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Element of the Host Response to InjuryEnhanced Regulatory T Cell Activity Is an

Thomas J. Murphy, John A. Mannick and James A. LedererNiamh Ni Choileain, Malcolm MacConmara, Yan Zang,

http://www.jimmunol.org/content/176/1/225doi: 10.4049/jimmunol.176.1.225

2006; 176:225-236; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/176/1/225.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 2006 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Enhanced Regulatory T Cell Activity Is an Element of theHost Response to Injury1

Niamh Ni Choileain, Malcolm MacConmara, Yan Zang, Thomas J. Murphy, John A. Mannick,and James A. Lederer2

CD4�CD25� regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controllingpotentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both ex-cessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using amouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity isapparent at 7 days after injury and is restricted to lymph node CD4�CD25� T cells draining the injury site. Moreover, we showthat this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surfaceTGF-�1 expression. To test the in vivo significance of these findings, mice were depleted of CD4�CD25� T cells before sham orburn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observedthat injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted ofCD4�CD25� T cells. Taken together, these observations suggest that injury can induce or amplify CD4�CD25� Treg function andthat CD4�CD25� T cells contribute to the development of postinjury immune suppression. The Journal of Immunology, 2006,176: 225–236.

A ccumulating clinical and experimental evidence indi-cates that serious injury promotes suppressed immunefunction predisposing the injured host to infectious com-

plications (1–3). In particular, severely injured patients exhibitclassical signs of impaired adaptive immune activity such as lossof delayed-type hypersensitivity responses, prolonged skin allo-graft survival, and reduced T cell proliferation to polyclonal andAg-specific stimulation (4–6). These changes in T cell responsesare also accompanied by lowered Th1-type cytokine productionand reduced Th1-dependent Ab isotype secretion (7). An associ-ation between suppressed adaptive immune function after majorinjury in patients and increased risk of developing nosocomial in-fections has been reported (8, 9). Furthermore, a causal relation-ship between suppressed Th1-type immunity after injury and low-ered resistance to sepsis has been demonstrated in animal studiesshowing that injured mice treated with the Th1-promoting cyto-kine IL-12 or with Ab to the Th2-type, counterinflammatory cy-tokine IL-10 manifested improved resistance to a septic challenge(10, 11).

In most studies, the injury-induced loss of T cell function ap-pears to be a progressive phenomenon, reaching a nadir severaldays to more than a week after the initial insult (12–14). Thissupports the idea that the development of suppressed T cell-me-diated immunity is a programmed response and may well representa compensatory host reaction to the intense inflammation induced

by tissue damage that accompanies serious injury. Several mech-anisms have been postulated to explain the gradual loss of T cellfunction after injury. These include the increased release of im-munosuppressive prostaglandins by innate immune cells, elevatedcirculating levels of corticosteroids, and the decreased productionof the Th1-inducing cytokine IL-12 (15–20). Although a role forso-called “suppressor T cells” in mediating this phenomenon wassuggested by studies performed �20 years ago, this explanationfor depressed adaptive immunity after injury disappeared from theliterature because of the failure to identify genetically or pheno-typically an unequivocal suppressor T cell subset (21–23).

Interest in suppressor T cells was rekindled when it was shownthat a minor population of thymic-derived CD4� T cells that co-express CD25, the �-chain of the IL-2R, was crucial for control-ling autoreactive T cells in vivo (24). These naturally occurringCD4� T cells were shown to potently inhibit TCR-driven T cellproliferation and were appropriately named regulatory T cells(Tregs)3 (25). More recent findings indicate that Tregs can be fur-ther distinguished from conventional CD4�CD25� T cells by theirCD45RBlow, L-selectin (CD62L)�, CD69�, glucocorticoid-in-duced TNFR-like protein 6 (GITR)�, TGF-�1�, and TLR4� cellsurface phenotype (26–28). In addition, the forkhead transcriptionfactor, Foxp3, has been shown to be expressed specifically inTregs and is required for their development in mice (29, 30). Athorough assessment of these cell surface markers and FoxP3 geneexpression can be used to judge whether populations of CD4�

CD25� T cells are natural Tregs vs induced Tregs.Beyond their ability to potently block the development of au-

toimmune disease and T cell proliferation, Tregs have been shownto suppress inflammatory responses in vivo by IL-10- or TGF-�-mediated mechanisms (31–33). It is this counterinflammatory na-ture of Tregs that prompted us to examine whether they might playa role in helping control the intense inflammatory reaction that

Department of Surgery (Immunology), Brigham and Women’s Hospital/HarvardMedical School, Boston, MA 02115

Received for publication April 29, 2005. Accepted for publication October 11, 2005.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by funding from National Institutes of Health GrantsGM57664 and GM35633. Additional support was provided by the Julian and EuniceCohen and Brook Family Funds for Surgical Research.2 Address correspondence and reprint requests to Dr. James A. Lederer, Departmentof Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115.E-mail address: jlederer@rics.bwh.harvard.edu

3 Abbreviations used in this paper: Treg, regulatory T cell; CD62L, L-selectin; GITR,glucocorticoid-induced TNFR-like protein 6; TNP, trinitrophenol; TNP-OVA, TNP-haptenated OVA; PD-1, programmed death-1.

The Journal of Immunology

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00

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occurs after severe injury, and we have recently reported that thisis indeed the case (34). Furthermore, we have been interested indetermining whether injury might increase Treg cell activity and,if so, whether these cells could be involved in mediating the knownprogressive loss of Th1 responses after injury. Using a mouse burnmodel that inflicts a controlled but significant level of tissue dam-age, we find that injury enhances the regulatory activity ofCD4�CD25� T cells purified from the lymph nodes draining theinjury site. This injury-induced augmentation of Treg activity isdetectable at 7, but not 1, day after injury, suggesting that it is aprogressively developed phenotype. Moreover, we show that thisinjury-induced increase in Treg function is cell-contact dependent,is mediated in part by TGF-�, and is associated with an increase incell surface TGF-�1 expression on lymph node CD4�CD25� Tcells. We find that the Tregs from injured mice more potentlysuppress T cell proliferation and IL-2 and IFN-� production thando Tregs from sham mice and that their in vivo depletion beforeinjury prevents the injury-induced inhibition of Ag-specific Th1-type reactivity. To our knowledge, these findings are the first toindicate that injury can enhance Treg activity and that Tregs con-tribute to the suppression of Th1-type immunity that occurs aftersevere injury.

Materials and MethodsMice

Male BALB/cJ mice were obtained from The Jackson Laboratory and weremaintained in an accredited virus-free animal facility in accordance withthe guidelines of the National Institutes of Health and the Harvard MedicalArea Standing Committee on Animals. The mice were acclimated for atleast 1 wk before being used in experiments at 6–9 wk of age.

Reagents

CD4�CD25� Regulatory T Cell Isolation kits were obtained from MiltenyiBiotec. Anti-mouse CD3Ab (clone 145-2C11) was obtained from R&DSystems. Culture medium for in vitro studies consisted of RPMI 1640supplemented with 5% heat-inactivated FCS, 1 mM glutamine, penicillin/streptomycin/Fungizone, 10 mM HEPES buffer, 100 �M nonessentialamino acids, and 2.5 � 10�5 M 2-ME, all purchased from Invitrogen LifeTechnologies. In some experiments 1% Nutridoma-SP (Roche MolecularBiochemicals) was substituted for 5% FCS in the culture medium. Bufferused for intracellular staining consisted of 0.01% saponin and 1% BSA(both purchased from Sigma-Aldrich) and 0.1% sodium azide (Fisher Sci-entific) dissolved in Dulbecco’s PBS (Invitrogen Life Technologies). FcBlock (Fc�III/II receptor (clone 2.4G2)); Cy5-labeled anti-CD4 Ab; FITC-labeled anti-CD25 Ab; PE-labeled CD28, CD45RB, CD152, and ICOSAbs; and mouse IgG1 � isotype control Ig were purchased from BDPharmingen. PE-conjugated anti-mouse CD62L and CD69 Abs were pur-chased from Caltag Laboratories. PE-conjugated anti-mouse TLR4-MD-2specific Ab was purchased from eBioscience, and PE-conjugated anti-mouse GITR was purchased from R&D Systems. PE-labeled mouse mono-clonal anti-human TGF-�1 Ab was purchased from IQ Products. Rat IgGwas purchased from Sigma-Aldrich. Purified PC61 Ab was purchased fromthe American Type Culture Collection and BioXpress.

Mouse injury model

The mouse thermal injury model, approved by the National Institutes ofHealth and the Harvard Medical Area Standing Committee on Animals,was performed as described (35). In brief, six mice per group were anes-thetized by i.p. injection of ketamine (175 mg/kg) with xylazine (6.5 mg/kg). The dorsal fur was shaved and the animal was placed in an insulatedplastic mold to expose 25% total body surface area. This part of the dorsumwas then immersed in 90°C (burns) or isothermic water (shams) for 9 s. Allgroups were resuscitated by i.p. injection with 1 ml of 0.9% pyrogen-freesaline. This protocol causes a well demarcated, full-thickness, anestheticinjury with �5% mortality.

