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

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

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD4⁺CD25⁺ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4⁺CD25⁺ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-1 expression. To test the in vivo significance of these findings, mice were depleted of CD4⁺CD25⁺ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4⁺CD25⁺ T cells. Taken together, these observations suggest that injury can induce or amplify CD4⁺CD25⁺ Treg function and that CD4⁺CD25⁺ T cells contribute to the development of postinjury immune suppression.

	Introduction

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Accumulating clinical and experimental evidence indicates that serious injury promotes suppressed immune function predisposing the injured host to infectious complications (1, 2, 3). In particular, severely injured patients exhibit classical signs of impaired adaptive immune activity such as loss of delayed-type hypersensitivity responses, prolonged skin allograft survival, and reduced T cell proliferation to polyclonal and Ag-specific stimulation (4, 5, 6). These changes in T cell responses are also accompanied by lowered Th1-type cytokine production and reduced Th1-dependent Ab isotype secretion (7). An association between suppressed adaptive immune function after major injury in patients and increased risk of developing nosocomial infections has been reported (8, 9). Furthermore, a causal relationship between suppressed Th1-type immunity after injury and lowered resistance to sepsis has been demonstrated in animal studies showing that injured mice treated with the Th1-promoting cytokine IL-12 or with Ab to the Th2-type, counterinflammatory cytokine IL-10 manifested improved resistance to a septic challenge (10, 11).

In most studies, the injury-induced loss of T cell function appears to be a progressive phenomenon, reaching a nadir several days to more than a week after the initial insult (12, 13, 14). This supports the idea that the development of suppressed T cell-mediated immunity is a programmed response and may well represent a compensatory host reaction to the intense inflammation induced by tissue damage that accompanies serious injury. Several mechanisms have been postulated to explain the gradual loss of T cell function after injury. These include the increased release of immunosuppressive prostaglandins by innate immune cells, elevated circulating levels of corticosteroids, and the decreased production of the Th1-inducing cytokine IL-12 (15, 16, 17, 18, 19, 20). Although a role for so-called "suppressor T cells" in mediating this phenomenon was suggested by studies performed >20 years ago, this explanation for depressed adaptive immunity after injury disappeared from the literature because of the failure to identify genetically or phenotypically an unequivocal suppressor T cell subset (21, 22, 23).

Interest in suppressor T cells was rekindled when it was shown that a minor population of thymic-derived CD4⁺ T cells that coexpress CD25, the -chain of the IL-2R, was crucial for controlling autoreactive T cells in vivo (24). These naturally occurring CD4⁺ T cells were shown to potently inhibit TCR-driven T cell proliferation and were appropriately named regulatory T cells (Tregs)³ (25). More recent findings indicate that Tregs can be further distinguished from conventional CD4⁺CD25^– T cells by their CD45RB^low, L-selectin (CD62L)⁺, CD69^–, glucocorticoid-induced TNFR-like protein 6 (GITR)⁺, TGF-1⁺, and TLR4⁺ cell surface phenotype (26, 27, 28). In addition, the forkhead transcription factor, Foxp3, has been shown to be expressed specifically in Tregs and is required for their development in mice (29, 30). A thorough assessment of these cell surface markers and FoxP3 gene expression 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 autoimmune disease and T cell proliferation, Tregs have been shown to suppress inflammatory responses in vivo by IL-10- or TGF--mediated mechanisms (31, 32, 33). It is this counterinflammatory nature of Tregs that prompted us to examine whether they might play a role in helping control the intense inflammatory reaction that occurs after severe injury, and we have recently reported that this is indeed the case (34). Furthermore, we have been interested in determining whether injury might increase Treg cell activity and, if so, whether these cells could be involved in mediating the known progressive loss of Th1 responses after injury. Using a mouse burn model that inflicts a controlled but significant level of tissue damage, we find that injury enhances the regulatory activity of CD4⁺CD25⁺ T cells purified from the lymph nodes draining the injury site. This injury-induced augmentation of Treg activity is detectable at 7, but not 1, day after injury, suggesting that it is a progressively developed phenotype. Moreover, we show that this injury-induced increase in Treg function is cell-contact dependent, is mediated in part by TGF-, and is associated with an increase in cell surface TGF-1 expression on lymph node CD4⁺CD25⁺ T cells. We find that the Tregs from injured mice more potently suppress T cell proliferation and IL-2 and IFN- production than do Tregs from sham mice and that their in vivo depletion before injury prevents the injury-induced inhibition of Ag-specific Th1-type reactivity. To our knowledge, these findings are the first to indicate that injury can enhance Treg activity and that Tregs contribute to the suppression of Th1-type immunity that occurs after severe injury.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

