JOURNAL OF VIROLOGY, Apr. 1994, p. 2598-2604 Vol. 68, No. 40022-538X/94/$04.00+0Copyright X 1994, American Society for Microbiology

Cytokine-Mediated Induction of Human ImmunodeficiencyVirus (HIV) Expression and Cell Death in Chronically Infected

Ul Cells: Do Tumor Necrosis Factor Alpha and GammaInterferon Selectively Kill HIV-Infected Cells?

PRISCILLA BISWAS,1* GUIDO POLI,1t JAN M. ORENSTEIN,2 AND ANTHONY S. FAUCI'Laboratory of Immunoregulation, National Institute ofAllergy and Infectious Diseases, National Institutes of Health,

Bethesda, Maryland 20892,1 and Department of Pathology, George Washington University Medical Center,Washington, D.C. 20007

Received 13 September 1993/Accepted 5 January 1994

Infection with several DNA or RNA viruses induces a state of increased sensitivity to cell lysis mediated bytumor necrosis factor (TNF), particularly in the presence of gamma interferon (IFN-y). Infection of humancells with the human immunodeficiency virus (HIV) may induce a similar phenomenon. However, TNF andIFN-'y are known upregulators of HIV replication, raising the question of the potential role of these cytokinesin the selective elimination of cells infected with this virus. The present study demonstrates that chronicallyinfected Ul cells were killed with much greater efficiency by costimulation with TNF-a and IFN-y than theiruninfected parental cell line U937. However, synergistic induction of viral expression also occurred in Ul cellsas a consequence of treatment with the two cytokines. Cell death in Ul cells was not caused by the massiveproduction of virions, in that costimulation with glucocorticoid hormones and TNF-ae or IFN-y resulted in highlevels of virion production without cytopathicity. To investigate the nature of the selective cytotoxic effectobserved in Ul cells costimulated with TNF-a plus IFN--y, a panel of uninfected cell clones was generated bylimiting dilution of U937 cells and tested for response to TNF-a and/or IFN-,y. In contrast to the uncloned bulkparental U937 cell line, most uninfected cell clones showed a very high susceptibility to being killed by TNF-oaand IFN-y. Similar findings were obtained when both infected Ul cells and several uninfected U937 cell cloneswere costimulated with an anti-Fas monoclonal antibody in the presence of IFN-'y, although, unlike cellsstimulated with TNF-a, cells treated with anti-Fas antibody did not express virus. Therefore, the increasedsusceptibility to cytokine-mediated lysis observed in cell lines infected with HIV is likely due to the selectionof preexisting cell clones rather than viral infection.

Several cytokines have been implicated in the immuno-pathogenesis of human immunodeficiency virus type 1 (HIV-1)infection, both as potential mediators of the immune dysfunc-tion typical of this disease and as direct modulators of virusexpression (22). In particular, tumor necrosis factor alpha(TNF-a) (13, 18) is known to induce HIV-1 transcription andexpression via activation of the cellular transcription factorNF-KB, which binds to the promoter-enhancer region of thevirus long terminal repeat (9, 15, 20). Interferon-gamma(IFN-y) can also activate HIV expression in several cells (17,22), including the chronically infected promonocytic cell lineUl (3). However, earlier reports indicated that treatment ofHIV-1-infected cell lines with TNF resulted in a decrease ofviral RNA synthesis, particularly in the presence of IFN--y (25).This possibility was consistent with earlier studies that demon-strated that infection of cells with several DNA or RNA virusesinduced a state of increased susceptibility to cell lysis by TNF(6, 14, 24). On the basis of these early findings, clinical trialsinvolving the individual and combined use of TNF-ot andIFN--y have been conducted with HIV-infected individuals,

* Corresponding author. Mailing address: Laboratory of Immuno-regulation, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Building 10A, Room 6A33 A, 9000Rockville Pike, Bethesda, MD 20892.

t Present address: AIDS Immunopathogenesis Unit, DIBIT, SanRaffaele Scientific Institute, Milano, Italy.

with inconclusive results in terms of whether cytokines werebeneficial or accelerated disease progression (2). Other in vitrostudies (19) confirmed that TNF could mediate lysis of severalHIV-infected cell lines but not of their parental uninfectedcounterparts. However, in contrast to an earlier report (25),cell lysis was associated with increased rather than decreasedvirus expression (19). Furthermore, the promonocytic U937cell line appeared to be more susceptible to TNF-induced lysisbefore rather than after infection with HIV (19).