CD4�CD25� and CD4�CD25� T cell purification

Spleens and lymph nodes (inguinal, axillary, and brachial) were harvestedfrom mice and cell suspensions were prepared by mincing tissues on wiremesh screens. Spleen cells were treated with ammonium chloride solutionfor 3 min to lyse RBCs and were washed twice before suspension in culture

medium. Lymph node cells were also washed twice before suspension inculture medium. CD4�CD25� and CD4�CD25� cells were purified usingthe regulatory T cell magnetic cell sorting kits (Miltenyi Biotec) accordingto the manufacturer’s instructions. Both CD4�CD25� and CD4�CD25� Tcell populations were collected and prepared for in vitro studies. Cell puritywas assessed by staining purified cell populations with Cy5-labeled anti-CD4 Ab and PE-labeled anti-CD25 Ab followed by analysis using aFACSCalibur Instrument (BD Biosciences), and results were processedusing the accompanying CellQuest Pro Software. The CD4�CD25� andCD4�CD25� T cells were consistently �95% pure using this approach(Fig. 1A).

Real-time PCR

CD4�CD25� or CD4�CD25� T cells were purified from the lymph nodesof sham or burn-injured mice at 7 days after injury. RNA was preparedfrom these cell suspensions using the TRIzol RNA isolation buffer follow-ing the protocol suggested by the manufacturer (Invitrogen Life Technol-ogies). We next synthesized cDNA by an oligo(dT) primed reverse tran-scriptase reaction using 0.5 �g of RNA from each sample. Real-time PCRswere performed in an Applied Biosystems 5700 gene detection instrumentusing murine GAPDH (F-CAGGTTGTCTCCTGCGACTT, R-CCCTGTTGCTGTAGCCGTA) and FoxP3 (F-CCATTGGTTTACTCGCATGT, R-GCTCTCCACTCGCACAAA) specific primers. SYBR green was used todetect changes in amplicon levels with each sequential amplification cycle.The level of FoxP3 gene expression was calculated by the 1/� Ct method(Ct is the cycle number where the fluorescence crosses a set thresholdlevel) using the following formula (1/CtFoxP3 � CtGAPDH).

In vitro cytokine production studies

Purified CD4�CD25� or CD4�CD25� T cells were cultured in Costarround-bottom 96-well plates at 2 � 105 cells/well in the absence or pres-ence of plate-bound anti-CD3� Ab (clone 145-2C11 added at 5 �g/ml).These cultures were performed in 1% Nutridoma-SP culture medium ratherthan in medium containing 5% heat-inactivated FCS to allow for accuratemeasurement of TGF-� production. After 24-h stimulation, culture super-natants were harvested and stored at 4°C. The cytokines IL-2, IFN-�, IL-10, and TGF-� were measured by cytokine-specific ELISAs. IL-10 andTGF-� were measured using ELISA kits purchased from R&D Systemsand used according to the manufacturer’s instructions. In the case ofTGF-�, 0.1 ml of 1 N HCl was added to 0.5 ml of sample, which wasincubated for 10 min at room temperature before neutralization by theaddition of 0.1 ml of 1.2 N NaOH/0.5 M HEPES to activate latent TGF-�1to its immunoreactive form. IFN-� levels were measured using Ab pairspurchased from R&D Systems, whereas IL-2 was measured using Ab pairspurchased from Caltag Laboratories. The ELISA results were analyzedusing an ELISA Plate Reader and the associated SoftMax Pro softwareprogram (Molecular Devices). Cytokine levels in culture supernatants werecalculated based upon cytokine standards included in each assay plate.

Assessment of CD152, CD28, ICOS, programmed death-1(PD-1), CD62L, CD69, CD45RB, TLR4-MD-2, GITR, andTGF-�1 expression on CD4�CD25� and CD4�CD25� T cells

Lymph node cells were prepared from sham or burn-injured BALB/cJmice. Cells were first incubated with Fc Block reagent in FACS buffer(Dulbecco’s PBS containing 1% BSA and 0.1% sodium azide) at 4°C for20 min to reduce nonspecific background staining and then were stainedwith Cy5-labeled anti-CD4 and FITC-labeled anti-CD25 Abs to identifythe CD4�CD25� and CD4�CD25� cell populations. Cells were stainedsimultaneously with PE-labeled anti-CD152, anti-CD28, anti-ICOS, anti-PD-1, anti-CD62L, anti-CD69, anti-CD45RB, anti-TLR4-MD-2, anti-GITR, or anti-TGF-�1 Abs. To detect intracellular CD152, cells that weresurface stained with anti-CD4 and anti-CD25 Abs were fixed for 10 min in1% paraformaldehyde solution and then permeabilized for 20 min in FACSbuffer containing 0.25% saponin (permeabilization buffer). The fixed andpermeabilized cells were then incubated for 10 min in permeabilizationbuffer containing 1 �g/ml normal rat IgG to block nonspecific staining,after which PE-labeled anti-CD152 Ab was added for an additional 30-minincubation. The cells were washed twice by centrifugation in permeabili-zation buffer and FACS analysis was performed as described above.

CFSE-based cell proliferation assays

Lymph node cell suspensions were prepared from three BALB/cJ mice andlabeled with CFSE using the Vybrant Cell Tracer Kit (Molecular Probes)following the manufacturer’s protocol. Briefly, lymph node cells were sus-pended in prewarmed (37°C) PBS containing CFSE at a 5 �M concentra-tion and then incubated for 15 min at 37°C. Cells were then centrifuged and

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resuspended in prewarmed (37°C) culture medium, incubated for an addi-tional 30 min to ensure complete modification of the probe, and then re-washed. The CFSE-labeled lymph node cells were added at 2 � 105 cells/well to individual wells of Costar round-bottom 96-well plates. PurifiedCD4�CD25� or CD4�CD25� T cells were then added to the CFSE-la-beled lymph node cells at 1:1, 1:2, 1:3, and 1:4 ratios in the absence orpresence of 5 �g/ml anti-CD3 Ab. Wells of CFSE-labeled cells without theadded CD4�CD25� or CD4�CD25� cells served as negative and positivecontrols. In some experiments, purified CD4�CD25� T cells from sham or

burn mice were CFSE-labeled and used in place of CFSE-labeled lymphnode cells to test the direct effects of CD4�CD25� T cells on anti-CD3-induced CD4� T cell proliferation. These cell mixes were cultured at 37°Cin 5% CO2 for 72 h, then pelleted by centrifugation, pretreated with FcBlock, and stained with Cy5-labeled anti-CD4 Ab. Flow cytometry wasperformed by gating on CD4� cells and determining CFSE signal intensityusing the FACSCalibur Instrument, and the results were analyzed usingCellQuest Pro software.

Transwell CFSE proliferation studies

Lymph node cells from three BALB/cJ mice were labeled with CFSE asdescribed. Cells were cultured in Costar Transwell (0.4-�m pore size) 24-well plates at 1 � 106 cells/well with 5 �g/ml anti-CD3 Ab (soluble) or noadditions. Lymph node CD4�CD25� and CD4�CD25� cell populationswere prepared from six mice at 7 days after sham or burn injury. In addi-tion, T cell-depleted APCs were prepared from the spleens of threeBALB/cJ mice by a double depletion approach using Pan-T (anti-Thy 1.2)Dynabeads (Dynal Biotech) according to the manufacturer’s instructions.CD4�CD25� or CD4�CD25� cells (3 � 105) along with T cell-depletedAPCs (3 � 105) were added to the well inserts, and the CFSE-labeledlymph node cells were added to the wells. These Transwell cultures wereincubated for 3 days at 37°C in 5% CO2 in the absence or presence of 5�g/ml anti-CD3 Ab. Control cultures included CFSE-labeled lymph nodecells cocultured with CD4�CD25� or CD4�CD25� cells in the absence orpresence of 5 �g/ml anti-CD3 Ab without well inserts. The levels of cel-lular proliferation were measured by flow cytometry as described above.

Anti-IL-10 or anti-TGF-�1 Ab blocking studies

Purified lymph node CD4�CD25� and CD4�CD25� T cells were pre-pared from 7-day sham or burn-injured BALB/cJ mice and mixed withCFSE-labeled naive BALB/cJ lymph node cells at a 1:3 ratio in the absenceor presence of 5 �g/ml anti-CD3 Ab. Anti-IL-10, anti-TGF-�1 Ab, or theappropriate IgG isotype control Abs (rat IgG1 or mouse IgG1, respectively)were added at 0.2, 2, and 20 �g/ml final concentrations. After 3-day in-cubation at 37°C in 5% CO2, anti-CD3-induced proliferation was measuredby flow cytometry and the results were analyzed as described.

