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21. A.Fire, U. Samuels, P. A.Sharp, J. Biol. Chem.

259, 2509 (1984); D. K. Hawley and R. G.

Roeder, ibid. 262, 3452 (1987).

22. B. Corthesyetal.,Science239, 1137 (1988).

23. S.K.Yoshinaga andL. P.Freedman, unpublished.

24. K.A. Jonesetal., Cell 42, 559(1985).

25. C.Scheidereit and M.Beato, Proc.Natl. Acad.Sci.

U.S.A.81, 3029 (1984).

26. H.M. Jantzenetal., Cell 49,29 (1987).

27. Wethank W. Soeller for embryoextractsand discus- sions; M. Biggin, B. England,D. Granner, J. La- Baer,and D.Picard for DNAreagents;S.Johnson andR. Myers for criticalreadings of the manuscript;

and K. Mulherin andB.Malerfor preparation of the

textandfigures, respectively. Supported bygrants

from the National Institutes of Health and the NationalScience Foundation; postdoctoralsupport wasfrom the American Cancer Society, California Division(S.K.Y.), and from the Bank of America- Giannini Foundation (L.P.F.).

18April 1989; accepted2June 1989

Monoclonal Antibody-Mediated Tumor Regression by Induction of Apoptosis

BERNHARD C. TRAUTH, CHRISTIANE KiAS, ANKE M. J. PETERS, SIEGFRIED MATZKU, PETER MOLLER, WERNER FALK,

KLAUS-MICHAEL DEBATIN, PETER H. KRAMMER*

To characterize cell surface molecules involved in control ofgrowth of malignant lymphocytes, monoclonal antibodieswereraisedagainst the human Blymphoblastcell

line SKW6.4. One monoclonal antibody, anti-APO-1, reacted witha 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocytelines, andonsomepatient-derivedleukemiccells.Nanogram quantities of anti-APO-l completely blocked proliferation ofcells bearing APO-1 in vitro in a manner characteristic ofa process called programmed cell death or apoptosis. Cell deathwas precededby changes in cell morphology and fragmentation of DNA. This

process was distinct from antibody- and complement-dependent cell lysis and was

mediatedby the antibody alone. A single intravenous injection of anti-APO-1 intonul numicecarryingaxenotransplant ofahuman Bcelltumorinducedregression of this

tumorwithin afewdays. Histological thin sections of theregressingtumorshowed thatanti-APO-1wasabletoinduceapoptosisinvivo.Thus, induction of apoptosisas aconsequenceofasignal mediated throughcellsurface molecules likeAPO-1maybea

usefultherapeutic approach intreatmentofmalignancy.

CEELLSURFACE MOLECULES ARE CRU-

cial in lymphocyte growth control.

Such molecules mayfunctionas re- ceptorsforgrowth-stimulating cytokines or

be associated with receptors and transmit signals essential for growth regulation. Re- ceptorblockadeor removal of the stimulat- ing cytokinescanleadtodecreasedlympho-

cyte growth. Withdrawal of interleukins slowhuman lymphocyte growthandfinally leads to a characteristic form of cell death called"programmedcell death"orapoptosis (1). Apoptosis isthemostcommonform of eukaryoticcell death andoccurs inembryo- genesis, metamorphosis,tissueatrophy,and

tumorregression (2). It is also induced by cytotoxic T lymphocytes and natural killer and killer cells; by cytokines like tumor

necrosis factor (TNF) and lymphotoxin (LT); and by glucocorticoids (1, 2). The

most characteristic signs of apoptosis are

segmentation of the nucleus, condensation ofthe cytoplasm, membrane blebbing, and DNA fragmentation into multimers of about 180 base pairs (called a"DNA lad- der") (1, 2). To analyze mechanisms of lymphocyte growth control andtointerfere withthereplication of lymphoidtumorcells we raised monoclonal antibodies (MAbs) against cell surface molecules involved in these processes.

We found one MAb (anti-APO-1) that blocks growth and induces apoptosis of SKW6.4 cells (3). Anti-APO-1 (IgG3, K, KD= 1.9 x 10-10)bound toapproximately 4 x 104sitesonthesurfaceof SKW6.4cells

(4). It specifically immunoprecipitated an

endogenously synthesized protein antigen (APO-1) from SKW6.4 cells which, under reducing conditions,wasobservedonSDS-

Fig. 1. Molecular weight of the cell surface antigen 97 -

APO-1: immunoprecip-

itation ofbiosynthetically la- 68- beledAPO-1 fromthesur-

face of SKW6.4 cells with f

either isotype-matchedcon- 43- _ trol MAb(left lane)oranti-

APO-1 (right lane). The numbersonthe left margin indicatethe positions of the 25.7

sizemarkers.Cells(3x 106)

werelabeled with60,uCi of 75Se-labeled methionine 18.4 (Amersham, Braunschweig,

FRG) in 6 ml ofmethionine-free culture medium (Biochrom,Berlin) for 48 hours. Afterwashing, the cellswereincubatedin either control MAbor

anti-APO-1 (1 ,ug/ml) at 4°C for 45 min. The

cells werewashed and resuspended in lysisbuffer (tris-buffered saline, pH7.3, 1% Nonidet P-40, 1 mMphenylmethylsulfonyl fluoride, 0.1% apro-

tinin) at room temperature for 30 min. The lysates were centrifuged and supernatants were

incubated with protein A-Sepharose beads (Phar- macia, Uppsala,Sweden) at4°C for hour.The immunecomplexeswerewashed four times with buffer(tris-bufferedsaline, pH 7.3,0.25% Noni- det P-40) andresuspended in SDS-PAGE sample buffer containing 5% SDSand 5% 2-mercapto- ethanol. Thesampleswereheatedto95°C,centri- fuged,andcountsperminuteof thesupernatants weredeterminedina-y-counter.A total of 15,000

cpmwereloadedineachlane andanalyzedbya

10% SDS-PAGE (18). The gel was dried and subjectedtoautoradiography.

polyacrylamide gel electrophoresis (SDS- PAGE) as amain band of 52 kD (Fig. 1).

