Immunology 1999 96 202–206

Synergistic action of stem-cell factor and interleukin-7 in a human immatureT-cell line

U. R. KEES & J. FORD Division of Children’s Leukaemia and Cancer Research, TVW Telethon Institute for Child HealthResearch, West Perth, Western Australia, Australia

SUMMARY

The thymus provides the microenvironment that is optimal for T-cell differentiation. The mostimmature cells in the human thymus express the stem-cell marker CD34 and they respond tocytokines, including stem-cell factor (SCF) and interleukin-7 (IL-7). For the normal progressionof T-cell development these two cytokines appear to be vital. We have established and characterizeda human pre-T-cell line, PER-487, which mirrors this requirement. This study shows that thesimultaneous presence of IL-7 and SCF produces a proliferative response far exceeding additiveeffects. Furthermore, providing these signals in succession did not achieve the effect observedwhen they were provided simultaneously. This finding suggests that the effect was not mediatedvia secretion of molecules or modulation of surface expression. The convergence of the signaltransduction pathways of the two cytokines is not known, thus cell line PER-487 provides aunique model for studying the synergistic interaction of IL-7 and SCF.

INTRODUCTION differentiation of these immature human thymocytes has beenanalysed by multiparameter flow cytometry. The sequence of

The thymus provides the microenvironment for T-cell differen-marker expression has been determined to be as follows:5,6tiation. The complex control of the differentiation steps haveCD34+, CD7++, CD1−, CD4−, CD8−, CD45RA+�CD34+,been extensively studied.1,2 Multipotent haemopoietic stemCD7++, CD1+, CD4+, CD8−, CD45RA±�CD34−, CD7++,cells undergo developmental changes before migration to theCD1+, CD4+, CD8±, CD45RO+�CD34−, CD7+, CD1++,thymus where expansion and selection of discrete populationsCD4+, CD8+, CD45RO+.of T-cell precursors takes place preceding rearrangement of

The control processes that regulate the T-cell differentiationthe T-cell receptor (TCR). Functionally competent matureare currently of great interest. Cytokines are thought toT cells are subsequently exported into the peripheral compart-provide some of the signals that mediate these events.ment. The individual steps of the murine intrathymic develop-Significant advances in this field have come from cytokine-ment have been delineated by defining five phenotypicallydeficient mice generated by homologous recombinationdistinct subpopulations.2 The first three of these subpopula-(reviewed in reference 7). Removal of most of the cytokinestions, denoted A to C, comprise the thymus-seeding precursorexamined does not significantly affect intrathymic T-cell devel-cells, showing TCR in germline state, and account for onlyopment; however, there appears to be an important require-0·1% of all thymocytes. The step to development stage D isment for interleukin (IL)-7 and stem-cell factor (SCF ) andcharacterized by TCR rearrangement and the transition fromboth appear to act on early thymocytes.8,9 Although IL-2,c-kithi to c-kitlo.IL-4, IL-7, IL-9 and IL-15 share the common c (cc) chain forThe developmental sequence in the human thymus hasbinding, IL-7 is unique and non-redundant in its capacity tobeen studied mainly by using surface markers. The earliestmaintain viability of early thymocytes. Studies of IL-7 andhuman thymocytes appear to be CD34+ CD7+ and mostIL-7-receptor deficient mice showed that IL-7 was an import-express cytoplasmic CD3. Their differentiation into matureant factor for proliferation of immature thymocytes.10,11T cells can be demonstrated using the Thy severe combined

immunodeficiency (SCID)-human mouse model3 or thymic Results from c-kit- and SCF-deficient mice revealed that SCFstroma in vitro model.4 CD34 is expressed on 1–2% of thymo- supports the expansion of early thymic immigrants.12 Thecytes in both paediatric and fetal human thymus.5,6 The immature human subset of thymocytes, expressing CD34+

and CD7+, could be shown to proliferate rapidly in thepresence of IL-7, alone or in synergy with SCF,6 while moreReceived 10 July 1998; revised 30 September 1998; accepted 30

September 1998. differentiated CD34− thymocytes did not show this response.We have studied a human cell line that is dependent on IL-7Correspondence: Dr Ursula R. Kees, TVW Telethon Institute forand SCF for growth to define more precisely the synergisticChild Health Research, PO Box 855, West Perth, Western Australia

6872, Australia. action of the two cytokines.

