of April 29, 2016.This information is current as

ImmunityCell Responses Enhances Antiparasitic

T+Receptor-Mediated Amplification of CD4Therapeutic Glucocorticoid-Induced TNF

Hill and Christian R. EngwerdaKuns, Vanessa Yardley, Shimon Sakaguchi, Geoffrey R.Fabian de Labastida Rivera, YongHong Zhou, Rachel D. Ashraful Haque, Amanda C. Stanley, Fiona H. Amante,

http://www.jimmunol.org/content/184/5/2583doi: 10.4049/jimmunol.0903080February 2010;

2010; 184:2583-2592; Prepublished online 5J Immunol 

Referenceshttp://www.jimmunol.org/content/184/5/2583.full#ref-list-1

, 26 of which you can access for free at: cites 55 articlesThis article

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is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Therapeutic Glucocorticoid-Induced TNF Receptor-MediatedAmplification of CD4+ T Cell Responses EnhancesAntiparasitic Immunity

Ashraful Haque,* Amanda C. Stanley,* Fiona H. Amante,* Fabian de Labastida Rivera,*

YongHong Zhou,* Rachel D. Kuns,† Vanessa Yardley,‡ Shimon Sakaguchi,x Geoffrey R. Hill,†

and Christian R. Engwerda*

Chronic infectious diseases and cancers are often associatedwith suboptimal effector T cell responses. Enhancement of T cell costim-

ulatory signals has been extensively studied for cancer immunotherapy but not so for the treatment of infectious disease. The few pre-

vious attempts at this strategy using infection models have lacked cellular specificity, with major immunoregulatory mechanisms or

innate immune cells also being targeted. In this study, we examined the potential of promotingT cell responses via the glucocorticoid-

inducedTNFreceptor (GITR)family-relatedproteininamurinemodelofvisceral leishmaniasis.GITRstimulationduringestablished

infectionmarkedly improved antiparasitic immunity. This required CD4+ T cells, TNF, and IFN-g, but crucially, was independent of

regulatory T (Treg) cells. GITR stimulation enhanced CD4+ T cell expansion without modulating Treg cell function or protecting

conventional CD4+ T cells from Treg cell suppression. GITR stimulation substantially improved the efficacy of a first-line visceral

leishmaniasis drug against both acute hepatic infection and chronic infection in the spleen, demonstrating its potential to improve

clinical outcomes. This study identifies a novel strategy to therapeutically enhanceCD4+ T cell-mediated antiparasitic immunity and,

importantly, achieves this goal without impairment of Treg cell function. The Journal of Immunology, 2010, 184: 2583–2592.

Costimulation of T cells is crucial for generating efficient Tcell responses (1–4). Many reports indicate that agonisticsignaling through T cell costimulatory receptors can en-

hance immunity to cancer (5–9). Glucocorticoid-induced TNF re-ceptor (GITR) family-related protein (10, 11) is one such receptorthat has received significant attention in recent years. GITR is ex-pressed at low levels by many immune cells (12). It is highly ex-pressed on CD4+Foxp3+ regulatory T (Treg) cells and is alsoupregulated on conventionalCD4+ andCD8+T cells upon activation(12, 13). Although GITR stimulation is thought to modulate Tregcells (13, 14), it is clear that GITR itself is not essential for Treg cellfunction, because GITR-deficient mice do not suffer spontaneous Tcell-mediated autoimmune diseases (15). Instead, GITR appears toplay an important role in conventional T cell activation, becauseGITR-deficient mice display impaired T cell responses in vivo (16),and in vitro studies show that signaling through GITR lowers thethreshold of CD28 costimulation required for efficient T cell acti-

vation (17). Stimulation of GITR via the agonistic AbDTA-1 breaksimmunological self-tolerance in mice (13). This Ab treatment wasoriginally reported to both impair regulatory CD4+ T cell functionand enhance conventional T cell responses (8, 9, 18, 19). More re-cent studies, however, have focused on the effects of GITR stimu-lation on conventional T cells, suggesting they are rendered moreresistant to suppression by regulatory CD4+ T cells (9, 20).Many infectious diseases are associated with suboptimal T cell

responses. One such disease is visceral leishmaniasis (VL) (21–24).An estimated 200million people are at risk forVLwith conservativeestimates suggesting that 500,000 new cases and 40,000 deathsoccur each year (25, 26). VL is caused by the protozoan parasitesLeishmania donovani and L. infantum (L. chagasi) (27, 28). There iscurrently no protective vaccine against VL, and first-line drugtreatments are imperfect because of long courses of treatment,significant toxicity, and emerging parasite drug resistance (25, 29,30). Therefore, new treatments against VL or strategies to improvethe safety and efficacy of current drugs are urgently needed. Ex-perimental L. donovani infection of genetically susceptible miceresults in many of the features of human VL and is characterized byan acute but resolving infection in the liver, whereas a lifelongchronic infection becomes established in the spleen (24, 31). Studiesin experimental VL have highlighted the importance of antiparasiticCD4+ T cell responses for hepatic granuloma formation, and para-site clearance in an IFN-g– and TNF-dependent manner (32–36).However, despite effective antiparasitic T cell responses beinggenerated in this model, they are suboptimal, as occurs in VL pa-tients (21–24). Previous attempts to stimulate conventional T cellresponses during infection (37–42) lacked specificity because theytargeted either critical Treg cell-associated molecules, such asCTLA-4 and IL-10, or exerted pleiotropic effects, for example,through CD40 or CD137 stimulation that target multiple leukocytepopulations. Thus, to our knowledge, there have been no reports todate of conventional T cell responses being specifically boosted

*Immunology and Infection Laboratory, Queensland Institute of Medical Researchand The Australian Center for Vaccine Development; †Bone Marrow TransplantationLaboratory, Queensland Institute of Medial Research, Herston, Queensland,Australia; ‡London School of Hygiene and Tropical Medicine, London, United King-dom; and xDepartment of Experimental Pathology, Institute for Frontier MedicalSciences, Kyoto University, Kyoto, Japan

Received for publication September 18, 2009. Accepted for publication December28, 2009.