Apart from actin (43 kD), whichwasnon-

specifically precipitated with IgG3, anti- APO-1 specifically immunoprecipitated a

minor band of25 kD. This 25-kD protein might eitherrepresent a degradation prod-

uctorbenoncovalently associated with the 52-kD protein.

There are two major modes of death in nucleated eukaryotic cells. Necrosis as a

result, for example, of complement attack is characterized by swelling of the cells and

ruptureofthe plasma membrane causedby

anincrease inpermeability. Cells that under-

go apoptosis, however, show a different biochemical andmorphologicalpattern (2).

Thispatterncorrespondstotheoneinduced by anti-APO-1: condensation of the cyto-

plasm, membrane blebbing (Fig. 2a), and endonuclease-induced DNA fragmentation (5) into multimers of approximately 180 bp

B.C.Trauth,C.Klas, A. M.J. Peters,W.Falk,P. H.

Krammer,InstituteforImmunologyandGenetics,Ger- manCancerResearchCenter,Heidelberg, Federal Re- publicofGermany.

S.Matzku,InstituteforRadiologyandPathophysiology, German Cancer Research Center, Heidelberg, Federal RepublicofGermany.

P.Molier,InstituteforPathologyof theUniversityof Heidelberg,FederalRepublicofGermany.

K.-M.Debatin,Oncology/Immunology Section,Univer- sityChildren'sHospital,Heidelberg,FederalRepublicof Germany.

*Towhomcorrespondenceshouldbe addressed:

ZnCI2). Typical yields obtained were 1 mg of

>90% pure T7EX556 perliter of starting culture.

13. F. Payvar etal., Cell35, 381 (1983).

14. W.C.Soelleretal., GenesDev.2, 68(1988).

15. U.Heberlein and R.Tjian,Nature331,410(1988).

16. J.LaBaer, thesis,University ofCalifornia,SanFran- cisco (1989).

17. R.Schule et al., Science 242, 1418 (1988).

18. DNAfragmentswithout GREswereinserted into the Xba I site(see Fig. 2) of -33GRE6, displacing the GRE cassette to -262, -366, and -1069bp from the start of transcription. Weakreceptor-medi- ated activation wasdetected when GREsweresitu- ated at-262, butnoeffectwasobserved from the more distal sites.

19. A.Sergeant,D.Bohman,H.Zentgraf,H.Weihler, W. Keller, J. Mol. Biol. 180, 577 (1984); P.

Sassone-Corsi,A.Wilderman,P.Chambon, Nature 313, 458 (1985); H. R. Scholer and P. Gruss, EMBO J. 4, 3005(1985); Y.-S. Lin, M.F.Carey, M.Ptashne, M. R. Green, Cell54, 659 (1988).

20. L. P.Freedman, unpublished.

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(Fig. 2b). Affinity-purified anti-APO-1 in- duced growth retardation and cell death (Fig. 2c), which was not observed with either an isotype-matched, control MAb (FII20) [anti-MHC (majorhistocompatabi- lity complex) class I antigens] or the non- binding MAb FI123. Abrogation of [3H]thymidineincorporation along with in- creased trypan blue uptake into dead cells were observed, andgrowthof104SKW6.4 cellsin200-,ulcultureswasblockedbymore than 95% byananti-APO-1 concentration ofonly 10ng/ml(Fig. 2c).Thespecificity of celldeathinducedby anti-APO-1 becomes evident from the fact that the following additional control MAbs were inactive for inductionof apoptosis: 18 nonbinding and 9binding MAbs of the IgG3 isotype (tested by immunofluorescence on SKW6.4 cells) and apanel of MAbs directedagainst known antigensonthecell surface ofSKW6.4cells including CD 19, CD20,CD22,MHC class

II, IgM (immunoglobulin M), and the SKW6.4idiotype (6).

Cell death induced by anti-APO-1 was complement-independent andoccurred un- derserum-free culture conditionsor incul- turemedium plusseruminactivatedat56°C for30min. Itdiffered from death mediated by complement-dependent lysis by: (i)mor- phology and formation of a DNA ladder (Fig. 2, a and b), (ii) exogenous Ca2+

independence (7), and (iii) delayed trypan blue uptake and delayed

5'Cr

release from radiolabeled target cells (8). These experi- ments indicate that cell death induced by anti-APO-1isfumndamentallydifferent from antibody- and complement-dependent cell lysis.

