Experimental Parasitology 94, 207–216 (2000)

doi:10.1006/expr.2000.4498, available online at http://www.idealibrary.com on

Trypanosoma cruzi: Roles for Perforin-Dependent and Perforin-IndependentImmune Mechanisms in Acute Resistance

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Steven P. Nickell1 and Divya Sharma

Department of Molecular Genetics and Microbiology, University of NeAlbuquerque, New Mexico 87131, U.S.A.

Nickell, S. P., and Sharma, D. 2000. Trypanosoma cruzi: Roles forperforin-dependent and perforin-independent immune mechanisms inacute resistance. Experimental Parasitology 94, 207–216. CD81 Tcells have been shown to be required for acute resistance to infectionwith the protozoan parasite, Trypanosoma cruzi, the causative agentof Chagas’ disease. However, to date, the mechanism by which CD81

T cells mediate protection in vivo has not been determined. WhileCD81 T cells can exhibit cytolytic function, they also secrete cytokinessuch as IFN-g, which is known to mediate protection against T. cruziinfections. To determine whether cytolysis is an important effectorfunction in vivo, we have compared outcomes of T. cruzi infection innormal and perforin-deficient mice. Our results indicate that whileperforin-dependent cytolytic mechanisms clearly make a major contri-bution to acute resistance to T. cruzi infection, this contribution maybe strain and challenge dose-dependent, since perforin-deficient micechallenged with lower doses of a less virulent strain survived and weresubsequently resistant to challenge with virulent organisms. In vivodepletion studies demonstrated that survival of perforin-deficient micechallenged with low doses of T. cruzi requires both CD41 and CD81

T cells and is dependent on IFN-g secretion. These studies documentthe participation of both perforin-dependent cytotoxic and perforin-independent, IFN-g-dependent immune mechanisms in acute resistance

to T. cruzi infection. q 2000 Academic Press

Index Descriptors and Abbreviations: T. cruzi, Trypanosoma cruzi;protozoa, parasitic; CD81 T cells; perforin; perforin-dependent cyto-toxicity; protection; survival. MHC, major histocompatibility complex;CTLs, cytotoxic T lymphocytes; B6, C57BL/6 mice; PFPKO, per-forin “knockout.”

1To whom correspondence should be addressed.

0014-4894/00 $35.00 207Copyright q 2000 by Academic PressAll rights of reproduction in any form reserved.

exico School of Medicine,

INTRODUCTION

A number of studies using mAb-depleted and knockoutmice have implicated class I MHC-restricted CD81 T cellsin acute resistance to challenge infection with Trypanosomacruzi, the causative agent of Chagas’ disease (Tarleton 1990;Tarleton et al. 1992; Rottenberg et al. 1993). However, themechanism(s) by which CD81 T cells protects mice hasnot been determined. One impediment has been the difficultyin establishing stable, T. cruzi Ag-specific CD81 effectorT cell lines for in vitro and in vivo characterization (Nickellet al. 1993), although recent success using parasite-derivedpeptides has been reported (Wizel et al. 1997).

CD81 T cells, which have been shown to exhibit protec-tive function against a variety of intracellular pathogens,including viruses (Zinkernagel and Doherty 1977), intracel-lular bacteria (Harty and Bevan 1992; Flynn et al. 1993; Kagiet al. 1994; Ladel et al. 1995), and protozoans (Schofield etal. 1987; Titus et al. 1987; Suzuki and Remington 1988;Weiss et al. 1988; Farrell et al. 1989), are capable of express-ing several distinct functions in vivo. In addition to theirparticipation in suppressor phenomena (Bloom et al. 1992),

activated CD81 T cells are capable of cytolysis, B cell help,and cytokine secretion (Mosmann et al. 1995; Mosmannand Sad 1996). While cytolysis of infected host cells mayconstitute the major mechanism by which CD81 T cellsprotect in vivo, this has been difficult to prove. Indeed, inseveral cases, cytokine secretion by CD81 T cells appears

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to mediate protection in vivo (Weiss et al. 1992; Cooper etal. 1997).

