Human immunodeficiency virus (HIV)-infected tumor xenografts as an in vivo model for antiviral therapy: role of alpha/beta interferon in restriction of tumor growth in nude mice injected with HIV-infected U937 tumor cells.

Copyright X 1991, American Society for Microbiology

Human

Immunodeficiency Virus (HIV)-Infected

Tumor

Xenografts

as an

In

Vivo Model for Antiviral Therapy: Role of Alpha/Beta

Interferon in Restriction of

Tumor

Growth

in Nude

Mice Injected

with

HIV-Infected

U937 Tumor

Cells

PATRIZIAPUDDU,1 CHIARA LOCARDI,1 PAOLA SESTILI,1FRANCO

VARANO,1

CARLO PETRINI,1 ANDREA MODESTI,2 LAURA MASUELLI,2 ION GRESSER,3 ANDFILIPPOBELARDELLIl*

Laboratory of Virology, Istituto Superiore diSanita, VialeReginaElena299, 00161 Rome, Italy';Departmentof Experimental Pathology, University ofRome,00161 Rome, Italy2; andInstitutdeRecherches Scientifiques surleCancer,

94802 Villejuif Cedex, Paris, France3 Received5 November1990/Accepted24January 1991

The host factors involvedin therestrictionoftumorgrowthwerestudiedin nudemicetransplanted witha

cloned line of chronically human immunodeficiency virus (HIV)-infected U937 cells. HIV-infected and uninfected U937 cellsexhibitedthe samegrowth patternsinculture. However, HIV-infected cells werenot tumorigenicwheninjected subcutaneously in nude mice, whereaslargesolidtumors wereobserved in mice

injected withuninfected U937cells. Injectionof nudemicewithantibody toalpha/betainterferon(IFN-a>)

enabled HIV-infected U937cells togrow progressively in approximately 90to 100% ofmice. HIV-infected U937 cells formed solid tumors in the majority (60 to 90%) ofeither immunosuppressed (splenectomized, irradiated,and

anti-asialo-GM,-treated)

orgeneticallyimmunodeficient(bg/nu/xid)nude mice. Inmicetreated

withantibodiestoIFN-alwithestablishedHIV-positive tumors,adirect correlationwasfoundbetween p24

antigenemiaand tumorsize. Treatmentofestablished HIV-positive U937 cell tumorswith human IFN-a or

mouseIFN-a$/ resultedinaclear-cut inhibition ofbothtumorgrowth and p24HIVantigenemia. Incontrast,

treatmentwith tumornecrosisfactor alpha markedly inhibitedtumorgrowth but didnotsignificantlydecrease

serum p24levels. 3'-Azido-3'-deoxythymidine treatment didnot affect either tumorgrowth orthe levelsof

serump24 antigen. Thesedataindicate thatendogenous IFN-oc$ isacrucialfactor in the restriction of both

tumorgrowth and p24 antigenemia in mice injected with HIV-infected tumor cells. Moreover, the results suggestthat thedevelopment of HIV-1 p24 antigenemiainathymicimmunosuppressedmicemayrepresentan

interestinginvivomodelforanti-HIVtherapy.

Althoughconsiderableprogress has been achievedinour

knowledge of the epidemiology of AIDS and its etiologic agent(humanimmunodeficiency virus[HIVI), little is known about the pathogenesis of infection. In particular, it is important to establish suitable in vivo model systems not onlytostudy someaspectsof thepathogenesisof AIDS but also to evaluate prophylactic measures, such as vaccines,

andtodefine effective antiviral therapies.

Genetically immunodeficient (SCID) mice transplanted withhematolymphoid cells from humanfetalliver, thymus, and lymph node (22, 25), adult human peripheral blood leukocytes(24),orhumanbonemarrowcontaining hemopoi-etic stemcells (17) have beenproposed as possible animal models for AIDS. The suppression of HIV infection in

3'-azido-3'-deoxythymidine (AZT)-treated SCID mice

en-grafted with human hematolymphoid organs has recently

been reported (23), suggesting that such models can be of someinterest instudyingnewantiviralcompounds.Inthese experimental systems, HIV expression is apparently

re-stricted to the virus-permissive transplanted human cells, and neitheramplification of HIV expressionnor viral anti-genemia is observed.

