(CANCER RESEARCH 49, 3568-3573. July I, 1989]

Immunoregulation and Production of Tumor Necrosis Factor a by HumanThymocytes1

Gabrielle H. Reem,2 Anne Duggan, and Michael Schleuning3

Department a/ Pharmacology anil the Kaplan Cancer Center, New York University Medical Center, New York, New York 10016

ABSTRACT

We have provided evidence that tumor necrosis factor a (TNF-a)enhances the proliferation and the state of activation of human thymo-cytes cultured with concanavalin A or interleukin 2 (IL-2), as evidencedby an increase in the expression of the c-myc gene and the gene of theIL-2 receptor (o-chain, Tac antigen) and by the expression of Tac antigenon the cell surface. Our observations suggest that TNF-a interacts withIL-2 and with another factor(s) which is induced in the course of activationby concanavalin A, since the immunosuppressant drug cyclosporin A-,which inhibits thymocyte activation, prevents the effect of TNF-a onthymocytes activated with concanavalin A, whereas anti-Tac, whichprevents the binding of IL-2 to its receptor without affecting the production of IL-2 or the expression of IL-2-specific mRNA, inhibits proliferation only partially. By contrast, anti-Tac inhibits the response to I M -a of thymocytes induced with IL-2 completely. These observations showthat TNF-a exerts a potentially important immunoregulatory effect insynergy with IL-2 on thymocytes, which could contribute to tumorrejection. In addition, we show that activated human thymocytes expressthe TNF-a gene and that the expression of this gene is inhibited bycyclosporin A and dexamethasone.

adherent cells can be induced to express TNF-a mRNA ifactivated with Con A or with Con A in combination with TPA.In addition, we found that TNF-a by itself, unlike IL-2, doesnot induce the proliferation of thymocytes in vitro. However,TNF-a increases the proliferation of thymocytes suboptimallyactivated with Con A and acts in synergy with IL-2 in inducingboth the expression of Tac antigen and its mRNA as well asthe proliferation of thymocytes. Freshly isolated thymocytes,unlike T-lymphocytes (peripheral blood lymphocytes), proliferate in medium in the absence of IL-2 or other inducing agentsfor at least 48 h and therefore express c-myc. However, TNF-aincreases the expression of mRNA specific for the protooncogene c-myc within 4 h, prior to an increase in proliferativeactivity. Studies carried out in our laboratory have shown thateven CD3" thymocytes can be induced to express IL-2 mRNA

and to proliferate in response to IL-2 (14). These findingstherefore support the notion that TNF-a acts as an immunoregulatory cytokine which can modulate the state of activationand the immune function of thymocytes.

INTRODUCTION

TNF-a,4 a cytokine which is produced by macrophages in

response to viral and microbial infections, has been reported toalso be produced by nonadherent peripheral blood mononuclearcells (1). TNF-a exerts pleiotropic effects which have beenreviewed recently (2, 3). It inhibits the growth of certain tumorcells, but promotes the growth of normal and transformed cellsin vitro (4) and, like IL-2, it has immunoregulatory effects onT- and B-cell functions in vitro (5-7). Recently, it has beenshown by Ranges et al. (8) to enhance the immune function ofmurine thymocytes. In vivo, it causes hemorrhagic necrosis,wasting, and tumor regression in mice (3).

The experiments reported here were designed to test whetherTNF-a synthesis could be induced in activated human thymocytes, and to study if TNF-a exerted immunoregulatory effectssimilar to those of IL-2 on human thymocytes in vitro. Earlierstudies carried out in our laboratory have shown that IL-2 actsas an immunoregulatory factor which stimulates thymocyteproliferation in vitro and induces the transcription of the a-chain of the IL-2 receptor gene (Tac gene) and the expressionof Tac antigen on the cell membrane (9-13). IL-2 acts in synergywith other inducing agents in activating thymocytes and T-cells.The present study shows that human thymocytes depleted of

Received 12/14/88; revised 3/16/89; accepted 3/30/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by NIH Grant RO 1 CA 33653.1To whom requests for reprints should be addressed, at Department of

Pharmacology, New York University Medical Center, 550 First Ave., New York,NY 10016.

