Switch from a type 2 to a type 1 T helper cell response and cure of established Leishmania major infection in mice is induced by combined therapy with interleukin 12 and Pentostam

Proc. Natl. Acad. Sci. USA Vol.92,

_pp.

3142-3146, April 1995 Immunology

Switch

from a

type 2 to a type 1 T helper cell response and cure

of

established

Leishmania

major

infection

in

mice

is

induced

by

combined

therapy

with

interleukin

12 and Pentostam

(T-cellsubsets/antimony/chronic infection)

GARY S. NABORS, Luis C. C.

AFONSO, JAY P. FARRELL,

AND

PHILLIP

SCOTr*

Department ofPathobiology, University ofPennsylvania, Philadelphia, PA 19104

Communicatedby BarryR. Bloom, AlbertEinstein College of Medicine,Brornx, NY,December 29, 1994

ABSTRACT Successful treatment in allergic,

autoim-mune, and infectious diseases often requires altering the nature of a detrimental immune response mediated by a particular CD4+ T helper (Th) cell subset. While several factors contribute to the development ofCD4+ Thi and Th2 cells, therequirements forswitchinganestablishedresponse are not understood. Here we use infection with Leishmania major as a model to investigate those requirements. We report that treatment with interleukin 12 (IL-12), in combination with the antimony-based leishmanicidal drug Pentostam, induces healing in L. major-infectedmice and that healing is associated with a switch froma

Th2

to a

Thl

response. The data suggest that decreasing antigen levels may be required for IL-12 to inhibit a

Th2

response and enhance a

Thi

response. These observations areimportant for treatment of nonhealing forms of humanleishmaniasis and also demonstrate that in a chronicinfectious disease aninappropriate

Th2

response can beswitched to aneffective

Thi

response.

Theprotozoan parasite Leishmaniamajor causes a disease with awide range of clinicalmanifestations. Experimental infec-tions in mice mimic to alargeextentthe humandisease. Strains

of mice that are highly susceptible to L. major, such as

BALB/c,

developpoorcell-mediated immunitytotheparasite andproduce cytokines associated with Thelper type 2(Th2) responses, such as interleukin 4 (IL-4), IL-5, and IL-10, following infection (1, 2). Similarly, enhanced levels of IL-4 and IL-10 have been reported in human patientswith non-healing forms of leishmaniasis (3-6). In contrast, resistant mouse strainssuch as C3Hdevelop

Thi

responses, character-ized by cell-mediated immunity and interferon

'y (IFN-.y)

production.Variousimmunologicalmanipulationscanbe used to render otherwise susceptible

BALB/c

mice resistant, in-cluding sublethal irradiation (7), B-cell depletion

(8),

and treatment with anti-IL-4 (9, 10), anti-transforming growth factor ,B

(TGF-13)

(11),anti-IL-2(12),anti-CD4(13),or, more

recently, recombinant IL-12 (14, 15). However, such

treat-ments areeffectiveonlyif administered at or near the time of parasite inoculation, suggestingthattheyinfluence theinitial developmentof

Thl

cellsfrom naivecells, rather thanreverse an established Th2response. It hasyet tobe demonstrated in

an infected

BALB/c

mousethat, once established, the

non-protective immune response can be sufficiently altered such that aprotective

Thi

responsepredominatesandhealing of the lesion occurs. Accomplishment of this task willbe of signifi-cance inthedevelopmentofimmunotherapies for controlling chronic infectious diseases suchasleishmaniasis.

IL-12 is a cytokine that induces IFN-ny production and cytotoxic activity bynaturalkiller and T cells(16, 17)and can initiate the differentiation of

_Thl

cells from naive T cells

Thepublication costs of this article weredefrayedinpart by page charge payment. This articlemusttherefore beherebymarked"advertisement"in

accordance with 18 U.S.C.§1734 solelytoindicate this fact.