Recently, the possibility of triggering lysis of TNF-sensitivetarget cells without inducing the regulatory effects associatedwith cell stimulation with this cytokine (such as activation ofNF-KB) has been demonstrated by use of monoclonal antibod-ies (MAbs) directed at the cell surface Fas antigen (27). Thisantigen belongs to the TNF-receptor family, and its expressionappears to play a major role in different models of immunedysfunction and cell death (7). With regard to HIV infection,it has been previously reported that anti-Fas MAbs can killHIV-infected cells without inducing viral expression (16).

In the present study, we have investigated the effects of thesimultaneous treatment of HIV-infected Ul cells and theirparental uninfected U937 cell line with TNF-ao and IFN--y. Ourresults support the interpretation that selection of cell cloneswhich were highly susceptible to cytokine-mediated cell deathand were already present in the bulk cell line before HIVinfection is the most probable explanation of the apparentlyincreased lysis of chronically infected Ul cells compared withthat of their uninfected counterpart.

2598

HIV-INFECTED CELL KILLING BY TNF-a AND IFN--y 2599

MATERUILS AND METHODS

Cell lines. Uninfected U937 cells were purchased from theAmerican Type Culture Collection (ATCC CRL 1593) andwere maintained at low density (1 x 105 to 5 x 105 cells per

ml) in RPMI 1640 (Whittaker M. A. Bioproducts, Walkers-ville, Md.) supplemented with 2 mM glutamine (Biofluids, Inc.,Rockville, Md.), 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (Biofluids, Inc.), and 10% fetal calfserum (Whittaker M. A. Bioproducts) (complete medium).U937 cells were cloned by limiting endpoint dilution as previ-ously described (11). Briefly, 0.2 cells per well in 0.2 ml ofcomplete medium were seeded in 96-well round-bottom platesfor tissue culture (Costar, Cambridge, Mass.) and were incu-bated at 37°C in 5% CO2. Cell clones, which began to bedetectable as microclusters of a few cells after approximately14 days, were further expanded, first in 1 ml of medium in24-well plates (Costar) and then in to tissue culture flasks.The chronically infected Ul cell line was originally derived

from the expansion of a single cell clone obtained by limitingdilution of U937 cells surviving an acute infection with HIV-1(11) and is characterized by a state of relative viral latency, as

defined by the constitutive presence of spliced, but not un-

spliced, mRNAs (23). These cells can be converted to highlevels of virus production after cell stimulation with phorbolmyristate acetate (11) or several cytokines (for a review, see

reference 22).No evidence of mycoplasma contamination of the different

U937 and Ul cell cultures was observed by both a commer-

cially available mycoplasma detection kit (Gene-Probe) and bytransmission electron microscopy.

Cytokines and reagents. Recombinant human (rHu) TNF-aand IFN--y were purchased from Genzyme (Genzyme Corp.,Boston, Mass.). The glucocorticoid hormone dexamethasone(DEX) was purchased from Sigma (Sigma Chemical Corp., St.Louis, Mo.). Anti-Fas monoclonal antibody (immunoglobulinM) was generously donated by Shin Yonehara (The TokyoMetropolitan Institute of Medical Science, Tokyo, Japan) andwas used in some experiments in the same range of concen-

trations previously shown to affect HIV-infected cells (16).Flow cytofluorometric analysis. The presence of cell surface

receptors for TNF and IFN-y was evaluated on U937 clones 17and 30, as well as on the Ul cell line, by cytofluorometricanalysis on an Epic profile (Coulter, Hialeah, Fla.). Fordetermination of IFN-y receptors, cells were stained with a

mouse anti-human IFN--y receptor MAb (Genzyme), washedand stained with a fluorescein isothiocyanate-conjugated sheepanti-mouse Ab. For determination of TNF receptors, cellswere directly stained with recombinant human TNF-a labeledwith phycoerythrin (Fluorokine, R&D Systems, Minneapolis,Minn.). Cells were also stained with the fluorescein isothiocya-nate-conjugated anti-CD14 MAb (MY4; Coulter).