In this study, we have investigated some host factors

involved in the restriction oftumor growth in nude mice transplanted withacloned line ofchronicallyHIV-infected

U937cells, andwehave defined theexperimentalconditions

* Correspondingauthor.

for obtaining p24 antigenemia in mice with established HIV-positivetumors. Thedatapresented demonstratethat injection of antibodies to mouse alpha/beta interferon

(mu-IFN-olp)in nudemicetransplantedwith HIV-infected U937 cells results in both tumor growth and p24 antigenemia. Moreover,these dataindicate thatnudeimmunosuppressed mice treated with anti-IFN-a/1 antibodies and transplanted withHIV-infected U937 cells mayrepresentauseful model

forstudyingthe action of anti-HIV substances in vivo. MATERIALS AND METHODS

Cells and culture conditions. The human histiocytic lym-phoma cell line U937(31) (kindly provided by A. Siccardi, Milan, Italy) was cultured in RPMI 1640 medium supple-mented with 10% heat-inactivated fetal calf serum. Cells

wereseededat2x 105/ml in fresh medium and reachedacell densityofapproximately2 x 106/ml 3to4days thereafter. Primary infection of U937 cells. A total of 3 x 106 U937 cellswere treated with Polybrene (2 ,ugIml)for 1 h at 37°C. Cellswerecentrifuged, and the cellpelletwasinfected with 50 jil of HIV (HLTVIII B strain), correspondingto 15,000

cpmofreversetranscriptase activity. Afterincubation for 1

hat37°C,the cellswereresuspendedataconcentrationof 5

x 105/ml in RPMI 1640 medium containing 20% fetal calf

serum. HIV-infected U937 cellswereclonedbythe dilution

methodapproximately80daysafter HIV infection. All of the 15 clones tested released HIV in the cell medium, as

determined by p24 assay, even though some differences

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were observed in the amounts of released virus (data not shown). These data indicated that virtually all U937 cells were HIV infected. In the experiments to be described, we used a cloned HIV-infected cell line (clone 10) capable of releasing greater amounts of HIV than can the uncloned HIV-infected cell population.

HIV-infected U937 cells exhibited a markedly lower

expression ofCD4, HLA-DR, and HLA-DP antigens than did uninfected U937 cells. No significant differences were observed in the expression of a panel of other membrane

antigens (OKM1, TecMG2, TecMG3, LeuMl, LeuM3,

OKM14, OKM13, OKM5, and HLA-DQ).

Mice. Male or female Swiss nude mice were purchased

fromCharles River Laboratories (Calco, Italy) at 4 weeks of ageand kept for 1 week before use. bg/nu/xid mice (17) were

obtained from Harlan Sprague-Dawley (Indianapolis,

Ind.).

Splenectomy and irradiation. The splenectomized,

irradi-ated, and anti-asialo-GM1-treatednude mice were prepared as described by Caretto et al. (4). Briefly, splenectomy was

performed via a 5-mm-long incision on the left upper flank. The mice received a sublethal total-body irradiation of 450 radsfrom a137Cs source providing a dose rate of 50 rads/min and were referred to as SI nude mice. Twenty-four hours after splenectomy and irradiation, a few mice were also

injected intravenously (i.v.) with 0.2 ml of

anti-asialo-GM1

antiserum (Wako Chemicals GmbH, Dusseldorf, Federal

Republic ofGermany) (SIA nude mice) (14). This treatment eliminates all natural killer cell activity (7, 11).

Reagents.Polyclonalsheep

anti-mu-IFN-odp

(sheep 1) was

prepared andpurified (13) and had a neutralizing titer of 6.4 x 106 against 4 to 8 U of

mu-IFN-ao/r

(13).

Partially purified

mu-IFN-a/p

(107 U/mg of protein) was

prepared asdescribed elsewhere (32). In some experiments, we used as control material a protein preparation derived from the culture supernatant of C243 mouse cells; this controlpreparationwassubjected to the same procedures as wereused toprepare the partially purified

mu-IFN-ot/&

(32). Human natural IFN-ot(hu-IFN-a; 5 x 106 U/mg of protein) was kindly provided by K. Cantell (Helsinki, Finland).

Recombinant human tumor necrosis factor alpha (hu-TNF-ot; specificactivity, 7 x 107 U/mg) was a generous gift of W. Fiers and J. Tavernier (Ghent, Belgium). Cytokines wereinjected peritumorally (0.2 ml per injection).

AZT waspurchased from The Wellcome Research Labo-ratories (Beckenham, Kent, England) through the courtesy of M. Martorelli (Wellcome Italia SpA, Pomezia, Italy). Mice were treated with AZT either by oral administration (1

mg/ml

in the drinking water) as described elsewhere (23) or

by peritumoral injection (480/mg/kg/day [maximal tolerated dose]).

Antigen capture assay. Sera were tested for HIV p24

antigen by an antigen capture enzyme-linked immunosor-bent assay (Cellular Products Inc., Buffalo, N.Y.).

AssayofIFN inserum. IFN was assayed by inhibition of thecytopathic effect ofvesicular stomatitis virus on L cells in monolayer cultures (0.2 ml per well) in Falcon

mi-croplates. Units are expressed in our laboratory units; 1 of our units is the equivalent of 4 mouse research reference units.