(18-22). IL-12 enhancesIFN-,y production by

Thl

clones (23) and promotes the proliferation ofThi but not Th2cells (18). However, inspite of its effects on established T-cell clones, IL-12 altered disease progression in leishmaniasiswhen

ad-ministered at the time of infection, but not 1 week after

infection(14, 15). Thesedatasuggest that IL-12 is ineffective atpromoting a Thi response in the presence of an ongoing Th2 response. We hypothesizedthatthe failureofIL-12 to influ-ence anongoingTh2 responsemightrelate to thehighantigen levels associated withanestablishedL. majorinfection, since

antigen dosecaninfluence thenatureofanimmuneresponse

(24-26). Therefore, we investigated whether the immune

response in L.major-infectedBALB/c mice,andthe outcome ofinfection,could be altered when IL-12 was administered to animals in which theparasiteload was reduced.

Pentavalent antimonycompounds such as Pentostam are

commonly used for thetreatmentof humanleishmaniasis, and

we chose thiscompound toreduce theparasite burdenin L.

major-infected mice. While Pentostam is active against L.

major, treatment of susceptible strains of mice, including

BALB/c, rarely leads to a cure, since once treatment is

terminated, the remaining parasite population almost invari-ably expands, leadingto afatal infection(27, 28).These data suggest that successful treatment in leishmaniasisrequiresnot onlyareduction inparasitenumbers butalsothedevelopment of aneffective cell-mediated immune response.Indeed,

suc-cessful drug treatment in human visceral leishmaniasis is

associated with the development of cell-mediated immunity

(29). We report that therapywith IL-12 and Pentostam can

inducehealing of establishedL.major lesionsin

BALB/c

mice and convert the anti-parasite T-cell response from a Th2 pattern to one characteristic of a

Thi

response, which is indicativeoflong-term resistance.

MATERIALS

AND

METHODS

Parasites and Mice. Five-week-old female

BALB/cByJ

(BALB/c)

and

C3HeB/FeJ

(C3H)micewere obtainedfrom The Jackson Laboratory. The clone of L. major (WHO

MHOM/IL/80/Friedlin)

usedinthis studywasmaintainedin Grace's insectcellculture medium

(GIBCO) containing

20% fetal bovineserum

(GIBCO),

2 mML-glutamine, potassium

penicillin G,

(100

units/ml),

and

streptomycin

sulfate

(100

lag/ml).

Infections and Treatment Protocols.Micewereinoculated in the right hind footpad with 105 metacyclic promastigotes

selected from stationary-phase cultures by use of Arachis

hypogaea agglutinin

(Sigma)

(30).

Lesion development was

monitoredwith a dialcaliper(Starrett, Athol, MA),and lesion sizewasexpressedasthedifferencebetween the infected and

Abbreviations: IFN,interferon; IL, interleukin; HPRT, hypoxanthine phosphoribosyltransferase.

*To whomreprint requestsshould be addressed.

contralateral footpad. The number ofparasitesintissueswas

determinedbylimiting-dilution analysis (31). Pentostam

(so-dium stibogluconate containing 100 mg ofpentavalent

anti-mony perml;proprietarynameofBurroughsWelcome)was

a gift from Max Grogl, Walter Reed, Washington, DC. Re-combinant murine IL-12was agift from StanleyWolf and Joe Sypek, Genetics

Institute,

Cambridge,MA. Inthe firstseries ofexperiments, miceweretreatedintramuscularly (i.m.)with Pentostam and intralesionally orintraperitoneally (i.p.)with 0.2,tgofrecombinant murine IL-12. Animals in thesestudies receivedten 5-mginjections of Pentostam (-250 mg/kgper day)over aperiod of2weeks, givenalternatelyintherightand left hindleg, beginning on day 14 of infection. Mice were treated with IL-12on days 14, 16, and 18 ofinfection. In a

secondprotocol,miceweretreatedintralesionally withvarious combinations ofphosphate-bufferedsaline(PBS), Pentostam, andIL-12,administered inatotalof50,ul,beginningonday 21of infection. Pentostam(2.5mg diluted inPBS),IL-12(0.2 ,ug in PBS), or Pentostamtogether with IL-12was adminis-teredondays 21, 23, 25, 28, 30, and 32 ofinfection.