Assessment of the effect of CD25� T cells on Th1-type cytokineproduction

CD4�CD25� and CD4�CD25� T cells were purified from lymph nodecell suspensions harvested 7 days after sham or burn injury (n � 6 mice/group). Lymph node cells were also prepared from naive BALB/cJ mice.The purified CD4�CD25� or CD4�CD25� T cells were added to lymphnode cells in ratios of 1:1 or 1:3 with or without 5 �g/ml anti-CD3 Ab.After 1-day incubation at 37°C in 5% CO2, culture supernatants were har-vested and IL-2 or IFN-� levels were measured by cytokine-specificELISAs.

Ag-specific T cell responses in Treg cell-depleted mice

Male BALB/cJ mice (n � 18–20/group) were treated with 0.25 mg ofpurified PC61 Ab or control rat IgG by i.p. injection. This Ab-mediatedCD4�CD25� T cell depletion protocol depletes CD4�CD25� T cells inmice by 3 days postinjection as judged by FACS (see Fig. 9A), as previ-ously described by others (36). At 3 days after PC61 Ab or rat IgG treat-ment, mice underwent sham or burn injury and were immunized s.c. with0.1 ml of CFA mixed 1:1 with trinitrophenol (TNP)-haptenated OVA(TNP-OVA; 0.1 mg/mouse). At 10 days after injury and immunization,blood, lymph nodes (axillary, brachial, and inguinal), and spleens wereharvested to prepare serum samples and cell suspensions from individualmice. The cells were stimulated in vitro with an optimal concentration ofOVA (0.1 mg/ml), and 48 h later culture supernatants were harvested tomeasure cytokine production levels. Serum TNP-specific Ig isotype levelswere measured by ELISA as described previously (7). In brief, 96-wellplates were coated with PBS containing 20 �g/ml TNP-haptenated BSA.Serial dilutions of serum samples prepared from individual mice wereadded and, after washing specific Ig isotypes, were measured using HRP-conjugated Abs specific for mouse IgM, IgG1, or IgG2a (Caltag Labora-tories). ELISA reactions were developed using a NBT substrate buffer, andthe plates were read at 450–570 nM using the ELISA plate reader.

Statistical analysis

Statistical analyses were performed by ANOVA using Tukey’s multiplecomparisons test or paired t tests. The p values for significance were setat 0.05.

FIGURE 1. CD4�CD25� and CD4�CD25� T cell purity and FoxP3gene expression levels. A, Purified CD4�CD25� and CD4�CD25� T cellsfrom day 7 sham and burn-injured mice were stained with Cy5-labeledanti-CD4 and PE-conjugated anti-CD25 Abs. CD25 expression levels onpurified CD4�CD25� and CD4�CD25� T cell populations are shown inthe upper right quadrant in these representative FACS plots. The resultsare representative of all purifications performed. B, Real-time PCR detec-tion of FoxP3 gene expression levels in RNA prepared from highly purifiedlymph node CD4�CD25� and CD4�CD25� T cells from day 7 sham orburn-injured mice. GAPDH gene expression level was used as an internalreference standard for each sample to determine FoxP3 mRNA expressionlevels as described in Materials and Methods. These data include the re-sults from three independent mouse injury studies using purified cells fromthree mice per group. �, p � 0.05 FoxP3 gene expression levels forCD4�CD25� vs CD4�CD25� T cells.

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Table I. Cell surface phenotype of lymph node CD4� CD25� and CD4� CD25� T cells at 1 and 7 days after injurya

CD45RBlow CD45RBhigh CD62L CD69 GITR TLR4-MD-2

CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25�

Sham (1 day) 77.2 (1.2) 2.8 ((0.01) 23.0 ((0.35) 97.2 (0.2) 54.4 (1.2) 25.8 (2.3) 7.3 (0.5) 18.5 (1.0) 90.3 (1.2) 25.0 (1.6) 6.6 (0.5) 0.17 (0.02)Burn (1 day) 71.9 (1.6) 3.4 (0.01) 28.2 (0.4) 96.6 (0.2) 53.5 (2.4) 22.2 (1.7) 9.7 (0.6) 22.7 (0.9) 86.1 (1.4) 19.6 (2.5) 6.9 (0.8) 0.2 (0.02)Sham (7 days) 73.7 (3.9) 2.7 (0.01) 26.5 (1.7) 97.3 (0.5) 60.0 (2.8) 23.2 (0.4) 6.1 (0.4) 14.2 (1.0) 84.9 (4.6) 15.5 (1.4) 4.2 (0.4) 0.6 (0.1)Burn (7 days) 78.2 (1.7) 3.8 (0.2) 21.6 (1.6) 95.9 (0.2) 59.5 (3.9) 28.9 (2.3) 5.4 (0.4) 16.3 (0.9) 85.0 (4.0) 24.5� (2.1) 3.8 (0.3) 0.1 (0.04)

a Lymph node cells were prepared from sham or burn mice at 1 or 7 days after injury. Cells were stained with Cy5-labeled anti-CD4 Ab and FITC-labeled anti-CD25 Aband then counterstained with PE-labeled Abs specific for CD45RB, CD62L, CD69, GITR, or TLR4-MD-2 as indicated. A total of 100,000 events was collected, and values shownrepresent the mean (SEM) percent positive of gated CD4� CD25� or CD4� CD25� T cells from six mice. The � indicates significant differences between sham and burn mice,p � 0.05 by ANOVA.

FIGURE 2. Injury enhances the Treg ac-tivity of lymph node CD4�CD25� T cells.Lymph node CD4�CD25� andCD4�CD25� T cells purified from six shamor burn-injured BALB/cJ mice were addedto 2 � 105 CFSE-labeled BALB/cJ lymphnode cells in ratios of 1:1 to 1:4. Cell mixeswere stimulated with or without soluble anti-CD3� Ab (5 �g/ml). After 3 days, cells wereharvested and proliferation levels were de-termined by CFSE staining intensity ofgated CD4� T cells. Representative FACSplots from 1-day (A) or 7-day (B) sham andburn mice demonstrate greater inhibition ofanti-CD3-stimulated CD4� T cell prolifera-tion mediated by CD4�CD25� T cells ascompared with CD4�CD25� T cells underall effector to responder ratios tested; p �0.05 by ANOVA. Seven-day burnCD4�CD24� T cells show greater Treg po-tency than do 7-day sham CD4�CD25� Tcells. The marker numbers indicate the per-centage of cells demonstrating reducedCFSE staining intensity. C, Plots showingthe combined CFSE proliferation assay re-sults measured at 1 and 7 days after injuryand representing the mean � SEM of threeindependent experiments. �, Differences be-tween groups at all cell ratios tested; p �0.05 by ANOVA.

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ResultsInjury augments the regulatory activity of CD4�CD25� T cells

It is known that severe injury suppresses T cell-mediated immuneresponses and that this induced change in immune function coin-cides with a decreased ability to control opportunistic infections(5). These observations, along with the discovery that a naturallyoccurring CD4�CD25� T cell subset plays a central role in con-trolling inflammatory responses convinced us to investigatewhether the injury response might prime these cell for increased Tregulatory activity (31). A well-established mouse burn model wasused as a clinically relevant in vivo injury (35). Prior studies usingthis model have documented that suppressed T cell function ismeasurable at 7 days, but not 1 day after injury (1). This feature ofthe injury response prompted us not only to characterize the in-fluence of injury on Treg function, but also to determine time-dependent changes in Treg activity after injury.

We first confirmed our ability to purify populations of CD25�

and CD25�CD4� T cells from the peripheral lymphoid tissues ofmice using a two-step magnetic bead cell sorting approach. Weconsistently obtained purity of �95% for CD4�CD25� andCD4�CD25� T cell populations (Fig. 1A). In addition, we wishedto characterize further the effects of injury on the cell surface phe-notype of CD25� and CD25�CD4� T cells. We measured by flowcytometry the levels of cell surface markers that are known to bedifferentially expressed on CD4�CD25� Tregs vs conventionalCD4� T cells. These included GITR, CD62L, CD45RB, CD69,and TLR4-MD-2. As listed in Table I, we found that theCD4�CD25� T cells were predominately CD45RBlow and ex-pressed higher levels of cell surface GITR, CD62L, and TLR4-MD-2 than did CD4�CD25� T cells. The CD4�CD25� T cellsalso expressed significantly lower levels of CD69 than didCD4�CD25� T cells. Interestingly, injury did not markedly alterthe cell surface expression levels of any of these molecules oneither CD4�CD25� or CD4�CD25� T cells at 1 or 7 days afterinjury. Furthermore, we did not observe a significant change in theoverall numbers of CD4�CD25� or CD4�CD25� T cells at 1 or7 days after burn injury (data not shown). We also wished to de-termine the FoxP3 gene expression levels in CD25� and CD25�

T cells purified from sham vs burn mice. Using a real-time PCRapproach, we showed that the purified CD4�CD25� T cells ex-pressed low levels of FoxP3, whereas purified CD4�CD25� Tcells expressed significantly higher levels of FoxP3 mRNA (Fig.1B). The results of these FoxP3 studies also showed that burninjury did not significantly alter FoxP3 gene expression levels ineither CD25� or CD25� CD4 T cells. Taken together, these resultssuggest that the purified CD4�CD25� T cells studied here mostlikely represent natural Tregs rather than induced Tregs as judgedby cell surface marker expression and FoxP3 gene expression.