3Supported in part by a grant of the Deutsche Forschungs GemeinschaftStipendium Schi 251/1-1.

4The abbreviations used are: TNF-n, tumor necrosis factor a (cachectin); IL-2, interleukin 2; Con A, concanavalin A; TPA, 12-O-tetradecanoylphorbol-13-acetate; CsA, cyclosporin A; Dex, dexamethasone; PE, phycoerythrin; FITC,fluoresccin isothiocyanate; IL-2R, interleukin 2 receptor; PBS. phosphate-buffered saline; cDNA, complementary DNA; IP, inositol phosphate; IFN-7, y-interferon.

MATERIALS AND METHODS

Isolation and Activation of Human Thymocytes. Thymocytes wereisolated as previously described from sections of thymus obtainedduring the course of cardiac surgery of infants and young children (9).Cell suspensions were prepared, and thymocytes were cultured (6 x IO6or 1 x 10' cells/ml) in 75-cnr tissue culture flasks or in 96-well

microtiter plates (Costar, Cambridge, MA) in RPMI 1640 medium(Grand Island Biological Co., Grand Island, NY) supplemented with5% heat-inactivated fetal bovine serum, 2 m\i glutamine, 100 units/mlof penicillin, 100 Mg/ml of streptomycin, and 250 ng/ml of fungizone(complete medium) at 37°Cin a humidified atmosphere of 5% CO2 in

air. Adherent cells were removed by incubation on a plastic surface for90 min and by passage through nylon-wool (14). The cultures depletedof adherent cells did not contain OKM*1 cells, and the majority ofthymocytes were T3+T6+(CD3+CD1+) as well as T4*T8+(CD4+CD8*).

Inducing Agents and Inhibitors. Concanavalin A (Sigma Biochemi-cals, St. Louis, MO), TPA (L. C. Service Corp., Woburn, MA), recombinant human TNF-a (Genentech, South San Francisco, CA), andpurified recombinant IL-2 (a gift from Hoffman-LaRoche, Nutley, NJ)were obtained. CsA was a gift from Dr. J.-F. Borei (Sandoz, Basel,Switzerland), and Dex was purchased from Sigma.

Monoclonal Antibodies. Anti-Tac was provided by Dr. Tom Wald-mann (NIH, Bethesda, MD), and anti-human TNF-a (TNF E) waspurified from ascitic fluid (neutralization titer, 6000 units/mg) by Dr.M. H. Shepard (Genentech). OKM*1, OKT3, and OKT6 were pur

chased from Ortho Diagnostics Systems, Inc., Raritan, NJ. The following PE-conjugated and FITC-conjugated antibodies were purchasedfrom Becton Dickinson, Mountainview, CA: monoclonal anti-humanIL-2 receptor antibody (PE IL-2R); PE Leu 4; PE Leu 2a; FITC Leu3a; FITC-OKT6; and PE T3.

Immunofluorescence. The phenotype and the expression of IL-2receptors were determined by direct immunofluorescence. Briefly, thymocytes (6 x IO6cells/ml) cultured in microwells (200 ^1)were washed

in Eppendorf microfuge tubes with buffer (PBS, pH 7.4, 5% (v/v) fetalbovine serum, and 0.1 % (w/v) NaN3) and resuspended in 50 ^1of buffer.Twenty of the appropriate monoclonal antibody were added to the cellsuspension. Cells were gently mixed and incubated for 30 min at 4°C

in the dark, washed twice with buffer, and stored in 1% paraformal-dehyde in PBS.

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TNF-<, AND HUMAN THYMOCYTES

The expression of the antigen recognized by OKM*1 and TNF-a onthe cell surface of thymocytes was determined by indirect immunoflu-orescence. Thymocytes were harvested as described and incubated withOKM*1 or, for the determination of TNF-«on the cell surface, withanti-TNF-a (1/1000) and FITC-conjugated rabbit anti-mouse F(ab'):

IgG (Cappel, Cochranville, PA). Fluorescence was assessed by fluorescent microscopy and with a FACSCAN cell sorter (Becton Dickinson,Mountainview, CA) after resuspending cells in PBS. Dead cells weregated out by forward scatter.