Immunological Assays. Popliteal lymph node and spleen cells

(5

x 106per

ml)

were cultured in Dulbecco's modified

Eagle'smedium(GIBCO)containing 10% fetal bovine serum,

potassium penicillin G

(100 units/ml),

streptomycin sulfate (100

,tg/ml),

25 mM Hepes, 2 mM L-glutamine, 50

p,M

2-mercaptoethanol,

1 mMsodiumpyruvateat37°C. After72 hr, supernatantswere harvested forcytokine analysis. Some cell cultureswerestimulated invitrowith soluble leishmanial antigen(50

,ug/ml) (32).

ELISAswereusedtomeasureIFN--y and IL-4

(33).

Standardsused incytokineassayswere

recom-binantIFN-,y

(a

giftfrom

Genentech),

and IL-4(a giftfrom

Robert Coffman,

DNAX).

IgE levels were measured by an

isotype-specific ELISA using a monoclonal anti-mouse IgE

(PharMingen) and a rat-anti-mouse IgE-horseradish

peroxi-dase conjugate

(Southern

Biotechnology Associates) as a

second antibody.

ReverseTranscription-PCRAnalysis.CytokinemRNA lev-els in tissues were analyzed

essentially

as described

(34).

Primer and probe sequences for IFN--y and hypoxanthine

phosphoribosyltransferase

(HPRT)

have been described (35), and the

following

sequenceswereused todetect IL-4cDNA:

sense

primer,

5'-ACGAGGTCACAGGAGAAGGGACGC-CATGCA-3';

antisense

primer,

5'-GCTCTTTAGGCTTTC-CAGGAAGTC-3'; probe,

5'-CTCACAGCAACGAAGAA-CACCACAGAGAGT-3'. After initial denaturation at 94°C

for2

min,

PCR consisted of 26

cycles (for

HPRTand

IFN-y)

or32

cycles

(for IL-4)

of94°Cfor 15 sec,54°Cfor 15 sec,72°C

for 1 min, followed

by

72°C for 7 min. After agarose

gel

electrophoresis

and Southern transfer, membranes were hy-bridizedwith the appropriate oligonucleotide probe labeled

with the ECLsystem

(Amersham).

Themethodfor

determin-ingfold increases incytokinegeneexpressionovernormal cell levels has been described

(34).

StatisticalAnalysis. Datawere analyzedwithanunpaired Studentt test.

RESULTS

Inourinitial

experiments,

micewereinoculated with L. major, and 2 weeks

later,

when

footpad

lesionswereapparent,were

treated with Pentostam alone

i.m.,

with IL-12 alone i.p. or

intralesionally,

with a combination of Pentostam i.m. and IL-12 i.p. or intralesionally or were untreated. Each of the

treatments

delayed

lesion

development

to some extent, al-thoughmicetreated with Pentostam i.m.orIL-12

intralesion-ally

(Fig. 1A),

as well as mice treated with IL-12 i.p., or

Pentostam i.m. plus IL-12

i.p.

(data

not

shown),

eventually

developed

progressivedisease. In contrast, 50% of the mice treatedintralesionallywith IL-12 incombinationwith

Pentos-tam

given

i.m. healed

(Fig. 1A).

Healing mice had lower

numbers oftissue parasites compared with all other groups (Fig. 1B). Additionally, of the four mice that healed, three had no detectable parasites in their spleens, indicating that the treatmentinhibited metastasis of parasites from the primary lesion site.

In a second series of studies, we investigated whether the local administration of both IL-12 and Pentostam would increase theefficacy of treatment. In these experiments, IL-12 andPentostam were administered together into the infected footpad,beginning 3 weeks after infection. The efficacy of the various therapies was initially assessed by quantifying the number of parasites in the footpad 1 week after treatment. Whereas footpads from PBS-treated mice hadaparasite titer of >1012, those from mice treated with Pentostam alone had

a titer of 106.4, demonstrating the effectiveness of the drug.