Cytotoxicity studies. Viability and proliferative capacity ofthe different cells were monitored by Trypan blue dye exclu-sion and 3H-labeled thymidine incorporation, respectively. ForTrypan blue dye exclusion determinations, cell suspensionswere diluted 1:1 (vol/vol) in a 0.4% solution of Trypan blue(Sigma) at different time points after the beginning of thecultures and counted in a hemocytometer chamber. Both totalcell numbers and percentage of viable cells (i.e., the fraction ofcells unstained by Trypan blue) were evaluated.For studies of [3H]thymidine uptake, cell cultures (1 x 105

to 2 x 105 cells per ml), carried out in duplicate or triplicatein 96-well flat-bottom microtiter plates, were pulsed overnightwith 0.5 ,uCi of [3H]thymidine (DuPont, NEN Products, Bos-ton, Mass.) per well and harvested by an automatic 96-well

plate harvester-washer (Tomtec, Orange, Conn.), and the glassfiber filters were counted in a liquid scintillation counter (1205Betaplate; Wallac Inc., Gaithersburg, Md.). The percentage of[3H]thymidine uptake was calculated by dividing the counts perminute of the samples by the counts per minute of unstimu-lated cells and then multiplying by 100 (100% being thearbitrary value given to the counts per minute of unstimulatedcells).

Reverse transcriptase (RT) activity assay. Culture superna-tants of Ul cells were collected at various time points afterstimulation and stored at - 70°C until tested. Five microlitersof supernatant was added in duplicate or triplicate to 25 ,ul ofa mixture containing poly(A), oligo(dT) (Pharmacia FineChemicals, Piscataway, NJ), MgCl2, and 32P-labeled dTTP(Amersham Corp., Arlington Heights, Ill.), and the mixturewas incubated for 2 h at 37°C. Six microliters of the mixturewas spotted onto DE81 paper, air dried, washed five times in2 x SSC buffer (1 x SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate) and twice in 95% ethanol. The paper was then dried,cut, and counted in a liquid scintillation counter (LS 5000;Beckman Instruments., Inc., Fullerton, Calif.). The variabilityof culture replicates was always less than 15%.Western blot (immunoblot) analysis ofHIV proteins. A total

of 107 Ul cells either unstimulated or stimulated with TNF-a,IFN--y, or TNF-a plus IFN--y for 72 h were pelletted and lysed,and the total protein content of each lysate was measured withthe use of a spectrophotometer (DU-64; Beckman) and re-ferred to a standard curve of plasma gamma globulin (4).Twenty-five microliters of each lysate, standardized at 10jig/pul, was added to each lane and subjected to verticalelectrophoresis through 3 to 27% gradient polyacrylamidegels (Integrated Separation Systems; Enprotech Co., Natick,Mass.) for 6 to 8 h. The migrated proteins were transferredfrom the gels onto nitrocellulose filters, which were thensaturated with a 5% milk solution for 1 h and incubated for 1h with a 1:500 (vol/vol) dilution of pooled AIDS patients'serum samples containing Abs recognizing most of the majorHIV proteins (3). The filters were then washed and incubatedfor 1 h with sheep anti-human immunoglobulin F(ab')2 frag-ments conjugated with horseradish peroxidase (Amersham).Finally, the filters were covered for 1 min with the detectionreagents (ECL, Amersham), dried, and exposed to X-ray filmfor 1 to 2 s.