To assessthespecificity ofanti-APO-1,a restricted panel of tumor cell lines was screened forexpression ofAPO-1 and sus- ceptibilitytogrowth inhibition andapopto- sis. APO-1wasexpressedonvarious human

B M 0 20 40 60 90 120

pK

Fig.2.Induction ofgrowth inhibition andapoptosis by anti- APO-1.(A)The T celllineCCRF-CEM.S2 (19)wascultured in the presenceofpurifiedMAb(1 ,ug/ml) inamicrotiterplate for 2hours before photography (left panel control MAb 13B1;

right panel anti-APO-1). (B) CCRF-CEM.S2 cells (106 per milliliter) were incubated with MAb (1 p.g/mi) in culture

medium at 37°C. At varioustimes, aliquots of106 cellswere 100- C removed and DNAwasprepared. M,marker;I, controlMAb

I3B1 for 2 hours; lanes 3 to 7, anti-APO-1 for the times .^ 00 indicated. (C) SKW6.4 cells were either incubated with the E

isotype-matched control MAb FII20 (0), FII23 (nonbinding ' MAb) (0),oranti-APO-1

(0)

in microcultures for 24 hours ° beforelabelingwith [3H]thymidineforafurther4hours. The 10i data represent themeanofduplicatecultureswithavariationof a) 1 less than 5%. Thecellswerecultured in RPMI 1640 medium X 1

(Gibco, Grand Island,NewYork), supplementedwith2mML- a 1 glutamine, streptomycin (100,ug/ml), penicillin(100 U/ml),20 \ mMHepesbufferpH7.3, and 10%heat-inactivated fetal bovine , 1

serum(ConcoLab-Division, Wiesbaden,FRG).Formicrocul- M tures, 1 x 104cells per wellwereculturedinduplicatesinflat- . \ bottom96-wellmicrotiterplates (Tecnomara,Fermwald,FRG) ' 1 (200 ,ulfinal volume perwell). After24hours,the cellswere -.

labeled with 0.5 ,uCi of [3H]thymidine (Amersham, Braun-

0o11

schweig, FRG) for 4hours. Before harvesting, the microcul- 1 01 102 103 104 tures wereexaminedbvmicroscopicinspection.DNAfragmen-

tation1 x 106 cellswerewashed with coldphosphate-buffered MAb(ng/ml) salineanddisruptedwith NTEbuffer,pH 8(100mMNaC1,10mMtris,1mMEDTA) containing1%

SDS andproteinaseK(0.2

mg/mi).

After incubation for 24 hoursat37°C,sampleswereextracted twice withphenolpluschloroform (1: 1, v/v) andprecipitated byethanol.The DNAwasdissolved in 38 ,ulof NTEbuffer anddigestedwith ribonuclease(1 mg/mi) for 30 minat37C. Toeachsample 10

RI1

of loadingbuffercontaining15%Ficoll 400(Pharmacia, Uppsala, Sweden),0.5%SDS,50mMEDTA, 0.05%bromophenol blue,0.05%xylene cyanolin TBEbuffer(2mMEDTA,89mMboricacid,89 mMtris,pH 8.4) were added. The mixture was loaded onto a 1% agarose gel and stained after electrophoresiswith ethidiumbromide(0.5,ug/ml).The size markerwasHind III+EcoRI-digestedX DNA.

2.5 E

0 2

(a oE : 1.5

0 a)

@ 1 -

E a)

0.5

0

S

0

* 0

* 0

*

S.0

. 0

0 00

0 0

0 0 0 A%

0 oo

A

0 14 0 14

Time afterinjection of antibodies (days)

Fig. 3. Anti-APO-1-induced regression of the EBV-negative Burkitt-like lymphoma BJAB in nu/nu mice. BJAB cells (4 x 107) were injected subcutaneously into the left flank ofnu/numice.

After 5 weeks (day 0) the micewereinjectedwith 500 ,ugof MAb into the tail vein. Control MAb FII20(]);FII23(0); I3B1 (A);and anti-APO- 1(0).Fourteendays later thesizeof thetumors was measured at the base of the tumor; the tumors from individual mice are represented by dots.

lymphoid B and T cell lines and was not foundon agibbonor mouseTcell lineora humanmonocytic cell line (Table 1). Anti- APO-1 blockedproliferationofthe APO-1- positivecell lines listedinTable 1via induc- tionofapoptosis, and formation ofaDNA ladderwasobservedineachcase(9).Expres- sionofAPO-1 was notrestrictedtocelllines in vitro but couldbe foundonleukemiccells freshly isolated from patients (Table 1).

Since APO-1was notfoundonallleukemic cells it may be possible that anti-APO-l definesa subpopulationof leukemias.

Wealso screened humanBandTcellsfor expression of APO-1. We did not detect APO-1onrestingBcells.However,APO-1 wasexpressedonactivatedBcells (Table 1) and IgM secretion was reduced approxi- mately fourfoldby3daysoftreatmentwith anti-APO-1 (10). PeripheralrestingTcells did not express APO-1. Activated T cells, however, expressed APO-1 and anti-APO- 1-inducedapoptosis andgrowth inhibition of these cells (Table 1). Thus, our data suggestthat APO-1is aspecies-specificanti- gen expressed on activated or malignant lymphocytes.

