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Deprivation MechanismEffects of Regulatory T Cells via an IL-2Effector T Cells Overcomes the Suppressive Microbial Infection-Induced Expansion of

YarovinskyBurnaevskiy, Reed Pifer, James Forman and Felix Alicia Benson, Sean Murray, Prashanthi Divakar, Nikolay

ol.1100769http://www.jimmunol.org/content/early/2011/12/04/jimmun

published online 5 December 2011J Immunol 

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The Journal of Immunology

Microbial Infection-Induced Expansion of Effector T CellsOvercomes the Suppressive Effects of Regulatory T Cells viaan IL-2 Deprivation Mechanism

Alicia Benson, Sean Murray, Prashanthi Divakar, Nikolay Burnaevskiy, Reed Pifer,

James Forman, and Felix Yarovinsky

Regulatory Foxp3+ T cells are a critical cell population that suppresses T cell activation in response to microbial and viral

pathogens. We identify a cell-intrinsic mechanism by which effector CD4+ T cells overcome the suppressive effects of regulatory

T (Treg) cells in the context of three distinct infections: Toxoplasma gondii, Listeria monocytogenes, and vaccinia virus. The acute

responses to the parasitic, bacterial, and viral pathogens resulted in a transient reduction in frequency and absolute number of

Treg cells. The infection-induced partial loss of Treg cells was essential for the initiation of potent Th1 responses and host

protection against the pathogens. The observed disappearance of Treg cells was a result of insufficiency in IL-2 caused by the

expansion of pathogen-specific CD4+ T cells with a limited capacity of IL-2 production. Exogenous IL-2 treatment during the

parasitic, bacterial, and viral infections completely prevented the loss of Treg cells, but restoration of Treg cells resulted in a

greatly enhanced susceptibility to the pathogens. These results demonstrate that the transient reduction in Treg cells induced by

pathogens via IL-2 deprivation is essential for optimal T cell responses and host resistance to microbial and viral pathogens. The

Journal of Immunology, 2012, 188: 000–000.

Foxp3+ regulatory T (Treg) cells exert pleiotropic immu-noregulatory effects essential for immune homeostasis,the prevention of autoimmunity, and the regulation of

pathogen-induced inflammatory reactions (1). The transcriptionfactor Foxp3 controls Treg cell development and function, anddeficiency in the Foxp3 gene results in hyperactivation of CD4+

T cells, overproduction of proinflammatory cytokines, and mas-sive multiorgan pathology (2, 3). It was initially thought that thefunctions of Treg cells were limited to the control of immuneresponses to autoantigens, but later studies demonstrated that Tregcells also exert suppressive effects on the immune responses totumors (4) and infectious Ags (5, 6). These and other studiesformally established that Treg cells have profound effects onimmunity toward pathogens (7). A number of distinct suppressivemechanisms by which Treg cells target APCs, in particular den-dritic cells (DC), and T lymphocytes have been identified (8). Thedominant suppressor functions of Treg cells present a serious ob-stacle in the establishment of robust protective immunity towardpathogens; several studies with bacterial, viral, or parasitic in-fections formally demonstrated that Treg cells restrict the effi-ciency and magnitude of T cell responses, which results in anincreased pathogen burden (9–11). Some studies have also re-vealed that by limiting immune responses to an infectious agent,

Treg cells minimize tissue damage and potentiate a rapid patho-gen clearance (12, 13). Importantly, the potential reciprocal in-teractions between effector and Treg cells during the initiation ofimmune responses are not completely understood.In this report, we establish a CD4+ T cell-intrinsic mechanism

for overcoming the suppressive effects of Treg cells upon the in-

duction of protective T cell responses in the context of three

distinct infections: Toxoplasma gondii, Listeria monocytogenes,

and vaccinia virus. We observed that the acute responses to the

investigated infectious agents resulted in the dramatic loss of Treg

cells. This infection-induced reduction of Treg cells was essential

for the initiation of potent Th1 responses and host protection

against the parasitic, bacterial, and viral infections. Furthermore,

we identified a mechanism responsible for the transient disap-

pearance of Treg cells. We observed that the pathogen-induced

expansion of effector T cells was associated with a limited amount

of available IL-2. The lack of this growth factor, which is known

to be essential for Treg cell development and survival (14),

resulted in the loss of Treg cells during the acute response to the

pathogens. The infection-induced Treg cell deficiency was com-

pletely prevented by treatment with exogenous IL-2 during the

course of the experimental diseases. However, the restoration of

Treg cells by IL-2 treatment had a negative impact on the in-

duction of Th1 responses and resulted in greatly enhanced sus-

ceptibility to the microbial and viral pathogens. Taken together,

our results establish that the pathogen-induced transient reduction

in Treg numbers caused by IL-2 insufficiency is essential for

optimal effector CD4+ T cell responses and host resistance to the

parasitic, bacterial, and viral pathogens. Furthermore, because the

observed loss of Treg cells was transient and incomplete, it en-

hanced the protective immune responses to pathogens, but it did

not result in severe immunopathology. Our results also suggest

that thymus-derived Treg cells, rather than those that develop

through peripheral conversion, have a major role in Treg recon-

stitution following the acute response to microbial pathogens.

Department of Immunology, University of Texas Southwestern Medical Center, Dal-las, TX 75390

Received for publication March 17, 2011. Accepted for publication November 7,2011.

This work was supported by National Institutes of Health Grant R01 AI085263.

Address correspondence and reprint requests to Dr. Felix Yarovinsky, Universityof Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX75390-9093. E-mail address: Felix.Yarovinsky@UTSouthwestern.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: ALDH, aldehyde dehydrogenase; DC, dendriticcell; Treg, regulatory T; UTSW, University of Texas Southwestern; WT, wild-type.