Proliferative Assays. Thymocytes (6 x IO6 cells/ml) cultured inmicrowells (200 ¿¿1)were pulsed with 1 ¿iCiof ['Hjthymidine (New

England Nuclear, Boston, MA; specific activity, 6.7 Ci/mmol) for thelast 6 h of culture, harvested on glass fiber filters (GM/C; Whatman,Clifton, NJ) precipitated with 10% (w/v) ice-cold trichloroacetic acid,and washed with methanol. Triplicate samples were counted in a liquidscintillation counter.

RNA Slot Blot Analysis. Thymocytes (5 x 10s cells) were cultured in50 ml of complete medium in 75-cm' tissue culture flasks (Corning,

Corning, NY). Total cellular RNA was extracted after specified culturetimes by a modification of the guanidine isothiocyanate method (15).Briefly, nucleic acids were isolated by guanidinium isothiocyanate lysis,and total cellular RNA was separated by gradient centrifugation incesium chloride. After extraction with phenol and quantification, RNAwas heat denatured in IM sodium chloride:50 HIMsodium phosphate(pH 7.0):6% formaldehyde at 60°Cfor 10 min, and specified amounts

of total cellular RNA were blotted onto 0.45-^m nitrocellulose filters(Schleicher & Schuell, Keene, NH) by using a Schleicher & Schuellslot-blot apparatus. Filters were air-dried, baked at 80°Cfor 2 h, and

subsequently used for hybridization. Hybridizations were performedaccording to established procedures (16) with appropriate nick-translated cDNA probes. TNF-a cDNA was provided by Dr. David V.Goeddel (Genentech) ( 17). The human c-myc cDNA, encompassing the3' exon (18c), was a gift of Dr. R. Dalla-Favera (NYU Medical Center,New York, NY). IL-2 receptor cDNA (a-chain) was provided by Dr.W. Greene (Duke Medical Center, Durham, NC) (19).

After hybridization, filters were washed under stringent conditions,air-dried, and exposed to X-ray films (Kodak X-Omat 5) intensified byDupont Cronex Quantra 3 screens at -70°C.

Accumulation of Inositol Phosphates. Freshly isolated thymocytes (Ix 108/ml) were labeled to equilibrium prior to activation with 100 nC\/ml of [/nyo-'Hjinositol (American Labeled Compounds, St. Louis, MO)

in FIO medium (Grand Island Biological Co, Grand Island, NY) for 42h in 25-cm2 tissue culture flasks (Corning). Subsequently, the mediumwas removed, and the cells (1 x 10s/rnl) were resuspended in completeRPMI 1640 medium. Dephosphorylation of inositol-1-phosphate wasblocked with 10 imi lithium chloride, and cells were induced for l h in2-ml 24-well flat-bottomed tissue culture plates (Costar, Cambridge,MA). After phase separation, aliquots of the aqueous phase were loadedonto Dowex AG-1-X8 formate columns (Bio-Rad, Richmond, CA),which had been equilibrated with 1 ml of 5 mvi unlabeled inositol. Freeinositol was eluted with 5 ml of H2O, glycerophosphoinositol with 4ml of 0.06 M ammonium formate and 5 HIMsodium tetraborate. andfinally IPs with 2 ml of 1 M ammonium formate and 0.1 Mformic acid.Aliquots of the eluted fractions were counted in Aquasol (New EnglandNuclear) in a liquid scintillation counter.

RESULTS

Induction of the Expression of TNF-a mRNA. The in vitrostimulation of thymocytes with Con A, which may be activatingthymocytes through the T3-T-cell antigen complex (20, 21),induces the accumulation of the TNF-a mRNA within 6 h, anda further increase in the expression of mRNA is apparent at 24h (Fig. 1). By contrast, stimulation with both TPA and Con Aresults in virtually maximal TNF-a mRNA accumulationwithin 6 h, and no further increase in mRNA is observed by 24h. Removal of adherent cells from the thymic culture by incubation on a plastic surface for 90 min and passage through anylon-wool column prior to incubation with Con A does not

6h 24h

Control

_ Con A

— — Con A + TPA

Fig. 1. Slot blot analysis of TNF-« specific mRNA isolated from humanthymocytes. Thymocytes were cultured in complete medium (lop), with Con A,10 up. ml (middle) and Con A. 10 Mg/ml. plus TPA 1 ng/ml (bottom), for 6 and24 h (left and right lanes), respectively. Twenty, 5. and 2 ng of total RNA wereblotted for each experimental condition.