This work was supported by grants from the Australian National Health and MedicalResearch Council.

Address correspondence and reprint requests to Dr. Christian R. Engwerda, Queens-land Institute of Medical Research, 300 Herston Road, Herston, Queensland 4006,Australia. E-mail address: christian.engwerda@qimr.edu.au

Abbreviations used in this paper: GITR, glucocorticoid-induced TNF receptor; IG,immature granuloma; iKC, infected Kuppfer cell; MFI, mean fluorescence intensity;MG, mature granuloma; MNC, mononuclear cell; p.i., postinfection; Sb(V), sodiumstibogluconate; Treg, regulatory T; VL, visceral leishmaniasis.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

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during infection using a strategy that avoids broader leukocytemodulation or Treg cell suppression. In this study, we demonstratefor the first time the therapeutic potential of agonistic signaling viaGITR during infection to enhance CD4+ T cell responses, withoutcompromising Treg cell function, to improve parasite clearance byTNF- and IFN-g–dependent mechanisms.

Materials and MethodsMice

Inbred female C57BL/6 were purchased from the Australian ResourceCentre (Canning Vale, Western Australia, Australia), and maintained underconventional conditions. Foxp3gfp/gfp mice (43) were backcrossed six toseven times onto the C57BL/6 background, bred, and maintained at theQueensland Institute of Medical Research Animal Facility along with IFN-gR–deficient C57BL/6 mice. All mice used were age matched (6–12 wk)and were housed under specific-pathogen free conditions. All animalprocedures were approved and monitored by the Queensland Institute ofMedical Research Animal Ethics Committee.

Parasites and infection of mice

L. donovani (LV9) was maintained by passage in B6.RAG-12/2 mice, andamastigotes were isolated from the spleens of chronically infected animals,as described previously (44, 45). Mice were infected by injecting 2 3 107

amastigotes i.v. via the lateral tail vein, killed at the times indicated in thetext by CO2 asphyxiation, and bled via cardiac puncture. Spleens and per-fused livers were removed, and parasite burdens were determined fromDiff-Quik–stained impression smears (Lab Aids, Narrabeen, New South Wales,Australia) and expressed in Leishman-Donovan units (the number ofamastigotes per 1000 host nuclei multiplied by organ weight in grams) (46).Liver and spleen tissue were also preserved in Tissue-Tek OCT Compound(Sakura, Torrance, CA). Hepatic mononuclear cells (MNCs) were isolatedimmediately following death as described previously (44, 45).

Abs and drugs for in vivo administration

All hybridomas (DTA-1 [anti-GITR], PC61 [anti-CD25], 145-2C11 [anti-CD3ε], YTS191.1 [anti-CD4], and 53-5.8 [anti-CD8b]) were grown in 5%(v/v) FCS, RPMI 1640 containing 10 mmol/l L-glutamine, 200 U/mlpenicillin, and 200 mg/ml streptomycin. Purified Ab was prepared fromculture supernatants by protein G column purification (Amersham Bio-sciences, Uppsala, Sweden), followed by endotoxin removal (Mustangmembranes, Pall, East Hills, NY). For in vivo stimulation of GITR, micewere injected i.p. with 0.5 mg DTA-1 mAb. For depletion of CD25hi Tregcells, mice were injected i.p. with 1 mg PC61 14 d preinfection. For de-pletion of CD4+ T cells or CD8+ T cells, mice were injected i.p. with 0.5mg YTS191.1 or 53-5.8, respectively, and were dosed every 3 d afterwardwith 0.2 mg of the same mAb. Control mice were administered the samequantities of control rat IgG (Sigma-Aldrich, St. Louis, MO) at the sametime points as the stimulatory or depleting mAbs. The pentavalent anti-monial, sodium stibogluconate [Sb(V); Burroughs Wellcome, U.K.], wasdissolved in 0.9% saline, and administered i.p.

TNF blockade in vivo

Mice were administered Enbrel etanercept, a human TNF blocking protein,that also blocks mouse TNF (47, 48). Mice were administered 100 mg every2 d from the start of treatment. Control mice were administered normalhuman IgG (Intragram P; CSL, Broadmeadows, Victoria, Australia).

BrdU incorporation

Mice were injected i.p. with 400 mg BrdU (BD Biosciences, FranklinLakes, NJ) on day +6, +9, and +12 postinfection (p.i.), in accordance withthe manufacturer’s guidelines. BrdU+ cell numbers were then assessed byflow cytometry.