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

Reagents

CD4⁺CD25⁺ Regulatory T Cell Isolation kits were obtained from Miltenyi Biotec. Anti-mouse CD3Ab (clone 145-2C11) was obtained from R&D Systems. Culture medium for in vitro studies consisted of RPMI 1640 supplemented with 5% heat-inactivated FCS, 1 mM glutamine, penicillin/streptomycin/Fungizone, 10 mM HEPES buffer, 100 µM nonessential amino acids, and 2.5 x 10^–5 M 2-ME, all purchased from Invitrogen Life Technologies. In some experiments 1% Nutridoma-SP (Roche Molecular Biochemicals) was substituted for 5% FCS in the culture medium. Buffer used for intracellular staining consisted of 0.01% saponin and 1% BSA (both purchased from Sigma-Aldrich) and 0.1% sodium azide (Fisher Scientific) dissolved in Dulbecco’s PBS (Invitrogen Life Technologies). Fc Block (FcIII/II receptor (clone 2.4G2)); Cy5-labeled anti-CD4 Ab; FITC-labeled anti-CD25 Ab; PE-labeled CD28, CD45RB, CD152, and ICOS Abs; and mouse IgG1 isotype control Ig were purchased from BD Pharmingen. PE-conjugated anti-mouse CD62L and CD69 Abs were purchased from Caltag Laboratories. PE-conjugated anti-mouse TLR4-MD-2 specific Ab was purchased from eBioscience, and PE-conjugated anti-mouse GITR was purchased from R&D Systems. PE-labeled mouse monoclonal anti-human TGF-1 Ab was purchased from IQ Products. Rat IgG was purchased from Sigma-Aldrich. Purified PC61 Ab was purchased from the American Type Culture Collection and BioXpress.

Mouse injury model

The mouse thermal injury model, approved by the National Institutes of Health and the Harvard Medical Area Standing Committee on Animals, was performed as described (35). In brief, six mice per group were anesthetized 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 insulated plastic mold to expose 25% total body surface area. This part of the dorsum was then immersed in 90°C (burns) or isothermic water (shams) for 9 s. All groups were resuscitated by i.p. injection with 1 ml of 0.9% pyrogen-free saline. This protocol causes a well demarcated, full-thickness, anesthetic injury with <5% mortality.

CD4⁺CD25⁺ and CD4⁺CD25^– T cell purification

Spleens and lymph nodes (inguinal, axillary, and brachial) were harvested from mice and cell suspensions were prepared by mincing tissues on wire mesh screens. Spleen cells were treated with ammonium chloride solution for 3 min to lyse RBCs and were washed twice before suspension in culture medium. Lymph node cells were also washed twice before suspension in culture medium. CD4⁺CD25⁺ and CD4⁺CD25^– cells were purified using the regulatory T cell magnetic cell sorting kits (Miltenyi Biotec) according to the manufacturer’s instructions. Both CD4⁺CD25⁺ and CD4⁺CD25^– T cell populations were collected and prepared for in vitro studies. Cell purity was assessed by staining purified cell populations with Cy5-labeled anti-CD4 Ab and PE-labeled anti-CD25 Ab followed by analysis using a FACSCalibur Instrument (BD Biosciences), and results were processed using the accompanying CellQuest Pro Software. The CD4⁺CD25⁺ and CD4⁺CD25^– T cells were consistently >95% pure using this approach (Fig. 1A).