Evaluation oftumor growthin nude mice transplanted s.c. with U937 cells. Nude mice were injected subcutaneously

(s.c.)in the shoulder with noninfected or HIV-infected U937 cells in 0.2 ml of saline. The diameters of tumor nodules were measured with calipers, and the mean surface area of the tumor perexperimentalgroup was determined.

Electron microscopy. The tissue fragments were fixed in

A

2

U

I1-3

ouninfected a HIV-

infected

in vitro

1 TI

1 2 3 4 5

days

B

I

4

2-ouninfected

* HIV- infected

invivo

10 20 30 40 50

days

FIG. 1. Growth of uninfected and HIV-infected U937 cells in culture and transplanted s.c. in nude mice. (A)Cells wereseededat 2x 105/mlin RPMI medium supplemented with10% fetal calfserum as described in Materials and Methods. At different days aftercell seeding, cells were counted and their viability was determined by the trypan blue dye exclusion method. Values represent the means of duplicate samples. The variability of replicate samples was always less than 10%. Cell viability was more than 90%. (B) Six-week-old male Swiss nude mice(eight per group) wereinjected s.c. with107 uninfected or HIV-infected U937 cells. Tumor growth was evaluated as described in Materials and Methods.

cacodylate-buffered 2.5% glutaraldehyde, postfixed in os-mium tetroxide, dehydrated in graded alcohols, and then embedded in Epon 812.

The cells were collectedby gentle centrifugation, and the pellet was resuspended in 2.5% glutaraldehyde in phosphate buffer (0.1 M) at pH 7.2 at room temperature and placed at

4°C

for 12 to 16 h. The cell suspension was pelleted at low speed, washed in the same buffer,

postfixed

with

1%

OS04,

rapidly dehydrated in a series of alcohols, and embedded in Epon 812.

Ultrathin sections were stained with uranyl acetate and lead hydroxide and observed under a Philips

CM10

electron microscope.

RESULTS

Growth properties of uninfected and HIV-infected U937 cells in culture and when

injected

s.c. in nude mice. Cloned HIV-infected U937 cells (clone 10) exhibited in vitro growth curves very similar to those of uninfected control cells (Fig.

1A).

These chronically infected cloned U937 cells released higher levels of HIV in the culture medium than did the parental uncloned HIV-infected cell population, as deter-mined by reverse transcriptase and p24 assays of the culture supernatants (data not shown). Large solid tumors devel-oped in mice transplanted with control uninfected U937cells

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(Fig. 1B). In contrast,s.c.tumorswerenotobserved innude mice after injection of107HIV-infectedU937cells (Fig. 1B). Some initialtumorgrowthwasobserved insomemice (10to

30%) injected s.c. with1.5 x 107 2x 107HIV-infected U937 cells, but these tumors generally regressed 3to4weeks after tumor cell injection (data not shown).

Effects of

injection

ofantibodies tomu-WN-a/j3 on tumor

growth in nude mice transplanted s.c. with uninfected or

HIV-infected U937 cells. It was of interest to determine whether the injection of antibodies to mu-IFN-a/o would result in any change oftumorgrowth in mice transplanted s.c. with uninfected and HIV-infected U937 cells. The injection ofantibodies to IFN-a/I in mice transplanted s.c.

with uninfected U937 cells resulted in significantly greater

tumor growththanin mice treatedwith control sheepgamma

globulin (Fig. 2A). Treatment with anti-IFN antibody of mice transplanted with HIV-infected U937 cells resulted in the developmentof rapidly growing solidtumors in allmice (Fig. 2B and C). Although small tumors were initially

de-tected afterinjection of large numbers ofHIV-infectedU937 cells (1.5 x 107) in some control treated mice, these s.c.

tumors regressedat alaterstagein this experiment(Fig. 2B). Development ofHIV p24 antigenemia in

anti-IFN-aIj8-treated nude mice transplanted s.c. with HIV-infected U937 cells and correlation withtumorgrowth. All ofthe anti-IFN-treated mice with established U937 HIV-positive tumors

exhibited detectable serumlevels of p24on day 40(Fig. 3).

p24 levelsranged between 800 and 3,600 pg/ml. Incontrast,

only 2 of10 mice treated with normal sheepgammaglobulin

and transplanted with HIV-infected cells developed solid tumors, and none of them exhibited detectable p24 antigen-emia. Evenwhen solid tumors did develop in mice treated with normalsheepgammaglobulin,nodirect correlationwas

observedbetweentumorgrowth and serump24levels (Fig.

4A).