Histological response to hepatic infection

Acetone-fixed liver sections (6 mm) were labeled with hamster L. donovani-specific antisera at a dilution of 1 in 1000. Labeling was detected witha biotinylated goat anti-hamster Ab (Vector Laboratories, Burlingame, CA).Sections were developed with Vector-Elite ABC kit, followed by 3,39-diaminobenzidine substrate kit (Vector Laboratories). Granuloma densitywas determined from 25 fields of view permouse liver (Olympus CX31 lightmicroscope,340/0.65 non-oil objective), and the maturation of granulomaswas scored around infected Kupffer cells, as described previously (37, 45).

Flow cytometry

For the staining of cell surface Ags, liver MNCs or splenocytes wereharvested andfirstpreincubatedwithCD16/32mAb(2.4G2;grown in-house)to avoid nonspecific binding ofAbs to FcgRs. Cellswere then incubatedwithfluorochrome-conjugated Abs on ice for 20 min. T cells, NKT cells, and NKcells were enumerated with Pacific Blue or FITC-conjugated anti-TCRb–chain (H57-597), PE-Cy5–conjugated anti-CD4 (GK1.5), PE-conjugatedanti-CD8a (H1.2F3), and allophycocyanin-conjugated anti-NK1.1 (PK136).B cells were enumerated using FITC-conjugated anti-CD19 (6D5) and Pa-cific Blue-conjugated anti-B220 (RA3-6B2). NKT cells were stained usingPE-conjugated a-galactosylceramide–loaded CD1d tetramers (a gift fromProf. D. Godfrey, Department of Microbiology and Immunology, Universityof Melbourne, Melbourne, Victoria, Australia). All mAbs were purchasedfrom Biolegend (San Diego, CA) or BD Biosciences. Intracellular stainingfor Foxp3 was performed using Alexa 647-conjugated anti-Foxp3 mAb(150D) (Biolegend) in conjunction with a Foxp3-staining buffer kit (eBio-science) used according to the manufacturer’s instructions. For intracellularcytokine staining, splenocytes were first incubated for 3 h at 37˚C/5%CO2 in5% (v/v) FCS and RPMI 1640 containing 10 mg/ml brefeldin A (Sigma-Aldrich) prior to FcgR blockade and cell surface labeling as detailed above.For intracellular Ab labeling of cytokines or Bcl-2, surface-labeled cellswere washed, fixed, and permeabilized using BD cytofix/cytoperm fixation/permeabilization kits according to the manufacturer’s instructions (BD Bi-osciences). Cells were incubated with PE-conjugated mAb against IFN-g orBcl-2 (or appropriate isotype control Abs) for 40 min on ice, extensivelywashed, and fixed in 1–2% (w/v) paraformaldehyde in PBS. Assessment ofBrdU incorporation was performed using a FITC-based BrdU detection kit(BD Biosciences) according to the manufacturer’s instructions. Flow cyto-metric analysis was performed on a FACSCanto II flow cytometer (BD Bi-osciences) and analyzed using FlowJo Software (Tree Star, Ashland, OR).

In vitro Treg cell suppression assay

CD4+TCRb+Foxp3+ Treg cells, CD4+TCRb+Foxp32CD44lo naive T cells,and CD4+TCRb+Foxp32CD44hi effector T cells from C57BL/6.foxp3gfp/gfp

micewere sorted using a FACSAria cell sorter (BDBiosciences). Cell purityafter sorting was confirmed to exceed 95%. Between 25,000 and 45,000naive or activated CD4+ T cells were incubated at 37˚C/5% CO2 in U-bottomed 96-well plates with CD4+TCRb+Foxp3+ Treg cells at ratios be-tween 1:1 and 128:1. Cells were stimulated with 1 mg/ml anti-CD3ε (145-2C11; made in-house) in the presence of 10,000 irradiated CD11c+ dendriticcells from naive C57BL/6 mice. Briefly, CD11c+ dendritic cells were pos-itively selected from splenocyte preparations using MACS with metallo-conjugated anti-mouse CD11c Abs (N418) and positive selection columns,according to the manufacturer’s instructions (Miltenyi Biotec, BergischGladbach, Germany). After 72 h of stimulation, wells were pulsed for 20 hwith 1 mCi of [3H]thymidine. Thymidine incorporation was then measuredusing a 1450 Microbeta plate reader (PerkinElmer, Boston, MA).

Measurement of serum cytokine levels

Serum harvested from blood was assessed for the presence of cytokinesusing flexset bead array kits and a FACSArray plate reader (BD Bio-sciences) according to the manufacturer’s instructions.

Statistical analysis

The statistical significance of differences between groups was determinedusing unpaired Student t test using GraphPad Prism version 4.03 forWindows (GraphPad Software, San Diego, CA), and p , 0.05 was con-sidered statistically significant. The distribution of hepatic histologicalresponse was compared using x2 analysis with Microsoft Excel software.All data are presented as the mean values 6 SEs unless otherwise stated.

