hematology,The University of Bergen and Haukeland University Hospital,Bergen, NorwayE-mail: [email protected].: +47-55-975000Fax: +47-55-972950

E. UlvestadDepartment of Microbiology and Immunology,The Gade Institute, Haukeland University Hospital andThe University of Bergen, Bergen, Norway

Cancer Immunol Immunother (2007) 56: 1324DOI 10.1007/s00262-006-0159-1


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The heterodimeric IFNcR (CD119) complex (re-viewed in [2, 10]) consists of the IFNc-binding R1 or achain (CDw119) and the signal-transducing R2 or bchain. Receptor ligation induces phosphorylation of Ja-nus kinases 1 and 2 that mediate activation of signaltransducer and activator of transcription (STAT) mole-cules as well as Stat1-independent pathways [2, 10]. In arecent study we demonstrated that downstream intra-cellular signalling events after ligation of CD119 can evenbe used in prognostic classification of AML patients [11].In vitro effects of IFNc on native human AML cells havealso been investigated previously, but in these studies (1)relatively low numbers of patients have been included; (2)patient selection has not been described; (3) the clinicaland biological characterization of the patients areincomplete; and (4) only the examination of proliferationand membrane molecule expression have been included[9, 1218]. In the present study we therefore characterizedCD119 receptor expression, intracellular events follow-ing CD119 ligation and a wide range of functional effectsof IFNc in native human AML derived from a largegroup of consecutive patients.

Materials and methods

Acute leukaemia patients

The study was approved by the local Ethics Committeeand samples collected after informed consent.

Acute leukaemia cells were derived from 87 consec-utive AML patients (39 females and 48 males; medianage 63 years with range 2684 years) and 9 acute lym-phoblastic leukaemia (ALL) patients [19] with highperipheral blood blast counts. Sixty-nine patients had de

novo AML, the remaining minority had AML relapse (4patients), chronic myeloid leukaemia in blast phase (2patients) or AML secondary to chemotherapy or pri-mary myelodysplasia (4 and 12 patients, respectively).The patients showed the following FAB classification:M0/M1 24 patients, M2/M3 28 patients, M4/M5 34patients and M6 1 patient. Forty-six patients had >20%CD34+ blasts. Cytogenetic analysis was performed for63 patients and the abnormalities classified as describedby Wheatley et al. [20]; 38 patients had normal chro-mosomes whereas 5 patients had low-risk, 10 patientshigh-risk and 10 patients intermediate-risk abnormali-ties. Seventy-eight patients were tested for genetic Flt3

abnormalities [21]; 27 patients had internal tandemduplications and 9 patients D835 mutations.Nine ALL patients were examined (five females and

four males; median age 24 years with range 1874 years). One patient had T ALL, and eight had B ALL(three pro-ALL, two pre-ALL, three common B ALL).

Preparation of native human leukaemia blasts

Leukaemic peripheral blood mononuclear cells (PBMC)were isolated by density gradient separation (Ficoll-

Hypaque; NyCoMed, Oslo, Norway; specific density1.077) from the peripheral blood of patients with >80%of AML blasts among blood leukocytes. Cells werestored frozen in liquid nitrogen [21]. The percentage ofblasts among leukaemic PBMC generally exceeded 95%[21, 22], the contaminating cells being mainly smalllymphocytes.

Nonleukaemic cells

Endothelial cells Human lung microvascular endothe-lial cells were obtained as frozen vials (Cambrex BioScience Walkersville, Walkersville, MD, USA) andstored in liquid nitrogen until used. The cells were de-rived from a healthy 16-year-old white male (productcode CC-2527, lot no. 3F1056). These cells (1) showed adoubling time in culture of approximately 18; (2) stainedpositive for acetylated LDL uptake stain, factor VIIIrelated antigen and PECAM staining; (3) stained nega-tive for alpha actin expression; and (4) were negativewhen tested for mycoplasma, human immunodeficiency

virus 1, hepatitis B and hepatitis C (polymerase chainreactions) (distributors information).