ConA+TPA ConA control

Fig. 2. Slot blot analysis of TNF-«-specific mRNA. Thymocytes were incubated at 37"C for 90 min on a plastic surface and passed through a nylon-woolcolumn in order to remove adherent cells. The nonadherent OKM*I~ cells were

incubated with Con A (10 Mg/ml) plus TPA (1 ng/ml) for 24 h (Lane I), withCon A (10 (jg/ml. Lane 2), and with medium (Lane 3). Total RNA (20. 10, and5 /ig) was blotted for each experimental condition.

prevent the induction of TNF-«mRNA by Con A and by ConA plus TPA (Fig. 2).

Effects of the Immunosuppressant Drugs Dex and CsA. Thesedrugs are known to inhibit lymphokine synthesis by thymocytesand T-cells (12, 22-24). Both CsA and Dex inhibit the accumulation of TNF-a mRNA in thymocytes cultured with Con Aplus TPA (Fig. 3).

TNF-a Is Synthesized by Thymocytes and Bound to the CellSurface. The data recorded in Fig. 4A show that thymocytesinduced with TPA plus Con A produce TNF-a which is boundto the cell surface (Fig. 4/4). Binding of anti-TNF-a can bedemonstrated by indirect fluorescence (solid line), whereas thymocytes cultured in unsupplemented medium, which do notexpress receptors for TNF-a, did not bind anti-TNF-a even inthe presence of exogenous TNF-a (Fig. 4B, solid line). Althoughmembrane-bound TNF-a could be detected on some, but notall thymocytes by direct immunofluorescence, the amount ofTNF-a in the medium was not adequate to be detectable byradioimmunoassay.

TNF-a Increases the Proliferation of Con A-activated Thymocytes. The effect of TNF-a as a comitogen was tested. Thedata recorded in Table 1 show that TNF-a alone does not

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Control

ConA+TPA+Dex

ConA+TPA+CsA

ConA+TPA

Fig. 3. Slot blol analysis of TNF-«-specific mRNA. Thymocytes were inducedfor 24 h with Con A (10 »ig/ml)plus TPA (1 ng/ml). CsA (I fig/ml) or Dex (10~6

MI was added as indicated. Thymocytes incubated with medium alone served ascontrol. Total RNA (15. 7.5, and 1.5 jig) was blotted for each experimentalcondition.

activate thymocytes, since thymocytes grown in completeRPMI 1640 medium supplemented with TNF-a (10 to 1000units/ml) cease to proliferate by 72 h of culture as do thymocytes cultured in the absence of TNF-a (Table 1, top). Bycontrast, cultures supplemented with suboptimal amounts ofCon A (2 and 5 ng/m\) incorporated ['Hjthymidine at a more

rapid rate when supplemented with TNF-a. The proliferativerate at 72 h was proportional to the concentration of Con Aand was higher in cultures supplemented with Con A plus TNF-<>:maximal stimulation was observed with approximately SOOunits/ml of TNF-a. At 24 h, thymocytes did not require ConA to proliferate, nor was their proliferative rate altered by theaddition of TNF-a (10 to 1000 units/ml) (Table 1, bottom).This is in agreement with our earlier observations (9), thatthymocytes do not increase their proliferative rate in responseto other activating agents during the first 24 h of culture,although by this time thymocytes express IL-2 receptors andsynthesize IL-2 (9, 22). Thymocytes preincubated with TNF-afor up to 4 h did not proliferate at a more rapid rate thanthymocytes cultured with Con A alone (data not shown), indicating that TNF-a alone does not prime thymocytes to respondmore vigorously to Con A.