Because resistance to T. cruzi infection has previouslybeen shown to be mediated, at least in part, by IFN-g (Reed1988; Silva et al. 1992), a cytokine which may be secretedby activated CD81 T cells (Mosmann et al. 1995), wedecided to examine test whether cytolytic effector mecha-nisms are required for acute resistance to this parasite byexamining outcomes in perforin-deficient mice (Kagi et al.1994). Cytolysis by CD81 CTLs can occur via two distinctmechanisms (Kagi et al. 1994). The dominant one is depen-dent upon perforin (Podack et al. 1985), a pore-formingprotein related to complement protein C9 (Lichtenheld etal. 1988) which polymerizes to form pores in target cells(Podack et al. 1985), allowing entry of CTL granzymesthat activate apoptosis (Hudig et al. 1993). The secondarymechanism, which is thought to be involved primarily inregulating T cell expansion during immune responses (Ju etal. 1995), depends upon Fas/FasL interactions, whereinFasL-bearing CTLs trigger apoptosis in target cells bearingFas (Kagi et al. 1994). Mice which are homozygous for atargeted disruption of the perforin gene (pore-forming pro-tein “knockout”, PFPKO) have normal levels of CD81 Tcells in lymphoid organs, which undergo normal expansionduring viral infections, but their CD81 T cells express mini-mal CTL activity and fail to clear some viruses (Kagi et al.1994) and tumors in vivo (Broek et al. 1996).

In this study, we investigated the role of perforin-depen-dent cytolysis in immunity to T. cruzi by comparing out-comes of infection in normal (B6) and perforin-deficientC57BL/6 (B6-PKO) mice. While supporting the view that

perforin-dependent cytolysis plays a significant role in pro-tecting mice from T. cruzi infection, our results also indicate that other immune mechanisms make an important contribu-tion since under conditions of low dose challenge dose,perforin-deficient mice do survive infection. Survival of B6-PKO mice is shown to depend on both CD41 and CD81T cells and secretion of IFN-g. The results are discussed inthe context of current views of anti-T. cruzi immunity.

MATERIALS AND METHODS

Mice. Homozygous breeder pairs of perforin-deficient, C57BL/6-Pfptm1Sdz (B6-PKO) (Kagi et al. 1994) mice were obtained from TheJackson Laboratory (Bar Harbor, ME) and bred in the Animal ResourceFacility at the University of New Mexico Health Sciences Center. Age-and sex-matched C57BL/6 (B6) control mice and immunodeficient

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C57BL/6J-Prkdc scid/SzJ (B6-scid ) (Bosma 1989) mice were also ob-tained from The Jackson Laboratory. In preliminary studies, we con-firmed that B6-PKO mice were defective in CTL potential, as evidencedby their inability to generate primary allospecific CTL activity in vitrousing standard induction and assay conditions (data not shown).

Parasites. The Y (Silva and Nussensweig 1953) and Peru strains(Wrightsman et al. 1986) of T. cruzi (generous gifts of Dr. JerryManning, U.C. Irvine, Irvine, CA, U.S.A.) were used in these studies.Trypomastigotes forms were grown in 3T3 fibroblasts cells (Sandersonet al. 1980), harvested from supernatants as previously described (Nick-ell et al. 1987), and washed several times in HBSS prior to injectionby the intraperitoneal route. Mice were age- and sex-matched andbetween 6 and 12 weeks of age. Challenged mice were monitored forsurvival and at intervals, parasitemias were determined by microscopicexamination of tail blood diluted in ammonium chloride as previouslydescribed (Nickell et al. 1987). For use as antigen, washed cell-culture-derived trypomastigotes were freeze-thawed three times as previouslydescribed (Nickell et al. 1993).