Footpadsfrom mice treated with IL-12 alone had a parasite

titer of1011-75atthistime, while those from mice treated with Pentostam plus IL-12 had a titer of 10144, indicating that

treatment with these two agents had a synergistic effect on

reducing the parasite burden.

Intralesional treatment with Pentostam delayed lesion de-velopment, as did treatment with IL-12 alone. However, neither treatment ledto healing of the infection. Incontrast, lesion growth wasmarkedlysuppressed when IL-12 and

Pen-tostamwerecombined(Fig. 1C).Four months afterinfection, 7 of 10 mice receiving this treatment had no visible lesion,

while lesions on the remaining mice measured <0.5 mm at

16-22 weeks ofinfection. Thesehealing micehad fewparasites

at the lesion site and no detectable parasites in the spleen, comparedwith thehighlevels ofparasitizationobserved in all other groups(Fig. 1D).Insixexperimentsinwhich mice with establishedinfectionsweretreated with Pentostam(giveni.m.

orintralesionally) in combination with intralesional injection ofIL-12,a 70%rate ofhealing (32 of46 mice)wasnoted.

Theabilityofintralesional treatmentwith IL-12 and

Pen-tostam to induce a healing response in L. major-infected

BALB/c

micewas associated with a switch in the dominant immune response observed in these animals. Prior to treat-ment, lymph node cells from infected mice produced high levels of IL-4 and low amounts of IFN--y (Fig. 2A). These resultsareconsistent withprevious studies demonstratingthat by 2 weeks of infection, BALB/c mice have developed a

dominantTh2response(36-38). Followingsuccessfultherapy with IL-12 and Pentostam, however, the cytokine profile observed by cells in the lymph node had switched to a

dominant Thi response (Fig. 2A). Whilewe were unable to

analyze lymph nodecytokine production in the control mice

aftertreatment, dueto the extentofparasitismand necrosis withinthelymphnode,acomparison ofresponses in thespleen of healed (IL-12 plus Pentostam-treated) and nonhealed (IL-12orPentostam

alone)

animalsconfirmedourfinding that

a

Thi

response haddevelopedinhealingmice.Thus,following treatment,spleencells from

BALB/c

micetreated intralesion-allywith IL-12plusPentostamproducedmoreIFN-,yand less IL-4 thanmice treated with eitheragent alone(Fig.2 BandC). Reverse transcription-PCR analysis of cytokine mRNA levelsconfirmed thathealingwasassociated withadominant

Thi

response (Fig. 3). Thus, Leishmania-reactive cells from healed mice contained more IFN-,y mRNA and.less IL-4

mRNAthan thosefrom control animals. Additionally,serum

IgElevels, indicativeof in vivo IL-4production,were

signifi-cantlylower inmicetreated with IL-12 and Pentostam

com-paredtothose in mice treated with PentostamorIL-12alone

(Fig.

2D).Similar resultswerenotedinthemice which healed

following treatment with IL-12 and Pentostam given i.m.,

describedinFig. 1A

(data

notshown).Takentogether,these data demonstrate that combined therapy with IL-12 and Pentostam inmice with established infectionspotentiates

Thl

Proc. Natl. Acad Sci. USA 92 (1995)

Weekofinfection

5

-

4-

3-

2-1

-2 4 6 8 10 12 14 22

Weekof infection

a)

ta

no

0

Untreated Pentostam IL-12 Pentostam Pentostam Pentostam

+IL-12 +IL-12 +IL-12

(nonhealing) (healing)