Northern (RNA) blot analysis of HIV mRNAs. Ul cells (0.5x 106 cells/ml) were either unstimulated or stimulated withTNF-a (100 U/ml) or IFN--y (1,000 U/ml) or costimulated withTNF-ot plus IFN--y for 12 and 24 h. Total RNA was extractedfrom 2 x 107 Ul cells by the guanidine thiocyanate phenolmethod with an RNA isolation kit (Stratagene, La Jolla,Calif.). Ten micrograms of the extracted total RNA wasseparated by 0.8% agarose formaldehyde gel electrophoresisand transferred to nitrocellulose filters which were baked,hybridized to a 32P-labeled HIV DNA fragment (SST-BssHII),washed, and exposed to X-ray film. The labeled probe wasstripped by washing the filters at 85°C for 10 min in 0.1 x SSCcontaining 0.1% sodium dodecyl sulfate. The filters were thenrehybridized with a 32P-labeled ,B-actin cDNA probe as acontrol (3).

Nuclear run-on analysis of HIV transcription. Nuclei from108 Ul cells were isolated by sucrose gradients and incubated30 min with 32P-UTP (Amersham) and a mixture of nucleo-tides (ATP, CTP, and TTP; Amersham) according to a pub-lished procedure (21). 32P-labeled RNA was then extractedfrom the nuclei and hybridized for 36 h to plasmid DNAprobes which were previously linearized and immobilized onnitrocellulose filters. The probes used were pUC19 (plasmid

VOL. 68, 1994

2600 BISWAS ET AL.

A. Cytolysis B. Cytostasis A.

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FIG. 1. Differential sensitivity of HIV-infected Ul cells and theirparental uninfected U937 cell line to the cytolytic and antiproliferativeeffects of TNF-oo plus IFN-y. Cell viability and proliferation in cellsstimulated with the two cytokines either alone or in combination were

evaluated by Trypan blue dye exclusion (A) and [3H]thymidine uptake(B) criteria. A higher susceptibility of HIV-infected Ul cells to thecytostatic and cytolytic effects of TNF-a plus IFN-y was observed as

compared to uninfected U937 cells. Three independent experimentswere performed with similar results.

control), pNL4-3 (which contains a full-length HIV genome[1]), and a human P-actin cDNA. The filters were thenextensively washed and exposed to X-ray film. Quantitation ofthe levels of RNA was obtained by laser scanning densitometryof the autoradiograms with a 2222-10 apparatus (LKB Instru-ments, Inc., Gaithersburg, Md.).

RESULTS

The combination of TNF-a and IFN-y induces death ofHIV-infected, but not uninfected, U937 cells. Both HIV-infected Ul cells and their parental uninfected cell line U937were exposed to TNF-ot (100 U/ml) and IFN--y (1,000 U/ml)either alone or in combination. Treatment of Ul cells withthese concentrations of individual cytokines was previouslyshown to be maximally effective in inducing HIV expression inthe absence of cytopathicity (3, 21). A reduction in theproliferative capacity of Ul cells in the presence of IFN-,yalone was noted, although without a decrease in cell viability(Fig. 1A and 1B). TNF-ot alone had no effect on either theviability or proliferative capacity of Ul cells. However, astriking cytopathic effect was observed when Ul cells werecostimulated with these two cytokines (Fig. 1). Ultrastructuralstudies by transmission electron microscopy confirmed that a

high degree of cytopathic effects occurred in Ul cells costimu-lated with TNF-ot plus IFN--y (data not shown). In contrast,uninfected U937 cells were not substantially affected by TNF-aLand IFN--y either alone or in combination (Fig. 1), despite thefact that these cells are usually considered susceptible toTNF-mediated cell lysis (19, 25). These results are consistentwith the hypothesis that HIV infection may have induced a

state of higher susceptibility to cytokine-dependent cell deathin U937 cells, as previously observed in other cell lines (6, 19,24, 25).TNF-a and IFN-y synergistically induce HIV expression in

Ul cells. Concomitant with cytopathicity, costimulation withTNF-ot and IFN-,y resulted in the expression of a greatermagnitude of viral proteins than that observed with IFN-,y or