Lymphocyte cultures. Pooled spleen cells from two to three mice/group were prepared and resuspended in T cell medium [45% RPMI1640, 45% Iscove’s modified Dulbecco’s medium, 25 mM Hepes, 100mM L-glutamine, 5 3 1025 M 2-mercaptoethanol, 10% FBS (Hyclone,UT, U.S.A.), 50 mg/ml gentamycin sulfate]. In some experimentsCD41 or CD81 T cells were depleted by “panning” (Wysocki andSato 1978) on anti-CD4 (GK 1.5, ATCC) or anti-CD8 (53-6.7, ATCC)mAb-coated tissue culture flasks prior to culture as described (Nickellet al. 1993). Spleen cells (3 3 106/well) were cultured in 24-well plateswith medium alone, 5 3 106 freeze-thawed Tc Y strain trypomastigotes,or Con A (2 mg/ml) in a total volume of 2 ml.

Cytokine measurement. Supernatants from stimulated spleen cellcultures were harvested at 24 or 48 h and frozen at 2208C untilassay. IFN-g levels in supernatants were determined using a standardsandwich ELISA as previously described (Nickell et al. 1993; Keane-Myers and Nickell 1995). To facilitate quantitation of IFN-g, a standardcurve of recombinant murine IFN-g (Biosource) was included (8200–128 pg/ml). IL-2 was quantitated in 24-h supernatants by bioassay usinggrowth-factor-dependent HT-2 cells as previously described (Nickell etal. 1993). Quantitation of IL-2 was aided by the construction of astandard curve of HT-2 proliferation using serial dilutions of recombi-nant human IL-2 (Tecin, Hoffmann–LaRoche).

In vivo depletion. Monoclonal Abs were produced as ascites inimmunodeficient B6-scid mice, partially purified by ammonium sulfateprecipitation, and frozen at 2208C until used. To deplete T cells invivo, groups of mice were injected intraperitoneally on day 21 andthen every 3–4 days thereafter for 4 weeks with 300 mg of anti-CD4(GK 1.5, rat IgG2b; ATCC), anti-CD8 (53-6.72, rat IgG2a, ATCC), bothanti-CD4 and anti-CD8, or the control anti-b gal (GL113, rat IgG1)mAb diluted in PBS. To deplete IFN-g, mice were injected on day 21and then at weekly intervals for 3 weeks with 1 mg/dose of anti-IFN-g (XMG1.2, rat IgG1) or control anti-b gal (GL113, rat IgG1) mAb

diluted in PBS. Hybridomas secreting XMG1.2 and GL113 were ob-tained from ATCC by kind permission of R. Coffman, DNAX.

Flow cytometry analysis. T cell subset depletion in mAb-treatedmice was confirmed by FACS analysis on day 16 postinfection. Briefly,2 3 105 washed spleen cells were resuspended in FACS medium(RPMI 1640, 20 mM Hepes, 0.3% BSA) and stained with a combinationof a FITC-conjugated rat anti-mouse CD8 mAb, a PE-conjugated anti-mouse CD3 mAb, and a RED613-conjugated anti-CD4 mAb (all fromBRL, Bethesda, MD, U.S.A.) for 45 min on ice. Cells were then

PERFORIN IN IMMUNITY TO Trypanosoma cruzi

washed twice in PBS and fixed with 1% formaldehyde in PBS priorto FACS analysis.

Histology. Experimental mice were sacrificed, and hearts wereexcised and fixed in neutral buffered formalin. Hearts were embeddedin paraffin and sectioned, and sections were stained with hematoxylinand eosin. H&E-stained heart tissue was examined under 203 powerfor Tc-infected cells.

RESULTS

To determine whether perforin-dependent cytolytic mech-anisms contribute to acute resistance to T. cruzi infection,we compared outcomes in age- and sex-matched B6-PKO

and normal B6 mice challenged with T. cruzi trypomastigotesof the Y strain (Silva and Nussensweig 1953). The Y strain

FIG. 1. Parasitemias (A) and survival (B) following challenge ofnormal B6 and perforin-deficient B6-PKO with 7 3 106 T. cruziY strain. B6 (n, —; n 5 6) and B6-PKO (▫, ---; n 5 9) mice wereinfected intraperitoneally with 7.5 3 106 cell-culture-derived trypomas-tigotes of T. cruzi Y strain. Mice were followed for survival and, atintervals, tail blood was examined for the presence of parasites asdescribed. This experiment was repeated twice with similar results.