Thestrikingeffect of anti-APO- 1 in vitro prompted us to test its effect on tumor

growth in vivo. Although the Epstein-Barr virus (EBV)-negative, Burkitt-likelympho- ma BJAB was the least sensitive to anti- APO-1 of the B cell panel inTable 1 and expressed only approximately 1.5 x 104 APO-1 epitopes per cell (4), we selected BJAB for our in vivo experiments. The reason for this choice was that only BJAB grew tolarge tumor massesin unirradiated nu/nu mice. Five weeks after injection of

SCIENCE,VOL. 24S 302

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Fig. 4. Localization of anti- A

APO-1 in xenografts ofBJAB 1 2 3

innu/nu mice and inductionof apoptosisof thetumor.(A)Up- - perrow:

uptake

of'251-labeled

MAb anti-APO-1 (50

p.g,

50 - -

,uCi

permouse)inthetumor at

12hours(1),48hours(2),and 96 hours (3) after intravenous injection of the MAb. Lower

row: uptake of I5-labeled MAb anti-APO-1 (500

,ug,

as used for therapy; 50 ,uCi per mouse) (4) and FII20 (control

MAb,bindingtoBJAB;50 ,tgr, 4 5 6

50,uCipermouse)(5)and125-

labeled FII23 (control MAb, nonbinding to BJAB; 50 ,ug, 50

p.Ci

per mouse) (6) in 48 hours, respectively. (B) Ten days after intravenous injection of 500 ,ugofMAbper mousethe remaining tumortissuewas removed andfixed with formalin. Paraffin sections of thetumorwerestained with haematoxylin/eosin. Left panel, tumor after

treatmnent

with control MAbFI120; right panel, tumoraftertreatmentwith anti-APO-1. Arrows

BJAB

cells the nu/nu mice carried tumors

withadiameterof

approximately

1.0to2.5

cm

(Fig. 3).

These micewere

injected

intra-

venously

with

purified anti-APO-l (500 p.g

per

mouse)

orthesame

quantities

of various

isotype-matched

control antibodies (FII20, anti-MHC class I

antigens, recognizing

5.8 x 105 sites per

cell;

or one of the two

nonbinding

MAbs F1123 and

13B1).

As a

control we also

injected

anti-APO-1

(500

,ug per mouse) into three nu/nu mice

carrying

the

APO-1-negative

B cell tumor OCILY1withtumordiameters of1.5, 1.8, and 3.4cm,

respectively

(11) (seealso Table

1).

Two

days

afteranti-APO-1

injection,

a

whitish discoloration of the

BJAB

tumors was observed that was followed

by rapid

tumor

regression. Macroscopic

tumor re-

gression

was seen in 10 of 11 treated mice within less than 14

days.

The control anti- bodies hadnoeffect

(Fig. 3).

In

addition,

no

tumor

regression

was observedin the mice

carrying OCI.LY1,

as

expected.

To demonstrate proper localization and enrichment of the

injected antibodies,

la- beled MAbswerevisualized

by autoradiog- raphy

ofsectionsof the

BJAB

tumortissue

(Fig. 4a).

These

autoradiographs

showed a

pronounced binding

ofanti-APO-1 inthe

periphery

but

only

sparse accumulation in thecenterof thetumor.The

binding

control MAb FII20 showed a

qualitatively

similar

binding pattern.

There wasno localization of the

nonbinding

control MAb FI123 above

background. Furthermore, paired

la- bel

experiments (12)

with labeled anti- APO-1 and FII23 revealedthat the

specific

enrichment of anti-APO-1 over FI123 in thetumorwasfour- and sixfold after48and 96

hours, respectively.

The main purposeofour

experiments

was toassesswhether anti-APO-1canalsoactin vivo.

Therefore,

the

tumor-bearing

mice

only

received one intravenous

injection

of anti-APO- 1 at adose in the rangeused in

21 JULYI989

indicatehost vessels. Finalmagnification, x 92.MAbswereradioiodinated accordingtotheIODO-Gen method (4). Labeled MAbswereinjected into thetail vein and animalswerekilled byether anesthesiaatthepredetermined timepoints.Thetumors wereexcised andembedded inmethylcelluloseand 20-,umcryotomesectionswereprepared. Lyophilizedsectionswereplaced onaKodak X-omat AR filmforautoradiography.

Table1.Reactivityofanti-APO-1with different cells.

Cells Relative Effects of MAbs on

Cells* positive fluorescence [3H]thymidine uptake

for intensity (103cpm)t

APO-1 (anti-APO-1/

Type Designation (%)t control) Control Anti-APO-1

Malignantcell lines

Hu B cells SKW6.4 98 11.1 30.0 0.02

CESS 95 12.0 23.0 0.1

BJAB 80 2.1 70.0 7.0

OCI.LY1 0 1 14.5 15.8

Hu Tcells Jurkat 83 2.3 20.3 8.1

Molt 91 2.4 35.7 0.6

CCRF-CEM 64 1.9 16.2 0.5

Hu myeloidcells U937 5 0.97 62.2 60.5

Gibbon T cells MLA 144 0 0.96 34.3 35.0

MouseTcells EL4 0 1 44.8 45.3

Leukemiccellsfrompatients§

PreT-ALL B.M. 54 4.4

T-ALL D.A. 53 3.2

CommonALL W.N. 72 5.0

Normalhumanlymphocytes

Tcellsll Resting 3 1.36

Activated 89 7.4 22.4 0.23

Bcells¶ Resting 0 0.9

Activated 91 1.1

*Hu,human; ALL,acute

lymphocytic

leukemia. tAliquotsof106 cellswereincubatedat4°C in 100 ,ul of medium with control MAb