TNF-ao alone (Fig. 2A). Furthermore, Ul cells costimulated

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FIG. 2. Costimulation of Ul cells with TNF-cx and IFN--y results ina synergistic induction of HIV proteins and RNA. (A) Ul cell lysateswere prepared after 72 h of incubation with the different stimuli. IFN--y(1,000 U/ml) and TNF-co (100 U/ml) alone induced detectable levels ofcell-associated HIV proteins which were increased in Ul cells costimu-lated with TNF-as and IFN-y. Enhanced viral proteins in TNF-o plusIFN-,y costimulated Ul cell lysates were observed also at earlier timepoints (24 and 48 h; data not shown). Three independent experimentswere performed with similar results. (B) Northern (RNA) blot analysiswas performed with total RNA extracted from Ul cells stimulated withIFN--y (1,000 U/ml), TNF-ot (100 U/ml), or TNF-ot plus IFN--y after 12and 24 h of incubation. The results shown are obtained from one

experiment representative of five independently performed. The syn-ergistic induction of HIV steady-state mRNAs by TNF-ot plus IFN--ywas present also at later time points (48 h) and with differentconcentrations of the two cytokines (TNF-ot [10 U/ml] plus IFN-,y[1,000 U/ml] and TNF-as [100 U/ml] plus IFN--y [100 U/ml]; data notshown). (C) Nuclear run-on analyses were performed on newly syn-thesized mRNA extracted from nuclei isolated after 24 h of incubationof Ul cells stimulated with IFN--y (1,000 U/ml), TNF-ot (100 U/ml) or

the combination of the two cytokines. The autoradiograms were thenanalyzed by laser scanning densitometry to obtain semi-quantitativevalues of the levels of HIV transcription.

with TNF-cx and IFN--y showed a clear synergy in terms ofaccumulation of HIV steady-state mRNAs as early as 12 hpoststimulation, when the inductive effect of IFN--y alone was

barely detectable (Fig. 2B). Stimulation of Ul cells withTNF-a alone induced an approximately fivefold increase ofHIV transcription over unstimulated cells as assessed bynuclear run-on analysis followed by laser scanning densitome-

Unstimulated TNF-a IFN-y TNF-a+lFN-y(10 U/ml) (1000 U/ml)

J. VIROL.

HIV-INFECTED CELL KILLING BY TNF-ot AND IFN-y 2601

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FIG. 3. The combinations of TNF-ot plus glucocorticoids andIFN-y plus glucocorticoids synergistically induce HIV production butare not cytopathic for Ul cells. Ul cells were stimulated with TNF-cs,IFN--y, or glucocorticoids alone and with the combination of TNF-aplus glucocorticoids (panel A and B) and IFN--y plus glucocorticoids(panel C and D). Culture supernatants were harvested 3 and 6 daysafter stimulation and tested for RT activity. At day 6, Ut cells underdifferent treatment conditions were also pulsed overnight with [3H]thy-midine. The results shown are obtained from one experiment of threeindependently performed.

try (Fig. 2C). Although IFN--y alone did not induce a cleareffect, the combination of TNF-ot and IFN--y resulted in a

synergistic effect on HIV transcription estimated to be an

approximately 15-fold increase over that of unstimulated cells(Fig. 2C). This synergistic increase of HIV transcription ob-served in Ul cells costimulated with TNF-ot and IFN--y wasconfirmed by studies in which a heterologous HIV-1 longterminal repeat linked to the reporter gene chloramphenicolacetyltransferase was transfected into Ul cells (data notshown).Death of Ul cells is selectively caused by costimulation with

TNF-a and IFN--y and is not a consequence of the massiveproduction of HIV particles. Because massive cell death wasassociated with the synergistic induction of HIV expression inUl cells costimulated with TNF-ot and IFN-y, we investigatedwhether the observed cytopathicity was caused by the highlevels of virus production, as suggested earlier in other in vitromodels of HIV infection (28). In this regard, glucocorticoidhormones such as DEX have been shown recently to synergizepotently with several cytokines in the induction of HIV expres-sion in Ul cells (5). Therefore, we investigated the correlationbetween levels of virus expression and cytopathicity in U1 cellscostimulated with either TNF-a or IFN--y plus DEX. Super-natant-associated RT activity was determined as a parameterof virion production (10), and [3H]thymidine incorporation ofUl cells under each condition was determined simultaneously.DEX alone had no substantial effect on virus expression in Ulcells but synergized with TNF-ca (Fig. 3A), as reported previ-ously (5). Despite high virus production, cell cultures showed

no cytopathicity as indicated by [3H]thymidine incorporation(Fig. 3B). Similar results were obtained when Ul cells werestimulated with IFN--y plus DEX (Fig. 3C and 3D). Thus, theprofound cytopathicity observed in Ul cells costimulated withTNF-ot and IFN--y was unlikely to be a consequence of highlevels of virus production synergistically induced by these twocytokines, since other similar synergistic costimulatory condi-tions did not substantially affect cell viability.