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second week of infection but continued to progress in B6-PKO mice, leading to death in all B6-PKO mice with amean survival time of 11.7 6 0.5 days (Fig. 1). These resultsindicate that perforin-dependent immunity contributes to re-sistance against T. cruzi infection. We also examined resis-tance in B6 and B6-PKO mice challenged with a low dose(103) of tissue-culture-derived trypomastigotes of the morevirulent Peru strain of T. cruzi (LD50 5 ,103) (Wrightsmanet al. 1986). When B6 and B6-PKO mice were infected withthe Peru strain, the infection was progressive—all miceexhibited increasing parasitemias and all mice in both groupsdied (data not shown). While no significant differences inparasitemias were observed, B6 mice survived significantlylonger than B6-PKO (mean survival time 5 19.5 6 1.0 vs15.0 6 0.2, P , 0.05), providing additional evidence thatperforin-dependent immune mechanisms contribute to resis-tance during virulent T. cruzi infections, as evidenced bysignificantly longer survival times in perforin-sufficient B6mice compared to perforin-deficient B6 mice.

To further explore perforin-dependent immunity to T.cruzi, we also challenged B6 and B6-PKO mice with a lowerdose of the less virulent Y strain of T. cruzi. In contrast tothe results obtained in mice challenged with the higher doseof 7 3 106 trypomastigotes (cf. Fig. 1), B6-PKO mice chal-lenged with 2 3 105 trypomastigotes developed parasitemiasdetectable at days 7 and 9 postinfection, which resolved byday 12, and all mice in both groups survived (data notshown). These findings, which we have repeated multipletimes, suggest that perforin-dependent mechanisms are notstrictly required to control T. cruzi growth and replicationin vivo.

One possible reason for the survival of B6-PKO mice wasthat the Y strain given lacked sufficient virulence to surviveand replicate in vivo when delivered at this low challengedose. To test this, we infected T- and B-cell-deficient B6-scid mice (Bosma 1989) with 2 3 105 cell-culture-derivedtrypomastigotes and followed them for parasitemias andmortality. As shown in Fig. 2, parasitemias increased pro-gressively and all challenged mice died by day 14, indicatingthat the perforin-independent resistance observed in low-dose T. cruzi Y-strain-challenged B6-PKO mice was proba-bly mediated by T and/or B cells.

was chosen because tissue-culture-derived trypomastigotesof this strain exhibit limited virulence in wild-type B6(LD50 5 .107, data not shown). As shown in Fig. 1, whenmice were challenged with 7 3 106 cell-culture-derivedtrypomastigotes, parasitemia levels in B6 and B6-PKO micewere initially comparable, and resolved in B6 mice by the

Since previous studies suggested that CD81 T cells fromperforin-deficient mice may have increased capacity to se-crete cytokines, including IL-2 and IFN-g (Sad et al. 1996),a cytokine with known protective function during T. cruziinfections (Reed 1988; McCabe et al. 1991; Silva et al.1992), we also compared proliferative responses (data notshown) and IL-2 (data not shown) and IFN-g production inspleen cells taken from T. cruzi-recovered B6 and B6-PKO

neutralizing anti-IFN-g mAbs would have on resistance ofB6-PKO mice to low-dose T. cruzi challenge infection. As

T. cruzi infection in mice. Perforin-deficient mice challenged

FIG. 2. Course of low-dose T. cruzi Y strain infection in immuno-deficient B6-scid mice. Four B6-scid mice were challenged ip with2 3 105 cell-culture-derived trypomastigotes of the Y strain of T.cruzi. Mice were followed for survival and, at intervals, tail blood wasexamined for the presence of parasites as described.

mice. To stimulate primarily T. cruzi antigen-specific CD41T cell responses, we stimulated spleen cells with freeze-thawed trypomastigotes. We also stimulated these spleencells with the T cell mitogen concanavalin A, which iscapable of activating both CD41 and CD81 T cells (Kup-pers et al. 1984). As shown in Fig. 3, IFN-g responses tofreeze-thawed trypomastigotes and to concanavalin A werecomparable in B6-PKO and B6 mice, although variable fromexperiment to experiment (cf. Figs. 3A–3C).