Copyright� 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1100769

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Materials and MethodsAnimals

C57BL/6 mice were obtained from the University of Texas Southwest-ern (UTSW) Medical Center Mouse Breeding Core Facility. B6.Cg-Foxp3tm2Tch/J (Foxp3EGFP) mice were obtained from The Jackson Lab-oratory. All animals used were age- and sex-matched and maintained in thespecific pathogen-free barrier facility at the UTSW Medical Center atDallas. All experiments were performed using protocols approved by theInstitutional Animal Care and Use Committees of the UTSW MedicalCenter.

Parasitic, bacterial, and viral infections

Mice were infected i.p. or orally with an average of 20 T. gondii (ME49strain) cysts as previously described (15). For infection of mice with L.monocytogenes, log-phase cultures of L. monocytogenes (10403 serotype)were washed twice and diluted in PBS to the desired concentration. L.monocytogenes was injected in the lateral tail vein at 104 CFU per mouse.Vaccinia virus was injected in the lateral tail vein at a dosage of 106 PFUper mouse.

IL-2 treatment

rIL-2 and the anti-mouse IL-2 mAb (clone JES6-1A12) were purchasedfrom eBioscience. The IL-2/anti–IL-2 complex was prepared as describedpreviously (16) with a few minor modifications: 50 mg anti–IL-2 Ab wasmixed with 1.5 mg IL-2 in 200 ml PBS 15 min prior to i.p. injection ondays 3 and 5 postinfection with T. gondii, L. monocytogenes, or vacciniavirus.

Flow cytometry and ELISA

On days 3, 5, 7, 10, 14, and 15 postinfection, the animals were necropsied,and their spleens and mesenteric lymph nodes were harvested for theanalysis of Treg cells by flow cytometry. Cells were stained in ice-coldFACS buffer (PBS supplemented with 2 mM EDTA, 1% heat-inactivatedFCS, and 0.02% sodium azide). Analyses of intracellular cytokine ex-pression were performed on splenocytes restimulated with 0.5 mg/ml anti-CD3 (BD Biosciences) for 5 h in the presence of GolgiPlug (brefeldin A;BD Biosciences). After in vitro restimulation, cells were washed once inFACS buffer, stained with PerCP-Cy5.5–labeled anti-CD4 mAb or anti-CD8 mAb, and fixed for 40 min in BD Cytofix/Cytoperm (BD Bio-sciences). Cells were then stained using fluorochrome-conjugated Absaccording to the manufacturer’s protocol (BD Biosciences). In short, afterincubation in the permeabilization buffer supplemented with anti-FcgRII/III mAb (2.4G2; 5 mg/ml) at 4˚C, cells were stained for 30 min with FITC-labeled anti-TNF, PE-labeled anti–IL-2, and allophycocyanin-labeled anti–IFN-g mAb (BD Biosciences), washed three times, and resuspended inPBS plus 1% FBS. Cell fluorescence was measured using an FACSCaliburor LSR II flow cytometer (BD Biosciences), and data were analyzed usingFlowJo software (Tree Star, Ashland, OR).

Flow cytometric analysis of CD4+Foxp3+ Treg cells was performedusing the mouse/rat Foxp3 Staining Set (eBioscience) as recommended bythe manufacturer.

The aldehyde dehydrogenase (ALDH)-positive cells were detected usingan ALDEFLUOR staining kit (StemCell Technologies). Splenocytes fromnaive and infected mice were incubated in the dark for 35 min at 37˚C inthe ALDEFLUOR assay buffer containing the activated ALDEFLUORsubstrate (StemCell Technologies), with or without the ALDH inhibitordiethylaminobenzaldehyde. Cells were subsequently stained using theCD11b-, CD11c-, and Gr1-specific Abs (BD Biosciences) and analyzed onan LSR II flow cytometer (BD Biosciences). Naive GFP-(Foxp32)CD4+

T cells were sort-purified from the spleen of the Foxp3EGFP (B6.Cg-Foxp3tm2Tch/J) mice. Sort-purified GFP-(Foxp32)CD4+ T cells (105

cells/well) were cocultured with ALDH+CD11b+CD11c2 or CD11c+ cells(2 3 104 cells/well) in the presence of an anti-CD3 mAb (0.5 mg/ml; BDBiosciences). In some experiments, ALDH+CD11b+CD11c2 and CD11c+

cells were mixed together with the GFP-CD4+ T cells. TGF-b (3 ng/ml)and IL-2 (5 ng/ml) were purchased from eBioscience. After 3 d of culture,Foxp3 expression was measured by flow cytometry. The data were ana-lyzed using FlowJo software (Tree Star).

IL-2 and IFN-g in culture supernatants were measured by ELISA usingcommercially available kits (R&D Systems for IFN-g and eBiosciences forIL-2).

Reverse-transcription PCR

RNA was isolated from splenocytes; sort or magnetic beads (MiltenyiBiotec) purified CD4+, CD8+, CD4+Foxp3EGFP cells with The PureLink

RNA Mini Kit (Invitrogen). cDNA was prepared using the SuperScript IIIkit (Invitrogen). Optimized primers targeting each gene (Foxp3: 59-TT-CATGCATCAGCTCTCCAC-39, 59-CTGGACACCCATTCCAGACT-39;il2: 59-ATGCAGCTCGCATCCTGT-39, 59-GGAGCTCCTGTAGGTCCA-TC-39; ifng: 59-ACTGGCAAAAGGATGGTGAC-39, 59-TGAGCTCATT-GAATGCTTGG-39; cd25: 59-AGAACACCACCGATTTCTGG-39, 59-AGC-TGGCCACTGCTACCTTA-39; and hprt 59-GCCCTTGACTATAATGAGTACTTCAGG-39, 59-TTCAACTTGCGCTCATCTTAGG-39) were designedusing the Primer 3 software. cDNAwas amplified with Power SYBR Greenmaster mix (Applied Biosystems). The MyiQ Real-Time PCR DetectionSystem (Bio-Rad) was used to obtain cycle threshold values. The relativeexpression of each sample was determined after normalization to hypo-xanthine phosphoribosyltransferase using the ddCt method.