Clonal variability in the susceptibility of U937 cells tocytokine-mediated killing. The observation that Ul cells aremuch more sensitive to cytokine-mediated killing than theiruninfected counterparts is in contrast to previous observationsindicating that HIV infection of U937 cells led to a decreasedsensitivity to the antiproliferative effect of TNF-a (19). There-fore, we investigated whether clonal variability within differentU937 cells could provide an explanation for this discrepancy.Uninfected U937 cells were cloned by a limiting-dilutiontechnique (11). Forty-five clones were obtained, expanded inculture for 14 days, and tested for their sensitivity to theantiproliferative effect of TNF-a and IFN--y, either alone or incombination. Surprisingly, despite the lysis-resistant phenotypeof the uncloned U937 cell line and a heterogenous suscepti-bility to cytokine-mediated antiproliferative effects that wasobserved among the different clones in response to eachcytokine alone, the vast majority of the clones were killed bythe costimulation of TNF-a plus IFN--y (Fig. 4A). To investi-gate this apparent paradox, reconstitution experiments inwhich one lysis-resistant clone (clone 30) was added in equalproportions to a panel of sensitive clones were performed. Themixture of these different clones showed a substantial resis-tance to the antiproliferative or cytotoxic effects of TNF-ax plusIFN-y (Fig. 4B). Similarly, mixing experiments in which Uland clone 30 cells were seeded in a 1:1 ratio resulted in theappearance of a culture phenotype that was relatively resistantto the antiproliferative effect of TNF-a plus IFN-y (data notshown). No evidence was obtained that the resistant phenotypeconferred by clone 30 was dependent upon the release of asoluble factor, in that the addition of supernatants from clone30 cultures to lysis-sensitive U937 clones or Ul cells did notalter their susceptibility to the antiproliferative effect of TNF-atplus IFN--y (data not shown).

Having observed the existence of cytokine-sensitive cloneswithin the bulk U937 cell line, we compared the susceptibilityof infected Ul cells to killing by TNF-a plus IFN--y to that ofa representative uninfected sensitive clone (clone 34). TNF-aand IFN--y alone at the highest concentrations used (100 and1,000 U/ml, respectively) showed a mild antiproliferative effect(ranging approximately from a minimum of 10% to a maxi-mum of 40% over the control level) on both cell lines (Fig. 5).The cytolytic effect of the cytokines at these concentrations was

also modest (ranging from 0 to 20%) (Fig. 1A). However,when TNF-ot and IFN--y were combined, a very strong andcomparable concentration-dependent antiproliferative effectwas observed in Ul cells (Fig. 5, upper panel) and clone 34(Fig. 5, lower panel).The lysis-resistant U937 clone 30 possesses cell surface

receptors for TNF or IFN--y. To rule out the possibility thatclone 30 was a trivial contaminant of the U937 cell line, studiesof the cell surface phenotype were conducted. No significantexpression of T-lymphocytic (CD3), B-lymphocytic (CD20), or

natural killer cell (CD56) markers were expressed on the cellsurface of either uncloned U937 cells, clone 30, or other clones(data not shown), whereas all the cells expressed comparablelevels of the CD14 antigen characteristic of the monocyticlineage (26) (Fig. 6). We also examined the possibility that theresistance to TNF-a-plus-IFN-y-mediated cell lysis manifested