(RF23

orI3B1)oranti-APO- 1for 30 min. Then the cellswerewashed and stainedwith fluoresceinisothiocyanate-coupledgoat anti-mouseIg F(ab')2(70iLg/ml)andanalyzed by acytofluorograph (Ortho DiagnosticSystems,Westwood,Massachusetts). tCells(104perwell)werecultured in the presence of MAb(500 ng/ml) for 24 hoursandlabeledwith [3H]thymidine for2 hoursbeforeharvest; thedata representthemeanof duplicate cultures with a variation of less than 5%. §Bonemarrow cells isolated from the patients were

mOrphologicay>95% blasts and showed thefoHowing phenotype: pre T-ALL, cytoplasmicCD3+, CD5+,CD7+, CD34,Tdt+, CD2-,surface CD3-, CD4-,andCD8-;T-ALLCD2',cytoplasmic CD3',CD5', CD7' and Tdt+, surface CD3-, CD4-,CD8-, andCD34-; common-ALLCD10+,CD19+, CD22+,CD24+,CD20-. The effect ofanti-APO-1 ontheseleukemic cellswas nottested, becausetheydied under normal culture conditions.

IIPeripheral blood mononuclear cells (PBMC) fromhealthyvolunteerswereisolatedbyFicollPaque(PharmaciaInc., Uppsala, Sweden) density centrifugation. Adherent cells were removed by adherence to plastic culture vessels overnight. T cellswereisolatedfrom PBMCbyrosettingwith 2amino-ethylisothyouronium-bromide (AET)-treated she red blood cells as described(20). Freshly preparedresting Tcells (2x10'permilliliter;96%OKT11+, 1%

Tac)wereactivated withphytohemagglutinin-M(50kg/mn)andPMA (10ng/nil)(SigmaChemicalCo.,Munich, FRG). Two, 7, and 12 days later the Tcellswerefed with 20to30U/miof recombinant humaninterleukin-2 (20to 30U/mi).Tcells (5 x 105permilliliter) activatedfor 12days (90% OKT11+;60%Tac+)werecultured in the presenceofFI123oranti-APO-1 (1 pLg/ml)intriplicatesfor 24 hours and then labeled with [3H]thymidinefora further 17 hours (see legend to Fig. 2). ¶RestingBcells(35.8%CD19')wereisolatedbytworoundsofrosetting asabove,followedb: separation viaaSephadexG-10columnasdescribed (21). For activatedBcells,PBMCwere adjusted to2 x 10 cells permilliliterand cultured in the presence ofpokeweedmnitogenat 10 j±g/ml (Serva, Heidelberg, FRG) for6days.Deadcellsand Tcellsweretheneliminatedby rosettingwit-hAET-treatedsheepred bloodcells andsubsequentcentrifugationoverFicollPaque.Theinterphasecellswereusedasactivated B cells(84%

sIgM+).

MAbtherapies.In othertherapy schedules, tumor wasobservedinthreeof thetenmice however,MAbsareinjectedrepeatedly(13). in which tumor regression had been ob- In our experiments regrowth of the BJAB served (Fig. 3). Regrowth was observed at

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the margin of the original tumor approxi- mately 3 months after the initial macroscop- ic tumor regression. One of these tumors was removed and found to express APO-1 byimmunofluorescence and to be sensitive toanti-APO-l in vitro at a MAb concentra- tion similar to the original in vitro BJAB tumorcell line (Table 1).

Todetermine the histology of the regress- ing BJAB tumors we prepared thin sections of tumors from MAb-treated nu/nu mice.

Ten days after intravenous injection of F1120, BJAB appeared as a solid tumor composed of densely packed large blasts withnumerous mitoses, some tumor giant cells,and rare apoptotic figures (Fig. 4b, left panel). The tumor was penetrated by host vessels. In contrast, almost all remaining BJABcellsof mice treated withanti-APO-1 (Fig. 4b, right panel) showed severe cyto- pathic changes including nudear pycnosis and cellular edema most pronounced in perivascular microareas. These morphologi- cal changes arecharacteristicofapoptosis.

Takentogether, these data strongly sug- gestthatapoptosis is inducedbyanti-APO- 1and is themechanismof death and regres- sionofBJAB tumorcells in vivo. The fact that FII20, which strongly binds to the cell surface of BJAB tumor cells, did not cause regression ofBJAB also precludes the possi- bility that killer cells or complement that might have bound toanti-APO-1mayhave been involved in thegrowth inhibition and tumorregression.

We showed that anti-APO-1 specifically blockedgrowth andtriggeredprogrammed cell death (apoptosis) ofa set of activated normal lymphocytes and cells from malig- nant lymphocyte lines after binding to the cellsurfaceproteinantigenAPO-1.Recent- ly, it has been shown that anti-CD3 induces apoptosis of immature thymocytes in vitro (14). Therefore, it has been suggested that CD3-triggered apoptosis might be responsi- ble for negative selection of T cells in the thymus. Since APO-1 is expressed on ma- ture activated lymphocytes, additional ex- periments will be needed to determine whether the antigen might play a similar role in the downregulation of the immune response and be involved in selection and eliminationoflymphocytes. It has previous- ly been shown that LT, TNF, and killercells with theireffectormoleculesinduce apopto- tic cell death (15). As anti-APO-1 also induces apoptosis a number of possibilities might be considered for the physiological roleoftheAPO-1antigen.APO-1mightbe a receptor for cytotoxic molecules or for autocrine growth factors. Alternatively, it could bea moleculeessential for verticalor lateral growth signal transduction. Thus, anti-APO-1 might triggerreceptorsforlytic

molecules or block receptors for growth signals.