Statistical analysis

All data were analyzed with Prism (version 5; GraphPad). Data wereconsidered statistically significant for p values ,0.05 using a two-tailed ttest.

ResultsAcute responses to parasitic, bacterial, and viral infectionsresult in the transient loss of Treg cells

Treg cells are involved in the potent regulation of the immuneresponses to a variety of microbial pathogens. For this reason, weinvestigated the interactions between the induction of protectiveT cells and that of regulatory Foxp3+CD4+ T cells in the context ofinfection with three distinct pathogens: the parasite T. gondii, theGram-positive bacteria L. monocytogenes, and vaccinia virus.

T. gondii is a protozoan parasite that stimulates potent and rapidTh1-biased CD4+ T cell immune responses (17). A protectiveimmune response to this pathogen requires a delicate balancebetween proinflammatory effector mechanisms, primarily regu-lated by the TLR-dependent activation of MyD88, and concomi-tant induction of an anti-inflammatory program (17, 18). Lackof either of these mechanisms results in high susceptibility to thisparasite, as is evident from the rapid mortality observed in T.gondii-infected MyD882/2 and IL-102/2 mice (19–21). BecauseTreg cells have an important role in the limitation of T cellresponses, we investigated the reciprocal interactions betweenIFN-g+CD4+ T cells and regulatory Foxp3+CD4+ T cells duringthe acute response to T. gondii. We observed that systemic in-fection with the parasite resulted in a dramatic reduction in thefrequency of Foxp3+CD4+ T cells by days 5–7 postinfection (Fig.1A, 1D). As is evident from Fig. 1A and 1D, the observed loss ofTreg cells during T. gondii infection was transient, and Treg cellsrecovered by the end of the acute response to the parasite. Inaddition, the remaining Treg cells expressed reduced levels ofFoxp3 when compared with naive controls (Fig. 1A).The relative loss of Foxp3+CD4+ T cells in the T. gondii-

infected mice was also observed during the analysis of Foxp3

mRNA expression in splenocytes and isolated CD4+ T cells (Fig.

1B). We observed a substantial reduction in the amount of Foxp3

mRNA seen in splenocytes and purified CD4+ T cells isolated

from T. gondii-infected mice when compared with their naive

controls (Fig. 1B). In addition, a reduction of Foxp3 mRNA levels

in the Treg cells themselves was observed in the T. gondii-infected

mice (Fig. 1B). The latter observation was consistent with the flow

cytometry data demonstrating that infection with the parasite

resulted not only in the loss of Treg cells, but also in reduced

levels of Foxp3 seen in the infected mice (Fig. 1A).To examine the possibility that the observed loss of Treg cells

was an artifact of the experimental i.p. infection with the parasite,

we next used a natural (oral) route of T. gondii infection. Using

the oral route of infection, we observed a similar progressive

and transient partial depletion of Treg cells (Fig. 1C, 1D). Thus,

2 INFECTION-INDUCED Treg CELL DEPLETION

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infection with the protozoan parasite resulted in the transient re-duction in frequency of Treg cells during the acute response to thepathogen independently of the route of infection (Fig. 1A–D).

Because the Treg cell loss closely coincided with the peak of theCD4+ T cell response against T. gondii, the observed decreasein the frequency of CD4+Foxp3+ Treg cells measured by flowcytometric or Foxp3 signals detected by PCR approaches couldbe explained by the expansion of pathogen-specific effector CD4+

T cells. To address this possibility, we performed an absolutequantification of CD4+Foxp3+ and CD4+IFN-g+ T cells during thecourse of the parasitic infection. The quantification of Treg cellsin mice infected i.p. or orally with the parasite revealed that Tregcells (Fig. 1E) were almost undetectable at the peak of the CD4+

T cell response against T. gondii (Fig. 1F), formally demonstratingthat T. gondii infection results in both absolute and relative loss ofTreg cells. In addition, we also observed that the disappearance ofTreg cells was not limited to the spleen but was also seen in alltissues examined (data not shown).We next investigated whether the observed transient depletion

of Treg cells is unique to the infection with the protozoan parasiteT. gondii or whether it is a common feature of acute immune

responses to microbial and viral pathogens. As a model bacterialpathogen, we selected L. monocytogenes, a Gram-positive bacte-rium that is responsible for severe disease in immunocompro-mised humans. Host resistance to this pathogen depends on anintact innate immune system and requires potent IFN-g productionby T cells (22). Similar to our observations postinfection withT. gondii, mice challenged with L. monocytogenes demonstrateda dramatic reduction of Treg cells, and this reduction had similarkinetics to those observed during the parasitic infection (Fig. 2A).Similar to the T. gondii-induced Treg cell loss, the depletion ofTreg cells triggered by infection with L. monocytogenes wastransient, and the Treg cell frequency returned to the levels typi-cally seen in naive animals within 10–14 d postinfection (Fig. 2A).Furthermore, we also observed that infection with vaccinia virus

also resulted in the transient and systemic depletion of Treg cells(Fig. 2B). The kinetics of the Treg cell disappearance and recov-ery closely resembled those observed in T. gondii- or L. mono-cytogenes-infected mice (Figs. 1, 2). In all of the experimentalinfections (with the viral, bacterial, or parasitic pathogens), weobserved that Treg cell depletion peaked by day 7 postinfection.Intriguingly, the loss of Treg cells was transient, and their fre-

FIGURE 1. The acute response to T. gondii results in the transient loss of Treg cells. WT mice (five animals per group) were infected i.p. with an average

of 20 T. gondii strain ME49 cysts per mouse, and the frequency of Treg cells defined as CD4+Foxp3+ cells was analyzed in the spleen of the infected and

control (d0) mice at the indicated number of days postinfection (A). B, Foxp3 expression levels were analyzed in splenocytes (left panel), isolated CD4+

T cells (center panel), or sort-purified CD4+Foxp3+ T cells (right panel) isolated from naive or T. gondii-infected mice (day 7 postinfection) by real-time

PCR. All of the data were normalized to the expression level seen in sort-purified CD4+CD442Foxp32 naive T cells. The data shown are the mean 6 SD.