In order to investigate the role of IL-2 in mediating thecomitogenic effect of TNF-a, we studied the binding of IL-2 toits receptor. As can be seen in Fig. 5, anti-Tac is only partiallyeffective in inhibiting the increase in ['Hjthymidine incorpora

tion by TNF-a of Con A-activated thymocytes, whereas anti-Tac completely inhibited proliferation induced by Con A alonein the absence of TNF-a. The effect of anti-TNF-a on thymocytes incubated with Con A (2 and 5 ng/m\) and varyingconcentrations of TNF-a is recorded in Fig. 6. Anti-TNF-a(10,000 units/ml) inhibits the increase in proliferation due toTNF-a of Con A-activated thymocytes completely, whereas itinhibits Con A-induced proliferation only partially, probablybecause it prevents synergy of endogenously secreted TNF-awith IL-2. The effect of TNF-a is specific, since it is abrogatedby anti-TNF-a. Taken together, these observations suggest that,in addition to IL-2, factors triggered by Con A act in concertwith TNF-a, and that these factors are not dependent on thebinding of IL-2 to its receptor.

TNF-a Enhances the Proliferation of Thymocytes Activatedwith IL-2. In order to explore whether IL-2 enhances theproliferative response to TNF-a, thymocytes were cultured withTNF-a in combination with varying concentrations of IL-2(Fig. 7). After 72 h of culture, IL-2-dependent ['Hjthymidineincorporation increases with increasing concentrations of IL-2.

TNF-o AND HUMAN THYMOCYTES

350

100 200

FL1

450

B

Q[IjiTTljTIlljlTTTj

100 2Q0FLi

Fig. 4. A, FACSCAN analysis of bound TNF-a. Thymocytes (6 x lO'/ml)

induced with TPA plus Con A were cultured for 48 h in microtiter plates, andthe binding of TNF-nr was determined by indirect immunofluorescence withmonoclonal antibody anti-TNF-<i as described in "Materials and Methods."

Bound TNF-« can be seen (solid line) by the binding of anti-TNF-« to the cellsurface. Thymocytes incubated in medium served as control (dolled line). B,FACSCAN analysis of uninduced thymocytes. Thymocytes (6 x 106/ml), which

were not activated, were cultured for 48 h in medium supplemented with exogenous TNF-« (1000 units/ml) (solid line). The binding of TNF-« was determinedby indirect immunofluorescence with monoclonal antibody as described in "Materials and Methods." No positive fluorescence was detected, since the solid line

is superimposed on the curve obtained from thymocytes cultured in unsupple-mented medium (dolled line) which served as control. Absence of fluorescenceindicates that TNF-« was not bound to uninduced thymocytes incubated inmedium supplemented with TNF-a.

This increase in incorporation is enhanced more than 3-fold bythe addition of TNF-a. However, proliferation induced by IL-2 alone or by IL-2 plus TNF-a is inhibited by anti-Tac, indicating that TNF-a enhances IL-2-induced proliferation, but is noteffective when IL-2 receptors are blocked by anti-Tac. In contrast as shown above (Fig. 5), anti-Tac was only partiallyeffective in inhibiting the increase in proliferative activity dueto TNF-a of Con A-activated thymocytes. Preincubation witheither IL-2 or TNF-«was not effective in enhancing the proliferative activity induced by IL-2 alone, indicating that primingcultures with one inducer was not adequate to induce an enhancement in proliferation.

CsA Abrogates the Proliferative Effect of TNF-a of Con A-activated Thymocytes. In an effort to further pinpoint the relationship between activation with Con A and the comitogenic

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TNF-tt AND HUMAN THYMOCYTES

Table 1 Effect of TNF-a on the proliferation of Con A-activated thymocytes

Human thymocytes were isolated as described (8). washed, resuspended (6 x10"cells/ml) in complete medium, and incubated in microtiter wells (total volume.200 n\) for 72 and 24 h, respectively, at 37*C in a humidified atmospherecontaining 5% CO;. Con A and TNF-a were added in the concentrations asindicated. Incorporation of [3H]thymidine. determined after a 6-h pulse, is expressed as cpm x 10~' per well. The mean of triplicate wells varied less than 10%.

All data recorded were obtained from the same thymic specimen.