Figure 4B shows the kinetics of the direct correlation betweentumor growth and p24 antigenemia inan

anti-IFN-treated mouse transplanted with HIV-infected U937 cells. These data suggested that endogenous IFN-a/, was

in-volvednot only intherestrictionoftumorgrowthbut also in thein vivo inhibition ofHIV expression.

Growth of HIV-infectedU937 cells inimmunosuppressedor

genetically immunodeficient nudemice. Itseemed of interest to determine the growth of HIV-infected U937 cells in immunosuppressed or genetically immunodeficient nude mice. As shown in Table 1, s.c. injection ofbglnulxid mice withHIV-infected U937cells resulted inthedevelopmentof s.c. tumors in 60% ofthe injected mice. Likewise, HIV-infectedU937 cellsformedsolid tumorsin90% of both SIA nudemice andanti-IFN-treated mice; 60%of the SIA nude mice transplanted with HIV-infected U937 cells exhibited detectable serumlevels of p24 antigen. In this experiment, treatment of SIA nude mice with antibodies to IFN-ac, resultedin a very rapid development of solidtumorsin100% of the mice injected with HIV-infected cells, and all mice exhibited a p24 antigenemia (Table 1). Figure 5 shows the kineticsof tumorgrowthin SIAnude micetransplanteds.c.

with controlandHIV-infected U937 cells. Therateoftumor

growthwaslower for HIV-infected cells than for uninfected U937 cells. Treatment of SIA nude mice with antibody to

IFN-aJp

resulted inahigherrateof growth ofHIV-infected U937 tumors than in mice not treated with antibody, al-thoughthe growthof thesetumorswasstill less rapid than in

mice injected with uninfected U937cells (Fig. 5).

Electron microscopy studies ofHIV-positive U937 cell

tu-mors. Figure 6A shows the morphology of HIV-infected

A

E

1. a

I0 o

IE

a c0-,

o normal serum

*anti-IFNri/u

10 20 30 40 50 60

days

B

a

120- 100-S

80-% 60-u

I.. 40-a V"

20-U

mrmalserum

*anti-IFNaip

(00/10)

(2/10)

\ ~r ~~~ I

10 20 30 40 50

days

c

E

a

c to

S.. U)

to

q

2-0

O no

* an

irmal serum I (10/10)

iti-IFNax/

(2/10)

10 20 30 40 50 60

days

FIG. 2. Effect of antibodies to

IFN-cx/p

on tumorgrowthinnude mice transplanted s.c. with uninfected orHIV-infected U937 cells. Five- to six-week-old female Swissnude mice (10 pergroup) were

injected s.c. with107 uninfected U937 cells (A) or 1.5 x107 HIV-infected U937 cells (B and C). Each mouse was treatedwith 0.2 ml of a 1:20 dilution of either antibody to

IFN-a/P

(320,000

neutralizing

units) or normal sheep gammaglobulin asfollows:

Timeoftreatment

3hbefore tumorcellinjection

time0,withtumorcells Day7

Day14

Day21

Day28

Route i.v.

S.C.

s.c. (peritumorally)

s.c. (peritumorally) s.c. (peritumorally)

s.c.(peritumorally)

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80- so

,

60 60

4,o40 40 ~.

20- 20

0 0

Control Anti-IFN

serum ct/p

FIG. 3. Correlation between tumor growth and HIV p24 antigen-emia in anti-IFN-treated nude mice transplanted s.c. with HIV-infectedU937cells. Five-week-old male nude mice (10 per group) were treated with either antibodies to

IFN-oi/,

or control sheep gammaglobulin as described in the legend to Fig. 2. Mice were injecteds.c. with 1.5x107HIV-infected U937 cells. The percentage ofmice with tumor was assessed 40 days after U937 cell injection. Sera were taken on day 40, and the levels of p24 antigen were determined as described in Materials and Methods.

U937 cells seen by electron microscopy after 3 days of

culture. Some HIVparticleswere found to be released from the cell membrane of in vitro-passaged U937 cells. Figures 6B and C illustrate some examples of the electron micros-copy analysis of an HIV-positive U937 cell tumor derived

from an anti-IFN-a/13-treated mouse. Extensive HIV

pro-ductionwith a large number of virus particles budding from

the U937 cell membrane was observed (Fig. 6C). Large

numbers ofHIVparticleswere detected in the intercellular spaces(Fig. 6B)and in close association with blood vessels

(datanotshown).

Effects of hu-IFN-aL,mu-IFN-aod,hu-TNF-a, and AZT on tumor growth and p24 antigenemia in immunosuppressed mice with established HIV-infected U937 cell tumors. The results presented above indicated that immunosuppressed nudemice injectedwith HIV-infected U937 cells and treated

with antibodies to IFN-a/, might represent a useful model

forthedevelopment ofHIV-1p24 antigenemia and therefore

A

E

Ch Q0

[image:4.612.93.275.75.190.2]

'-Q.