C, WT mice were infected orally with an average of 20 T. gondii strain ME49 cysts per mouse, and the frequency of Treg cells was analyzed in the spleen of

the infected mice at the indicated number of days postinfection. D, Average frequency of Foxp3+ cells in the spleens of mice infected i.p. (filled circles) or

orally (open circles). E, Absolute quantification of Treg cells in the spleens of mice infected i.p. (filled circles) or orally (open circles) with the parasite. The

data shown are representative of five independent experiments. F, Absolute quantification of CD4+ and CD4+IFN-g+ T cells in the spleens of naive (d0) and

T. gondii-infected mice (d7). The data shown are representative of at least six independent experiments. *p , 0.05, **p , 0.01.

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quency was largely restored within 2 wk of the infection (Figs. 1,2).Quantifications of Treg cells in L. monocytogenes- and vaccinia

virus-infected mice formally established that both infectionsresulted in absolute reduction of Treg cells (Fig. 2C). Becauseboth L. monocytogenes and vaccinia virus result in the expan-sion of effector CD4+ and CD8+ T cells (Fig. 2C), both infec-tions caused a profound Treg cell insufficiency during the acuteresponses to these pathogens.

Activation of TLRs not required for Treg cell disappearance

It has been previously established that the innate recognition ofpathogens by TLRs can overcome the suppressive effects of Tregcells. The activation of TLRs on APCs results in the production ofsoluble factors, including IL-6, which render effector CD4+ T cellsresistant to the suppressive effects of Treg cells (23). TLR acti-vation on Treg cells can result in the elimination of the immu-nosuppressive effects of this cell population (24–26). TLR ac-tivation in effector T cells modulates CD4+ and CD8+ T cellresponses (27–29). During T. gondii infection, we previously es-tablished that TLR11 is a major innate immune sensor for theparasite that coordinates CD4+ T cell responses to the pathogen(18). We thus investigated whether TLR11 activation was requiredfor the depletion of Treg cells during acute toxoplasmosis. Weobserved that similar to their wild-type (WT) counterparts, micedeficient in TLR11 demonstrated a progressive loss of Treg cellsduring both the systemic and mucosal responses to the parasite(Supplemental Fig. 1). The only differences between WT andTLR11-deficient mice were the slightly delayed kinetics and re-duced magnitude of the disappearance of Treg cells in TLR112/2

mice. Furthermore, the analysis of T. gondii-infected TLR2-,

TLR4-, TLR2 3 TLR4-, and TLR9-deficient animals revealedthat none of the examined TLRs were responsible for the loss ofTreg cells during parasitic infection (data not shown). Taken to-gether, our results suggest that TLR activation is dispensable forthe infection-driven loss of Treg cells.

Pathogen-specific CD4+ T cells produce limited amounts ofIL-2

We next determined the mechanism that was responsible for thedisappearance of Treg cells during the course of parasitic, bacterial,and viral infections. We observed that the loss of Treg cells wasindependent of their proliferation status. During T. gondii infec-tion, only a small fraction of Foxp3+ T cells was also positive forthe proliferation marker Ki67 in infected WT mice, whereas Tregcells actively divided in mice infected with L. monocytogenes orvaccinia virus (Supplemental Fig. 2 and data not shown). Never-theless, all three infections resulted in a similar loss of Foxp3+

T cells. We thus hypothesized that the infection-driven depletionof Treg cells was a result of a lack of a survival factor(s) essentialfor Treg cell maintenance.Among various growth factors, IL-2 has an important role in the

regulation of T cell function (30). Although IL-2 is essential forT cell proliferation in vitro, a central function for IL-2 in vivo isthe regulation of Treg cell development and survival (14). Thelack of IL-2 or of the components of the IL-2R results in a dra-matic reduction in Treg cells in the thymus and in the periphery(14). We thus analyzed the effects of the parasitic, bacterial, andviral infections on the production of IL-2 by pathogen-specificCD4+ T cells.WT animals were infected with T. gondii using an i.p. or oral

route, and intracellular staining for IFN-g, TNF, and IL-2 was

FIGURE 2. Acute responses to L. mono-

cytogenes and vaccinia virus caused loss of

Treg cells. WT mice (five animals per group)

were infected i.v. with L. monocytogenes (104

CFU per mouse) (A) or vaccinia virus (106 PFU

per mouse) (B), and the frequency of splenic

Treg cells was analyzed at the indicated num-

ber of days postinfection. C, Absolute quanti-

fication of Treg cells (CD4+Foxp3+), CD4+,

CD8+, CD4+IFN-g+, and CD8+IFN-g+ cells

was performed in naive (black bars), L. mono-

cytogenes (open bars), or vaccinia virus (gray

bars) infected mice on day 7 postinfection. The

data shown are the mean 6 SD. For identifi-

cation of IFN-g+ cells splenocytes were re-

stimulated with 0.5 mg/ml anti-CD3 for 5 h in

the presence of GolgiPlug (BD Biosciences).

The data shown are representative of four in-

dependent experiments. **p , 0.01.