Having optimized a purification approach for CD25� andCD25�CD4� T cells, we next performed a series of studies to testinjury effects on CD4�CD25� T cell-mediated regulatory func-tion, focusing on the capacity of this subset to inhibit T cell pro-liferation. In these studies, purified CD4�CD25� andCD4�CD25� T cells from sham or burn-injured mice were addedto suspensions of CFSE-labeled lymph node cells at varying cellratios (1:1 to 1:4). These cell mixtures were then stimulated withsoluble anti-CD3� Ab. After 3 days in culture, CD4� T cell pro-liferation was visualized as a sequential halving of CFSE fluores-cence intensity by FACS analysis. Our results confirmed that theCD4�CD25� T cell populations from sham or burn-injured micecould effectively suppress anti-CD3-induced CD4� T cell prolif-eration, whereas CD4�CD25� T cells demonstrated low antipro-liferative activity under identical conditions (Fig. 2). We also

found that the Treg activity measured in these assays was cellconcentration-dependent (Fig. 2C).

Experiments comparing Treg activity at 1 vs 7 days after injuryshowed no significant difference between sham and burn mice at 1day after injury, whereas at 7 days post-injury we observed amarked increase in Treg activity by lymph node CD4�CD25� Tcells from burn mice (Fig. 2C). Interestingly, spleen-derivedCD4�CD25� T cells did not exhibit a similar injury effect (Fig. 3).This finding indicates that burn injury triggers enhanced Treg cellactivity in a tissue-compartmentalized fashion.

Next, to determine whether a similar injury effect on Treg ac-tivity might be seen in a purified cell system, experiments wereperformed with purified CD4�CD25� T cells as CFSE-labeledresponder cells. To compensate for the absence of APCs in thesecultures, the CFSE-labeled CD4�CD25� T cells were stimulatedwith plate-bound anti-CD3� Ab. Using this approach, we foundthat sham or burn lymph node CD4�CD25� T cells prepared at 7days after injury could suppress CD4�CD25� cell proliferation.This direct effect of CD4�CD25� T cells on CD4�CD25� T cellproliferation occurred in a cell concentration-dependent fashionand was significantly greater with burn as compared with shamCD4�CD25� T cells (Fig. 4). Interestingly, we found that thesource of the CD4�CD25� responder T cells, sham vs burn mice,had no bearing on the regulatory activity mediated by theCD4�CD25� T cells added to these cultures. These results con-firm that injury augments CD4�CD25� Treg function by 1 wkpost-injury and suggest that the enhanced Treg activity can occurby direct interaction between CD4�CD25� and CD4�CD25� Tcells.

Effects of injury on cytokine production by CD4�CD25� andCD4�CD25� T cells

Severe injury skews polyclonal or Ag-specific CD4� T cell cyto-kine responses toward a Th2 phenotype (35, 37). This influence ofinjury on the peripheral T cell pool does not occur early, but it iseasily detected 7–10 days later. The temporal overlap betweeninjury-enhanced Treg activity displayed by lymph node

FIGURE 3. Compartmentalization of the effects of injury on Treg ac-tivity: injury fails to augment the Treg activity of spleen CD4�CD25� Tcells. Purified CD4�CD25� and CD4�CD25� T cells purified from thespleens of sham and burn BALB/cJ mice at 7 days post-injury were addedin ratios of 1:1 to 1:4 to CFSE-labeled lymph node cells. Cells were stim-ulated with soluble anti-CD3� Ab (5 �g/ml). After 3 days in culture, cellswere harvested and proliferation levels were determined by CFSE stainingintensity of gated CD4� T cells by FACS. Results shown represent themean � SEM of three independent experiments; n � 6 mice/group. Therewas no significant difference between sham and burn Treg activity at allcell ratios examined.

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CD4�CD25� T cells and the injury-biased Th2-type cytokine re-sponse suggested to us that there might be a link between these twoinjury-induced changes in host immune responses. To address thispossibility, we directly compared Th1, Th2, and Treg-type cyto-kine production profiles by anti-CD3-stimulated purified popula-tions of CD4�CD25� and CD4�CD25� T cells prepared from thelymph nodes or spleens of mice at 1 and 7 days after sham or burninjury. The results of these studies revealed some significant dif-ferences in the levels and types of cytokines produced by CD25�

and CD25� CD4� T cells. We found that purified CD4�CD25�

cells prepared from either the lymph nodes or spleens producedsignificantly higher levels of IFN-� than did CD4�CD25� T cells,whereas CD4�CD25� T cells released low levels of IL-2 andIFN-� and higher levels of IL-10 and TGF-� (Fig. 5). Burn injurydid not significantly alter IL-2, IFN-�, or TGF-� production levelsby either CD4� T cell subset at 1 or 7 days post-injury; however,at 7 days after burn injury, we observed a significant increase inIL-10 production by both lymph node and spleen CD4�CD25� Tcells (Fig. 5).

The enhanced Treg function observed after injury is cell-contactdependent and not mediated by IL-10

The increased IL-10 production by highly purified CD4�CD25� Tcells detected at day 7 after injury led us to question whether IL-10might mediate, at least in part, the injury-induced increase in Tregactivity. We also wished to learn whether CD4�CD25� T cellsacted through a cell-contact-dependent mechanism as previouslydescribed (26). To address these questions, we first used a con-ventional Transwell approach to test whether the measured Tregactivity was mediated by a soluble factor. We added purifiedCD4�CD25� or CD4�CD25� lymph node T cells from 7-daysham or burn mice along with T cell-depleted splenic APCs to oneside of the semipermeable membrane and CFSE-labeled lymphnode responder cells to the other side at a 1:3 purified T cell tolymph node responder cell ratio. After 3-day incubation in thepresence of soluble anti-CD3 Ab, proliferation levels were as-sessed by FACS analysis. As shown in Fig. 6A, preventing cellcontact by separating the CFSE-labeled lymph node respondercells from CD4�CD25� T cells eliminated the Treg activity nor-mally displayed by these cells. The purified CD4�CD25� T cellsfrom sham or burn mice also did not alter the proliferation of theCFSE-labeled lymph node responder cells under identical experi-mental conditions. As a positive control, purified CD4�CD25�

lymph node T cells from 7-day sham or burn mice cultured in thesame well with CFSE-labeled lymph node responder cells at a 1:3

FIGURE 4. CD4�CD25� T cells can directly inhibit CD4�CD25� Tcell proliferation. Lymph node CD4�CD25� and CD4�CD25� T cellswere harvested and purified from sham and burn BALB/cJ mice at 7 daysafter injury. The pure CD4�CD25� T cells were labeled with CFSE andmixed with pure CD4�CD25� T cells at the indicated ratios. Cells werestimulated with plate-bound anti-CD3� Ab. After 3-day incubation, cellswere harvested and assessed for proliferation by FACS analysis. Resultsshown represent the mean � SEM of three independent experiments; n �3 mice/group. �, p � 0.05 by ANOVA for differences between burnCD25� and sham CD25� at all ratios tested.

FIGURE 5. CD4�CD25� T cellsproduce increased IL-10 at 7 days af-ter injury. Purified CD4�CD25� orCD4�CD25� T cells (2 � 105) fromthe lymph nodes (A) or spleens (B) ofday 1 or day 7 sham and burn micewere stimulated with plate-bound anti-CD3� Ab. At 1 day, culture superna-tants were harvested and tested forIL-2, IFN-�, IL-10, and TGF-� lev-els by cytokine-specific ELISA. Theresults shown represent the mean �SEM of triplicate wells and representthree independent experiments in-volving six mice/group. �, p � 0.05for sham vs burn groups by ANOVA.

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ratio again showed a marked suppression of anti-CD3-induced Tcell proliferation. These findings indicate that cell contact is re-quired for CD4�CD25� T cells to mediate their Treg activity andthat IL-10 or other soluble mediators are not likely to contributedirectly to the observed injury-dependent increase in Tregfunction.

To fully rule out a role for IL-10 in mediating the enhanced Tregactivity exhibited by CD4�CD25� T cells at 7 days after burninjury, we attempted to block Treg activity by adding anti-IL-10Ab at several concentrations to cultures of purified CD4�CD25�

or CD4�CD25� lymph node T cells harvested from 7-day sham orburn-injured mice mixed at a 1:3 ratio with CFSE-labeled lymphnode responder cells. The results of these studies indicated thatIL-10 does not mediate the in vitro Treg activity by sham or burnCD4�CD25� T cells because all concentrations of anti-IL-10 Abor isotype-matched control IgG tested had indistinguishable andnegligible effects on the measured Treg cell function (Fig. 6B).