Apoptosis is found inall tissues and also in cells from lower organisms (16). It is conceivable, therefore, that several distinct cell surface antigens with a different tissue distribution are involved in the induction of apoptosis. Elucidation of the structure of APO-1, its possible connection to the cyto- skeletonand the molecular eventsfollowing anti-APO-1 binding might resolve some of theseissues.

Our data might also have clinical rele- vance. APO- 1 was found on somelymphoid tumor cells freshly isolated from patients.

Thus, anti-APO-1 might be usefufl as a diagnostic tool to define subsets of normal and malignant lymphocytes. In addition, induction of apoptosis may have implica- tions for anti-tumor therapy. Antibodies have frequently been used as heteroconju- gateswith toxins ordrugs to destroy tumor cells (17). Our data, however, show that MAb alone can be lethal to target cells.

Anti-APO-1 and related MAbs might, therefore, be considered for ex vivo or in vivotherapy, under conditions where reac- tivity with vitalnormalcellscanbeexcluded ortolerated. Finally, the molecular investi- gationof cell death inducedbyanti-APO-1 might lead to a general understanding of apoptosis. In this case, the useof modified ornormal physiological ligands tothe cell surface antigen initiating apoptosis or of chemicalsinterfering with the apoptotic sig- nalmightbeenvisaged.

REFERENCESAND NOTES

1. E.Duvall andA. H.Wylie,Immunol. Today 7, 115 (1986).

2. A. H.Wylie,J. F. R.Kerr, A. R.Currie,Int. Rev.

Cytol. 68, 251 (1980).

3. BALB/c micewereimmunizedonce perweekovera 4-week period by intraperitoneal injection of 1 x107 SKW6.4 cells. Four days after the last injection,spleencells from immunizedanimalswere fusedwith theP3.X63.Ag8.653myeloma[G.Koh- ler and C. Milstein, Nature 256, 495 (1975)].

Twelve daysafterfusionculturesupematants from wellspositivefor growthwere testedfortheirability toinhibitgrowth of SKW6.4 cells.Hybridomas that producedblocking MAbswerecloned three times bylimitingdilution at a concentrationof0.5cells perwell.MAbswerepurifiedfromserum-freecul- turesupernatantbymeansofaproteinA-Diasorb column (Diagen,Dusseldorf,FRG).Bound MAbs wereelutedwith 0.1M NaCl and 0.1Mglycine,pH 2.8,dialyzedagainstphosphate-bufferedsalineand sterilized.Theisotypeofthe MAbs wasdetermined byenzyme-linked immunosorbentassay[S. Kiesel, etal., Leuk. Res. 11, 1119, 1987) with isotype- specificgoatanti-mouseIg that hadbeenconjugated withhorseradish peroxidase(Dunn,Asbach,FRG).

4. Affinityandnumber ofanti-APO-1 bindingsites percellwere determined by Scatchard analysis as described[I. von Hocgen, W.Falk,G.Kojouharoff, P. H.Krammer,Eur.J. Immunol.19, 329 (1989)].

Briefly, MAbs were iodinated bytheIODO-Gen method [P. J. Fraken and J. C. Speck, Biochem.

Biophys. Res. Commun. 80, 849 (1980)].Aliquotsof 5 x10' cellswere resuspendedin 200,ulofculture medium containing 0.1% NaN3anddifferentcon-

centrations of "LI-labeledMAbs.After incubation at 4°Cfor4hours, two95-pAl portions were re- moved andcentrifuged asdescribed aboveby von Hoegenetal.

5. A.H.Wyllie,Nature284, 555 (1980).

6. Monoclonal anti-CD19 (HD37) and anti-CD22 (HD39)were kindly providedby B.Dorken(Poli- clinic ofthe University, Heidelberg, FRG) and monoclonal anti-CD20 by G. Moldenhauer (IV Leukocyte typing workshopandconference,Vien- na,Austria, 1989),respectively. The18nonbinding and9bindingMAbsoftheIgG3isotype(testedby inmmunofluorescence on SKW6.4 cells) and the MAbsdirected against MHC class II, IgM, and SKW6.4Igidiotypes were raisedin our own labora- tory.

7. The kinetics of membrane blebbing induced by anti-APO-1 (within 30 min; Fig. 2a) was not influenced by the presence of10 mM EDTA or EGTA. In addition,endonudease-mediated DNA fragmentationinduced by anti-APO-1 was not in- hibitedbytheCa2" channel blockersFuramicin(50 FM) orNifedipin(50;.M).

8. When51Cr-labeled SKW6.4cells were incubated with anti-APO-1 (1pg/ml) for 2, 4, 8, and 24 hours, the specific 5"Crrelease [R. C. Duke, R.

Chervenak, J. J. Cohen,Proc.Nati. Acad. Sci.U.S. A.

80, 6361 (1983)] wasfoundto be 2.9%, 7.6%, 21.3%,and32.5%,respectively.Trypanblueuptake wasmeasuredatthesametimepoints: 2.5%, 4.7%, 10.6%,and 73.6%,respectively, of the cellswere trypanblue-positive.Incontrast, 2 hoursafterthe addition ofMAbs, plus complement the specific 51Cr release was 108.7% and 92.7% ofthe cells stainedwithtrypan blue.

9. Twohours after addition of MAbs(1 Rg/ml)the genomicDNAofeach tumorlinewasisolatedand analyzed on agarose gels as described (Fig. 2).