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used to identify fully activated T. gondii-specific T cells. As pre-viously reported (15, 17), T. gondii infection resulted in the rapidinduction of CD4+ T cells producing high amounts of IFN-g andTNF independently of the route of infection (Fig. 3A, Supple-mental Fig. 3). In striking contrast, T. gondii-specific CD4+ T cellswere largely deficient in the production of IL-2 (Fig. 3B, 3C,Supplemental Fig. 3A–C). Real-time PCR-based gene expressionanalysis also revealed a reverse correlation between expression ofIL-2 and IFN-g genes. We observed that T. gondii infectionsresulted not only in induction of IFN-g (Fig. 3D), but also in thedramatic loss of IL-2 signals measured in purified T cells or bulksplenocytes isolated from infected mice (Fig. 3E). These obser-vations were also confirmed by an ELISA assay that demonstrateda reduced amount of IL-2 detected in splenocytes from theinfected mice (Fig. 3F, Supplemental Fig. 3D). Furthermore, T.gondii infection resulted in the upregulation of CD25, a high-affinity subunit of IL-2R, on the activated T cells (Fig. 3G, Sup-plemental Fig. 3E). These results suggest that the acquisition ofIFN-g secreting ability during T. gondii infection was associated

with increased consumption of IL-2 coupled with reduced pro-duction of this cytokine.Intracellular staining was next used to detect IFN-g and IL-2

proteins during the course of L. monocytogenes and vaccinia virusinfections. Similar to infection with the parasite, infection with thebacterial or viral pathogen also resulted in a high frequency ofCD4+IFN-g+ T cells (Fig. 4A, 4B). At the same time, only a smallfraction of activated CD4+ T cells produced high levels of IL-2(Fig. 4A, 4B). Real-time PCR (Fig. 4C) and ELISA (Fig. 4D)methods also confirmed the flow cytometric observation regardingthe loss of IL-2 expression and acquisition of IFN-g productionduring the acute responses to the pathogens.Taken together, our results suggest that the effector phenotype

during acute responses to T. gondii, L. monocytogenes, and vac-cinia virus is associated with a limited ability to produce IL-2.

Rescue of Treg cells by IL-2 negatively regulates the hostresistance to microbial and viral infections

We next investigated whether the limited production of IL-2,coupled with the expansion of pathogen-specific T cells, is re-sponsible for the infection-induced loss of Treg cells. This hy-pothesis is based on previous experiments that revealed high sen-sitivity of Treg cells to the decreased availability of IL-2, as well as

FIGURE 3. T. gondii-specific Th1 T cells produce limited amounts of

IL-2. WT animals (five mice per group) were either left untreated (A) or

infected with T. gondii i.p. (B) or orally (C), and 7 d later, the ability of

splenic CD4+ T cells to produce IFN-g, TNF, and IL-2 was analyzed.

Analyses of intracellular cytokine expression were performed on spleno-

cytes restimulated with 0.5 mg/ml anti-CD3 for 5 h in the presence of

GolgiPlug (BD Biosciences). Expression levels of IFN-g (D) and IL-2 (E)

were analyzed by real-time PCR in splenocytes, CD4+, CD8+, and CD4+

Foxp3+ T cells isolated from naive or T. gondii-infected mice (day 7

postinfection). F, IL-2 in cell-culture supernatant was measured by ELISA

on splenocytes isolated from naive (d0) or T. gondii-infected mice at the

indicated time points after restimulation with 0.01 mg/ml anti-CD3 for 48

h. G, Expression levels of CD25 were analyzed by real-time PCR in naive

or T. gondii-infected mice (day 7) on the same samples as shown in D

and E. The data shown are representative of five independent experiments.

*p , 0.05, ***p , 0.001.

FIGURE 4. L. monocytogenes- and vaccinia virus-specific CD4+ T cells

produce limited amounts of IL-2. WT mice (five animals per group) were

infected with L. monocytogenes (104 CFU per mouse) (A) or vaccinia virus

(106 PFU per mouse) (B), and the ability of CD4+ T cells to secrete IFN-g

and IL-2 was analyzed by flow cytometry after restimulation with 0.5 mg/

ml anti-CD3 for 5 h in the presence of GolgiPlug (BD Biosciences). C,

Expression levels of IFN-g (left panel) and IL-2 (right panel) were ana-

lyzed by real-time PCR in naive (black bars), L. monocytogenes- (open

bars), and vaccinia virus-infected (gray bars) mice on day 7 postinfection.

D, IFN-g and IL-2 were measured by ELISA in unstimulated (media) or

0.01 mg/ml anti-CD3–restimulated splenocytes isolated from naive (black

bars), L. monocytogenes- (open bars), and vaccinia virus-infected (gray

bars) mice. The data shown are representative of four independent

experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

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our observation regarding limited amounts of IL-2 producedby pathogen-specific CD4+ T cells (Figs. 3, 4). To directly ex-amine this possibility, we determined whether exogenous treat-ment with IL-2 would prevent the disappearance of Treg cellscaused by the microbial and viral infections.Animals infected with T. gondii, L. monocytogenes, or vaccinia

virus were additionally treated with an IL-2/anti–IL-2 complex,which is known to increase the biological activity of the cytokine(16). As is evident in Fig. 5, IL-2 supplementation during thecourse of all of these infections prevented the disappearance ofTreg cells. IL-2 treatment completely prevented the loss of Tregcells triggered by T. gondii infection (irrespective of the route ofinfection) in the spleen (Fig. 5A–C) and in all of the examinedtissues (data not shown). Similarly, IL-2 treatment of L. mono-cytogenes- or vaccinia virus-infected mice prevented the loss ofTreg cells (Fig. 5D–F). These results suggested that insufficiencyof IL-2 is a major cause of the loss of Treg cells triggered by theparasitic, bacterial, and viral pathogens.We next investigated the effects of Treg cell reconstitution on the