ResultsGITR stimulation reduces liver parasite burdens when givenafter the establishment of infection

To assess the effect of GITR stimulation during experimental VL,we treated L. donovani-infected C57BL/6 mice with the agonisticanti-GITR mAb DTA-1, either on the day of infection (day 0), or 5d p.i. We chose these two time points to model two scenarios;firstly, one in which parasite-specific T cells were naive (day 0),and secondly, where parasite-specific T cells had been activatedthrough interaction with APCs displaying cognate Ag. Liverparasite burdens at day 14 p.i. were significantly reduced in mice

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administered DTA-1 at 5 d p.i., compared with control rat IgG-treated mice, but not in mice administered DTA-1 at the time ofinfection (Fig. 1A). Thus the positive effect of DTA-1 on parasitecontrol was dependent on the timing of administration. We furtherassessed the effect of DTA-1 treatment on day 5 p.i. on parasiteburdens in the liver and spleen 14 and 28 d p.i. (Fig. 1B). We notedthat DTA-1 treatment reduced the peak level of infection in theliver, although it is interesting to note that the rate of parasiteclearance from days 14–28 p.i. was similar for both DTA-1 andcontrol treated mice. DTA-1 also exerted positive effects in thespleen, by significantly delaying, although not preventing, estab-lishment of infection in this organ. These data demonstrate thatDTA-1 treatment after the establishment of infection enhancedparasite control both in the liver and spleen.

GITR stimulation enhances liver granuloma assembly andfunction

We next examined the effect of DTA-1 treatment 5 d p.i. on liverleukocyte numbers and granuloma formation after L. donovaniinfection. The formation of granulomas around infected Kupffercells is a critical event in resolution of hepatic infection in thisdisease model (35). We observed a substantial increase by day 14p.i. in the number of leukocytes in the livers of DTA-1–treatedmice, compared with control rat IgG-treated mice (Fig. 2A).However, by day 28 p.i. similar granulomatous responses wereevident in both DTA-1 and control treated mice (Fig. 2A). Thesedata suggested that DTA-1 treatment had accelerated the process of

granuloma formation over the first 14 d of infection. A detailedassessment of granuloma formation and maturation at day 14 p.i.(Fig. 2B) indicated that DTA-1 treatment reduced the total numberof infected foci, compared with control infected mice (Fig. 2C).The foci present in the livers of control rat IgG treated mice werepredominantly infected Kupffer cells, with relatively few immatureor mature granulomas present. In contrast, the majority of infectedfoci in the livers of DTA-1–treated mice were immature and maturegranulomas. These data indicate that DTA-1 treatment at day 5 p.i.reduced parasite burden by increasing hepatic leukocyte numbersand enhancing granuloma formation in the liver.

Increased GITR expression by conventional CD4+ T cells,CD8+ T cells, and Treg cells in the liver following infection

We hypothesized that the importance of DTA-1 treatment timingcould be explained by changes in GITR expression on specific celltypes over the first 5 d of infection. GITR expression by all celltypes analyzed in the spleen changed very little over this time (Fig.3A). However, GITR expression significantly increased on Foxp3+

CD4+ Treg cells, conventional CD4+ T cells and CD8+ T cells inthe liver (Fig. 3B). In contrast, GITR expression by liverNKT cells, NK cells, and B cells was unchanged over this time-frame, suggesting that despite being targets for DTA-1 from theday of infection, GITR stimulation of these cells was unlikely tocontribute to DTA-1–mediated enhanced immunity (Fig. 3B). In-stead, these data suggested that increased targeting of DTA-1 toliver CD4+ Treg cells, conventional CD4+ T cells, and CD8+

T cells at day 5 p.i. might account for the enhanced immunitygenerated by GITR stimulation.

DTA-1 enhances CD4+ T and CD8+ T cell responses

We next examined the effect of DTA-1 treatment on hepatic T cellresponses, because of their importance in controlling parasitegrowth, and in the case of CD4+ T cells, their critical role in gran-uloma formation (34, 35).We found a significant increase (p, 0.01)at day 14 p.i. in the number of conventional CD4+ and CD8+ T cellsin the livers of mice treated with DTA-1 on day 5 p.i. compared withthose treated with control rat IgG (Fig. 4A). Importantly, however,DTA-1 treatment did not induce changes in the small number ofTregcells found in the liver (Fig. 4A), as previously reported in otherexperimental systems (8). The number of IFN-g–producing CD4+

T cells was dramatically increased by DTA-1 treatment (Fig. 4B,4C). In contrast, the number of IFN-g+CD8+ T cells was onlyslightly enhanced byDTA-1 treatment (Fig. 4C). Relatively fewNKor NKT cells produced IFN-g at this time, either in control or DTA-1–treated infected mice (Fig. 4C). These data demonstrate thatDTA-1 treatment had a dramatic effect on CD4+ T cell responses,a moderate effect upon CD8+ T cell responses, and no effect on thenumbers of CD4+ Treg cells in the liver.

DTA-1–mediated immunity is dependent on CD4+ T cells butnot CD8+ T cells

To determine the relative roles of CD4+ and CD8+ T cells in DTA-1-mediated enhanced antiparasitic immunity, both cell types were in-dependently depleted from the time ofDTA-1 administration on day5p.i. Depletion of CD4+ T cells completely abrogated DTA-1–medi-ated enhanced immunity, whereas depletion of CD8+ T cells had noeffect (Fig. 5). Importantly, CD4+ or CD8+ T cell depletion in theabsence of GITR activation had little impact on parasite burdens atday14p.i. (Fig. 5), because the effects ofT cell absenceare not readilyapparent until later in infection (34). Therefore, although DTA-1treatment moderately enhanced CD8+ T cell responses, GITR stim-ulation improved parasite control via a mechanism requiring en-hanced CD4+ T cell activation but not CD8+ T cell activation.