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MATERIALS AND METHODS sodium orthovanadate, 2 m EDTA, 10 m iodoacetamide,25 mg/ml p-nitrophenyl–guanidinobenzoate, 10 mg/ml leupep-

Establishment of cell line PER-487tin, 10 mg/ml aprotinin (Sigma, St. Louis, MO) and 0·5%Cell line PER-487 was established from a paediatric patientTriton-X-100 (Pharmacia, Uppsala, Sweden) was added atdiagnosed with acute lymphoblastic leukaemia, using a pre-100 ml/107 cells. Cells were vortexed and left on ice for 15 min.viously published method.13 The cells were initially placed inThe lysates were cleared by centrifugation (14 000 g at 4°) forIscove’s medium (Flow Laboratories, Irvine, UK), sup-15 min. Protein content was measured using the protein assayplemented with supernatant (SN ) from phytohaemagglutininfrom Bio-Rad (Hercules, CA) and equal amounts of lysates(PHA)-activated human peripheral blood cells (5%) and 20%were loaded per lane of an 8% sodium dodecyl sulphatefetal calf serum (FCS) from CSL Biosciences (Parkville,(SDS)–polyacrylamide gel and electroblotted to a Hybond-CAustralia). After 11 weeks the cells were adapted toSuper membrane (Amersham). After blocking in 2% bovineRPMI-1640 medium (ICN Biomedicals, Costa Mesa, CA)serum albumin (BSA) at 4° overnight, the membranes weresupplemented with 20% FCS and 20% SN. In order towashed in Tris-buffered saline (20 m Tris, 150 m NaCl )determine their requirements for growth in tissue culture,with 0·05% Tween and 0·1 m Na3VO4 and incubated withseveral cytokines, supplied as human recombinant products,TUGh4 antibody (151000), specific for the cc chain, for 1 hrwere tested for activity. These cytokines included IL-2, IL-3,at room temperature.IL-5, IL-6, IL-7, SCF, transforming growth factor-b (TGF-b)

Horseradish peroxidase-conjugated secondary antibodiesand granulocyte–macrophage colony-stimulating factorwere used for 1 hr at a concentration recommended by Santa(GM-CSF) (see Results). Based on these studies, the cells wereCruz Biotechnology (Santa Cruz, CA). After extensive wash-further propagated in RPMI-1640 supplemented with IL-2ing, the membranes were developed by the enhanced chemi-(100 ng/ml ), IL-7 (6 ng/ml ), SCF (75 ng/ml ) and 20% FCS.luminescence method (ECL, Amersham, Little Chalfont,The recombinant cytokines were kindly supplied as follows:England) according to the manufacturer’s instructions. TheIL-2 by Cetus Corporation (Emeryville, CA); IL-7 and SCF

by Amgen (Thousand Oaks, CA); and GM-CSF by Genetics signals obtained for bands of interest were quantified using aInstitute (Cambridge, MA). The studies reported here were Kodak digital camera, Adobe Photoshop NIH Image. Forperformed using PER-487 cells that had been in culture for Fig. 3, the levels are normalized to the signal obtained in8–16 months. The cell line was repeatedly shown to be free of lane 1, which was assigned a band density of 1.Mycoplasma.