induction of host resistance to T. gondii, L. monocytogenes, andvaccinia virus. We observed that IL-2 treatment of T. gondii-infected mice dramatically reduced the ability of CD4+ T cells toacquire a Th1 phenotype, a hallmark of the immune response tothis parasite (Fig. 6A). Furthermore, the reconstitution of Tregcells resulted in increased mortality of the infected mice comparedwith WT animals without IL-2 treatment (Fig. 6B). These resultsstrongly suggest that the infection-triggered depletion of Tregcells is essential for establishing a protective Th1 response duringthe acute response against the parasite. We also observed that inWT mice that survived the acute phase of toxoplasmosis, a shorttreatment with IL-2 at the initial phase of the adaptive immuneresponse to the parasite that resulted in an enhanced brain cystload during the chronic phase of the infection (Fig. 6B).Similar to the results obtained with T. gondii-infected mice, we

observed that in vivo IL-2 treatment impaired Th1 responses to L.monocytogenes and enhanced susceptibility to the pathogen (Fig.6C). Whereas mock or untreated WT animals completely clearedL. monocytogenes by day 7 postinfection, animals that maintained

the Treg cell population as a result of IL-2 treatment containedhigh numbers of bacteria (Fig. 6C). IL-2 treatment and Treg cellreconstitution in vaccinia virus-infected animals also had a majornegative impact on the clearance of the virus (Fig. 6D).

Regeneration of Treg cells during microbial infection

Deficiency in IL-2 or Treg cells results in a fatal autoimmunedisease affecting multiple organs (1, 14). In contrast, infection-induced loss of Treg cells was transient and even beneficial for theinduction of pathogen-specific immune responses (Fig. 6). Theseobservations suggest that mechanisms are in place that regulateTreg cell recovery following acute responses to pathogens. Be-cause Treg cells can either originate from the thymus or developas a result of peripheral conversion from naive T cells (1), weinvestigated the relative contribution of these mechanisms in therestoration of Treg cells.The de novo induction of Treg cells in the periphery critically

depends on the vitamin A metabolite retinoic acid, and cells ex-pressing the ALDH enzymes are known to have a major role inthe generation of inducible Treg cells (31). We used flow cyto-metry to analyze ALDH-expressing cells during the course of T.gondii infection. Whereas only a small number of cells expressedALDH under steady-state conditions, we observed a rapid accu-mulation of ALDH+ cells in the spleen of infected animals duringthe acute response to the parasite (Fig. 7A, Supplemental Fig.4). The ALDH-positive cells also expressed CD11b and Gr1, butnot CD11c, surface markers (Supplemental Fig. 4 and data notshown). Intriguingly, the highest numbers of ALDH+ cells weredetected in the T. gondii-infected mice at the start of Treg cellreconstitution (Fig. 7, Supplemental Fig. 4). To examine whetherthe infection-induced ALDH+ cells were responsible for the res-toration of the Treg cell pool, we evaluated the capacity ofCD11b+ALDH+ cells to generate Treg cells in vitro from Foxp3-negative CD4+ T cells. Non-Treg naive CD4+ T cells were isolatedfrom Foxp3-GFP mice and incubated with sorted ALDH+CD11b+

CD11c2 cells alone or with the addition of rIL-2 and TGF-b (Fig.7B). For comparison, we also isolated CD11c+ DC from the samemice and analyzed the Treg cell conversion ability of these cells

FIGURE 5. Exogenous IL-2 treatment during para-

sitic, bacterial, and viral infections prevented the loss

of Treg cells. WT mice (n = 5) were infected i.p. (A) or

orally (B) with T. gondii and additionally treated with

IL-2 (1.5 mg of IL-2 plus 50 mg of anti–IL-2 Ab per

mouse). The Treg cell frequency was analyzed by flow

cytometry on day 7 postinfection. C, Average fre-

quency of Foxp3+ cells in the spleens of control (2),

i.p. (IP), or orally (O) infected mice treated with IL-2

plus anti–IL-2 Ab (+IL-2). The data shown are the

mean 6 SD. Animals were infected with L. mono-

cytogenes (104 CFU per mouse) (D) or vaccinia virus

(106 PFU per mouse) (E) and, where indicated, were

treated with exogenous IL-2 as described above. Treg

cells were analyzed in IL-2–treated (+) and untreated

(2) mice on day 7 postinfection. F, The data shown are

the mean frequency of Treg cells 6 SD in L. mono-

cytogenes- (LM) or vaccinia virus-infected (VV) mice.

The data shown are representative of three independent

experiments. ***p , 0.001.

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alone or in the combination with ALDH+ cells. We observed thatALDH+ cells alone or those cocultured with DC failed to induceTreg cells in the absence of TGF-b. The addition of this cytokineto the culture of DC (CD11c+MHC class II+ALDH2) or ALDH+

CD11b+CD11c2 cells dramatically enhanced the ability of bothcell populations to induce Treg cells (Fig. 7B). The addition ofexogenous IL-2 resulted in a further increased generation of Tregcells. Nevertheless, under all tested conditions, ALDH+ cells werenot superior to ALDH2 DC in the conversion of Foxp3-negativeCD4+ T cells into Foxp3-positive lymphocytes (Fig. 7B). Fur-thermore, the efficient induction of Treg cells by ALDH+CD11b+

and CD11c+ cells was in part dependent on IL-2 (Fig. 7B), arguingagainst the possibility that the peripheral induction of Treg cellscould compensate for the loss of Treg cells that results frominfection-induced IL-2 insufficiency.We next analyzed whether an increased production of Treg cells

in the thymus could have a role in the reconstitution of Treg cellsafter the acute response to parasitic infection. We first observedthat whereas the frequency of thymic Treg cells was reduced inT. gondii-infected mice (Fig. 8A, 8B), their loss was less promi-nent when compared with Treg cells in the periphery (Fig. 1). In-triguingly, at the peak of peripheral Treg cell loss (day 7, Fig. 1),we observed a relative expansion of thymic Foxp3+ cells that alsoexpressed Ki67, a marker of recently divided cells (Fig. 8A, 8C).Importantly, the thymic Treg cells rapidly recovered in both theirabsolute number and their frequency after the parasitic infection(Fig. 8B, 8D), suggesting that thymus-derived Treg cells, ratherthan the peripheral induction of Foxp3+CD4+ T cells, have a major

role in Treg cell recovery following the acute response to thepathogen.