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VOL. 68, 1994

2602 BISWAS ET AL.

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FIG. 4. Heterogeneous sensitivity of U937 clones to the cytostatic or cytolytic effects of TNF-ox and/or IFN-'y. Cell proliferation was measuredby [3H]thymidine incorporation 72 h after cytokine treatment. (A) The relative insensitivity of the bulk U937 cell line is not reflected in the majorityof the U937 clones obtained (a selected and representative panel of a total of 45 clones is shown). In particular, the simultaneous addition of TNF-caplus IFN--y resulted in the lysis of all clones but clone 30, which was essentially unaffected in its proliferative ability by the presence of TNF-ot and/orIFN--y. (B) Mixing of clone 30 (arrow) with different lysis-sensitive clones partially reconstitutes a lysis-resistant phenotype to the cell culturefollowing costimulation with TNF-ot and IFN--y. These results are representative of four (A) and three (B) experiments, respectively, that wereindependently performed.

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by clone 30 was due to a lack of surface receptors for thesecytokines. However, both lysis-resistant (clone 30) and lysis-sensitive (clone 17) uninfected clones of U937 showed levels ofcell surface TNF and IFN-,y receptors comparable to thosepresent on the plasma membrane of Ul cells (Fig. 6).

Anti-Fas MAb synergizes with IFN-y in triggering cell deathin Ul cells and several uninfected U937 clones without induc-ing HIV expression. It has been previously reported thatanti-Fas MAb can induce lysis of TNF-sensitive target cells,including HIV-infected cells, without transducing a cell activa-tion signal such as the activation of NF-KB (7, 16, 27). Anti-FasMAb alone did not cause substantial cytopathicity in eitherHIV-infected Ul cells or in several uninfected U937 cell clones(Fig. 7). However, the combination of anti-Fas MAb andIFN-,y resulted in a synergistic antiproliferative effect both inUl cells and in the majority of the uninfected U937 clones(Fig. 7). As previously observed in cells costimulated withTNF-ot and IFN-,y, anti-Fas plus IFN--y did not cause substan-tial cytopathicity in uninfected bulk U937 and clone 30 (Fig. 7).Consistent with previous reports (16), and in contrast toTNF-ox or IFN-y, anti-Fas MAb did not cause upregulation ofHIV expression in Ut cells when used either alone or incombination with IFN-y or TNF-o (data not shown). Thesedata further emphasize that the death of Ut cells observedwith stimulation by TNF-ot and IFN-y was not the consequence

of massive production of newly synthesized virions.

DISCUSSION

The present study has demonstrated that costimulation ofUl cells with the HIV-inductive cytokines TNF-ox and IFN-,y

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J. VIROL.

HIV-INFECTED CELL KILLING BY TNF-ot AND IFN-y 2603

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FIG. 6. The insensitivity of U937 clone 30 to the anti-proliferativeeffect of TNF-a plus IFN--y does not depend on the lack of the cellsurface cytokine receptors. Fluorescence-activated cell sorter analyseswere performed twice on Ul, U937, and a panel of U937-derivedclones. Ul and clone 17 are sensitive to TNF-ac plus IFN--y, whereasU937 bulk cell line (not shown) and clone 30 are resistant. All four celllines and clones expressed the monocytic marker CD14 (althoughfluctuations of the levels of expression of this marker have beenobserved from experiment to experiment) and comparable levels ofIFN-y and TNF receptors. Dashed line, isotype Ab control. Solid line,specific fluorescence.

resulted in profound synergistic effects on virus expression aswell as lysis of persistently infected Ul cells. Since cytokinessuch as TNF-a and IFN-y which induce lysis of HIV-infectedcells in vitro have been used in clinical trials (2) because oftheir potential ability to eliminate HIV-infected cells in vivo, itis important to determine whether such cytokines, either aloneor in combination, also induce substantial virus production inthe process of inducing cytolysis.Our findings are in contrast with earlier observations by