To further investigate the immunological basis of resis-tance to low-dose challenge in B6-PKO mice, we examinedthe effect of T cell subset depletion. Separate groups of B6-PKO were administered a control mAb (anti b-gal), anti-CD4, anti-CD8, or both anti-CD4 and anti-CD8 mAbs duringinfection with 2 3 105 Y strain trypomastigotes. While T.cruzi-infected B6-PKO mice treated with control mAbs wereable to control parasitemias (Fig. 4) and mostly survive(11/13), parasitemias in B6-PKO mice treated with bothanti-CD4 and anti-CD8 mAbs increased progressively(Fig. 4) and all mice died (11/11) (Table I). Similarly,B6-PKO mice treated with either anti-CD4 or anti-CD8mAbs during infection also succumbed to infection (Table1, 11/11 and 12/13, respectively), although parasitemias in

these single T cell subset-depleted mice did not increaseprogressively (Fig. 4), indicating partial but insufficient con-trol of parasite replication. The effectiveness of T cell deple-tion was confirmed by FACS analysis of spleen cells ofsentinel mice on day 16 (data not shown).

Because previous studies provided evidence that IFN-gis an important cytokine in the protective immune responseto T. cruzi (Plata et al. 1984; Reed 1988; McCabe et al. 1991;

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Torrico et al. 1991), we also tested what effect treatment with

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shown in Fig. 5, treatment of B6-PKO mice with anti-mouseIFN-g mAbs abrogated their resistance to low-dose T. cruzichallenge infection. Histologic examination of hearts fromthese mice confirmed that anti-IFN-g mAb-treated B6-PKOmice had increased tissue parasitosis compared to controlmAb-treated B6-PKO mice (Fig. 6, Table II).

DISCUSSION

The data presented confirm the importance of perforin-dependent cytolytic effector mechanisms in control of acute

with either high doses of tissue-culture-derived trypomasti-gotes of the relatively avirulent Y strain or low doses ofthe virulent Peru strain suffered either increased or earliermortality when compared to their perforin-sufficient coun-terparts (Fig. 1, data not shown). Since CD81 T cells com-prise the major lymphocyte population expressing perforinactivity (Garcia-Sanz et al. 1987; Kawasaki et al. 1990),

FIG. 3. In vitro IL-2 and IFN-g production by spleen cells fromlow-dose Tc Y-recovered (n) and naive (▫) perforin-deficient B6-PKOand normal B6 mice. Pooled spleen cells prepared from mice at 40days postinfection (three mice/group) were cultured in 24-well plates(3 3 106 cells/well) with medium alone, freeze-thawed cell-culture-derived Tc Y strain trypomastigotes (5 3 106/well), or concanavalinA (2 ug/ml) as described under Materials and Methods. Supernatantswere harvested at 24 (IL-2) and 48 (IFN-g) h and cytokines weremeasured as described under Materials and Methods.

6-e Cat

FIG. 4. Parasitemias in T cell subset-depleted perforin-deficient Bmice (4–7/group) were treated with mAbs (300 mg/dose) against mousboth anti-CD4 and anti-CD8, or b-galactosidase as a control (GL113, r

Mice were infected ip with 2 3 105 cell-culture-derived trypomastigoteintervals, tail blood was examined for the presence of parasites as describ

a Age-matched B6 and B6-PFPKO mice were infected ip with2 3 105 cell-culture-derived trypomastigotes of the Y strain of T. cruzi.Groups of mice were injected ip every 3–4 days beginning on day 21before challenge through day 20 with 300 mg of GL113 (anti-b-gal,rat IgG1), GK1.5 (anti-CD4, rat IgG2b), 53-6.7 (anti-CD8, rat IgG2b),or both GK1.5 and 53-6.7 (anti-CD4/CD8) mAbs. Data presented aremortality, mean survival times, and standard error of the means ofexpired mice. Data from two separate experiments are presented. Para-sitemias from these experiments are presented in Fig. 4.