The enhanced CD4�CD25� Treg activity after injury isassociated with increased cell surface TGF-�1 expression

Because we observed that the CD4�CD25� Treg activity was cell-contact dependent, we wished to investigate whether membraneTGF-� might be responsible for CD4�CD25� T cell-mediatedTreg function detected in our studies. Using an approach similar tothe anti-IL-10 experiments, we tested the effects of adding a mono-clonal anti-TGF-�1 Ab or its isotype control at a range of con-centrations to cultures of purified CD4�CD25� or CD4�CD25�

lymph node T cells harvested from 7-day sham or burn-injuredmice mixed at a 1:3 ratio with CFSE-labeled lymph node re-sponder cells. As shown in Fig. 6C, we observed a significantdose-dependent effect of the anti-TGF-�1 Ab on CD4�CD25� Tcell-mediated Treg activity. At the highest dose tested, 20 �g/ml,we observed a complete inhibition of Treg function by burnCD4�CD25� T cells and a significant 2-fold reduction in Tregfunction mediated by sham CD4�CD25� T cells. Taken together,these results indicate that the counterproliferative activity dis-played by the CD4�CD25� T cells in our experiments is mediatedin part by TGF-�1.

Because we did not detect increased TGF-� production by anti-CD3-stimulated CD4�CD25� T cells purified from 7-day burnmice, we reasoned that changes in cell surface TGF-� expressionlevels might be responsible for the augmented Treg activity de-tected at 7 days after burn injury. To test this possibility, we per-formed three-color FACS studies using a PE-labeled anti-TGF-�1Ab to detect cell surface expression of TGF-�1 on lymph node andspleen CD4�CD25� and CD4�CD25� T cells prepared from 1-or 7-day sham or burn mice. We found a significant increase inTGF-�1 expression on 7-day burn lymph node-derivedCD4�CD25� T cells (Fig. 7). A similar injury-induced increase incell surface TGF-�1 was not detected on CD4�CD25� T cellspurified from the spleens of burn-injured mice, and we did notdetect any cell surface TGF-�1 on the CD4�CD25� T cell sub-sets. The fact that expression of cell surface TGF-�1 was restrictedto the CD4�CD25� T cell subset in mice fully agrees with severalpublished reports (26, 38, 39). However, the observed up-regula-tion of cell surface TGF-�1 expression on CD4�CD25� T cellsafter injury is novel. Moreover, the association between the tissue-compartmentalized influence of injury on CD4�CD25� Treg func-tion and changes in TGF-�1 expression suggests that the enhancedTreg function expressed by lymph node CD4�CD25� T cells at 7days after burn injury is likely mediated by increased cell surfaceTGF-�1 expression.

CD4�CD25� T cells inhibit Th1-type cytokine production

The results of our proliferation studies, which showed enhancedTreg function by CD4�CD25� T cells at 7 days after injury, sug-gested that these cells might also be able to suppress Th1-typecytokine production. To explore this possibility, we tested the ef-fects of CD4�CD25� or CD4�CD25� T cells on anti-CD3 Ab-induced IL-2 and IFN-� production by naive lymph node cells. For

FIGURE 6. CD4�CD25� T cell-mediated inhibition of CD4� T cellproliferation is cell contact and TGF-� dependent, but does not involveIL-10. For the results shown in A, CD4�CD25� and CD4�CD25� T cellspurified from the lymph nodes of sham or burn mice were cultured with1 � 106 CFSE-labeled BALB/cJ lymph node cells at a 1:3 ratio. TheCFSE-labeled lymph node cells in the bottom well were separated from thepurified cells and T cell-depleted spleen cells (APCs) in the Transwellinsert by a semipermeable membrane. All cell mixtures were stimulatedwith anti-CD3� Ab (5 �g/ml). Controls included sham or burnCD4�CD25� T cells added to CFSE-labeled lymph node cells at a 1:3ratio, but without separation. After 3-day incubation, the cells were har-vested and tested for anti-CD3-induced proliferation by FACS. The resultsshown represent the mean � SEM of three experiments with six mice/group. To address the role of IL-10 (B) and TGF-� (C) in mediating theCD4�CD25� Treg activity, anti-IL-10 or anti-TGF-�1 Abs, along with theappropriate IgG isotype control Abs, were tested at the indicated concen-trations for their ability to block CD4�CD25�-mediated Treg activity. Forall these experiments, CD4�CD25� and CD4�CD25� T cells purifiedfrom the lymph nodes of sham or burn mice were added to CFSE-labeledBALB/cJ lymph node cells at a 1:3 ratio and were stimulated with anti-CD3� Ab (5 �g/ml). The proliferation of gated-CFSE-labeled CD4� Tcells was assessed by FACS. The results represent the mean � SEM ofthree independent experiments using four mice per group; �, p � 0.05 byANOVA for differences between burn CD25� and all other groups at thehighest Ab concentration tested.

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these experiments, purified CD4�CD25� or CD4�CD25� lymphnode T cells from 7-day sham or burn mice were added at 1:3ratios to lymph node cells, which were then stimulated with anti-CD3� Ab. As shown in Fig. 8, we found that the addition ofCD4�CD25� T cells caused a significant reduction in CD3-stim-

ulated IL-2 and IFN-� production, whereas the addition ofCD4�CD25� T cells did not suppress the production of eithercytokine. Moreover, CD4�CD25� T cells from 7-day burn micewere more effective at suppressing IL-2 and, in particular, IFN-�production than were sham CD4�CD25� T cells. Similar resultswere observed when experiments were performed using a 1:1 pu-rified T cell to lymph node cell ratio, except that the CD4�CD25�

T cell-mediated suppression of Th1-type cytokine production waseven more profound (data not shown).

Injury effects on costimulatory or regulatory receptor expressionon CD4�CD25� and CD4�CD25� T cells

To further investigate the influence of injury on CD4�CD25� Tcell phenotype, we measured injury effects on several T cell co-stimulatory and immune regulatory receptors (CD28, CD152,ICOS, and PD-1) that are known to be associated withCD4�CD25� T cell activation and function (Table II) (40). At 1day after injury, we detected increased expression of CD28 andICOS on lymph node and spleen CD4�CD25� T cells but notCD4�CD25� T cells. By 7 days after injury, we found a signifi-cant increase in intracellular CD152 expression by lymph node andspleen CD4�CD25� T cells, whereas ICOS was significantly in-creased only on lymph node CD4�CD25� T cells. Collectively,these observations suggest that injury provides an early CD4� Tcell-activating signal that may cause a sustained increase in theexpression of CD152 and ICOS, two regulatory receptors associ-ated with inhibiting proinflammatory immune responses.

FIGURE 7. Injury increases cellsurface TGF-�1 expression on lymphnode, but not spleen CD4�CD25� Tcells at 7 days after injury. Spleen andlymph node cells harvested from shamand burn mice at 1 or 7 days after in-jury were stained with Cy5-conjugatedanti-CD4 Ab, FITC-conjugated anti-CD25 Ab and PE-conjugated anti-TGF-�1 Ab, or PE-conjugated IgG1isotype control Abs. The representativeFACS plots of TGF-�1 staining ongated CD4�CD25� (A) andCD4�CD25� (B) T cells illustrate thatTGF-�1 is selectively expressed onCD4�CD25� T cells. As shown in C,injury caused a significant increase incell surface TGF-�1 expression onlymph node CD4�CD25� T cells. �,p � 0.001 by ANOVA. No significantinjury-induced changes in cell surfaceTGF-�1 expression were observed onday 1 post-injury lymph node or spleenCD4�CD25� T cells or on day 7spleen-derived CD4�CD25� T cells.Results shown represent the mean �SEM of six individual mice.

FIGURE 8. CD4�CD25� T cells inhibit Th1-type cytokine production.At 7 days after injury, purified CD4�CD25� or CD4�CD25� T cells (2 �105) from sham or burn mice (n � 6/group) were added to BALB/cJ lymphnode cells at a 1:3 ratio. Controls included BALB/cJ lymph node cellswithout purified cell additions. These cell mixes were stimulated overnightwith anti-CD3� Ab (5 �g/ml), after which supernatants were harvested andtested for IL-2 and IFN-� levels by ELISA. Both sham and burnCD4�CD25� T cells caused a significant reduction in IL-2 and IFN-�production by anti-CD3-stimulated lymph node cells. �, p � 0.001 byANOVA; ��, significant difference in IL-2 and IFN-� production mediatedby CD4�CD25� T cells from burn as compared with sham mice (p � 0.05by ANOVA).