Human fibroblasts The cell line HFL1 (ATCC, Va-nessa, no. CCL-153) was derived from the lungs of a 16to 18-week-old fetus, has a typical adherent growthpattern and a diploid karyotype (distributors informa-tion). Its functional characteristics have been describedpreviously [2325].

Human osteoblasts The cell line Cal72 (DeutscheSammlung von Zellkulturen und Mikroorganismen,Braunschwaig, Germany) has previously been charac-terized in detail [2528]. It has a phenotype close to

normal osteoblasts with an adherent growth pattern anda broad cytokine release profile [21].

Normal human bone marrow stromal cells These cellswere delivered in frozen vials (Cambrex) and stored inliquid nitrogen until use in the coculture assay. The cellswere derived from a healthy 20-year-old white female.Bone marrow mononuclear cells were then derived bygradient separation (specific density 1.077) and culturedin Myelocult growth medium (BioWhitacker) for4 weeks. The stromal cells represent the adherent cellpopulation of the cultured cells and are a heterogeneouspopulation of fibroblasts, reticulum cells, endothelialcells, macrophages and fat cells. The cells showed a

purity of 95% and tested negative for mycoplasma,human immunodeficiency virus 1, hepatitis B and hep-atitis C (polymerase chain reaction) (distributorsinformation).

Flow cytometric analysis of membrane moleculeexpression

Membrane molecule expression was analysed byflow cytometry (FCM) using either PE, FITC or

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APC-conjugated anti-CD119, HLA-DR, CD40, CD80and CD83 monoclonal antibodies (Becton Dickinson,San Jose, USA). The cut-off for positive cells was de-fined as a fluorescence corresponding to 1% positivecells when using an isotypic control antibody.

Analysis of STAT and Erk1/2 phosphorylationin AML cells

The method has been described in detail previously [11].Briefly, AML blasts were incubated for 15 min withor without IFNc in medium. STAT and Erk1/2phosphorylation was analysed by FCM using Alexa647-conjugated anti-phospho-Stat1 (pY701), anti-phospho-Stat5 (pY694), anti-phospho-Erk1/2 (T202/Y204) andAlexa488-conjugated anti-phospho-Stat3 (pY705)monoclonal antibodies (BD, Biosciences, Norway).

In vitro culture of native human AML blasts

Reagents Unless otherwise stated the culture mediumStem Span SFEM (StemSpan; Stem Cell Technologies,Inc., Vancouver, BC, Canada) supplemented with100 lg/ml of gentamycin was used for culture of AMLcells. In cocultures containing nonleukaemic cells themedium was also supplemented with 10% heat-inacti-vated fetal calf serum (BioWhitacker) [25]. Culturescontaining endothelial cells were prepared in the endo-thelial cell EBM-2 medium supplemented with EGM-2MV single quots (Cambrex Biosciences); preliminaryexperiments demonstrated that this medium could alsobe used for in vitro culture of AML blasts. The fol-lowing exogenous cytokines were used at 50 ng/ml:

IFNc, TNFa, IL1RA, IL1b, IL3, IL4, IL6, IL8, IL10,IL13, Flt3L, SCF, GM-CSF and G-CSF (PeproTech ECLtd, London, UK). The cytotoxic drug idarubicin(Zavedos, Pfizer, Inc., NY, USA) was used at 0.1 and0.01 lM, while cytarabine (Pfizer, Inc.) was used at 1and 0.1 lM concentrations.

Proliferation in suspension cultures As described pre-viously [28], 5104 cells/well were cultured in 150 llmedium in flat-bottomed microtiter plates (Costar 3796;Cambridge, MA, USA). Cultures were incubated at37C in a humidified atmosphere of 5% CO2. After6 days 20 ll of 3H-thymidine (37 kBq/well; TRA 310,Amersham International, Amersham, UK) in 0.9%NaCl solution was added to each well and nuclearradioactivity assayed 18 h later by liquid scintillationcounting.