DiscussionTreg cells expressing the transcription factor Foxp3 contribute tothe dominant control of self-reactive T cells, thus contributing tothe maintenance of immunologic self-tolerance (1, 8). Solid evi-dence from experimental animals and clinical observations havedemonstrated that the expansion and accumulation of these im-munosuppressive cells correlates with advanced tumor growth andpredicts poor prognosis during infectious diseases. Treg cells notonly prevent the induction of tumor- or pathogen-specific CD4+

and CD8+ T cell responses but also limit the efficacy of vacci-nations and other therapeutic interventions (1).The effect of Treg cells in the context of host–pathogen inter-

actions has been particularly extensively studied during chronicinfectious diseases. In a model of cutaneous leishmaniasis, Tregcells are responsible for the suppression of effector T cells and theestablishment of a persistent parasitic infection (5). The removalof Treg cells during malaria infection limited the expansion of theparasite and prevented the host from developing cerebral malaria(6, 9). The immunosuppressive effects of Treg cells are not limitedto parasitic diseases. The depletion of Treg cells greatly enhancedantiviral responses during HIV and SIV infections (32–34). Sim-ilarly, failure to control hepatitis B and C viruses correlated witha high frequency of Treg cells (35). An important role for Tregcells in the progression of mycobacteria infection has also beendemonstrated (10).

FIGURE 6. Exogenous IL-2 treatment during

parasitic, bacterial, and viral infections results in

enhanced susceptibility to the pathogen. A, T. gon-

dii-infected animals (five mice per group) were

treated with the IL-2/anti–IL-2 complex, and Th1

polarization was analyzed on day 7 postinfection. B,

The cumulative survival and cyst burden on day 30

postinfection are shown for untreated and IL-2–

treated mice. L. monocytogenes- (C) or vaccinia

virus-infected (D) animals were additionally treated

with IL-2 (+), and Th1 polarization and pathogen

loads were analyzed on day 7 postinfection. The

data shown are representative of five independent

experiments for T. gondii and three independent

experiments for L. monocytogenes and vaccinia vi-

rus infections. ***p , 0.001.

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A role for Treg cells during the initiation of acute responses topathogens is less clear. The profound inhibitory effects of Treg cellson the priming of pathogen- or model Ag-specific T cells have beenwell characterized (8). The experimental depletion of Treg cellsresulted in greatly enhanced Th1 and Th2 responses and formallydemonstrated a major role for Treg cells in the prevention of ef-fector T cell activation (36, 37). What is not clear is how T cellresponses are initiated in the presence of Treg cells in vivo. Onemechanism of the modulation of the suppressive effects of Tregcells depends on the activation of TLRs on DC and other APCs(38). Because TLRs are involved in the recognition of all knowngroups of pathogens, including viruses, bacteria, fungi, and pro-tozoa, TLR-dependent inhibition of the suppressive effects ofTreg cells can coordinate the immune responses to all groups ofmicroorganisms. More recent studies revealed that in addition toAPC, murine and human Treg cells express high levels of severalTLRs, but the effects of TLR activation on Treg cells remaincontroversial. For example, TLR5 activation by flagellin increasesthe suppressive activity of Treg cells (39). However, triggering ofTLR8 on Treg cells results in the abrogation of their suppressivefunctions (24). The positive and negative effects of TLR2 acti-vation on Treg cells depend on the nature of the TLR2 agonist(29). Similarly, TLR4 can both enhance and suppress Treg cellfunctions (40, 41). In this report, we identified a distinct self-

regulated mechanism that allows effector T cells to avoid thesuppressive effects of Treg cells. Importantly, this mechanismdoes not require TLR-mediated recognition of the pathogen butdepends on the tight regulation of IL-2 availability.We observed that the acute immune response to three distinct

pathogens—T. gondii, L. monocytogenes, and vaccinia virus—resulted in the transient and systemic partial loss of Treg cells. Thedisappearance of Treg cells did not require TLR activation butrather correlated with the degree of effector T cell activation andthe inability of the activated CD4+ T cells to produce IL-2. Weobserved that the rapid expansion of pathogen-specific Th1 CD4+

T cells resulted in a transient insufficiency of IL-2. Because IL-2has a dominant role in Treg cell development and in the regulationof the suppressive functions of Treg cells (14), infection-inducedinsufficiency in IL-2 assures the loss of Treg cells during theinitiation of pathogen-specific T cell responses. Our observationsestablished that in contrary to the previous models in whichdeprivation of IL-2 was proposed to be a mechanism by whichTreg cells could control effector T cells (42), limited amounts ofIL-2 have more profound effects on Treg cells. Most importantly,we observed that the transient loss of Treg cells was essential foroptimal host resistance to all of the tested pathogens: T. gondii,L. monocytogenes, and vaccinia virus. Prevention of the transientloss of Treg cells by treating the infected animals with IL-2

FIGURE 7. Peripheral Treg cell conversion by

ALDH+CD11b+ and CD11c+ DC cells. A, The relative

(left panel) and absolute (right panel) counts of

ALDH+CD11b+ cells at the indicated time points after

T. gondii infection. The appearance of ALDH+ cells in

response to T. gondii infection was analyzed by flow

cytometry on days 0, 7, 10, 14, 21, and 28 postinfection

shown in Supplemental Fig. 4. B, ALDH+CD11b+ and

ALDH2CD11c+ cells were sort-purified from spleens

of T. gondii-infected mice on day 7 postinfection and

were mixed with sort-purified Foxp3GFP- CD4+

T cells in the presence of anti-CD3 alone or in com-

bination with IL-2 and TGF-b. T cell Foxp3 expression

was examined by flow cytometry after 3 d of culture.