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Effect of Treg depletion on Ag-specific T cell-dependentresponses in vivo

To directly measure the functional influence of Treg cells on thedevelopment of Ag-specific T cell responses in vivo, we examinedwhether sham and burn mice immunized with the T cell-dependentAg TNP-OVA respond differently after in vivo Treg depletion. In

these studies, mice were given 10 mg/kg anti-CD25 Ab or controlIgG at the same concentration by i.p. injection. As illustrated inFig. 9A, this single i.p. injection of anti-CD25 Ab reduces the levelof CD4�CD25� T cells in the lymph nodes and spleen of mice to�1% by 3 days after treatment. Accordingly, at 3 days after anti-CD25 Ab or rat IgG treatment, mice underwent sham or burn

Table II. Costimulatory receptor expression levels on lymph node CD4� CD25� and CD4� CD25� T cellsa

CD152 IC-CD152 CD28 ICOS PD-1

CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25� CD25�

Sham (1 day) 2.4 (0.3) 0.6 (0.07) 14.7 (1.6) 2.6 (0.5) 33.4 (2.3) 5.57 (0.07) 44.9 (3.2) 23.1 (1.3) 32.6 (6.0) 3.3 (0.5)Burn (1 day) 2.6 (0.21) 0.4 (0.04) 14.2 (0.8) 1.8 (0.1) 51.7� (2.9) 5.24 (0.04) 55.8� (1.5) 25.1 (0.6) 40.2 (2.3) 3.3 (0.4)Sham (7 days) 2.4 (0.7) 0.1 (0.01) 16.9 (2.1) 0.7 (0.1) 24.4 (3.4) 1.3 (0.4) 52.1 (4.0) 17.5 (1.0) 27.9 (2.1) 1.0 (0.1)Burn (7 days) 2.7 (0.3) 0.1 (0.02) 33.1� (3.2) 2.0 (0.1) 19.1 (2.4) 0.5 (0.1) 64.4� (1.3) 17.5 (0.7) 23.0 (1.6) 1.3 (0.1)

a Lymph node cells were prepared from sham or burn mice at 1 or 7 days after injury. Cells were stained with Cy5-labeled anti-CD4 Ab and FITC-labeled anti-CD25 Aband then counterstained with PE-labeled Abs specific for CD152, CD28, ICOS, or PD-1. An additional stain for intracellular CD152 (IC-CD152) was performed as indicatedin Materials and Methods. Each value represents the mean (SEM) percent positive events for gated CD4� CD25� or CD4� CD25� T cells from six mice. The � indicatessignificant differences between sham and burn, p � 0.05 by ANOVA.

FIGURE 9. In vivo depletion of CD25� cells enhances and restores Th1 responses in sham and burn-injured mice. Mice were given 0.25 mg ofanti-CD25 Ab by i.p. injection 3 days before sham or burn injury. Mice were immunized s.c. at the time of injury with 0.1 mg of TNP-OVA emulsified1:1 in CFA. Ten days later, mice were sacrificed to harvest blood, spleen, and regional lymph nodes to prepare serum and cells. A, FACS plots showingthe level of CD4�CD25� T cells remaining at 3 days after anti-CD25 Ab or rat IgG treatment. The numbers shown in the upper right quadrants indicatethe percent of gated CD4� T cells expressing cell surface CD25. The data shown are representative of n � 6 mice. B, Lymph nodes and spleen cells werestimulated in vitro with OVA (0.1 mg/ml), and 48 h later culture supernatants were assayed for IFN-� levels. Results shown are the mean � SEM of ninemice per group. �, p � 0.05 by paired t test for anti-CD25 Ab vs rat IgG-treated sham mice; ��, p � 0.05 by paired t test for anti-CD25 Ab vs rat IgG-treatedburn mice; #, p � 0.05 by paired t test for rat IgG-treated sham vs burn mice. C, Serum samples were tested at the indicated dilutions for TNP-specificAb isotype levels by ELISA as described in Materials and Methods. �, p � 0.05 by ANOVA for anti-CD25 Ab vs rat IgG-treated sham mice; ��, p �0.05 for anti-CD25 Ab vs rat IgG-treated burn mice; #, p � 0.05 for rat IgG-treated sham vs burn mice; n � 9 mice per group.

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injury and were immunized s.c with 0.1 mg of TNP-OVA emul-sified 1:1 in CFA. After 10 days, blood, spleen, and regional lymphnodes were harvested to measure TNP-specific Ab isotype levelsand OVA-induced IFN-� production. As shown in Fig. 9B, Ag-stimulated IFN-� production by lymph node cells was markedlyincreased in sham or burn mice that were Treg-deficient at the timeof injury. A similar, though less dramatic, effect of Treg depletionon OVA-induced IFN-� production was observed in spleen cellcultures. These findings indicate that Tregs control the develop-ment of Ag-specific IFN-� responses in vivo and that Treg deple-tion restores Ag-driven IFN-� responses in burn-injured mice tonormal levels.

We next measured the influence of Treg depletion on Ag-spe-cific Ab isotype formation by measuring the levels of TNP-specificIgM, IgG1, and IgG2a in serum prepared from TNP-OVA-immu-nized burn and sham mice. We found that Treg depletion markedlyand significantly increased titers of the Th1-dependent Ab isotypeIgG2a in both sham and burn mice, whereas Treg depletion did notsignificantly alter IgM or IgG1 Ab isotype levels (Fig. 9C). Theeffect of Treg depletion on Ag-specific IgG2a Ab production wasmost dramatic in the sham group, which showed a supernormalIgG2a response. Nonetheless, we found that Treg-depleted burnmice displayed a significantly higher IgG2a Ab response than didcontrol Ig-treated sham or burn mice, suggesting that Tregs con-tribute to suppressed Th1-type Ab responses after injury. Takentogether with the observation that Treg depletion boosted Ag-spe-cific IFN-� production (Fig. 9B), these findings support the ideathat Tregs play a central role in suppressing Th1 responses afterburn injury.

DiscussionThe stress and tissue damage associated with severe injury appearto initiate a programmed immune response that involves both in-nate and adaptive immune cell types and mediators (5, 9). Inves-tigations into how the immune system compensates for the initialinflammatory response to injury have led us and other investigators(9, 12) to conclude that T cells play a central role. For example, werecently reported evidence that Rag1�/� mice, which lack an adap-tive immune system, acquire a more proinflammatory phenotypeafter injury than do wild-type mice, suggesting that the adaptiveimmune system must play an active role in modulating innate im-mune reactivity after injury (41). Further investigations into theadaptive immune cell types that mediate this regulatory responseshowed that CD4�CD25� T cells were the primary cell populationinvolved in controlling the inflammatory reactivity of innate im-mune cells after injury (34). Other studies have shown that injury-induced alterations in T cell function include a deviation towardTh2-type reactivity and suppressed Th1-type immune function (7,35). We report here a potential mechanism involved in the devel-opment of suppressed adaptive immunity after severe injury. Spe-cifically, we show that injury primes CD4�CD25� T cells forenhanced T regulatory activity. It is noteworthy that this enhancedTreg function is detected at a time coincident with the expressionof injury-induced immune suppression, as judged by reduced re-sistance to bacterial or viral infections in mice and man (3, 42, 43).Another interesting aspect of these findings is the relative restric-tion of the enhanced Treg activity to the lymph nodes draining theinjury site. This compartmentalized effect of injury on Treg cellfunction suggests that tissue injury-associated Ags or lymph node-specific APC types might play an important role in initiating oramplifying the injury-associated increase in Treg activity.

We demonstrate that the Treg activity measured in this study iscell-contact dependent and mediated in part by TGF-�1. This con-clusion is supported by the observations that anti-TGF-�1 Ab ab-

rogates the ability of purified CD4�CD25� T cells to inhibit anti-CD3-induced CD4� T cell proliferation. However, the finding thatCD4�CD25� T cells from sham and burn-injured mice secreteequivalent amounts of TGF-� and the fact that injury significantlyup-regulates cell surface TGF-�1 expression on regional lymphnode but not splenic CD4�CD25� T cells leads us to conclude thatthe enhanced regulatory function of the CD4�CD25� T cell is notattributable to TGF-� release, but rather to an injury-induced in-crease in cell surface TGF-�1 expression. Yet, some reports con-clude that TGF-� activity is not responsible for CD4�CD25� Tregfunction because anti-TGF-� Ab failed to block CD4�CD25�

Treg activity (44). A possible explanation for this is that cell-to-cell contact may be required to convert the latent TGF-� to activeTGF-�, and anti-TGF-� Ab levels may need to be high enough torapidly block TGF-� activity during this conversion process (32).The fact that we observed significant abrogation of Treg activity atthe highest anti-TGF-�1 concentration tested (20 �g/ml) but not atthe lower concentrations (0.2 or 2 �g/ml) supports this possibility.An additional report suggests that TGF-�1 cannot be solely re-sponsible for CD4�CD25� Treg activity because Treg activitypersists in TGF-�-deficient mice (44). Although the results ob-tained using these mice are convincing, it is unclear what com-pensatory pathways or mechanisms might have evolved in TGF-�-deficient mice that may not be present in wild-type mice.

Another question relates to whether Treg activity is mediated byTGF-� alone or whether other factors such as IL-10 may also playa contributing role. We verified in this study that CD4�CD25� Tcells produced higher levels of IL-10 than did CD4�CD25� Tcells. Moreover, we found that anti-CD3 Ab-stimulatedCD4�CD25� T cells from 7-day burn mice produced significantlyhigher levels of IL-10 than did CD4�CD25� T cells from shammice. Nevertheless, we were unable to link Treg activity in vitro toIL-10 as judged by anti-IL-10 Ab blocking studies. Despite thesefindings, it is still possible that IL-10 contributes to CD4�CD25�

T cell-mediated activities in vivo. For example, it was shown thatthe transfer of CD4�CD45RBhigh T cells into SCID mice led to thedevelopment of colitis, but the transfer of these cells along withCD4�CD45RBlow T cells prevented colitis, whereas anti-IL-10 oranti-IL-10R treatment blocked the CD4�CD45RBlow Treg effect(45). The results of that study suggest that IL-10 may be an im-portant factor in mediating in vivo Treg activity. Therefore, it ispossible that IL-10 may play a significant in vivo role in the in-crease in Treg activity observed in the present report. Other studiesaddressing the function of IL-10 during the injury response haveindicated that blocking its activity early after injury promoted Th1-type immunity and prevented the development of postinjury im-mune suppression, suggesting that IL-10 does indeed influence thehost immune response to injury (7, 11).