RESULTSImmunofluorescence studies

Cytokine requirement for growth of PER-487 cellsCells were labelled with the indicated monoclonal antibodies(mAbs) or isotype-matched control mAbs, using a microplate The establishment of cell line PER-487 is detailed in themethod for indirect immunofluorescence, as previously

Materials and methods. The phenotype of the line was deter-described.13 Bound mAb was detected using a biotinylated

mined to be CD5+, CD6+, CD7+, CD56+, CD113+ (c-kit)sheep antibody, followed by streptavidin–fluorescein fromand the cells expressed cytoplasmic CD3. The cell line wasAmersham (Little Chalfont, Bucks, UK). YB5.B814 is aninitially propagated in medium supplemented with 20% SN.immunoglobulin G1 (IgG1) mAb that identifies the extracellu-In order to examine the dependence on cytokines in morelar domain of P145c-kit. mAb RFT2 (CD7) was obtained fromdetail, PER-487 cells were maintained for 16 hr in the absenceDr G. Janossy, Royal Free Hospital, London, the Tac antibodyof cytokines and then used in short-term proliferation experi-(CD25) was a gift from Dr T. Waldmann, National Cancerments (48 hr) in the presence of individual cytokines, includingInstitute and the TUGh4 antibody,15 which identifies the ccIL-2, IL-3, IL-5, IL-6, IL-7, SCF, TGF-b and GM-CSF. Thechain, was a gift from Dr K. Sugamura (Tohoku Universityproliferation rate of the cell line was increased by only threeSchool of Medicine, Sendai, Japan). Flow cytometry analysiscytokines: IL-2, IL-7 and SCF. Based on these findings, thewas performed using an Epics Elite instrument (Coultercell line was adapted to medium supplemented with these threeElectronics, Hialeah, FL).cytokines and cells were propagated in this medium for4 months. During this time the cells appeared to have becomeProliferation assaysIL-2 independent, hence this was assessed in a further set ofPER-487 cells were incubated for 20 hr in medium not sup-proliferation assays.plemented with any cytokine. The cells were then washed

thoroughly and resuspended in culture medium supplementedas indicated below. Quadruplicate cultures of cells, in the wells IL-7 and SCF together enhance proliferation of PER-487 cellsof round-bottom microtitre plates, contained 1×105 cells in

The growth requirement of cell line PER-487 was determined0·2 ml of culture medium. Cultures were incubated for 48 hrfor each cytokine, individually and in combination. Cellsand [3H ]thymidine incorporation was determined during acultured for 16 hr in the absence of cytokines were used for6-hr period. The samples were counted for 5 min in a Matrixthese experiments. The studies revealed that IL-2 did not9600 Direct Beta Counter (Packard, Meriden, CT). Valuesincrease proliferation of the cell line above background levels.shown are means of quadruplicate cultures and, as standardIn contrast, low levels of proliferation were observed in theerrors were less than 5% of the mean values, they are notpresence of either IL-7 or SCF, and proliferation was consist-included in the figures.ently higher with SCF than with IL-7 (Fig. 1). We next studiedthe effect of the two cytokines in combination by maintainingImmunoblot analysisone of them at a constant concentration that achieved plateauWhole cell extracts were analysed in immunoblots for the

presence of the cc chain. Ice-cold lysis buffer containing 1 m levels of proliferation (SCF at 75 ng/ml and IL-7 at 10 ng/ml )

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U. Kees & J. Ford204

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Figure 1. Panel (a): proliferation of PER-487 cells cultured withinterleukin (IL)-7 (titrated from 0·5 ng/ml to 12 ng/ml ) in mediumalone (open symbols) or in medium containing 75 ng/ml stem-cellfactor (SCF ) (closed symbols). Panel (b): proliferation of PER-487cells cultured with SCF (titrated from 50 ng/ml to 400 ng/ml ) inmedium alone (open symbols) or in the presence of 10 ng/ml IL-7(closed symbols). Proliferation was measured as [3H]TdR incorpor-

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ation (expressed as c.p.m.×10−2) after culturing the cells for 48 hr. Figure 2. Proliferation of PER-487 cells after priming in cytokine-containing medium for 16 hr and subsequent culture of the cells foran additional 48 hr, measured as [3H]TdR incorporation (expressedas c.p.m.×10−2). The conditions for priming (preincubation of cellsand adding the other one at a different concentration, rangingfor 16 hr) and addition of cytokines during the subsequent 48 hr arefrom 0·5 ng/ml to 12 ng/ml for IL-7 and from 50 ng/ml toindicated on the Figure. Stem-cell factor (SCF) was used at 75 ng/ml400 ng/ml for SCF, A representative result is shown in Fig. 1,and interleukin (IL)-7 at 10 ng/ml. Panel (a): cells cultured in the