TABLE 1. Subcutaneous growthofHIV-infected U937cellsin immunosuppressed(SIA) orgeneticallyimmunodeficient

(bg/nu/xid) nude micea

No. ofmice withtumorsc/total No. of mice

Anti-IFN- with

detect-Mice

e.Xsa/pb

Uninfected HIV-infected ableserum U937cells U937 cells p24levelsd

Nude - 10/10 0/10 0

bglnulxid - 8/8 6/10 NDe

Nude + 10/10 9/10 9

SIA nudef - 8/8 9/10 6

SIAnude + ND 10/10 10

aFive-tosix-week-oldmale mice wereinjecteds.c.with107uninfectedor

HIV-infectedU937cells.

bTreatments withanti-IFN antibodieswereperformedasdescribed in the legendtoFig.2

cCalculated35 to 40days after U937 cellinjection.

dSerafrom10miceinjected with HIV-infectedU937cellswereharvested

onday 28 and tested for p24 levelsasdescribed in Materialsand Methods. Detectable p24serumlevels were those >100pg/mnl.

IND, Notdetermined.

fForpreparation of SIA mice,seeMaterials and Methods. The micewere

injecteds.c.withU937cells1day after i.v.injection ofanti-asialo-GMI.

for studies to determine the relative value of anti-HIV therapies.

We investigated the effects of hu-IFN-a, mu-IFN-ao/,

hu-TNF-a, and AZT on the p24 antigenemia in mice with established HIV-infected U937 cell tumors. Preliminary in vitro studies were performed to determine the possible effectsofthesesubstances on HIVproduction inchronically infectedU937cells under cultureconditions. Figure7shows

theeffects of hu-IFN-a, mu-IFN-a/,, hu-TNF-a, and AZT on HIV production in chronically infected U937 cells in

culture, as determined by measuring the p24 levels in the culture supernatants. Hu-IFN-a exhibiteda marked

inhibi-tory effect on HIV yield, whereas hu-TNF-a significantly increased HIV production. Neither AZT nor

mu-IFN-ac/p

affected HIV production in vitro (Fig. 7). Hu-IFN-a and TNF-a exhibited someinhibitoryeffect on cellmultiplication (i.e.,a 20to30% inhibitionofcellgrowth in the presence of

B

U E Cm

0

t-4, :E

a

S.

U)

0 10 20 30 40 50

days

6E

I-C 0. a

E

z

4'

I-a I0

IU)

days

FIG. 4. Analysis oftherelationship between tumor growth and HIV p24 antigenemia in nude mice transplanted s.c. with HIV-infected U937 cells andtreated withnormal sheep serum (A) or antibodies toIFN-a/,(B). Five-week-old male nude micewere treated with either control sheep gammaglobulin or antibodies to

IFN-a/p

as described in the legend to Fig. 2. Mice were injected s.c. with 1.5 x 107 HIV-infectedU937 cells. At different times after tumor cell injection, orbital venous blood was taken from tagged individual mice and serum samplesweretested for the p24 antigen as described in Materials and Methods. Values represent the surface area of each tumor and the serum p24antigenlevels ofonerepresentative tumor-bearingmousetreated as indicated above.

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E44

I,

11 3 3-L.

E 2

a_i

Co

- 1

c

O uninfected U937 cells

* HIV-infected

*HIV-infected+

anti-IFN a/p7

//

i (7/7)

.., (6/7)

..

..

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

0 10 20 30 40

days

FIG. 5. Subcutaneous growth of uninfected and HIV-infected U937 cells in SIA nude mice and effect of antibodies toIFN-a/,.

Five-week-old male nude mice (seven pergroup) were

splenecto-mized, irradiated,andtreated with

anti-asialo-GM,

antibodies(4)as

described in Materials and Methods. At 24 h after i.v. injection of

anti-asialo-GM,,

themicewereinjecteds.c. with 107uninfectedor

HIV-infected U937 cells. Onegroupof micewasalsotreated with

antibodiestoIFN-a/, asdescribed in the legendtoFig. 2. Numbers in parentheses indicate the numberof mice with tumor/totalnumber of mice injected (determined40days aftertumorcellinjection).

100 to 500 U/ml), whereas mu-IFN-a/, and AZT were

ineffective (datanotshown).