Inhibitionof[3H]thymidine uptakebyanti-APO-1 was paralleled by fragmentation of the genomic DNA. This was notobserved after treatment with control MAb(13B1).

10. Activated Bcells(106 per milliliter)wereincubated inthepresenceof MAbFI123oranti-APO-1 at 1

ug/mi. After3days theculturesupematants were collectedand the IgM concentration measured with ahuman IgM-specific ELISA containingHRPO- conjugated goat anti-human IgM (Medac, Ham- burg, FRG). IgM secretion after treatmentwith FII23 or anti-APO-1 was 2100 and 550 ng/ml, respectively.

11. OCI-LY1wasobtained fromH.Messner,Ontario CancerInstitute,Toronto, Canada.

12. D. Pressman etal, CancerRes. 17, 845(1957).

13. S. L. Brown etal.,Blood73,651(1989).

14. C. A.Smithetal.,Nature337,181 (1989).

15. D.S.Schmid, J.P.Tite,N. H.Ruddle,Proc.Natl.

Acad.Sci. U.S.A.83,1881(1986);G. B.Dealtry, M. S. Naylor, W. Fiers, F. R. Balkwill, Eur. J.

Immunol.17,689 (1987);M. M.Donetal.,Aust.

J. Exp. Biol. Med. Sci. 55, 407 (1977); C. J.

Sanderson,Biol.Rev.56,153(1981); J.H.Russell and C. B.Dobos,J. Immunol.125,1256(1980);D.

M.Howell andE.J. Martz,Immunology140,689 (1988); J. C. Hiserodt, L. J. Britvan, S. R.Tag Targan, J. Immunol. 129, 1782 (1982); J. D.-E.

Young and C.-C. Liu, Immunol. Today 9, 140 (1988).

16. F.Giorgi andP.J.Deri,Embryol.Exp.Morphol.35, 521(1976).

17. E.S.Vitettaetal., Science219,644(1983).

18. V. K.Laemmli,Nature277,680(1970).

19. The CCRF-CEM.S2 subclone was obtained by doning cells underlimitingdilution conditions from the CCRF-CEMTcell lineatonecellperwellin96- wellmicrotiterplates.CCRF-CEM.S2wasselected because ofitshigh sensitivitytoprogrammedcell death inducedbyanti-APO-1(500ng/ml)asmea- suredby microscopic inspectionin a4-hourculture.

20. M. Madsen et al., J. Immunol. Methods 33, 323 (1980); M. A. Pellegrino et al., Clin. Immunol.

Immunopathol. 3,324(1975).

21. T. R.Jerrells,J.H.Dean,G. L.Richardson,D. B.

Hcrberman,J.Immunol. Methods32,11(1980).

22. WethankK.Hexel,J.K6llner,R.Kuihnl,C.Mandl, andW.Muller for excellenttechnicalassistance;H.

Sauter for excellentsecretarialassistance;G.Ham-

merlingandG.Moldenhauer for theircriticism;B.

SCIENCE,VOL. 245

304-

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gie, Bonn, theMinisterium fuir Wissenschaftund Kunst, Stuttgart, and theTumor Center Heidel- berg/Mannheim, FRG. A.M.J.P.wassupported by the Boehringer IngelheimFonds,Stuttgart, FRG.

3February 1989; accepted19 May 1989

The Reservoir for HIV-1 in Human Peripheral Blood Is

a

T Cell That Maintains Expression of CD4

STEVEN M. SCHNITrMAN,* MILTIADES C. PSALLIDOPOULOS, H. CLIFFORD LANE, Louis THOMPSON, MICHAEL BASELER, FERDINANDMASSARI, CECIL H. Fox, NoRMAN P. SALZMAN, ANTHONY S. FAUCI

Human

immunodeficiency

virustype 1(HIV-1) selectively infects cells expressing the CD4molecule, resultinginsubstantialquantitative and qualitative defects in CD4+ T lymphocyte function in patients with acquired

immunodeficiency

syndrome (AIDS).

However, onlyaverysmall number of cells intheperipheral blood of HIV- 1-infected individualsareexpressing virusatanygiven time. Previous studies have demonstrated that in vitro infection ofCD4+ T cells with HIV-1 results in downregulation of CD4 expression such that CD4 protein isnolonger detectableonthesurface of the infected cells. In the present study, highly purified subpopulations of peripheral blood mononuclear cells (PBMCs) from AIDS patients were obtained and purified by fluorescence-automated cellsorting. Theywereexamined with themethodologies of virusisolationbylimitingdilutionanalysis, in situ hybridization, immunofluorescence, andgeneamplification. WithinPBMCs,HIV-1 wasexpressed in vivo predominantly inthe T cell subpopulation which, incontrast tothe in vitro observations, continuedto express CD4. The precursor frequency of these HIV-l1-expressing cells was about 1/1000 CD4+ T cells. The CD4+ Tcellpopulation contained HIV-1 DNA in all HIV- 1-infected individualsstudied and thefrequencyinAIDS patientswasatleast 1/100

cells.Thishigh level of infectionmaybe theprimarycausefor the progressivedecline in numberandfinctionofCD4+ T cells in patients with AIDS.

THHE HUMAN IMMUNODEFICIENCY

virus type 1 (HIV-1), the etiologic

agent ofthe acquired immunodefi- ciency syndrome (AIDS), selectively infects

cells expressing the CD4 molecule, includ- ing T lymphocytes and cells of the mono-

cyte/macrophage lineage (1). In vitro infec- tion of cells with HIV-1 results in a de- creasedexpression of the CD4 moleculeon

the surface of theinfected cells (2).