showing that IL-7 (panel a) and SCF (panel a) alone inducedpresence of IL-7 (0·5 ng/ml to 12 ng/ml ). Panel (b): cells cultured in

low levels of proliferation when supplied in medium, while the the presence of SCF (50 ng/ml to 400 ng/ml ).presence of both cytokines resulted in a much enhancedproliferation. In more than 10 repeats of this experiment itwas established that three to 10-fold higher plateau levels were (group 3) did not increase proliferation to a level higher than

that obtained by IL-7 alone (group 1). Identical results werereached in the presence of both cytokines, compared with theplateau levels obtained by individual cytokines; this far obtained for the SCF titrations, shown in Fig. 2(b). These

findings show that the synergistic effect was dependent on theexceeded additive effects. PER-487 cells cultured long term inmedium supplemented with the two cytokines showed corre- simultaneous presence of both cytokines. Prior incubation

with one of the cytokines did not provide the required signals,sponding increases in cellularity.as shown in this functional assay.

Synergistic effect of IL-7 and SCF is dependent on thesimultaneous presence of the cytokines Regulation of the cc chain by IL-7 and SCF

To assess whether the synergistic effect of IL-7 and SCF maySynergistic effects between cytokines can be caused by theinduction of respective cytokine receptors. To address whether involve cytokine-receptor regulation, examination of surface

markers by flow cytometry was performed. PER-487 cells weresuch a mechanism was operating, the expression of functionalcytokine receptors was examined in a cell culture system. analysed, after culture for 48 hr, either in IL-7/SCF-sup-

plemented medium or in medium without cytokines. ThesePER-487 cells were preincubated for 16 hr with only oneof the cytokines and subsequently incubated with the second experiments revealed down-regulation of cc chain expression

on cells cultured in the absence of IL-7/SCF (data not shown),cytokine in order to determine whether prior exposure to thecytokines produced induction of the cytokine receptors under while all other surface molecules analysed showed the same

level of expression or only moderately lower expression.test. Representative results are shown in Fig. 2. The cytokinespresent during the initial 16 hr of the experiment are listed on This observation was followed-up by studies designed to

determine whether IL-7 and SCF were involved in the regu-Fig. 2 under ‘Priming’ and are numbered 1–5. The cytokinespresent during the subsequent culture for 48 hr are listed on lation of the cc chain. Immunoblot analyses were performed

using PER-487 cells that had been cultured for 16 hr in theFig. 2 under ‘Addition’. In all cases, the cytokines were addedto the same final concentrations as in the previous experiments. absence of any cytokine, followed by culture for 48 hr in the

presence of either IL-7 or SCF, alone or in combination.In addition, during the 48-hr culture phase, IL-7 and SCFwere added at different concentrations as in the previous Figure 3 shows an immunoblot of total cell extracts stained

with the mAb, TUGh4, which binds to cc. Each cytokine usedexperiments. The titrations of IL-7 in Fig. 2(a) clearly showthat enhanced proliferation was obtained under only two individually induced cc in a reproducible fashion. In three of

four identical experiments, SCF alone induced a strongerconditions, namely 2 and 4; in both of these, SCF was presentduring the second stage of the experiment. There was no signal than IL-7 alone. However, in all four experiments the

presence of both cytokines yielded a significantly enhancedsignificant difference caused by the absence (i.e. group 2) orpresence (i.e. group 4) of SCF during the priming phase. effect compared to the induction observed by the individual

cytokines.Importantly, SCF supplied during the priming phase only

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Synergism between SCF and IL-7 205

were provided simultaneously. This finding suggests that theeffect was not the result of secretion of molecules or modulationof surface expression. Such mechanisms would be expected tooperate under conditions where the signals are delivered atdifferent times; however, the preincubation study shown inFig. 2 clearly showed this not to be the case. In addition, wecould demonstrate that one effect of the synergistic interactionof the two cytokines was the enhanced expression of the ccchain. Whether the simultaneous presence of the cytokinesinduces a qualitatively different signal compared to signalsinduced by SCF and IL-7 individually remains to be explored.