We then performeda series ofin vivo experiments with

these substances. SI nudemice weretreatedwith

anti-IFN-al

antibody and subsequently injected s.c. with

HIV-infected U937 cells. On day 14, allof themice haddeveloped solid tumors and were treated with hu-IFN-a or AZT or

were leftuntreated. Treatmentwith hu-IFN-axresulted in a

marked reduction oftumorgrowth and even tumor

regres-sion in comparison with controlmice (Fig. 8A). Incontrast,

AZTtreatment (1 mg/ml in thedrinkingwaterfor 3 weeks) did not inhibit tumorgrowth. On day 38, mice were

sacri-ficed, solidtumorswere removed,and serumsamples were

taken for p24 titration. None of the mice treated with hu-IFN-a exhibited detectable serum p24 levels (<100 pg/

ml), whereas three ofthefourcontrol mice and three of the four AZT-treated mice exhibited serum p24 levels ranging

between 400 and 1,350 pg/ml. One control and one

AZT-treated mouse exhibited very small tumors (mean surface

area, <150 mm2) and had no detectable serum p24 levels

(<100pg/ml). Inasubsequentexperiment, AZTwas

admin-istered peritumorally eachday for 2weeks (480 mg/kg/day [maximal tolerated dose]). No significant change in tumor growth or in p24antigenemia was observed inAZT-treated

miceincomparison with controlmice (datanotshown). Inanotherexperiment, establishedHIV-positive U937cell

tumors were treated with mu-IFN-a/,, hu-TNF-a, or a

control preparation. Both mu-IFN-a/, and hu-TNF-a stronglyinhibited tumorgrowth (Fig. 8B). Allof the TNF-treated tumors exhibited detectable levels ofp24

antigene-mia, whereas none of the mice treated with mu-IFN-a/,B

exhibiteddetectablelevels ofserump2438daysaftertumor

cellinjection. Thesedatasuggestedthat p24antigenemia did

notsimplyreflecttumorgrowth and thatcytokines such as IFN and TNFmay exhibitopposite effects onHIV produc-tion inthis invivomodel.

The electron microscopy analysis of HIV-infected U937

tumors 7 and 18 days after the initiation of therapy (two

tumors per group) revealed a clear-cut reduction of HIV

particles intumorstreatedwitheitherhu-IFN-axor

mu-IFN-a/,1. Areasoftumornecrosis weredetectedinHIV-infected

U937 tumors treated with

hu-TNF-a,

hu-IFN-a,

and

mu-IFN-a/,B intheabsence of obvious host cell infiltrates

(data

notshown).

DISCUSSION

The results ofthese

experiments

demonstrate that

injec-tion of antibodiesto

mu-IFN-a/1

innude mice

transplanted

s.c.with HIV-infected U937 cells resultsinthe

development

of HIV-positive U937 cell tumors and

p24

antigenemia.

Usually, neither tumor

growth

nor

p24

antigenemia

was observed in control mice

transplanted

with HIV-infected U937cells.In

anti-IFN-a/W-treated

mice,

adirectcorrelation

was observedbetweentumor

growth

and HIV

p24

antigen-emia,

indicating

that

endogenous

IFN-&x/I

represents an

importantfactorin therestriction of bothtumor

growth

and HIVproductionin this system.

Although

HIV-infectedU937 cellsdidgrowto someextentin

immunosuppressed

(SIA)

or genetically immunodeficient

(bg/nu/xid)

nude

mice,

treat-mentwith antibodiesto

IFN-a/1

seemed essentialtoobtain progressive tumor growth and HIV

production.

Treatment

ofSIA nudemice withantibodiestoIFNresults in the

rapid

development

(latency

time,

8 to 10

days)

of

HIV-positive

U937 cell tumors and in

p24

antigenemia

in 100% of the

tumor-injected mice.

We have previously shown that

injection

ofantibodies to

IFN-a/p

markedly increased tumor

growth

in mice

trans-planted with different

syngeneic

tumors

(9)

and

abrogated

the resistance to the

multiplication

ofFriend

erythroleuke-mia cells in the livers of

allogeneic

mice

(10,

12).

In nude

mice, it had been

previously

shownthat

injection

of antibod-iesto

mu-IFN-a/,3

markedly

increasedtumor

growth

inmice transplanted with tumor cells

persistently

infected with different viruses (28,

29)

as well as with human

prostatic

tumors(28). Itis alsoof interestthattreatmentof mice with

antibody

to

IFN-oa/,

abrogated

theresistance of X-irradiated

F1hybrid and

allogeneic

mice to bonemarrow

grafts (1).

Inview oftheinterest in in vivo murine models for

AIDS,

it is reasonable to suggest that

HIV-positive

U937 cell tumors

implanted

in

anti-IFN-a/,-treated

nude mice with

established p24

antigenemia

may represent a useful model

forthedefinition ofnew

protocols

for antiviral

therapies.

In

particular,

this system allowed usto compare the effects of hu-IFN-aandAZT

(Fig. 8A).