This report does not conclusively demonstrate whether burn in-jury enhances the regulatory function of natural Tregs or inducesa naive population of Treg cells that are responsible for the en-hanced Treg activity at 7 days after injury. However, detailed ex-amination of the cell surface phenotype of CD25� vs CD25� CD4T cells suggests that injury did not significantly alter those markersthat identify CD25� CD4 T cells as a natural Treg cell population(Table I). Moreover, we did not detect a significant change inFoxP3 gene expression by CD25� CD4 T cells from burn as com-pared with sham mice. Taken together, these findings suggest thatthe purified CD25� CD4 T cells examined in this study are not amixture of induced and natural Tregs. This evidence leads us tofavor the idea that injury enhances the regulatory activity of nat-ural Treg cells rather than inducing CD4 T cells with Treg cellproperties. However, a clearer picture of whether or not injuryinduces a unique population of Treg cells will emerge from the

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results of future studies addressing the contribution of factors suchas IL-10, TGF-�, or LPS on Treg function after burn injury (28,36, 46, 47).

To explore the functional significance to up-regulated Treg ac-tivity after injury, we examined whether Tregs might contribute tothe injury-induced suppression of Ag-specific Th1-type immunereactivity because this is an established feature of the mammalianinjury response. Because prior studies have shown that mice im-munized at the time of injury with a T cell-dependent Ag displayeda significant decrease in Th1-type Ab isotype formation, we usedthis observation to determine how Treg cell depletion might influ-ence the development of Th1 and Th2 responses (7). We found thatmice treated with a Treg-depleting dose of anti-CD25 Ab showeda marked increase in Th1-type reactivity. Most importantly, burnmice that were depleted of CD4�CD25� T cells at the time ofinjury did not show suppressed Th1 Ab responses, indicating thatinjury in the absence of Tregs did not result in suppression of Th1immunity. However, the most dramatic effect of Treg depletion onTh1 Ab production was observed in TNP-OVA-immunized shammice, which displayed extremely high levels of serum TNP-spe-cific IgG2a. Another recent report shows a similar consequence ofTreg depletion on Ag-specific Th1-type Ab responses (48). Inter-estingly, burn-injured mice that were rendered Treg deficient didnot show this abnormally high Th1-type reactivity, but did showhigher Th1-type Ab production than did sham or burn mice thatwere not CD4�CD25� T cell depleted. This finding indicates thatTregs do play a role in suppressing Th1-type reactivity after injurybut that other mechanisms may prevent the development of thesupranormal Ag-driven Th1 response seen in sham mice. The ideathat Tregs control the level of Th1 reactivity in vivo is furthersupported by our finding that lymph node and spleen cells fromimmunized, Treg-depleted mice produced significantly higher lev-els of Ag-specific IFN-� than did cells from control Ig-treatedmice. The latter finding also suggests that injury induces a signif-icant increase in Treg activity in the spleen even though such anincrease was not observed in our in vitro studies of antiprolifera-tive function.

Although we demonstrate that mice given anti-CD25 Ab to de-plete CD25� T cells are able to respond to immunization with a Tcell-dependent Ag, there was concern that residual anti-CD25 Abmay influence the emergence of activated T cells after injury andimmunization. Therefore, we performed an additional immuniza-tion study in sham and burn mice using a concentration of anti-CD25 Ab (1 mg/kg) that was found to be the minimally effect doseneeded to deplete mice of CD25� CD4 T cells but that allows forthe repopulation or emergence of CD25-expressing CD4 T cells by14 days after anti-CD25 Ab treatment. In this immunization study,we found that burn mice showed enhanced Ag-specific Th1-typeAb formation as was reported in Fig. 9C, suggesting that transientdepletion of Treg cells also restores Th1 responses in mice afterburn injury (data not shown).

The detection of augmented regional lymph node, but notspleen, CD4�CD25� Treg activity suggests that the exposure oflymph node cells to endogenous Ag(s) released at the injury sitemay be responsible for this phenomenon. Other factors that areknown to enhance CD4�CD25� Treg activity include the C3bproduct of complement activation (which is a ligand for CD46expressed on CD4� T cells), TGF-�, and bacterial LPS or poten-tial endogenous agonists activating TLR4 uniquely expressed onCD4�CD25� T cells (28, 49–51). Other endogenous activatingagents may include uric acid or a TLR2-activating Ag that is re-leased by cells after necrotic cell death (52, 53).

However, further investigations will be required to identify thecontribution of these and other potential factors to the increase in

Treg activity after injury. The identification of such factors or Agswould be of great interest to the immunological community and,since loss of Th1 immune function after injury is associated withdiminished resistance to infection, a more complete understandingof injury-induced alterations in Treg function may be of majorsignificance for developing therapeutic strategies for the benefit ofinjured patients in the future.

AcknowledgmentsWe thank Drs. Peter Sayles and Mohammed Sayegh for their critical read-ing of this manuscript.

DisclosuresThe authors have no financial conflict of interest.

References1. Moss, N. M., D. B. Gough, A. L. Jordan, J. T. Grbic, J. J. Wood, M. L. Rodrick,

and J. A. Mannick. 1988. Temporal correlation of impaired immune responseafter thermal injury with susceptibility to infection in a murine model. Surgery104: 882–887.

2. Cheadle, W. G., M. Mercer-Jones, M. Heinzelmann, and H. C. Polk, Jr. 1996.Sepsis and septic complications in the surgical patient: who is at risk? Shock6(Suppl. 1): S6–S9.

3. Moore, F. A., A. Sauaia, E. E. Moore, J. B. Haenel, J. M. Burch, andD. C. Lezotte. 1996. Postinjury multiple organ failure: a bimodal phenomenon.J. Trauma 40: 501–510.

4. Ninnemann, J. L., J. C. Fisher, and H. A. Frank. 1978. Prolonged survival ofhuman skin allografts following thermal injury. Transplantation 25: 69–72.

5. Lederer, J. A., M. L. Rodrick, and J. A. Mannick. 1999. The effects of injury onthe adaptive immune response. Shock 11: 153–159.

6. Faist, E., C. Schinkel, and S. Zimmer. 1996. Update on the mechanisms of im-mune suppression of injury and immune modulation. World J. Surg. 20:454–459.

7. Kelly, J. L., C. B. O’Suilleabhain, C. C. Soberg, J. A. Mannick, and J. A. Lederer.1999. Severe injury triggers antigen-specific T-helper cell dysfunction. Shock 12:39–45.

8. Baker, C. C., C. L. Miller, and D. D. Trunkey. 1979. Predicting fatal sepsis inburn patients. J. Trauma 19: 641–648.

9. Angele, M. K., and E. Faist. 2002. Clinical review: immunodepression in thesurgical patient and increased susceptibility to infection. Crit. Care 6: 298–305.

10. O’Suilleabhain, C., S. T. O’Sullivan, J. L. Kelly, J. Lederer, J. A. Mannick, andM. L. Rodrick. 1996. Interleukin-12 treatment restores normal resistance to bac-terial challenge after burn injury. Surgery 120: 290–296.

11. Lyons, A., A. Goebel, J. A. Mannick, and J. A. Lederer. 1999. Protective effectsof early interleukin 10 antagonism on injury-induced immune dysfunction. Arch.Surg. 134: 1317–1323.

12. Kupper, T. S., and D. R. Green. 1984. Immunoregulation after thermal injury:sequential appearance of I-J�, Ly-1 T suppressor inducer cells and Ly-2 T sup-pressor effector cells following thermal trauma in mice. J. Immunol. 133:3047–3053.

13. Singh, H., D. N. Herndon, and M. D. Stein. 1986. Kinetics of lymphoproliferativeresponses following scald injury in a rat burn model. Clin. Immunol. Immuno-pathol. 40: 476–484.

14. Teodorczyk-Injeyan, J. A., B. G. Sparkes, R. E. Falk, and W. J. Peters. 1986.Polyclonal immunoglobulin production in burned patients: kinetics and correla-tions with T-cell activity. J. Trauma 26: 834–839.

15. Jeffries, M. K., and M. L. Vance. 1992. Growth hormone and cortisol secretionin patients with burn injury. J. Burn Care Rehabil. 13: 391–395.

16. Schrader, K. A. 1996. Stress and immunity after traumatic injury: the mind-bodylink. AACN Clin. Issues 7: 351–358.

17. Ninnemann, J. L., and A. E. Stockland. 1984. Participation of prostaglandin E inimmunosuppression following thermal injury. J. Trauma 24: 201–207.