FIGURE 1. GITR stimulation using DTA-1 mAb enhances parasite

clearance if given on day 5 p.i. A, C57BL/6 mice (n = 5 mice/group) were

infected with L. donovani and treated either with nonspecific control rat IgG

or DTA-1 on the day of infection (day 0) or 5 d p.i. Fourteen days p.i., liver

parasite burdens were determined. ppp , 0.01. Data presented is repre-

sentative of .10 independent experiments. B, C57BL/6 mice (n = 5 mice/

group/time point) were infected with L. donovani and treated either with

nonspecific control rat IgG or DTA-1 5 d p.i. Liver and spleen parasite

burdens were determined at time points indicated. pppp, 0.001; pp, 0.05.

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GITR stimulation boosts CD4+ T cell proliferation but does notenhance protection against apoptosis

To determine whether the increased number of activated CD4+

T cells in the livers of GITR-stimulated mice was due to enhanced

proliferation, we assessed the incorporation of the synthetic nu-

cleotide BrdU into the DNA of CD4+ T cells. By day 14 p.i., the

number of BrdU+CD4+ T cells in the livers of infected mice was

significantly greater in mice administered DTA-1 on day 5 p.i.,

compared with mice administered control rat IgG (p , 0.05) (Fig.

6A). In contrast, the number of BrdU+CD8+ T cells in the liver was

not significantly increased by the administration of DTA-1 (Fig.

6A). In addition to boosting proliferation, it was also possible that

GITR stimulation could have been enhancing protection of CD4+

T cells from apoptosis. To examine this, we measured expression of

Bcl-2, an important antiapoptotic protein, in both CD4+ T cells and

CD8+ T cells. We observed that ∼90% of CD4+ T cells in the livers

of naive mice expressed detectable levels of Bcl-2 (Fig. 6B). Thispercentage dropped to ∼45% of CD4+ T cells harvested from thelivers of infected mice treated with control rat IgG, suggesting thatduring L. donovani infection, CD4+ T cells in the liver becomemore susceptible to apoptosis by day 14 p.i. (Fig. 6B). Furthermore,the proportion of Bcl-2+CD4+ T cells in the livers of day 5 p.i.,DTA-1–treated mice was significantly lower than that of controlinfected mice (Fig. 6B). Taken together, the BrdU incorporationand intracellular Bcl-2 expression data demonstrate that GITRstimulation specifically stimulated CD4+ T cell proliferation but didnot provide them with enhanced protection from apoptosis.

DTA-1–mediated immunity during L. donovani infection isindependent of modulation of Treg cell function

GITR stimulation has been reported to reduce the suppressive ca-pacity ofTregcells. TodeterminewhetherDTA-1 treatmentwas alsoacting directly on these cells to reduce suppression and enhance

FIGURE 2. GITR stimulation on day 5 p.i. enhances liver granuloma formation. A, H&E-stained liver tissue sections from rat IgG-treated and DTA-1–

treated infected mice on day 14 p.i. (top two panels) and day 28 p.i. (bottom two panels) were analyzed by light microscopy using a 310 objective to

delineate dense areas of lymphocytes among hepatocytes. Areas enclosed by dotted lines indicate areas dense with lymphocytes. Graph enumerates liver

MNC numbers 14 d p.i. from four pooled experiments. pppp , 0.001. B, Frozen liver tissue sections were stained with anti–L.donovani-specific polyclonal

Ab sera, followed by counterstaining of nuclei with hemotoxylin. Representative examples of an infected Kuppfer cell, an immature granuloma, and

a mature granuloma are presented (original magnification 3100). C, The number of infected Kuppfer cell, immature granuloma, and mature granuloma per

25 fields of view for infected mice (n = 5 mice/group) given either control rat IgG or DTA-1. Data presented are representative of two independent ex-

periments. ppp , 0.01 comparison by x2 analysis. IG, immature granuloma; iKC, infected Kuppfer cell; MG, mature granuloma.

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immunity, we depleted CD25hi Treg cells from naive mice 14 dpreinfection with L. donovani. We have previously shown that thisregimen depletes CD25hiFoxp3+CD4+ Treg cells but does not de-plete activated, conventional CD4+ T cells generated during in-fection (49, 50). Control and CD25hi Treg cell-depleted mice wereinfected and treated with control rat IgG or DTA-1 on the day ofinfection, or alternatively, with DTA-1 or control rat IgG on day 5p.i. As shown above (Fig. 1), DTA-1 treatment enhanced parasitecontrol in the liver when administered at day 5 p.i. but had no effectif given on the day of infection (Fig. 7A). This pattern was alsoobserved in mice depleted of CD25hi Treg cells prior to infection(Fig. 7A). These data indicate that GITR stimulation enhancesimmunity to L. donovani via a mechanism that is independent ofCD25hi Treg cells.To further analyze any potential effects of GITR stimulation on