Hu-IFN-awas

highly

effective in

inhibiting

both tumor

growth

and

p24

antigenemia,

whereas AZT treatment did notaffect the

growth

of estab-lished

HIV-positive

U937 cell tumors

(Fig. 8A)

and did not

inhibitp24

antigenemia.

Hu-IFN-a

probably

acts

directly

on established

HIV-positive

tumors, as

hu-IFN-a

showsavery low

cross-reactivity

on mouse cells

(2).

In this context, it is

of interest that addition of

hu-IFN-a

to these

chronically

infectedU937 cells in culture resulted inamarkedinhibition of HIV

production,

whereas AZT did not affect HIV

pro-duction

(Fig. 7).

These data are in agreement with those

reported by Poli and co-workers

using

other

chronically

HIV-infected cell lines

(27).

Although HIV-infected U937 cells in culture were resis-tant to the

antiproliferative (data

not

shown)

and antiviral (Fig. 7)effectsof

mu-IFN-a/,, injection

of

mu-IFN-a/p

was

highly effective in

inhibiting

both tumor

growth

and

p24

antigenemia. This

finding

suggests that host antitumor and antiviral mechanisms were also most

likely

activated

by

mu-IFN-a/,B in

immunosuppressed

nude mice.

Although

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utilized for in vivo

investigation

ofmany human tumors, there is

ample

evidence that

they

display

residual

immunity

against

xenotransplants

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FIG. 6. Electron microscopy of HIV-infected U937 cells in vitro and when transplanted s.c. in anti-IFN-a/p-treated nude mice. (A)

Morphology of HIV-infected U937 cells in culture. Cellswere harvested 3 days after cellseeding and processed for electron microscopy

analysis asdescribed inMaterials and Methods. Some HIV particlesareobserved in association with the cell membrane and foundtobe

released inthe extracellularspace. Magnification, x8,820. (B) HIV-infected U937 celltumorestablished inan

anti-IFN-a/P-treated

mouse.

Thetumorwasdissected 40 days aftertumorcell injection and processed for electronmicroscopy analysis asdescribed in Materials and Methods.Invivotreatmentswereperformedasdescribed in thelegendtoFig.2.Note the cluster of virusparticles in the extracellularspace. Magnification, x14,400. (C) Typicalpatternof extensive virusproduction inanHIV-infected U937 celltumordissected froman

anti-IFN-a/a-treatedmouse.Notethe differentstagesof virus formation in theU937 cell plasma membrane. Budding with crescent-shaped nucleoid surroundedbyanenvelopewithfuzzysurfaceprojectionsandimmaturevirus withring-shapednucleoidprofilesandmatureforms sectioned

longitudinally andperpendiculartothecoreaxis canalso beseen. Magnification, x50,000. 2250

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hu-IFNa mu-IFNa/13 AZT TNFa

FIG. 7. Effects ofhu-IFN-a,

mu-IFN-a/p,

hu-TNF-a,and AZT on HIV production in chronically infected U937 cells in culture. HIV-infectedU937cellswereseededat2 x 105/ml inthe presence orabsence of different substances. On day 3 of culture, cells were diluted 1:2 with fresh medium with or without IFN or AZT to maintainthe initial concentration. Supernatants werecollected on day6of culture and testedforp24. Treatments wereperformedas follows: hu-IFN-a, 100U/ml; mu-IFN-a/1, 100U/ml; andAZT, 1

F±M. Values represent the means of duplicate samples. Standard deviations did not exceed10%.

immunosuppression (reference 30 and references herein). In fact, despite their nominal athymic state, nude mice have

some residual Tcells and exhibit immune reactions (16, 18, 19, 33). The numbers of B cells, natural killer cells, and macrophages are similar to or even higher than those in

euthymicmice of thesamestrain(5, 15). Recovery of natural

killer cell and B-cell activity has been described after the initial suppression induced either by whole-bodyirradiation

orby injectionof anti-asialo-GM1 antibody. Likewise,

mac-rophage functions appear to be unaltered in SIA or

geneti-cally immunodeficient nude mice. In this regard, Balkwill

and Proiettipreviously reportedthatmu-IFN-a/ exerteda

potentantitumoractivityin nudeandnude/beigemice trans-planted with human tumors (3). The fact that SI nude mice with establishedHIV-positive U937 cell tumors respond to

mu-IFN-o/ treatmentby reducedtumorgrowthand inhib-itedp24 antigenemia is inaccord with these findings.

The question still to be answered concerns the antiviral

action ofmu-IFN-a/,BonHIV-infected humanU937tumors.