20

TNF-a at the followingunits/mlPnnAGjg/ml)

0 10 100 250 500750cpmx ]0~3 at 72h0

0.9 0.9 0.8 0.6 0.50.7218.3 33.6 31.2 55.1 58.255.7549.5 51.0 83.0 87.3 93.098.9cpm

x 10~s at 24h0

16.7 18.3 15.0 13.4 ND13.7217.4 19.9 15.6 14.5 12.713.0518.5 19.1 19.4 18.4 18.416.350-40-ro0

30'*~XI

20-o10-oi

o.cO

O nosupplementsAA +TNF-aA-

••-A + TNF-a +anti-TNF-a^* + TNF—a + anti—Tac^^^•

--•+ anti-TNF-a^"•— •+ anti-Tac^",A-^'s

'^_^__—/

^^^~^T/

^^T^//*

^^^^/B=~—-H00

1.000 2.000 3.000 4.00010000.460.796.012.312.717.3T„

A^••^i^—

-—T0*5.000

ConA (/¿g/ml)Fig. 5. Enhancement of thymocyte proliferation by TNF-a (1250 units/ml)

and its inhibition by either anti-TNF-a (10,000 neutralizing units/ml) or anti-Tac (1/1000 dilution). Thymocytes were induced with the concentrations of ConA as indicated. Experimental conditions were as described for Table 1. Bars, SE.

to0Xno90-

80-

70-60-50-40-20^10,in.A

A ConA 2/¿g/ml• •ConA 5fj,g/m\A A ConA 2/¿g/ml+ anti-TNFaO O ConA 5¿¿g/ml+anti-TNFa•//

„-^'A>T ^^T.

I ,'i>---^C'skT*~~~~^-

A

0 100 200 300 400 500 600 700 800 900 1000TNF-a (U/ml)

Fig. 6. Inhibiting effect of monoclonal antibody anti-TNF-a (10.000 neutralizing units/ml) on the enhancement of thymocyte proliferation induced by ConA and increasing concentrations of TNF-a. experimental conditions were asdescribed for Table 1. Bars, SE.

effect of TNF-a, the effect of CsA on the induction of proliferation by Con A and Con A plus TNF-a was investigated (Fig.8). We show that CsA inhibits the proliferation not only ofthymocytes induced with Con A, but also of thymocytes inducedwith con A plus TNF-a, indicating that thymocyte activation isa prerequisite for the effect of TNF-a. By contrast, CsA is noteffective in inhibiting thymocytes proliferation induced by IL-

10-

£5

O O no supplementsA A + TNF-a• •+ anti-Tac* * + TNF-a + anti-Tac

.\L-2 (U/mlJ

Fig. 7. Enhancement of proliferation by TNF-a (1000 units/ml) of IL-2-induced thymocytes and the inhibition of proliferation by anti-Tac (1/1000dilution). Thymocytes (6 x 106/ml) were cultured for 72 h as described above.

Bars, SE.

n_o

15-

10-

5-

CD -CsAES +CsA I

n ÉMedia ConA ConA + TNF-a

Fig. 8. Antiproliferative effect of CsA (1 fig/ml) on thymocytes cultured withCon A (10 /Jg/ml) or con A plus TNF-a (1000 units/ml). Thymocytes cultured incomplete medium served as control. Thymocytes were cultured for 72 h asdescribed above. Bars, SE.

2 and by IL-2 plus TNF-a, since CsA is ineffective in inhibitingthe effects of IL-2 (25).

TNF-a Does Not Increase the Accumulation of Inositol Phosphates. The effect of TNF-a on the early transduction signalswas investigated, since we have demonstrated that Con A increases the formation of IPs (25) which serve as early transducing signals in the activation cascade from phosphoinositides inhuman thymocytes (Fig. 9). We found that TNF-a causes nofurther increase in the accumulation of IPs induced by Con A.The effect of TNF-a is apparently not transmitted by thispathway.