Coculture with nonleukaemic cells Cultures were pre-pared in transwell culture plates (Transwell 3401; Co-star) where cells in the lower large compartment wereseparated from the cells in the upper small chamber ofthe same well by a semipermeable membrane with porediameter of 0.4 lm [29]. Nonleukaemic cells were seededin the lower compartment (104 cells in 1 ml). Leukaemia

blasts (106 cells in 0.5 ml) were added to the upperchamber and the cultures thereafter incubated for7 days. Cultures were always ended before the nonle-ukaemic cells were confluent.

Analysis of cell proliferation Cultures were prepared asdescribed above and incubated for 6 days before 3H-thymidine (280 kBq/well in 150 ll saline) was added andcultures incubated for an additional 18 h. The leukaemic

cells were then resuspended and nuclear radioactivityassayed for 50 ll aliquots by liquid scintillation count-ing. The adherent nonleukaemic cells were washed inisotonic saline before 300 ll/well of trypsinEDTAsolution (Stem Cell Technologies) was added and nu-clear radioactivity assayed in 70 ll aliquots.

T cell activation in the presence of cocultured AMLand bone marrow stromal cells

The cultures were prepared in transwell cultures (Costar3401 Transwell plates). Native human AML cells

(1106

cells) were cultured in the lower chamber in directcontact with 1104 normal bone marrow stromal cells.Normal PBMC derived from a healthy individual(5104 cells) were incubated in the upper chamber andactivated by anti-CD3 antibody (Central Laboratory ofthe Netherlands Red Cross Blood Transfusion Services,Amsterdam, The Netherlands, final dilution 1:500) [30].The cultures were incubated for 4 days before IFNclevels were determined.

Analysis of AML cell viability during in vitro culture

AML blasts were incubated for 24 and 48 h (24 wellCostar 3524 culture plates; 2106 cells in 2 ml StemSpan medium per well) before the percentages of via-ble/apoptotic/necrotic cells were determined by FCManalysis of AnnexinV-FITC and propidium iodide po-sitive cells (Nexins Research, Kattendjike, The Nether-lands) [24].

Analysis of AML cell cytokine secretion

As described previously [28], 1106 AML blasts/mlwere cultured in 24 wells tissue culture plates (Costar

3524; 2 ml cell suspension/well) for 48 h before super-natants were harvested. ELISA analyses were used todetermine the levels of IL1b, IL6, TNFa (Pelikinecompact ELISA kits; Central Laboratory of the Neth-erlands Red Cross Blood Transfusion Services),CXCL811, G-CSF and GM-CSF (Quantikine ELISAkits; R&D Systems) in the supernatants. The minimaldetectable levels were IL1b 0.8 pg/ml, IL6 0.8 pg/ml,TNFa 1.0 pg/ml, CXCL8 3.5 pg/ml, CXCL9 3.8 pg/ml,CXCL10 1.7 pg/ml, CXCL11 13.9 pg/ml, GM-CSF3 pg/ml and G-CSF 8 pg/ml.

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Presentation of the data

Cell proliferation was assayed by 3H-thymidine incor-poration and the mean counts per minute (cpm) oftriplicate determinations were used in all calculations.Detectable 3H-thymidine incorporation was defined as>1,000 cpm. A significant alteration of 3H-thymidineincorporation was defined as a difference correspondingto (1) an absolute value of at least 2,000 cpm and (2) thisabsolute value being >20% of the corresponding con-trol. For statistical analysis the Wilcoxons signed ranktest was performed. Differences were regarded as sig-nificant when P


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showed a uniform distribution with no evidence for dualpopulations. The percentage of positive cells showed asignificant correlation with MFI (Pearson correlation;r=0.64; P


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icant effects on pStat3, pStat5 or pErk1/2 were observed(data not shown).