Mu-IFN-a/, exertednoeffectonthese cells inculture(Fig. 7), and there is no evidence to suggestthat the important species specificity of mu-IFN-a/3 isnotconserved in vivo. Thus, it seems unlikely to us that mu-IFN-oa/, directly inhibitedHIV replicationin these humantumors. The anti-viral effectof mu-IFN-a/3 maybeindirect and secondaryto theIFN-inducedreductionintumorgrowth.Thefindingthat TNFinhibitedtumorgrowthand didnotinhibitp24 antigen-emia would seem to argue against this hypothesis, but the conditionsmaynotbesimilar,since TNFalsoenhances HIV multiplicationin thesecells(Fig. 7; 6, 20, 26). Lastly,itmay

be thatIFN-a/, induces theproduction bymousetissuesof

another cytokine that is not species specific and exerts

anti-HIV activity.

Afew mouse models for AIDS have beenproposed (17, 22-25), and the advantages and disadvantages of these

Days

FIG. 8. Effects ofcytokines (hu-IFN-a, mu-IFN-a/,, and

hu-TNF-a) andAZTon tumorgrowthinnude mice withestablished HIV-positive U937 cell tumors. Five-week-old female nude mice weresplenectomized and irradiated (SI nude mice)as describedin

Materials andMethods.Micewereinjectedi.v.with 0.2 mlofa1:20 dilution (containing 320,000 neutralizing units)ofanti-IFN-a/p

an-tibody.Three hourslater, the micewereinjecteds.c.with1.5x 107

HIV-infected U937cellsin 0.2ml of salinecontaininga1:20dilution ofanti-IFN-a/, antibody. The mice were further treateds.c. with

thesameantibodydilutiononday5. (A)Onday 14, themicewere

divided into three groups and treated with AZT (1 mg/ml in the

drinkingwaterfor3weeks)orhu-IFN-a(105U permousein 0.2 ml

s.c.; five daily injections per week; 3 weeks oftreatment) or left

untreated(NT). Onday 38,the mice weresacrificed, solidtumors

wereremoved, andserum samples were tested forp24 antigenas

described in Materials and Methods. Theserump24levelswereas

follows:

Treatment

None hu-IFN-a AZT

Serump24(pg/ml), 4mice/group 1,350, 1,300,550, <100 <100, <100, <100, <100

500, 400,600, <100

(B)On day21 oftumorgrowth, the mice were divided into three

groupsand treateds.c.with 0.2ml of controlpreparation (five daily injections perweek; 2 weeks oftreatment), hu-TNF-a (2 ,ug per mouse ondays21 and 22),ormu-IFN-a/,B (105Uper mouse; five

daily injections perweek; 2 weeks oftreatment). On day 38, the

mice were sacrificed, solid tumors were removed, and serum

sampleswere tested forp24 antigenas describedin Materialsand

Methods. Theserump24levelswere asfollows: Treatment

Controlpreparation mu-IFN-ap

hu-TNF-a

Serump24(pg/ml), 4mice/group 2,800, 1,400, 1,000,100 <100, <100, <100, <100 4,100, 500, 500, 100

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models have recently been discussed (8, 21). In particular, the SCID-hu mouse has been found to be permissive for replication of HIV after direct injection of virus into the engrafted human lymph node or thymus (22). HIV expres-sion was apparently restricted to some transplanted human cells (22). In this system,virus replicationwassuppressed if AZT was administered before virus inoculation (23). There-fore, thismodel seems to be very useful for therapy aimed at preventing infection after acute virus exposure. One major differencebetween the modeldescribed in this report and the existing SCID models is the fact that we have directly injected micewithchronically HIV-infected tumorcells that arecapable of rapidly growing in anti-IFN-treated mice and ofproducing high amounts of HIV in vivo. Both tumor size and serum p24 levels can be easily evaluated in individual mice, both beforeand in the course ofan antiviraltherapy. In conclusion, our results suggest that HIV-infected U937 cell tumors transplanted in anti-IFN-a/,-treated mice con-stitute a simple and useful model for the therapy of chronic viremia and for evaluating the in vivo effects of different substances on HIV-infectedcells.

ACKNOWLEDGMENTS

This work was supported in part by grants from Ministero della Sanita/ISS (IIProgetto diRicherche sull'AIDS 1989, contract 4207/ 01), bytheAssociazione Italiana per laRicerca sulCancro, and by theAgence Nationale deRecherches sur leSida/C.N.R.S.(contract 89N60/0811).

We are gratefulto M. Martorelli(WellcomeSpA, Pomezia, Italy) and to L. A. Shute (The Wellcome Research Laboratories, Beck-enham, Kent, England) for providing AZT. We thank G. Forni (Institute of Microbiology, University of Turin, Turin, Italy) for helping to establish in our laboratory the optimal experimental conditions for preparation of SIA nude mice.

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