18. Strong, V. E., P. J. Mackrell, E. M. Concannon, H. A. Naama, P. A. Schaefer,G. W. Shaftan, P. P. Stapleton, and J. M. Daly. 2000. Blocking prostaglandin E2

after trauma attenuates pro-inflammatory cytokines and improves survival. Shock14: 374–379.

19. Goebel, A., E. Kavanagh, A. Lyons, I. B. Saporoschetz, C. Soberg, J. A. Lederer,J. A. Mannick, and M. L. Rodrick. 2000. Injury induces deficient interleukin-12production, but interleukin-12 therapy after injury restores resistance to infection.Ann. Surg. 231: 253–261.

20. Schwacha, M. G., C. S. Chung, A. Ayala, K. I. Bland, and I. H. Chaudry. 2002.Cyclooxygenase 2-mediated suppression of macrophage interleukin-12 produc-tion after thermal injury. Am. J. Physiol. 282: C263–C270.

21. Dorf, M. E., and B. Benacerraf. 1984. Suppressor cells and immunoregulation.Annu. Rev. Immunol. 2: 127–157.

22. Dorf, M. E., V. K. Kuchroo, and M. Collins. 1992. Suppressor T cells: someanswers but more questions. Immunol. Today 13: 241–243.

23. Kupper, T. S., C. C. Baker, T. A. Ferguson, and D. R. Green. 1985. A burninduced Ly-2 suppressor T cell lowers resistance to bacterial infection. J. Surg.Res. 38: 606–612.

235The Journal of Immunology

by guest on August 19, 2018

http://ww

w.jim

munol.org/

Dow

nloaded from

24. Asano, M., M. Toda, N. Sakaguchi, and S. Sakaguchi. 1996. Autoimmune diseaseas a consequence of developmental abnormality of a T cell subpopulation. J. Exp.Med. 184: 387–396.

25. Suri-Payer, E., A. Z. Amar, A. M. Thornton, and E. M. Shevach. 1998.CD4�CD25� T cells inhibit both the induction and effector function of autore-active T cells and represent a unique lineage of immunoregulatory cells. J. Im-munol. 160: 1212–1218.

26. Nakamura, K., A. Kitani, and W. Strober. 2001. Cell contact-dependent immu-nosuppression by CD4�CD25� regulatory T cells is mediated by cell surface-bound transforming growth factor �. J. Exp. Med. 194: 629–644.

27. Fu, S., A. C. Yopp, X. Mao, D. Chen, N. Zhang, M. Mao, Y. Ding, andJ. S. Bromberg. 2004. CD4� CD25� CD62� T-regulatory cell subset has optimalsuppressive and proliferative potential. Am. J. Transplant. 4: 65–78.

28. Caramalho, I., T. Lopes-Carvalho, D. Ostler, S. Zelenay, M. Haury, andJ. Demengeot. 2003. Regulatory T cells selectively express Toll-like receptorsand are activated by lipopolysaccharide. J. Exp. Med. 197: 403–411.

29. Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs thedevelopment and function of CD4�CD25� regulatory T cells. Nat. Immunol. 4:330–336.

30. Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell de-velopment by the transcription factor Foxp3. Science 299: 1057–1061.

31. Maloy, K. J., L. Salaun, R. Cahill, G. Dougan, N. J. Saunders, and F. Powrie.2003. CD4�CD25� T(R) cells suppress innate immune pathology through cy-tokine-dependent mechanisms. J. Exp. Med. 197: 111–119.

32. Nakamura, K., A. Kitani, I. Fuss, A. Pedersen, N. Harada, H. Nawata, andW. Strober. 2004. TGF-�1 plays an important role in the mechanism ofCD4�CD25� regulatory T cell activity in both humans and mice. J. Immunol.172: 834–842.

33. Zhang, X., D. N. Koldzic, L. Izikson, J. Reddy, R. F. Nazareno, S. Sakaguchi,V. K. Kuchroo, and H. L. Weiner. 2004. IL-10 is involved in the suppression ofexperimental autoimmune encephalomyelitis by CD25�CD4� regulatory T cells.Int. Immunol. 16: 249–256.

34. Murphy, T. J., N. N. Choileain, Y. Zang, J. A. Mannick, and J. A. Lederer. 2005.CD4�CD25� regulatory T cells control innate immune reactivity after injury.J. Immunol. 174: 2957–2963.

35. Guo, Z., E. Kavanagh, Y. Zang, S. M. Dolan, S. J. Kriynovich, J. A. Mannick,and J. A. Lederer. 2003. Burn injury promotes antigen-driven Th2-type responsesin vivo. J. Immunol. 171: 3983–3990.

36. Levings, M. K., R. Bacchetta, U. Schulz, and M. G. Roncarolo. 2002. The roleof IL-10 and TGF-� in the differentiation and effector function of T regulatorycells. Int. Arch. Allergy Immunol. 129: 263–276.

37. Mack, V. E., M. D. McCarter, H. A. Naama, S. E. Calvano, and J. M. Daly. 1996.Dominance of T-helper 2-type cytokines after severe injury. Arch. Surg. 131:1303–1308.

38. Green, E. A., L. Gorelik, C. M. McGregor, E. H. Tran, and R. A. Flavell. 2003.CD4�CD25� T regulatory cells control anti-islet CD8� T cells through TGF-

�-TGF-� receptor interactions in type 1 diabetes. Proc. Natl. Acad. Sci. USA 100:10878–10883.

39. Peng, Y., Y. Laouar, M. O. Li, E. A. Green, and R. A. Flavell. 2004. TGF-�regulates in vivo expansion of Foxp3-expressing CD4�CD25� regulatory T cellsresponsible for protection against diabetes. Proc. Natl. Acad. Sci. USA 101:4572–4577.

40. Powrie, F., S. Read, C. Mottet, H. Uhlig, and K. Maloy. 2003. Control of immunepathology by regulatory T cells. Novartis Found. Symp. 252: 92–98.

41. Shelley, O., T. Murphy, H. Paterson, J. A. Mannick, and J. A. Lederer. 2003.Interaction between the innate and adaptive immune systems is required to sur-vive sepsis and control inflammation after injury. Shock 20: 123–129.

42. Kobayashi, M., H. Takahashi, A. P. Sanford, D. N. Herndon, R. B. Pollard, andF. Suzuki. 2002. An increase in the susceptibility of burned patients to infectiouscomplications due to impaired production of macrophage inflammatory protein1�. J. Immunol. 169: 4460–4466.

43. Oberholzer, A., C. Oberholzer, and L. L. Moldawer. 2001. Sepsis syndromes:understanding the role of innate and acquired immunity. Shock 16: 83–96.

44. Piccirillo, C. A., J. J. Letterio, A. M. Thornton, R. S. McHugh, M. Mamura,H. Mizuhara, and E. M. Shevach. 2002. CD4�CD25� regulatory T cells canmediate suppressor function in the absence of transforming growth factor �1production and responsiveness. J. Exp. Med. 196: 237–246.

45. Asseman, C., S. Mauze, M. W. Leach, R. L. Coffman, and F. Powrie. 1999. Anessential role for interleukin 10 in the function of regulatory T cells that inhibitintestinal inflammation. J. Exp. Med. 190: 995–1004.

46. Roncarolo, M. G., S. Gregori, and M. Levings. 2003. Type 1 T regulatory cellsand their relationship with CD4�CD25� T regulatory cells. Novartis Found.Symp. 252: 115–127.

47. Foussat, A., F. Cottrez, V. Brun, N. Fournier, J. P. Breittmayer, and H. Groux.2003. A comparative study between T regulatory type 1 and CD4�CD25� T cellsin the control of inflammation. J. Immunol. 171: 5018–5026.

48. Pasare, C., and R. Medzhitov. 2004. Toll-dependent control mechanisms of CD4T cell activation. Immunity 21: 733–741.

49. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, andS. M. Wahl. 2003. Conversion of peripheral CD4�CD25� naive T cells toCD4�CD25� regulatory T cells by TGF-� induction of transcription factorFoxp3. J. Exp. Med. 198: 1875–1886.

50. Kemper, C., A. C. Chan, J. M. Green, K. A. Brett, K. M. Murphy, andJ. P. Atkinson. 2003. Activation of human CD4� cells with CD3 and CD46induces a T-regulatory cell 1 phenotype. Nature 421: 388–392.

51. Fantini, M. C., C. Becker, G. Monteleone, F. Pallone, P. R. Galle, andM. F. Neurath. 2004. Cutting edge: TGF-� induces a regulatory phenotype inCD4�CD25� T cells through Foxp3 induction and down-regulation of Smad7.J. Immunol. 172: 5149–5153.

52. Shi, Y., J. E. Evans, and K. L. Rock. 2003. Molecular identification of a dangersignal that alerts the immune system to dying cells. Nature 425: 516–521.

53. Beg, A. A. 2002. Endogenous ligands of Toll-like receptors: implications forregulating inflammatory and immune responses. Trends Immunol. 23: 509–512.

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