IFNc affects spontaneous and cytokine-dependentin vitro AML cell proliferation

AML blasts derived from 65 consecutive patients werecultured in medium alone or in medium containing

IFNc 50 ng/ml. Detectable proliferation correspondingto at least 1,000 cpm was observed only for 21 patients.IFNc caused no statistically significant alteration ofspontaneous in vitro proliferation. AML blasts derivedfrom the same patients were also cultured in the presenceof exogenous cytokines, and 3H-thymidine incorpora-tion was assayed after 7 days of culture. The effect ofadding IFNc was examined in the presence of 50 ng/mlof IL1b, IL3, Flt3L, SCF, GM-CSF or G-CSF. Ourstatistical analyses included the results only for thosepatients that showed detectable proliferation (corre-sponding to >1,000 cpm) for either the cytokine controlor the corresponding IFNc-containing culture. IFNc

caused a statistically significant inhibition of cytokine-dependent AML blast proliferation in the presence ofIL1b, SCF, GM-CSF and G-CSF (Fig. 4, Table 1); formany of these patients the inhibitory effect exceeded2,000 cpm and 20% of the IFNc-free control. In con-trast, only borderline significance was observed for IL3and no significant effect for Flt3L (Table 1).

IFNc has divergent effects on AML blast apoptosis

The effect of IFNc on AML blast apoptosis was inves-tigated for 16 randomly selected patients. The percent-ages of viable/apoptotic/necrotic cells were determined

after 24 and 48 h of in vitro culture with and withoutIFNc, and for 10 patients the results were reproduced inindependent experiments. The fraction of viable cellsdecreased gradually during culture and was accompa-nied by a corresponding increase in the percentage ofapoptotic cells. The percentage of viable cells after 48 hshowed a wide variation (median 46%, range 1586%)and an inverse correlation with the percentage ofapoptotic cells. IFNc had reproducible divergent effectson the percentage of viable cells after 48 h of culture. AnIFNc-induced difference in cell viability exceeding 5%was observed for 8 of the 16 patients; increased viabilitywas detected for 5 patients and decreased viability for 3

patients. The same divergence was observed after 24 h ofculture, although the percentages of viable cells weregenerally higher both for cultures with and withoutIFNc (data not shown).

Effects of IFNc in combination with cytotoxic drugson AML cell apoptosis and proliferation

We investigated the effect of IFNc on AML cell apop-tosis in the presence of cytarabine 0.1 and 1.0 lM. Ten

consecutive patients were investigated, and the numberof viable cells determined after 24 and 48 h. The overallresults are summarized in Fig. 5. The presence of cyt-arabine decreased the percentage of viable cells, but thedivergent effects of IFNc on AML cell apoptosis wasalso detected in the presence of cytarabine. When anal-ysing the overall results there was a significant correla-tion between IFNc effects on apoptosis for cultures withand without cytarabine (Pearson correlation; r=0.760.90; P1,000 cpm) with or without IFNc were included in eachcomparison. Line denotes the median. IFNc inhibited cytokine-dependent proliferation (P=0.0013 for IL1b, P


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Effects of IFNc on the constitutive AML cell releaseof IL1b and GM-CSF

The levels of GM-CSF and IL1b were determined forAML cells derived from 65 consecutive patients, whencells were cultured in medium alone and together withIFNc 50 ng/ml. Constitutive IL1b release was detectedonly for 14 patients (range 4300 pg/ml) and GM-CSFrelease for 10 patients (range 3.12,313 pg/ml). IFNccaused no significant alteration of these levels. In addi-tion, the effect of IFNc on AML cell proliferation wasinvestigated when cells were cultured in the presence of

exogenous IL1RA. IL1RA had no significant effect onthe spontaneous AML blast proliferation in our in vitromodel, and IFNc did not alter blast proliferation in thepresence of IL1RA (Table 1).

Effects of IFNc on the constitutive AML cell releaseof angioregulatory chemokines

Angiogenesis is probably important both for leukemo-genesis and chemosensitivity in AML, and the constit-utive release of angioregulatory mediators by theleukaemia cells [34] is probably important for the local

regulation of bone marrow angiogenesis in AML. Wetherefore investigated the effect of exogenous IFNc onthe constitutive release of pro- and antiangiogenicchemokines by AML cells. Leukaemia cells derived from65 consecutive patients were cultured in vitro in mediumalone or together with IFNc for 48 h before the levels ofangioregulatory chemokines were determined (Fig. 6).Constitutive release of proangiogenic CXCL8 was ob-served for most patients (57/67), and CXCL8 levels wereslightly decreased in the presence of IFNc (P=0.0004).In contrast, constitutive increased levels of antiangio-