TNF-a Increases the Accumulation of IL-2R a-Chain (TacAntigen)-specific mRN A and the Expression of Tac Antigen onthe Cell Membrane of Con A-activated Thymocytes. To furtherelucidate the effects of TNF-a on the thymocyte activation, thebinding of PE-labeled anti-IL-2 receptor antibody to Con A-activated thymocytes was determined. TNF-a induced a moderate increase in the expression of Tac* thymocytes within 24

h as shown in Fig. 10; this increase was sustained for 48 h (datanot shown). The increase in the expression of Tac antigen onthe cell surface was reflected by a concurrent increase in theaccumulation of Tac mRNA. This effect of TNF-a was apparentat 4 h (data not shown) and was more marked by 7 h (Fig. 11).TNF-a alone did not induce the expression of Tac antigenmRNA, nor did it induce a detectable increase in the expressionof Tac antigen (data not shown).

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TNF-a AND HUMAN THYMOCYTES

to ici -TNFa[X] +TNFa

6-4-2-ri-LLS1?çX1$

Fig. 9. ['Hjlnositolphosphate content of thymocytes labeled to equilibriumwith [myo-'Hjinositol and activated with the indicated concentrations of Con Afor l h in the absence and presence of TNF-or (1,000 units/ml) was determinedby ion exchange Chromatograph). The results are expressed as the percentage oftotal radioactivity recovered (20,000 cpm). The experiment was done in triplicate.Bars, SE.

80-,

70-

60-

50-

40-

30-

20-

10-

0-

-TNF-a+TNF-«

0 ConA

Fig. 10. Expression of Tac antigen on thymocytes in response to varyingconcentrations of Con A and TNF-a (1000 units/ml). Thymocytes were culturedin complete medium for 18 h, and Tac antigen expression was determined bycytofluorometry as described in "Materials and Methods".

ConA

ConA TNF Control

sion of c-myc mRNA therefore reflects the activation of thymocytes. As seen in Fig. 12, Con A increases c-myc expressionwithin 4 h, prior to any increase in the proliferative rate, andthis increase is further enhanced by addition of TNF-a, indicating that TNF-a amplifies signal(s) generated by Con A,whereas it does not affect c-myc expression in cultures notsupplemented with Con A (data not shown).

DISCUSSION

In this study we demonstrate that TNF-a exerts immunoreg-ulatory functions on human thymocytes apparently by an autocrine mechanism. We found that thymocytes cultured withCon A plus TPA and depleted of adherent cells express mRNAspecific for TNF-a (Figs. 1 and 2). The majority of the thymo-cyte population was CD3+CD1 + (>70%), and depending on thespecimen, 40 to 70% were CD4+CD8+, indicating that the

majority of the populations studied were not fully maturethymocytes. The phenotype was not significantly different after72 h of incubation (data not shown). These depleted culturesdid not contain OKM*1 cells, and it is therefore highly unlikelythat the TNF-a-specific message was generated by adherentcells or by contaminating T-cells. We showed that the inductionof mRNA accumulation was inhibited by both CsA and Dex(Fig. 3). Earlier studies from our laboratory have shown thatthese drugs inhibit IL-2 and IFN-7 synthesis by thymocytes(22, 23). These observations indicate that the regulation of theexpression of TNF-a mRNA in thymocytes is similar to thatof the genes of other lymphokines. TNF-a can be detected onthe cell membrane of thymocytes (Fig. 4) only following activation. Since only activated thymocytes bind TNF-a, it isreasonable to postulate that TNF-a is bound to TNF-a receptors. The amount of TNF-a produced was not adequate to bedetected in the medium. Other investigators have shown thatTNF-a is secreted by monocytes and bound to their cell membrane (27). Only if large amounts of TNF-a were synthesizedby monocytes was TNF-a detected in the medium.

Additionally, we present evidence that TNF-a enhances thestate of activation of thymocytes suboptimally activated withCon A, as reflected by an increase in the proliferative activity(Table 1), by the expression of the gene coding for Tac antigen(Fig. 10) and the expression of Tac antigen (Fig. 9), and by anincrease in the expression of the protooncogene c-myc (Fig.11). Our earlier studies have shown that the expression of c-

ConA+

ConA TNF Control

Fig. 11. Slot-blot analysis of thymocyte total cellular RNA hybridized to II2R a-chain (Tac) cDNA. Thymocytes (1 x 107/ml) were cultured in the presenceof Con A (2 ¿ig/ml)or Con A (2 >ig/ml) plus TNF-n (1000 units/ml) as indicated.Control cells were cultured in complete medium with no additions. Twenty, 10,5, and I fig of RNA were applied for each condition.