._

CA

0~

ct

to 0

Untreatd PBS IL-12 Pentostam Pentostam

+IL-12

FIG. 1. Course of infection andparasiteburdens in treated and untreatedBALB/c mice infected withL. major.(A)Infected miceeitherwere

untreated(0),receiveddailyintramuscularinjectionsofPentostam duringthethird andfourthweekof infection (0),received IL-12 in the infected footpadondays 21, 23,and 25 of infection (o),orreceived both Pentostam and IL-12(m).Datarepresentmeant SE of 8 micepergroup(or

of 4 of 8mice,at16weeks)andarerepresentative ofthree experiments. (B) Parasite burdensinfootpadtissue(hatched bars) and spleens (open bars) from thegroupsof mice shown inA,at 11 weeks(untreated mice)or16 weeks(allothergroups)of infection. Barsrepresentmean + SD of thepositiveendtitration of tissue from8micepergroup.Barstotherightof thedotted line depict themean+ SD ofthepositive endtitration oftissue from fourhealingmice and fournonhealingmicefollowingtreatmentwith IL-12plus Pentostam giveni.m. Resultsarerepresentative ofthree experiments.Asterisks indicatesignificant differences comparedwithvalues shownfor untreatedmice, micetreated withPentostamor

IL-12alone,andmice that didnothealaftertreatmentwithIL-12andPentostam(P<0.05). (C) Courseofinfectioninuntreated mice (0)and inmicereceivingintralesionalinjectionsof PBS(O),IL-12(A),Pentostam(0),orIL-12plusPentostam(A)ondays 21, 23, 25,28, 30, and 32 of

infection. Datarepresentmean±SEfor 10 miceper group.(D)Parasiteburdens in footpads (hatched bars) and spleens (open bars)in thegroups

of mice described inC,at10 weeks of infection(untreatedandPBS-treated mice)or14weeks of infection(other groups).Data shownarethe

positive end titrations (mean + SD) ofhomogenized tissue fromS mice pergroup. *,Significantly different compared with all othergroups

(P<_0.05).

DISCUSSION

This study hasdemonstratedthat BALB/c mice, which would otherwise succumbtofatal L. major infection,canbe clinically cured of disease by combined treatment with the anti-leishmanial drug Pentostam and recombinant IL-12. That the mice treated with Pentostam and IL-12 wereindeed healing was shown by decreased footpad lesion size and decreased parasite numbers in the footpadsandspleens. This therapywas effective inmice with ongoing infections in whicha

nonpro-tective Th2responsehadbeen established. Aftertreatmentand

the resulting resolution of the lesion, the cytokine profile in thesemice indicated aswitch toaprotective

Thl

pattern.

We showed that a Thi response was potentiated in mice

treated with Pentostam plus IL-12 by demonstrating that lymph node and spleen cells from treated mice which healed produced high levels of IFN-y and low levels of IL-4 and that the concentration of IgE in serum from these mice was

significantly lowerthanthat foundinnonhealing mice. Merely transiently reducing the parasite burden bya shortcourseof

treatmentwithantimony didnotresultin healingor aswitch

to a

Thl-like

cytokine production profile; rather, the mice apparently continued to preferentially expand the

IL-4-producing cell population following treatment. These data

agree with previous studies in which lesion development

resumedfollowing cessation of drug treatmentin susceptible mice infected with L. major, despite the ability of the drugto

reduce the parasite burden (27, 39). Our findingmayexplain

why treatment of Leishmania donovani-infected mice with Corynebacteriumparvum and antimonywas found to be sig-nificantly more effective than treatment with either agent

alone (40), since C. parvum is a potent inducer of IL-12 (unpublishedobservation).

Several protocols combining Pentostamand IL-12 therapy

were used for the treatment of infected mice. The most

effective treatment involved intralesional injections of both

agents,after which 85% of all treated mice healed in multiple experiments. In addition, intramuscular injection of Pentostam combined with intralesional IL-12 injection also promoted healing in 50% of the mice. That 50%, and not a greater

proportion, of mice treated i.m. with Pentostam and intrale-sionally with IL-12 healed maybe due to slight variations in infective doses and doses of Pentostam and IL-12 adminis-tered. Nolong-lastingbenefitoftreatmentwasobserved when infected micewere_{treated with IL-12 i.p. alone,}orIL-12 i.p.