Treg cell function, we adopted a routine in vitro suppression assay,in which titrated numbers of CD4+ Treg cells are able to blockproliferation of anti–CD3ε-stimulated naive CD4+ T cells. To sort

viable Foxp3+CD4+ Treg cells, we used C57BL/6.foxp3gfp/gfp

mice, which express eGFP under the control of the foxp3 promoter(43). The eGFP+ (Foxp3+) CD4+ Treg cells were sorted from naivemice and infected mice 4 d after administration of either controlrat IgG or DTA-1 (administered on day 5 p.i.). These cells werethen assessed in vitro for their ability to suppress proliferation ofFoxp32CD4+CD44lo naive T cells (Fig. 7B). We observed thatCD4+ Foxp3+ Treg cell function was not altered by DTA-1treatment. GITR stimulation of Ag-specific CD4+ T cells has alsobeen reported to render these cells refractory to Treg cell-mediatedsuppression (9, 20). To explore this possibility during L. donovaniinfection, we assessed the ability of sorted CD4+CD44hiFoxp32–activated T cells from infected control mice, and infected DTA-1–treated mice, to proliferate in the presence of naive CD4+Foxp3+

Treg cells. We observed no difference between activated CD4+

T cells from control infected mice or DTA-1–treated mice in theirability to resist Treg cell-mediated suppression (Fig. 7C). Takentogether, these data demonstrate that during L. donovani infection,

FIGURE 3. Upregulation of GITR by liver T cell

subsets by 5 d p.i. C57BL/6 mice (n = 5 mice/group)

were infected with L. donovani. Five days later,

lymphocytes harvested from spleens (A) and perfused

livers (B) of infected and naive mice were assessed for

GITR expression by flow cytometry. pp, 0.05; ppp,0.01. Data are representative of two independent ex-

periments. MFI, mean fluorescence intensity.

FIGURE 4. GITR stimulation enhances liver CD4+ T

cell responses. A, MNCs harvested from the livers of

naive, infected control rat IgG-treated and DTA-1–treat-

edmice 14 d p.i. (n= 5mice/group)were phenotyped and

enumerated by flow cytometry. B, Representative plots

gated on CD4+TCRb+ cells depict direct ex vivo IFN-g

production by CD4+ T cells harvested from livers. Boxes

indicate IFN-g+ events on the basis of isotype control Ab

staining.C, The number and cellular phenotypes of IFN-

g+ cells in the livers ofmice. Cells inA–Cwere identified

as follows: B cells (CD19+B220+), NKT (TCRb+CD1d2

tetramer+), CD4+ T (CD4+TCRb+), CD8+ T (CD8+

TCRb+), NK (NK1.1+TCRb2), and CD4+ Treg (CD4+

Foxp3+TCRb+). ppp , 0.01. Data presented are repre-

sentative of two independent experiments.

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GITR stimulation does not modulate either Treg cell function,or the susceptibility of conventional CD4+ T cells to Treg cell-mediated suppression. Hence, GITR stimulation enhances im-munity via a mechanism that is independent of CD25hi Treg cells.

DTA-1–mediated immunity is dependent upon TNF and IFN-g

We next examined which cytokines are important in DTA-1–mediated, CD4+ T cell-dependent antiparasitic immunity. We notedsignificantly enhanced levels of both TNFand IFN-g at day 14 p.i. inthe serum of infected mice treated with DTA-1, compared withcontrol infected mice (Fig. 8A). Both these cytokines are crucial forcontrolling parasite numbers in the liver (32, 33, 36). To determinethe requirement for IFN-g in DTA-1–mediated enhanced immunity,IFN-gR–deficient C57BL/6 mice were infected and treated withcontrol rat IgG or DTA-1 on day 5 p.i. Wild-type C57BL/6 micewere also infected and treated in the samemanner. Although DTA-1treatment reduced parasite burden in wild-type mice as expected,this reduction in parasite burden was not observed in IFN-gR–deficient mice (Fig. 8B), indicating a critical role for IFN-g in DTA-1–mediated antiparasitic immunity. To determine the role of TNF inDTA-1–mediated enhanced immunity, infected mice were givencontrol rat IgG or DTA-1 on day 5 p.i. and treated with controlhuman IgG or Enbrel etanercept, a human TNF blocking moleculethat also blocks TNF in mice (47, 48). The enhanced protectionelicited byDTA-1 treatment of control human IgG-treatedwild-typemice was completely abrogated in mice treated with Enbrel eta-nercept (Fig. 8C), indicating an important role for TNF in DTA-1–mediated immunity against L. donovani. Taken together, these datashow that enhanced production of both IFN-g and TNF are requiredfor the antiparasitic immunity elicited by GITR stimulation.

Therapeutic GITR stimulation enhances drug-mediatedparasite clearance

Apentavalent antimonial drug, Sb(V) (also known as Pentostam), isone of themost widely used treatments for VL in humans, despite itstoxicity, requirement for several weeks of daily dosing, andemerging parasite drug resistance (25, 29, 30). Therefore, an im-portant clinical goal is to clear parasites using lower, less frequentdoses of Sb(V). Because Sb(V) relies on the host immune responsefor its efficacy (51, 52), we used a well-validated system for testingadjunctive therapies in VL (38, 39, 51–53) to investigate whether

DTA-1 could improve the efficacy of a supoptimal dose of Sb(V)during an established L. donovani infection.To assess the potential for DTA-1 to target conventional CD4+