Table 1 The effect of IFNc on cytokine-dependent proliferation by native human AML cells: a summary of the results for 67 consecutivepatients

Exogenouscytokine

Number of patientswith detectable proliferationin IFNc-free controlsa

Statistical comparison of proliferative response(mean cpm standard error)

Number of patients with>20% inhibitionb

Cultures without IFNc Cultures with IFNc P valuec

None 21 5,8851,376 9,6923,456 NS IL1RA 25 5,7681,113 5,1691,519 NS IL1b 40 11,9113,353 6,0601,679 0.001 16/40

IL3 49 17,5433,661 12,0482,015 0.052 22/49SCF 50 22,4914,553 11,0782,510 1,000 cpm) either in the IFNc-containing or corresponding IFNc-free control were included in the statistical analysisbThe column indicates the number of patients who showed an IFNc-induced alteration corresponding to at least 2,000 cpm and exceeding20% of the control responsecThe two-tailed Wilcoxons signed rank test was used for the statistical analysis

Fig. 5 The effect of IFNc on cytarabine-induced AML cellapoptosis. Leukaemia cells derived from 10 consecutive patientswere examined. The median percentage of viable cells was 40%(variation range 1784) after 24 h and 29% (range 1783) after48 h for cells cultured in medium alone, and viability was decreasedboth by cytarabine 1.0 lM (24 h: median viability 26%, range 1857; 48 h: median 18; range 930) and 0.1 lM (24 h: medianviability 37%, range 1782; 48 h: median 25; range 1780). Thefigure compares the effect of IFNc 50 ng/ml on AML cell viabilityfor cells cultured in the medium alone ( horizontal axis) and mediumwith cytotoxic drugs (vertical axis). The results are presented as thedifference in percentage of viable cells for cultures with and withoutIFNc, and we present the overall results for both concentrationsand both time intervals (*P=0.02; **P=0.008; ***P


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genic CXCL9 (P


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of these cells. Cultures were prepared in medium alone

and medium containing various exogenous growth fac-tors (IL1b, IL3, IL6, IL8, GM-CSF, G-CSF, TNFa) andproliferation assayed by 3H-thymidine incorporation.Exogenous IFNc did not alter the proliferation ofendothelial cells, but IFNc in combination with G-CSF,IL6 or IL8 increased the proliferation of these cells.Exogenous IFNc caused a weak inhibition of Cal72osteoblast proliferation and decreased HFL1 fibroblastproliferation by at least 70% both for cultures with andwithout exogenous cytokines.

Effects of IFNc on cytokine-dependent proliferation

by native human ALL blasts

Acute lymphoblastic leukaemia blasts derived from 12consecutive patients were cultured in medium alone andin medium with various exogenous cytokines (IL4, IL10,IL13, SCF, GM-CSF, Flt3L) [38]. ALL cells did notproliferate in medium alone and for three patientsdetectable proliferation was not observed with anyexogenous cytokine. Flt3L was the only cytokine thatinduced detectable proliferation for all the other ninepatients, and IFNc caused a strong inhibition of this

proliferation for five patients (Table 2). Detectableproliferation was only observed for a minority of pa-tients with the other cytokines, and either unaltered ordecreased proliferation was then observed with IFNc(data not shown).

Discussion

IFNc is a cytokine released by most CD4+ and CD8+ Tcell clones, and this is true for healthy individuals, acuteleukaemia patients with chemotherapy-induced cytope-nia and bone marrow transplant recipients [6, 7]. IFNc isreleased by T cells activated in vitro in the presence ofnative human AML cells [31, 39]. Taken together theseexperimental observations strongly suggest that localIFNc release will be a part of antileukaemic T cell

Fig. 7 Effect of IFNc on in vitro proliferation of AML cellsincubated in transwell culture together with either microvascularendothelial cells (EC), HFL1 fibroblasts (FB), Cal72 osteoblasticsarcoma cells (OB) or normal bone marrow stromal cells (BMSC).The figure compares leukaemia cell proliferation for cultures with(+) and without () IFNc 50 ng/ml. Only those samples showingdetectable proliferation (i.e. >1,000 cpm) for at least one of the invitro models (+/) are presented