Cytokines promote the survival and proliferation of haemo-poietic stem and progenitor cells and are thought to play arole in differentiation. Synergistic effects between cytokineshave been reported for many combinations of cytokines. SCF,in particular, is a potent co-stimulating cytokine that acts insynergy with a range of other cytokines, including GM-CSF,IL-3, IL-6 and erythropoietin.9,20 The receptor for SCF, c-kit,is a receptor-type protein tyrosine kinase (RTK) that is amember of the platelet-derived growth factor receptor family.Binding of SCF to the receptor induces dimerization of thereceptor, followed by transphosphorylation and subsequentRTK activation. The resulting tyrosine-phosphorylated c-kitreceptor binds SH-2-containing proteins such as phospholipasec-c, phosphatidylinositol 3-kinase, Syp and HCP, which are,in turn, phosphorylated by the c-kit RTK. In contrast, thereceptor for IL-7 does not possess intrinsic kinase activity;

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Figure 3. (a) Immunoblot using TUGh4 antibody to cc on PER-487 however, activation of cytoplasmic tyrosine kinases occurs incells. Cells were incubated for 48 hr as follows: lane 1, medium; lane both B and T cells, involving the Jak–Stat pathway in T cells.212, interleukin (IL)-7 (10 ng/ml ); lane 3, stem-cell factor (SCF) Mechanisms underlying the synergy of SCF and other cyokines(75 ng/ml ); lane 4, IL-7 and SCF. The band of 64 kDa is indicated are largely unknown. Of critical importance is the elucidationby arrow. (b) Results of densitometric scanning of the 64 kDa band; of the integration of the distinct pathways for IL-7 and SCF.see the Materials and methods for further details.

There are several possible mechanisms underlying synergisticinteractions of cytokines and they are not mutually exclusive.

DISCUSSIONFirst, the signal transduction pathways of two cytokines mayconverge to enhance the response or trigger a response notIL-7 and SCF have been demonstrated to play a role in

intrathymic T-cell development. The function of IL-7 appears achieved by either cytokine alone. Second, the signalling byone cytokine may result in modulation or induction of theto be in supporting the viability and expansion of early

thymocytes. Results from several experimental systems suggest expression of cytokine receptors (for the same or for othercytokines) or alterations in the expression and secretion ofthat SCF/c-kit interaction is involved in thymocyte differen-

tiation.12,16 Expression of the c-kit receptor is associated with cytokines. Receptor expression may be modified, for instance,by dimerization, or association with other cell surface compo-the earliest steps of thymocyte development.17 These immature

thymocytes proliferate optimally when SCF and IL-7 are nents and cytokines may trigger an autocrine/juxtacrine cycle.PER-487 appears to be the first cell line to show synergisticcombined.17,18 Stimulation by the combined presence of these

cytokines seems to be vital for the normal progression of interaction of SCF and IL-7. Hence it provides a unique modelsystem for studying the synergistic interaction of these twointrathymic T-cell development. It is possible that the signals

provided play a role in the lineage commitment process. A cytokines.c-kit+ maturation pathway has been described in the mouse.19At present, the precise contribution to the proliferation of

ACKNOWLEDGMENTSimmature thymocytes by the two cytokines is not known.Cell line PER-487 investigated here is dependent on IL-7 We thank Dr L. Ashman, Dr K. Sugamura, Dr G. Janossi and Dr

and SCF and thus mirrors the requirements of immature Waldmann for providing antibodies, and Cetus Corporation, Geneticshuman thymocytes. Freshly isolated human thymocytes, selec- Institute and Amgen for supplying cytokines. This work was supported

by the Children’s Leukaemia and Cancer Research Foundation ofted on the basis of surface marker expression, have beenWestern Australia and by the National Health and Medical Researchreported to require IL-7 plus SCF for optimal proliferation.6Council of Australia.The long-term culture of our immature T-cell line, PER-487,

is dependent on medium supplemented with both cytokines.Importantly, our study shows that the simultaneous presence

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