T cells at later time points during infection, we first analyzedGITR expression on conventional CD4+ and CD8+ T cells, as wellas Treg cells in mice infected for 14 d with L. donovani, a timewhen liver parasite burdens are maximal, and infection has be-come established in the spleen (Fig. 9A) (24, 31). We observedmoderate upregulation of GITR on Foxp3+CD4+ Treg cells andCD8+ T cells in the liver and spleen at this time point, and sim-ilarly, minimal upregulation of GITR on splenic conventionalCD4+ T cells. However, substantial upregulation of GITR wasobserved on conventional CD4+ T cells in the liver, indicating thatactivated, conventional CD4+ T cells were potential targets forDTA-1 treatment during established infection.We administered DTA-1 at day 14 p.i. either alone or with

a suboptimal dose of Sb(V). One week after administration of Aband/or drug (on day 21 p.i.), liver and spleen parasite burdens weredetermined (Fig. 9B, 9C). DTA-1 treatment alone enhanced parasiteclearance in the liver, although this was not statistically significant,but did not affect splenic parasite burdens. A suboptimal dose of Sb(V) (50 mg/kg), partially reduced liver and spleen parasite burdens,relative to mice administered a full therapeutic dose of Sb(V) (500mg/kg). Importantly,DTA-1 substantially improved liver and spleen

FIGURE 5. GITR stimulation enhances immunity via a CD4+ T cell-

dependent, CD8+ T cell-independent mechanism. Infected C57BL/6 mice

were treated on day 5 p.i. with control rat IgG or DTA-1. Some groups of

mice (n = 5 mice/group) were also treated with either anti-CD8b Abs or

anti-CD4 Abs from day 5 onward, as indicated, and liver parasite burdens

were determined at 14 d p.i. ppp , 0.01. Data presented are representative

of two independent experiments.

FIGURE 6. GITR stimulation enhances CD4+ T cell proliferation but

does not increase protection against apoptosis. A, Naive mice and infected

mice (n = 5 mice/group) treated with either control rat IgG or DTA-1 were

administered BrdU from day 6 p.i. At day 14 p.i., liver CD4+ T cells and

CD8+ T cells were assessed for incorporation of BrdU by flow cytometry.

B, Naive mice and infected mice treated with either control rat IgG or

DTA-1 (n = 5 mice/group) were assessed at day 14 p.i. for intracellular

expression of the antiapoptotic protein Bcl-2 by liver CD4+ T cells and

CD8+ T cells using flow cytometry. Data presented are representative of

two independent experiments. ppp , 0.01.

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parasite clearance in mice treated with the suboptimal dose of Sb(V)in both the liver and spleen. In fact,DTA-1 combinedwith supoptimaldrug treatment was as successful at clearing parasites from the liverand spleen, as the full therapeutic dose of Sb(V). These data indicatethat GITR stimulation has potential as an adjunct to conventionalchemotherapy for the treatment of VL by clearing parasites from theliver and spleen, the two main infected organs in mice and humans.

DiscussionManipulation of T cell activation via T cell costimulatory receptorshas received relatively little attention in the context of infectiousdisease, despite its reported efficacy in improvingantitumor immune

responses (5–9, 18, 54, 55). We employed a murine model of VL toassess the effect of GITR stimulation during infection. We dis-covered that GITR stimulation enhanced parasite clearance viamechanisms requiring CD4+ T cells, IFN-g, and TNF but not CD8+

T cells or Treg cells. GITR stimulation specifically enhanced CD4+

T cell proliferation, IFN-g and TNF production but did not renderCD4+ T cells refractory to CD4+ Treg cell-mediated suppression orincrease levels of the antiapoptotic molecule Bcl-2. In fact, giventhat GITR stimulation appeared to further activate CD4+ T cells andreduce their Bcl-2 levels, it is possible that activation induced celldeath may have been accelerated among this cell population. Mostimportant, however, is the fact that GITR stimulation did not alterthe number or suppressive capacity of Treg cells. Thus, activation ofGITR during experimental VL specifically enhanced a suboptimal

FIGURE 7. GITR stimulation does not modulate CD4+ Treg cell

function during L. donovani infection. A, CD25hiCD4+Foxp3+ T cell-de-

pleted and control mice were infected and treated with DTA-1 on the day

of infection or 5 d p.i. Control mice received rat IgG. Liver parasite bur-

dens were determined 14 d p.i. pppp , 0.001; ppp , 0.01. These data are

representative of two independent experiments. B, CD4+Foxp3+ Treg cells

were sorted 9 d p.i. from pooled spleens of infected C57BL/6.foxp3gfp/gfp

mice treated with DTA-1 or control rat IgG on day 5 p.i. Treg cells were

titrated into wells containing naive CD4+CD44loFoxp32 T cells from

C57BL/6.foxp3gfp/gfp mice and stimulated with anti-CD3ε Ab and irradi-

ated CD11c+ APCs. CD4+ T cell proliferation was measured by [3H]

thymidine incorporation. Data are representative of two independent ex-

periments. C, CD4+ Treg cells sorted from uninfected C57BL/6.foxp3gfp/gfp

mice were titrated against effector CD4+CD44hi Foxp32 T cells from

pooled spleens of infected C57BL/6.foxp3gfp/gfp mice treated either with

DTA-1 or control rat IgG on day 5 p.i. Effector cells were stimulated and

proliferation assessed as in B. Data presented here are representative of

three independent experiments.