Fig. 8 The effect of IFNc on in vitro proliferation of nonleukaemic

cells cocultured with AML blasts in transwell cultures. Microvas-cular endothelial cells (EC), HFL1 fibroblasts (FB) or Cal72osteoblastic sarcoma cells (OB) were cultured together with AMLblasts in the presence (+) or absence () of IFNc 50 ng/ml. Onlythose patient samples showing detectable proliferation (i.e.>1,000 cpm) for at least one of the in vitro culture models (+/) are presented

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responses, and observations from clinical studies ofIFNc therapy have demonstrated that IFNc has bio-logical effects in vivo on human AML cells [8]. In thiscontext we have investigated the effects of IFNc on thefunctional characteristics of native human AML cells.Our results demonstrate that IFNc can affect prolifera-tion, cytokine release and regulation of apoptosis forAML cells, and in addition IFNc seems to inhibit theproliferation of various nonleukaemic cells in the AMLmicroenvironment.

Even though the effects of IFNc on native humanAML cells have been investigated previously [9, 1218,32, 33], our recent study [11] describing a possibleprognostic impact of IFNc-induced intracellular signal-ling in AML cells justifies a more detailed character-ization. The previous reports have several limitations.First, they usually include a low number of patients(often less than 1015), consecutive patients are notexamined and the criteria for selection of patients arenot given. Secondly, the clinical and biological charac-teristics of the selected patients are usually incomplete ormissing. Finally, only effects on proliferation and

membrane molecule expression have been examined,and the experimental models differed. These previousreports have generally described (1) divergent effects ofIFNc on AML colony formation and (2) IFNc induc-tion of various membrane molecules (i.e. HLA class IImolecules, HLA-G, CD54, CD80, CD86, CD95) forsubsets of patients. In the present study we thereforeinvestigated the effects of IFNc for a large group ofconsecutive and well-characterized patients, and ourstudies included receptor expression, intracellular sig-nalling events, cytokine release and a detailed charac-terization of the effects on leukaemia cell proliferationincluding coculture with stromal cells.

We used well-characterized and standardized experi-mental models with regard to culture medium and AMLcell preparation [2129]. Our patients represent a con-secutive group with high peripheral blood blast countsso that highly enriched AML cells could be preparedwithout extensive cell separation procedures and therebythe risk of procedure-induced functional alterations [19].We therefore emphasize that our results may be repre-sentative only for this subset of patients, but we avoidedfurther selection by investigating randomly selected orconsecutive patient subsets.

We first investigated whether IFNc could be releasedwhen T cells were activated in the presence of prolifer-

ating AML cells cocultured in direct contact with nor-mal bone marrow stromal cells. This in vitro model willmimic the natural in vivo microenvironment, and weused an activation signal directed against the TCR/CD3complex to mimic antigen-specific activation. Our resultsdemonstrated that T cells can be activated to releaseIFNc when sharing the microenvironment with AMLcells that show constitutive release of several immuno-regulatory mediators.

CD119 expression was examined for 39 randomlyselected patients, and AML blast expression was de-

tected for all of them and a majority showed strongexpression with at least 70% of CD119+ cells. Thus,high expression of CD119 is common and independentof morphology (FAB classification), cytogenetic aber-rations or genetic abnormalities of the Flt3 gene. Fur-thermore, CD119 expression seems to be downregulatedafter in vitro exposure to IFNc; this may be due toreceptor internalization following receptor ligation.However, the majority of cells are still CD119+ evenafter the in vitro exposure.

Previous studies have demonstrated that IFNc canincrease AML cells expression of HLA-DR, CD40,CD54, C80, CD95 and possibly CD86 [32, 33]. How-ever, in our cocultures of AML cells and IFNc secretingT cells only HLA-DR expression was increased whereasthe expression of the immunostimulatory moleculesCD40 and CD80 and the dendritic cell marker CD83was not significantly altered. The most likely explana-tion for the difference between our present and theseprevious observations [32] is that IFNc is only a part of abroad T cell cytokine response in our cocultures, and theIFNc levels reached in our cultures are relatively low

compared with the concentrations of exogenous IFNcused by Costello et al. [32].