PKO infected with a low dose of T. cruzi Y strain. Groups of B6-PKOD4 (GK1.5, rat IgG2a, ATCC), mouse CD8 (53-6.7, rat IgG2b, ATCC),IgG1, ATCC) at 3- to 4-day intervals beginning 1 day before challenge.s of the Y strain of T. cruzi. Mice were followed for survival and, ated. The results from two independent experiments are presented (C, ●).

cells. Of course, since NK cells also require perforin forcytolytic function (Kagi et al. 1994) and appear to contributeto anti-T. cruzi immunity (Rottenberg et al. 1988; Cardilloet al. 1996), we cannot formally exclude a contribution byNK cells to this effect, although recent data suggest that NKcells may contribute to protection mainly via IFN-g secretion(Cardillo et al. 1996).

The involvement of cytolytic effector mechanisms in theprotective immune response to this parasite is entirely con-sistent with the parasite’s life cycle in vivo. Parasite replica-tion takes place in the cytoplasm of invaded host cells,subsequent to escape from endosomal compartments (Leyet al. 1990). Induction of CD81 T cells is facilitated by thiscytosolic location, since the class I-associated Ag processingmachinery resides there (Germain 1986). Furthermore, de-struction of Tc-infected host cells by specific CD81 CTLeffectors constitutes an effective host defense mechanism

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these data suggest that one mechanism by which CD81 Tcells protect in vivo (Tarleton 1990; Tarleton et al. 1992) isvia perforin-dependent cytolysis of T. cruzi-infected host

TABLE I

Mortality in T. cruzi-Infected B6-PKO Mice Depleted in Vivo ofCD41, CD81, or both CD41 and CD81 T Cellsa

Expt. Treatment Mortality MST 6 SEM

1 Control mAb 0/6 —Anti-CD4 4/4 15.5 6 0.5Anti-CD8 5/6 17.2 6 0.9Anti-CD4/CD8 4/4 15.5 6 0.7

2 Control mAb 2/7 (17, 18)Anti-CD4 7/7 19.1 6 2.1Anti-CD8 7/7 16.0 6 1.0Anti-CD4/CD8 6/6 14.6 6 0.3

since the intracellular replicating form of the parasite ispoorly infectious (Ley et al. 1988) and release to the extracel-lular environment would mean swift elimination.

While these results clearly support an important role forperforin-dependent cytotoxicity in immunity against T. cruzi,we also demonstrate that this mechanism is not strictly re-quired for host survival. Thus, we found that perforin-defi-cient B6-PKO mice challenged with a low dose of Y strain

tedanithin

hist

FIG. 5. Parasitemias (A) and survival (B) of anti-IFN-g mAb-treaT. cruzi Y strain. Groups of 10 B6-PKO mice were treated with eitheron days 21, 7, and 14 postinfection. Mice were infected ip on day 0 wMice were followed for survival and, at intervals, tail blood was examwere sacrificed on day 16, and their hearts were fixed in formalin for

trypomastigotes (23105) experienced only transient para-sitemias and survived indefinitely (.60 days). The abilityof B6-PKO mice to control parasite growth and survivewas clearly immune-mediated and was not attributable toinsufficient parasite growth potential or virulence since T-cell-, B-cell-deficient B6-scid mice given the same dose ofY strain trypomastigotes developed progressive parasitemiasand experienced 100% mortality (Fig. 2). Surviving B6-PKO mice also exhibited acquired immunity as evidencedby their consistent ability to resist challenge infection with.100 LD50 of the virulent Peru strain of T. cruzi (data notshown). These findings document the existence of perforin-independent immune mechanisms in vivo which are capableof limiting parasite growth sufficient to allow survival andcapable of rendering mice resistant to subsequent lethal chal-lenge. The finding that perforin-deficient mice are able tosurvive avirulent T. cruzi infections and resist subsequent