FIGURE 8. GITR stimulation enhances immunity via IFN-g– and TNF-

dependent mechanisms. A, Analysis of serum IFN-g and TNF levels in day

14-infected mice treated 5 d p.i with DTA-1 or control rat IgG (n = 5 mice/

group). Horizontal bars indicate median values per group. Data presented

are pooled from three independent experiments. B, Wild-type and IFN-gR–

deficient C57BL/6 mice were infected and treated with DTA-1 or control

rat IgG on day 5 p.i. (n = 5 mice/group). Liver parasite burdens were

determined at day 14 p.i. Data presented are representative of two in-

dependent experiments. C, C57BL/6 mice were infected and then treated

with DTA-1 or control rat IgG on day 5 p.i. Also from day 5 p.i. onward,

mice were treated with control human IgG or the TNF blocking protein,

Enbrel etanercept (n = 5 mice/group). Liver parasite burdens were de-

termined at day 14 p.i. Data presented are representative of two in-

dependent experiments. pppp , 0.001; ppp , 0.01.

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antiparasitic CD4+ T cell response, without impairing Treg cellfunction. Furthermore, therapeutic administration of GITR duringan established infection significantly enhanced the efficacy ofsuboptimal drug treatment. This last observation highlights thepotential for GITR stimulation to shorten the long, toxic, chemo-therapy regimens currently used, and to improve parasite clearancein VL patients.Three recent studies demonstrated that GITR stimulation could

enhance antitumor immune responses in mice (8, 9, 18). Thesestudies are consistent with our data in that CD4+ T cells mediatedenhanced protection. Others have reported that GITR activationstimulates proliferation of Treg cells (8, 9). We did not observethis phenomenon during L. donovani infection. The reasons forthis are unclear at present but could be related to the immune cell

and cytokine environment that exists during this infection. We didnot observe any change in the suppressive capacity of Treg cellsin vitro after GITR stimulation in vivo, which is consistent withthe observations of others (9). Critically, our in vivo Treg celldepletion studies clearly demonstrate that GITR stimulation doesnot enhance immunity via direct modulation of their function.Previous studies suggested thatGITR stimulation protectedCD4+

T cells from suppression by Treg cells (9, 20).We did not find this tobe the case during L. donovani infection. Again, the reasons for thisare unclear but could be due to differences in experimental designin at least one case (9). We specifically purified Foxp32CD4+

CD44hi T cells as the responder CD4+ T cell population, whereasthe previous study classified responders as being CD4+CD252

cells. CD4+CD252 cells would have contained naive CD4+ T cells,

FIGURE 9. GITR stimulation enhances the efficacy of suboptimal drug therapy. A, Analysis of GITR expression by indicated cell types from livers and

spleens of mice infected 14 d previously with L. donovani. Lines indicate GITR expression by individual mice from a group of five infected mice compared

with a representative naive mouse (solid gray histogram). Liver (B) and splenic (C) parasite burdens in mice 7 d after being given various indicated

treatments on day 14 p.i. Data presented are representative of two independent experiments. ppp , 0.01.

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activated CD4+ T cells, and CD252Foxp3+CD4+ T cells, the rela-tive proportions of which may have varied between treatmentgroups. Therefore, this previous study did not directly compareresponder CD4+ T cells from control and DTA-1 treated mice butinstead studied mixed populations of CD4+ T cells.In the current study, increases in CD8+ T cell responses were

modest in comparison with that of CD4+ T cell responses. Thispattern was similar to observations made using murine antitumormodels (8, 9). The reasons for this pattern are unclear, given thatboth CD8+ and CD4+ T cells express intermediate levels of GITR,and both upregulate its expression during infection. The signalingpathways triggered by GITR stimulation of CD4+ T cells in vivoremain to be fully elucidated but could provide an explanation forthe preferential effects of GITR activation on CD4+ T cells and notCD8+ T cells.Finally, we observed that the enhanced antiparasitic immunity

mediated by GITR stimulation was more effective if administeredp.i., rather than at the time of infection. This observation is con-sistent with others, who observed that antitumor immunity wasboosted by GITR stimulation if administered the day after tumorswere implanted, but not the day before or on the day of implan-tation. The reasons for this are not linked to Treg cells in oursystem, because we observed this phenomenon in the presence orabsence of CD25hiCD4+ Treg cells in vivo. Instead, we hypothe-size that GITR signaling pathways are inefficiently triggered inCD4+ T cells during initial encounters with parasite Ag but thatincreased GITR expression on CD4+ T cells following activationmakes them amenable to enhanced GITR-mediated costimulation.In conclusion, the data presented in this study demonstrate that

CD4+ T cell responses during L. donovani infection are suboptimaland can be enhanced by GITR stimulation to improve antiparasiticimmunity. Importantly, GITR stimulation did not affect the ca-pacity of Treg cells to suppress immune responses, suggesting thismajor regulatory mechanism for preventing autoimmunity wasunaffected. Therefore, GITR stimulation is a viable strategy, par-ticularly in the context of adjunctive immunotherapy, for specifi-cally boosting endogenous T cell responses during infection.

AcknowledgmentsWe thank the staff of the Queensland Institute of Medical Research animal

facility for animal husbandry, Paula Hall and Grace Chojnowski for assis-

tance in cell sorting, Dale Godfrey and Dan Pellicci for providing CD1d tet-

ramer, and Alexander Y. Rudensky for making available foxp3gfp/gfp mice.

DisclosuresThe authors have no financial conflicts of interest.

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