in combination with Pentostam. These results suggest that optimaltreatmentof establishedL.major infections withIL-12

a;

N CA

._

0

ce

a;

N

._

0 rA

U,)

Proc. Natl.Acad. Sci USA 92 (1995) 3145

Before treatment Aftertreatine]t

C

-_

mom

mlim~~~~~~~~~~~~~~~~~

IL- 12llcntustam Pentostam~~~~~~ l -1

IL-12 Pentostam Pentostam

+IL-12

D

7.T

.,n,,E~

IL-12 Pentostam Pentostam

+11-12

10

7 FIG. 2. Immunological analysis of L. major-E _{infected BALB/c mice. (A) IFN-,y and IL-4}

pro-, ductionby popliteal lymph node cellswasanalyzed

5 _{in three untreated BALB/c mice at 2weeks of}

infection (before treatment)and infive miceat16 weeks of infection, following intralesional

treat-2.5 _{mentwith IL-12 and Pentostamasin Fig.1C(after}

treatment). Forcomparison, -cells from resistant C3H mice infected for2 weeksproduced IFN--yat

° 29.33±7.99ng/ml andIL-4at0.71±0.72unit/ml

(datanotshowninfigure).Dataarerepresentative

v0 of three experiments. (B-D) After intralesional

treatmentof infected mice withIL-12, Pentostam,

or both agents, spleen cells from five mice per

8 group were similarly analyzed for IL-4 (B) and

IFN-y (C) production 16 weeks after infection.

- 6 q Serum fromsimilarlytreated micewasanalyzedfor

= IgE levels 22 weeks after infection (D). Normal

4 BALB/cserumcontainedIgEat0.3,ug/ml.

PBS-treated mice hadto be sacrificedat 11weeks of infection duetotheextentoftheir disease. Spleen

- 2 cells from theseanimalsproducedIFN-,yat11.21 ±3.77 ng/mland IL-4at4.11+0.34units/ml,and

L

o serumfrom these mice contained

_IgE

at6.23 ±

1.29,ug/mlatthetimeof sacrifice.Barsrepresent

mean + SE;*,significance atP< 0.05.

maynecessitate local application of the cytokine. In addition

tovarying theroutesof administration of IL-12 and

Pentos-tam,wehave also varied the number of injections required for successful therapy. Preliminary results suggest thatthree

in-tralesionaIf

injections of IL-12 plus Pentostamaresufficientto

renderhealing, whereasoneinjection is insufficient (datanot

shown).

The Thi effectorcells that mediatecurefollowing IL-12 and Pentostam treatmentmayoriginate from eitherapopulation of uncommitted cellsor

Thl

cells, since in additiontoTh2cells, both Leishmania-reactive naive and

Thl

cells havebeen iden-tified in L. major-infected BALB/c mice (41-43). Alterna-tively, IL-12mayhave switchedTh2 cellsto a

Thl

phenotype, although thisseemslesslikely, since IL-12canonlytransiently alter cytokine production by cloned Th2 cells (22). In either

case, in our experiments, IL-12 alone appeared unable to

enhance in vivoThi expansion duringaprogressive infection. Thus, while IL-12 promotes healing in BALB/c mice when given atthe time ofinfection (14, 15), it is ineffective when given after infection. Similarly, during asecondary infection withNippostrongylus, IL-12 didnotmarkedly affect cytokine production (44). In thepresentstudy, only whenwecombined IL-12treatmentwithareduction intheparasite burden-asa result of Pentostamtherapy-didwenoteaconversion from aTh2to aThi response.