The effect of IFNc ligation on Stat1, Stat3, Stat5 andErk1/2 phosphorylation status was examined. Increasedphosphorylation was observed only for Stat1. CD119ligation can also induce signalling through Stat1-inde-pendent pathways, including Erk1/2-mediated effects[10], but we did not observe any effects on Erk1/2, Stat3or Stat5 phosphorylation.

IFNc did not affect spontaneous or IL3- and Flt3L-dependent AML cell proliferation, whereas statisticallysignificant inhibition was observed in the presence ofIL1b, SCF, GM-CSF and G-CSF. Both these results

and the coculture experiments demonstrated that thegrowth-inhibitory effect of IFNc depends on the localcytokine network, and it is not an indirect effect medi-ated through reduction of IL1b or GM-CSF levels. Fi-nally, IFNc could also affect AML cell proliferation inthe presence of cytarabine and idarubicin, two drugsthat are commonly used in AML therapy [3].

We investigated the effects of IFNc on both stress-induced (spontaneous) and cytarabine-induced AMLcell apoptosis, and IFNc then had divergent effects inboth experimental models. Thus, the divergent IFNceffects represent a true biological variation.

IFNc caused an increase in the levels of the antian-

giogenic chemokines CXCL911 and a slight reductionof proangiogenic CXCL8 levels. CXCL9 and CXCL10expressions are Stat1 dependent, at least in macrophages[10], thus the increased levels are probably caused by theStat1-mediated signalling. This altered balance betweenpro- and antiangiogenic signalling may contribute to theinhibition of endothelial cell proliferation in the presenceof IFNc (see below), but it should be emphasized thatCXCL8 was still released at relatively high levels even.

We investigated the effects of IFNc on AML blastproliferation when the cells were cocultured with various

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nonleukaemic stromal cells. These experiments furtherdemonstrated that the IFNc effect on blast proliferationwas dependent on the local cytokine network. IFNcinhibited AML blast proliferation significantly onlyduring coculture with microvascular endothelial cellswhereas AML cell proliferation was inhibited only for asubset of patients in the presence of fibroblasts, osteo-blasts and normal stromal cells. Exogenous IFNc had anantiproliferative effect on several nonleukaemic cells (i.e.microvascular endothelial cells, osteoblasts, fibroblasts)when added to cocultures of AML blasts and nonle-ukaemic cells. However, only osteoblastic Cal72 cellsand HFL1 fibroblasts but not endothelial cells were di-rectly inhibited by IFNc when cells were cultured withexogenous IFNc alone. Taken together our observationstherefore suggest that IFNc can affect the proliferationof nonleukaemic bone marrow stromal cells both di-rectly (fibroblasts, osteoblasts) and indirectly via effectson cocultured AML cells. All these nonleukaemic cellssupport leukemogenesis [23, 24, 26], and the antiprolif-erative effects may then indirectly affect the AML cells.

IFNc can affect the proliferation of various normal

cells [4049], and an important question is then whetherIFNc affects the proliferation of other transformed cellsthan native human AML cells. AML and ALL are bothaggressive malignancies characterized by rapid accumu-lation of immature and morphologically similar leukae-mia blasts in the bone marrow, and for this reason ALLblasts are possibly the most closely related cells to AMLblasts. Our results demonstrated that IFNc could affectthe proliferation of different malignant haematopoieticcells, i.e. the effects are not specific for AML blasts.

To conclude, IFNc can affect AML cell proliferationboth when cells are cultured alone and in the presence ofnonleukaemic stromal cells. It can also alter the balance

between pro- and antiangiogenic chemokine release bythe AML cells. These effects may contribute to antil-eukaemic T cell reactivity after allotransplantation andpossibly also in antileukaemic immunotherapy.

Acknowledgement The work was supported by the NorwegianCancer Society and Helse-Vest. The technical assistance of LailaMentzoni and Kristin Paulsen is gratefully acknowledged.

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Effects of Interferon Gamma on Native Human Acute Myelogenous

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