lethal challenges was recently reported (Kumar and Tarleton1998). In that same study, similarly infected CD81 T-cell-deficient mice surviving avirulent challenge were not ableto survive secondary challenge with more virulent parasites,suggesting a possible perforin-independent role for CD81T cells in resistance to secondary challenge. However, inanother study (Rottenberg et al. 1995), CD8-deficient miceinfected with avirulent Tc were resistant to subsequent lethal

perforin-deficient B6-PKO mice infected with a low challenge dose ofanti-IFN-g mAb (C, ---) or a control anti b-galactosidease mAb (●,—)2 3 105 cell-culture-derived trypomastigotes of the Y strain of T. cruzi.ed for the presence of parasites as described. Two mice in each groupologic examination.

challenge, as were Tc-immune mice depleted of CD81 Tcells during lethal challenge infection (Tarleton 1990). Takentogether, these studies indicate that immunity to secondaryTc challenge can be generated in the absence of perforin-dependent cytotoxicity and possibly in the absence of CD81T cells altogether.

The survival of perforin-deficient B6-PKO mice subjectedto low-dose challenge with the Y strain of T. cruzi wasfurther investigated and shown to depend on the combinedfunction of both major T cell subsets, since depletion ofeither CD41 or CD81 T cells led to increased mortalityin challenged mice (Table 1). T cell subset depletion alsoinfluenced levels of parasitemia. Parasitemias were progres-sive in B6-PKO mice depleted of both T cell subsets, whereasdepletion of single subsets (CD41 or CD81) led to persis-tence of parasitemias that were nonprogressive (Fig. 4).While previous studies in class-II-deficient (Tarleton et al.

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1996), CD4-knockout (Rottenberg et al. 1993), and CD4-depleted (Araujo 1989) mice had clearly established thatCD41 T cells are necessary for parasite clearance and sur-vival of mice following T. cruzi infection, the nature ofthis CD4 dependence was not entirely clear, since in manysystems, CD41 T cells potentiate CD81 T cell responsesvia a helper effect. These current results, however, argueagainst a strict helper role for CD41 T cells in promoting

t tisson weads

FIG. 6. Photomicrographs of hematoxylin- and-eosin-stained hear105) challenged perforin-deficient B6-PKO mice treated during infectitaken at 203 magnification and B and D were taken at 403. Arrowsh

effector cytotoxic CD81 T cell responses in vivo, sinceCD41 T cells clearly contribute to protection in the absenceof cytotoxic potential. The finding that perforin-deficientCD81 T cells were also required for survival (Table I)suggested the likely involvement of cytokines in this protec-tive effect.

In vivo depletion studies also demonstrated that IFN-gwas necessary for survival of low-dose Y-strain- challengedB6-PKO (Fig. 5). This was not altogether unexpected since

a number of studies have demonstrated that IFN-g plays animportant role in host defense to T. cruzi (Plata et al. 1984;Reed 1988; McCabe et al. 1991; Torrico et al. 1991; Silvaet al. 1992). Since both CD41 and CD81 T cell subsetswere required for survival (Table I) and both are able tosecrete IFN-g (Mosmann et al. 1995; Mosmann and Sad1996), it is likely that these T cell subsets mediate resistancein B6-PKO mice via IFN-g secretion. The requirement for

ue sections obtained on day 16 from T. cruzi Y strain low-dose (2 3ith either control (A, B). or anti-IFN-g mAbs (C, D). A and C weremark T. cruzi-parasitized myocardial cells/“pseudocysts.”