Wong, who observed a suppressive effect on viral RNA syn-thesis in a panel of cell lines infected with HIV and exposed toTNF-oa, either alone or in combination with IFN--y (25).However, U937 cells were not used in that study because oftheir known susceptibility to TNF-mediated lysis. Further-more, very high concentrations of TNF were used, suggestingthe possibility that cell toxicity occurred before viral mRNAsand proteins could accumulate (25).Matsuyama first described the phenomenon of increased

virus expression concomitant with cell lysis in HIV-infectedcells after stimulation with TNF-a (19). However, no informa-tion was provided on the potential role of IFN--y in thoseculture conditions. Furthermore, it was reported in the samestudy that certain cell lines differed from this general patternand, in particular, HIV-infected U937 cells were less suscepti-ble to TNF-mediated killing than their uninfected counterparts(19). Our comparative analysis of the cytopathic effects in-duced by TNF-a and/or IFN-y on HIV-infected Ul cells,uninfected U937 cells, and uninfected U937 clones indicatesthat a high level of heterogeneity exists within the populationof U937 cells. In this regard, it is important to underscore thata strong selection process occurs during the acute phase ofHIV replication in both primary and tumor CD4+ target cells(such as U937 cells) where a substantial proportion of cells arekilled and survivor cells emerge thereafter which containintegrated proviral DNA in their genome (11, 12, 28). Themechanisms underlying this active clonal selection process are

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+

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FIG. 7. Anti-Fas MAb kills both Ul cells (without inducing viralexpression) and several uninfected cell clones in synergy with IFN-y.The proliferative capacity of HIV-infected Ul cells and uninfectedU937 and related cell clones was assessed after 72 h of treatment withIFN--y alone (1,000 U/ml), anti-Fas MAb (1 ,ug/ml), or the combina-tion of these two agents. The antiproliferative effects were confirmedin two independent experiments and by comparative analysis of cellproliferation and viability determined by Trypan blue dye exclusioncriteria. Neither induction of RT activity nor enhancement of TNF-oa-or IFN-y-mediated upregulation of virus expression was observed inthe presence of anti-Fas MAb.

currently unknown. Therefore, functional or phenotypicchanges in persistently infected cells may not necessarily reflectthe influence of HIV genes, as in the case of adenovirusinfection (6, 8, 14), but may be the result of the selectiveprocess imposed on target cells during the acute phase of virusreplication. Experiments in which anti-Fas MAb (7, 16, 27)instead of TNF-ot was used in combination with IFN-y con-firmed that both infected Ul cells and the majority of unin-fected U937 cell clones were highly susceptible to cytokine-mediated lysis. The presence of such clonal variability dictatesthat caution should be exercised in comparative analyses ofinfected and uninfected cell lines. In this regard, the decreasesusceptibility to the lytic effect of TNF-ot observed by Mat-suyama in infected U937 cells versus their uninfected counter-parts (19), which is in contrast with our findings (Fig. 1), maybe explained by clonal variability within the bulk U937 cells.Finally, it is very unlikely that lysis of Ul cells was theconsequence of high levels of virus expression induced by

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2604 BISWAS ET AL.

TNF-a plus IFN--y since costimulation of Ul cells with TNF-atplus glucocorticoids or IFN--y plus glucocorticoids resulted inthe synergistic induction of virus expression that was dissoci-ated from substantial cytopathicity. Furthermore, treatmentwith IFN--y plus anti-Fas MAb resulted in death of Ul cellswithout synergistic induction of HIV expression.

In conclusion, these data demonstrate that TNF-at andIFN--y in combination can induce substantial expression ofHIV from infected cells despite the fact that they may ulti-mately lead to the elimination of these cells. Hence, cautionshould be exercised in the design and implementation oftherapeutic protocols involving the administration of poten-tially HIV-inductive cytokines.

ACKNOWLEDGMENTS

We thank D. Cohen, G. Pantaleo, and E. Vicenzi for helpfulsuggestions and critical reading of the manuscript; C. Muro-Cacho forstudies of cytokine-induced apoptosis; M. Baseler and J. Adelsbergerfor performing cytofluorometric analysis; E. Hardy for technicalassistance; and Janet S. Bey for typing the manuscript.

Priscilla Biswas is funded by a fellowship for AIDS research from theIstituto Superiore di Sanita (I.S.S.), Rome, Italy. This project waspartly supported by the I.S.S./A.I.D.S. project no. 8204-99.

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