Plots are gated on CD4+ cells, and the percentages of

Foxp3+ cells are shown. The data shown are repre-

sentative of three experiments. *p , 0.1, **p , 0.01.

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resulted in impaired pathogen-specific responses and, in the caseof parasitic infection, was responsible for the high mortality of theinfected mice. Although Treg cell reconstitution did not result inmortality of vaccinia virus-infected mice, and only a small frac-tion of L. monocytogenes-infected animals died after Treg cellreconstitution, a dramatic elevation of the pathogen loads wasobserved in all of the experimental infections. This enhancedsusceptibility to viral and microbial infection correlated withimpaired IFN-g production by effector T cells.A recent report from Belkaid and colleagues (13) revealed that

the mucosal responses to T. gondii are characterized by the dis-appearance of Treg cells, which causes lethal intestinal and liverpathology. Although we also observed that Treg cell reconstitutionby IL-2 reduced the damage caused by the exaggerated immuneresponses to the parasite, several lines of evidence suggest that themain function for the transient loss of Treg cells is to enhance Th1responses to pathogens. First, we established that T. gondii is nota unique pathogen in its ability to cause the loss of Treg cells.Mice infected with L. monocytogenes or vaccinia virus demon-strated a similar loss of Treg cells, but these bacterial and viralinfections do not cause severe intestinal damage. Furthermore, weobserved that systemic (i.p.) infection with T. gondii also re-sulted in the IL-2–dependent disappearance of Treg cells, similarto that observed during the mucosal responses to the parasite. Be-cause the intestinal pathology is triggered during mucosal, but not

systemic, responses to the parasite, we conclude that althoughthe infection-induced transient loss of Treg cells contributes to thepathological response, it is not sufficient for the initiation of theintestinal immunopathology. Indeed, we previously reported thatTLR11-deficient animals are protected from T. gondii-inducedileitis (15), but TLR11 activation was not required for the infec-tion-induced loss of Treg cells. These results suggest that theobserved transient loss of Treg cells caused by microbial infectionhas a distinct biological function. We propose that the loss of Tregcells caused by the limited ability of the pathogen-specific CD4+

T cells to produce IL-2 is an important regulatory mechanism thatis essential for host resistance to microbial infections.Ablation of Treg cells is not always beneficial for the acute

response to pathogens. In the context of mucosal HSV infection, theablation of Treg cells accelerates fatal infection associated withincreased viral loads in the mucosa and CNS (12). This is becausea complete lack of Treg cells results in uncoordinated responses tothe virus, characterized by an exaggerated inflammatory reactionwithout the sufficient number of NK cells, DC, and T cells re-quired for protection of the host against the HSV infection. Sim-ilarly, the complete depletion of Treg cells results in impairedrespiratory syncytial virus clearance in the lung, which is asso-ciated with a delay in the recruitment of respiratory syncytialvirus-specific CD8+ T cells (43). These studies clearly demon-strate that the complete and prolonged ablation of Treg cells can

FIGURE 8. Thymic Treg cells are relatively resistant to infection-induced Treg cell loss. A, WT mice (five animals per group) were infected i.p. with an

average of 20 T. gondii strain ME49 cysts per mouse, and the frequency of Treg cells, among all CD+ T cells, was analyzed in the thymus of the infected and

control mice at the indicated number of days postinfection. Analysis of thymic Treg cell proliferation was performed by intracellular staining for the nuclear

Ag Ki67. Average frequency of Foxp3+ cells (B) and Foxp3+Ki67+CD4+ T cells (C) in the thymus of T. gondii-infected mice. D, Absolute quantification of

Treg cells in the thymus of mice infected with the parasite. The data shown are representative of three independent experiments. *p , 0.05.

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be detrimental not only because of fatal autoimmune reactions,but also because of uncoordinated pathogen-specific immune re-sponses.Our results revealed that T. gondii, L. monocytogenes, and

vaccinia virus caused a transient and incomplete infection-inducedTreg cell depletion. The rapid recovery of Treg cells correlatedwith the enhanced replication of thymic Foxp3+CD4+ T cells,suggesting that thymus-derived Treg cells have a major role in therestoration of Treg cells in the periphery. In addition, we observedthat thymic Treg cells were more resistant to the infection-inducedIL-2 deprivation, and our results are consistent with previouspublications that established that in the absence of an IL-2 signal,the thymic production of Foxp3+CD4+ T cells was only partiallyreduced, indicating that thymic Treg cells are less sensitive toIL-2 deficiency (44, 45). In summary, we observed that parasitic,bacterial, and viral pathogens caused a prominent and transientloss of Treg cells. The infection-induced expansion of effectorT cells was associated with a limited amount of IL-2 produced byactivated CD4+ T cells, and the deficiency in this cytokine wasresponsible for the infection-triggered loss of Treg cells. Ourresults revealed that the naturally occurring partial depletionof Treg cells is essential for establishing protective immunity toT. gondii, L. monocytogenes, and vaccinia virus and suggest amechanism that regulates adaptive immunity to pathogens thatdepends on the homeostatic IL-2–based interactions between ef-fector and regulatory CD4+ T cells.

AcknowledgmentsWe thank Ellen Vitetta and Julie Mirpuri for carefully reading the manu-

script.

DisclosuresThe authors have no financial conflicts of interest.

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