both T cell subsets may reflect a cytokine dosage require-ment or a perhaps a need for both class-I- and class-II-restricted effector T cells to target cytokines to distinct in-fected cell types. A possible dosage effect is suggested bythe observation that B6-PKO mice depleted of either T cellsubset, e.g., CD41 or CD81, showed partial ability to con-trol parasitemias compared to mice depleted of both subsets(Fig. 4). The mechanism by which IFN-g, mediates protec-tion in these B6-PKO mice is likely via induction of NO-

PERFORIN IN IMMUNITY TO Trypanosoma cruzi 213

dependent trypanocidal activity in infected macrophages(Gazzinelli et al. 1992) and possibly other cell types (Wisse-man and Waddell 1983; Goldman et al. 1996; Castanos-Velez et al. 1998). Increased susceptibility to either virulentor mild T. cruzi infection in both CD4- and CD4/CD8-deficient mice has been shown previously to correlate withdiminished IFN-g and iNOS mRNA levels and reduced NOexpression (Rottenberg et al. 1995).

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Anti-IFN-g 8/8 (100) 15.9 6 0.4 49/123

a Two mice from each experimental group described in Fig. 6 were

sacrificed on day 17 postinfection, and hearts were excised and fixedin neutral buffered formalin. Hearts were then embedded in parrafinand sectioned, and sections were stained with hematoxylin and eosin.Stained tissue was examined blindly at 203 power for the presenceof T. cruzi pseudocysts.

b Number of T. cruzi pseudocysts observed/No. of 203 fieldsexamined.

Since it had previously been reported that CD81 T cellsfrom perforin-deficient mice have increased potential to pro-duce IFN-g and IL-2 in vitro (Sad et al. 1996; Cooper etal. 1997; Laochumroonvorapong et al. 1997), we consideredit possible that the survival of low-dose Y-strain-challengedB6-PKO mice was aided by compensatory increases in cyto-kine production by CD81 T cells. However, this appearsunlikely as we were unable to detect any consistent differ-ences in cytokine levels when whole spleen cells from Tc-recovered B6 or B6-PKO mice were stimulated either withfreeze-thawed trypomastigotes or the mitogen Con A (Fig.3),which is capable of activating both CD41 and CD81 Tcell subsets (Kuppers et al. 1984).

In summary, our results indicate that perforin-dependentcytotoxicity, while clearly playing an important role in im-munity to T. cruzi, is not strictly required for survival, espe-cially during challenge with less virulent parasite strains.Under these circumstances, survival appears to depends uponthe production of IFN-g by both CD41 and CD81 T cellsubsets. These data strongly suggest that the extent of earlytissue invasion may determine the relative importance ofperforin-dependent cytotoxic effector mechanisms vs per-forin-independent, IFN-g-dependent mechanisms. Chal-

lenge of mice with a more virulent T. cruzi strain or highernumbers of less virulent parasites probably may lead to moreextensive early tissue invasion, necessitating the participa-tion of cytotoxic effector mechanisms in parasite clearanceand mouse survival. Infections with lower doses of lessvirulent T. cruzi strains, on the other hand, may lead toless extensive tissue invasion, perhaps more restricted tomacrophages and other IFN-g-responsive host cell types, so

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TABLE II

Levels of Tissue Parasitosis in Anti-IFN-g and Control mAb-Treated Perforin-Deficient B6-PKO Mice Infected with a LowDose of T. cruzi Y Straina

MyocardialMortality parasitosis

Treatment (%) MST 6 SEM (IC/total fieldsb)

Control mAb 0/8 (0) — 0/105

that IFN-g-dependent induction of intracellular killing issufficient to limit parasite growth. These data confirm theparticipation of both perforin-dependent cytotoxicity andIFN-g mediated mechanisms in protective immunity to T.cruzi infection.

ACKNOWLEDGMENTS

This work was supported in part by the Dedicated Health ResearchFunds of The University of New Mexico School of Medicine andby AI37233 from NIAID. We thank Cherese Bellino for excellenttechnical assistance.

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Received 18 February 1999; accepted with revision29 February 2000