An association between high antigen loads and the

devel-opment ofTh2 responses has beenpreviously described (24,

25). Our data stronglysuggestthatantigen levels contributeto

the inability of infected mice to expand a

Thl

population followingtreatmentwith IL-12alone, although the possibility that Pentostam is influencing the immune response in an unknown fashion cannot be excluded. Other studies with L. majorsupport the hypothesis that high antigen levels inhibit Thi expansion. For example, infection withverylownumbers

of parasites elicited resistance and a Thi response in the

normally susceptible BALB/cmousestrain (25). In addition, when cells from L. major-infected BALB/c micewere

trans-ferred toscid/scid mice, the cellpopulation could be shifted from a dominant Th2 to a

Thl

phenotype with anti-IL-4

treatment, while donor mice, containing large numbers of parasites,wereunaffected by suchtreatment (43). IL-12 can switch theTh cell subset balance fromaTh2to

Thl

phenotype

following immunization with Schistosoma mansonieggs,where the levelofantigen will be low when compared withanactive

IL-12

.

IFN-y

IL-4 I

S..

HPRT I

#

1 1

Pentostam

IL-12

+ Pentostam

0 _;,:

ItSIS~~~~~~~.."

I::

I ..

*-

_I..l

.

.

I.1 ti li

* IFN-Y 0 IL-4

IL-12 Pentostan IL-12+Penitostam

FIG. 3. Reverse transcription-PCR analysis ofIFN-,y, IL-4, and HPRT gene expression by spleen cells from groups of three mice

treatedintralesionallyas inFig. 1CwithIL-12, Pentostam, or

Pen-tostamplusIL-12. Spleencellswereharvested frommice 22 weeks

after infection andwerestimulated in vitro with soluble leishmanial

antigenfor18 hrpriortoRNA isolation.Shownareresultsusing1:10

(upper band) and 1:100 (lower band)dilutions of cDNA. Levels of HPRTexpressionwereusedtocontrol forRNAcontentandintegrity. Fold increasesinIFN-,yand IL-4geneexpressionincells from treated

miceovertheamountobserved insimilarlystimulated normal cellsare

shown at the bottom. Cytokine PCR sampleswerenormalized for

HPRTsignal priortodeterminingfoldincreases ingeneexpression.

*,Significance atP<0.05.

20

--

15-E.

r-W.

5-60

-50

-S

40-01)

3->- 30

z

L

20-10 -

0-Immunology:

NaborsetaL

Proc. Natl. Acad Sci USA 92 (1995)

infection (45). The mechanism bywhich antigen load

influ-encesThcellsubset dominance remains unclear.However,one

factor may involve the capacity of high antigen levels to

selectivelytolerize CD4+

Thl

cells (26).

In summary,we have found that a Th2-mediated immune

responsecanbeovercomeduringaprogressive infection using

IL-12,

indicatingthatatthepopulation level,the dominance of

aparticular Thcellsubsetcanbe altered.Further,sinceIL-12 waseffectiveonlywhen theparasiteburden had beenreduced,

ourresultssuggest thatlowering antigenlevels maybecritical

forimmunotherapies directed toward reducing a deleterious

Th2response. Thatan established, inappropriate

Th

response

canbe switched to an appropriate response has implications

not onlyfor the treatment fornonhealing leishmaniasis, but also for the treatmentof other chronic infectious diseases.

Note Added inProof.The presence ofaThl-type cytokine profilein mice treatedwithIL-12plusPentostam(asdescribed inFig. 1C)would

predictthatthese animals should be resistanttoreinfection.In fact,

whenchallengedwith 1x 105metacyclic promastigotes15 weeks after treatment, IL-12 plus Pentostam-treated mice were immune toL. majorinfection, asevidencedbythe absence of lesiondevelopment. Additionally, these cured mice exhibited a positive delayed-type

hypersensitivityreaction in responsetothechallengeinoculum. We are grateful to Leslie Taylor and Dr. Jian Li for technical assistance. We thank Dr. Giorgio Trinchieri for comments on the

manuscript. This study was supported byresearch grants from the National Institutes of Health (AI27828, AI30073, AI35914, and

CA09140) and from the United Nations Development Program/

World Bank/World Health Organization Special Program for Re-search andTraining inTropical Diseases. P.S. is the recipient ofa BurroughsWellcome New Investigator Award in Molecular

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