c-myc mRNA Expression Is Increased by TNF-a in Con A-activated Thymocytes. Studies carried out earlier in our laboratory have shown that c-myc mRNA is expressed in freshlyisolated thymocytes and is increased within 4 h in thymocytesactivated with inducing agents (26). An increase in the expres-

Fig. 12. Slot-blot analysis of thymocyte total cellular RNA hybridized to c-myc cDNA. Thymocytes (1 x 10'/ml) were cultured for 4 h in the presence ofCon A (2 jig/mi) or Con A (2 ng/ml) plus TNF-«(1000 units/ml) as indicated.Control cells were cultured in complete medium with no additions. Twenty, 10,5, and 2.5 /jg of RNA were applied for each condition.

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TNF-«AND HUMAN THYMOCYTES

myccan be used as an index of thymocyte activation (26). Thus,TNF-a does not merely enhance the proliferation of activatedthymocytes, but enhances the expression of other genes important in immunoregulation. In this respect its action on thymocytes is similar to its effect on activated B-cells, which secreteIgM and to a lesser extent IgG in response to TNF-a (6).

The effects of TNF-a are specific, since they are abrogatedby anti-TNF-a antibody (Figs. 4 and 5), and they are dependenton thymocyte activation. The effect of TNF-a is not reflectedby an increase in the accumulation of IPs in Con A-activatedthymocytes (Fig. 8). The proliferative effect of TNF-a appearsto be dependent on a factor(s) induced in the course of activationwith Con A, since it is completely inhibited by the immunosup-pressant drug CsA (Fig. 7), which is known to inhibit geneexpression and production of lymphokines in activated T-cellsand thymocytes without affecting the generation of IP.<(22, 23,25, 29, 30). Furthermore, anti-Tac (Fig. 4), which blocks thebinding of IL-2 to both high- and low-affinity receptors (31),only partially inhibits the increase in proliferation of Con A-activated thymocytes stimulated with TNF-a. The proliferativeeffect of TNF-a is also dependent on IL-2, since TNF-a acts asa comitogen with IL-2 in the absence of Con A (Fig. 6).Induction of proliferation by IL-2 and the increase in theproliferative rate of thymocytes cultured with IL-2 and TNF-aare completely inhibited by anti-Tac (Fig. 6) and, therefore,dependent on the activation of thymocytes by IL-2. Our observations are in agreement with the findings of other investigatorswho have found that TNF-a enhances the activation of murinethymocytes (8) and augments IL-2-dependent IFN-7 synthesisand the expression of IL-2 receptors of phytohemagglutinin-activated T-cells (32). Our present observations illustrate someof the similarities in the spectrum of activity of IL-2 and TNF-a. Unlike IL-2, TNF-a acts only on activated human andmurine thymocytes (8), whereas activation is not a prerequisitefor response to IL-2 by thymocytes and certain T-cell subsets

(13).In conclusion, activated human thymocytes express the mes

sage for the TNF-a gene and produce TNF-a. TNF-a increasesthe state of activation and thus acts as an immunoregulatorylymphokine on both human and murine (8) thymocytes. Recentstudies by other investigators on the effect of TNF-a on thegrowth of murine sarcoma in vivo provided evidence that TNF-a exerts its tumoricidal effect by activating T-cell functions,since mice depleted of CD4+ or CD8+ T-cells failed to rejecttumors when treated with TNF-a, whereas TNF-a inhibitedtumor growth in control mice (33).

ACKNOWLEDGMENTS

We thank Dr. Grace Wong (Genentech, South San Francisco, CA)for the hybridization of our filters with the TNF-«probe, Julia Cohenand Janice Small for the preparation of the manuscript, and Mary LynMonson for expert assistance with the FACSCAN.

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1989;49:3568-3573. Cancer Res   Gabrielle H. Reem, Anne Duggan and Michael Schleuning  by Human Thymocytes

αImmunoregulation and Production of Tumor Necrosis Factor

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