Patients with AIDS have severe depres- sion of the normal cell-mediated immune mechanisms that ispartially attributedtothe considerabledepletion of CD4 lymphocytes (3). Despite this, examination ofcells from lymph nodes and peripheral blood from patients with AIDS and AIDS-relatedcom-

plex (ARC) has revealedaverylowfrequen-

cyof viral RNAsynthesis, generally occur-

ring in 1/100,000 to 1/10,000 of total mononuclearcells(4). However, it is possi- ble thata larger proportion ofcellsmay be latently infected (containing proviral DNA butnotexpressing viral mRNAorprotein).

Until thedevelopmentofgeneamplification [polymerasechain reaction (PCR)]method- ology (5, 6), HIV-1-infected cells not ex-

pressing viruswerenotreadily detectable by

availabletechniques.

Inthepresentstudy, bloodwasobtained from HIV-1 culture-positive patients with AIDS either directly in heparinized syringes

or via apheresis and subjected to Ficoll- Hypaque separation (7). First, peripheral blood mononuclear cells(PBMCs) frompa-

tientswerestained with fluorescein isothio-

cyanate (FITC)-conjugated antibody to

CD3 and sorted bya fluorescence-activated

cell sorter (FACS) into CD3+ and CD3- populations. Sorted cells were cocultivated with anexcess of normalphytohemaggluti- nin (PHA)-stimulated blast cells and we

determined the time to peak viral expres-

sion, a highly consistent and reproducible

parameter of viral expression. A predom- inance ofHIV-1 expression in the >98%

enriched CD3+ population, as determined bythe timetopeak syncytiaformation(Fig.

1A) andreverse transcriptase (RT) activity (Fig. 1B), was seen. Similar results were

obtained insevenadditional AIDSpatients.

Delayed expression of HIV-1 in cells that

wereinitially99%CD3-cells(Fig. 1B)was

duetooutgrowthof the fewcontaminating CD3+ cells. Phenotypic analysis ofnonco-

cultivated enriched CD3- cellsgrownwuder

thesameconditionsrevealed that 35to65%

ofthe cellswere CD3+by day10inculture.

In the second series of experiments, PBMCs from AIDS patients were double- stained with FITC-conjugated anti-CD3 and anti-CD4 and sorted by FACS into CD3+/CD4+ and CD3+/CD4- popula- tions. These sorted cells were cocultivated with an excess ofnormal PHA-stimulated blast cells and showed a predominance of HIV- 1expression in thehighly enriched(98 to99%) CD4+ T cellpopulation as deter- minedby the time to peak syncytiaforma- tion (Fig. IC) and RT activity (Fig. ID).

Similar resultswereobtained inseven addi- tional AIDSpatients. The phenotypic analy- sis of freshly sorted CD3+/CD4+ cells re- vealed a greater than 98 to 99% CD4+

purity in most experiments when stained with the monoclonal antibody to Leu 3a.

Again, the delayed expression ofHIV-1 in cells that were initially 99% CD4- (Fig.

ID) was mostlikely duetooutgrowth ofa few contaminatingCD4+Tcells.Phenotyp- ic analysis of non-cocultured enriched CD4- Tcellsgrownunder thesamecondi- tions revealed that 30 to 55% of the cells wereCD4+ by day 10 in culture.

In situ hybridization for HIV-1 viral RNAwas then performed at time zero on the highly enriched CD3+/CD4+- and CD3+/CD4--sorted PBMCs. There was a predominance of viral expression in the CD4+ T cell population at a frequency of about 1/1000cellsinfourAIDSpatients

(X

± SEM per 1000 cells was 0.95 ± 0.21) (Fig. 2A). Thisis incomparisonto alevel of viralexpression in the CD4-Tcellpopula- tionof<1/100,000 cells(Fig. 2B), whichis equivalenttobackground signal in controls.

The frequency ofin situ-positive CD4+ T cells remained unchanged in three of the patients reexaminedat6 to 12months after the initial studies.

Indirect immunofluorescence studies for HIV-1viralantigenswas alsoperformedat timezero onhighlyenrichedCD3+/CD4+- and CD3+/CD4--sorted PBMCs. These demonstrateapredominanceof viralexpres- sion in the CD4+ T cell population at a

frequency of about 1/1000 cells in four AIDS patients (X ± SEM per 1000 cells was 1.10 ± 0.35 (Fig. 2C). This isin com- parison to alevel of viralexpression inthe CD4- Tcellpopulation of<1/10,000 cells

S.M.Schnittman,H. C.Lane,F.Massari,C.H.Fox,A.

S. Fauci, Laboratory of Immunoregulation, National InstituteofAllergy and Infectious Diseases, National InstitutesofHealth,Bethesda,MD20892.

M. C. Psallidopoulos, L. Thompson, N. P. Salzman, Division of Molecular Virology and Immunology, Georgetown UniversitySchool ofMedicine, Washing- ton,_DC20007.

M. Baseler, ProgramResources, Incorporated, Freder- ick, MD 21701.

*Towhomcorrespondenceshould be addressed.

Dorken and G. Moldenhauer for providing MAbs, andDr.H. Messnerfor providing tumor cells. We also thank D.Scheppelmann and H.-P. Meinzer for the computerized image analysis of normal and apoptotic cells. Supported by grants from the Bundesministerium firForschung und Technolo-

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