View larger version (25K): [in this window] [in a new window]   FIGURE 1. CD4+CD25+ and CD4+CD25– T cell purity and FoxP3 gene expression levels. A, Purified CD4+CD25+ and CD4+CD25– T cells from day 7 sham and burn-injured mice were stained with Cy5-labeled anti-CD4 and PE-conjugated anti-CD25 Abs. CD25 expression levels on purified CD4+CD25+ and CD4+CD25– T cell populations are shown in the upper right quadrant in these representative FACS plots. The results are representative of all purifications performed. B, Real-time PCR detection of FoxP3 gene expression levels in RNA prepared from highly purified lymph node CD4+CD25– and CD4+CD25+ T cells from day 7 sham or burn-injured mice. GAPDH gene expression level was used as an internal reference standard for each sample to determine FoxP3 mRNA expression levels as described in Materials and Methods. These data include the results from three independent mouse injury studies using purified cells from three mice per group. *, p < 0.05 FoxP3 gene expression levels for CD4+CD25– vs CD4+CD25+ T cells.

CD4⁺CD25^– or CD4⁺CD25⁺ T cells were purified from the lymph nodes of sham or burn-injured mice at 7 days after injury. RNA was prepared from these cell suspensions using the TRIzol RNA isolation buffer following the protocol suggested by the manufacturer (Invitrogen Life Technologies). We next synthesized cDNA by an oligo(dT) primed reverse transcriptase reaction using 0.5 µg of RNA from each sample. Real-time PCRs were performed in an Applied Biosystems 5700 gene detection instrument using murine GAPDH (F-CAGGTTGTCTCCTGCGACTT, R-CCCTGTTGCTGTAGCCGTA) and FoxP3 (F-CCATTGGTTTACTCGCATGT, R-GCTCTCCACTCGCACAAA) specific primers. SYBR green was used to detect 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 threshold level) using the following formula (1/Ct_FoxP3 – Ct_GAPDH).

In vitro cytokine production studies

Purified CD4⁺CD25⁺ or CD4⁺CD25^– T cells were cultured in Costar round-bottom 96-well plates at 2 x 10⁵ cells/well in the absence or presence of plate-bound anti-CD3 Ab (clone 145-2C11 added at 5 µg/ml). These cultures were performed in 1% Nutridoma-SP culture medium rather than in medium containing 5% heat-inactivated FCS to allow for accurate measurement of TGF- production. After 24-h stimulation, culture supernatants were harvested and stored at 4°C. The cytokines IL-2, IFN-, IL-10, and TGF- were measured by cytokine-specific ELISAs. IL-10 and TGF- were measured using ELISA kits purchased from R&D Systems and used according to the manufacturer’s instructions. In the case of TGF-, 0.1 ml of 1 N HCl was added to 0.5 ml of sample, which was incubated for 10 min at room temperature before neutralization by the addition of 0.1 ml of 1.2 N NaOH/0.5 M HEPES to activate latent TGF-1 to its immunoreactive form. IFN- levels were measured using Ab pairs purchased from R&D Systems, whereas IL-2 was measured using Ab pairs purchased from Caltag Laboratories. The ELISA results were analyzed using an ELISA Plate Reader and the associated SoftMax Pro software program (Molecular Devices). Cytokine levels in culture supernatants were calculated 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, and TGF-1 expression on CD4⁺CD25⁺ and CD4⁺CD25^– T cells

Lymph node cells were prepared from sham or burn-injured BALB/cJ mice. 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 for 20 min to reduce nonspecific background staining and then were stained with Cy5-labeled anti-CD4 and FITC-labeled anti-CD25 Abs to identify the CD4⁺CD25⁺ and CD4⁺CD25^– cell populations. Cells were stained simultaneously 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 were surface stained with anti-CD4 and anti-CD25 Abs were fixed for 10 min in 1% paraformaldehyde solution and then permeabilized for 20 min in FACS buffer containing 0.25% saponin (permeabilization buffer). The fixed and permeabilized cells were then incubated for 10 min in permeabilization buffer containing 1 µg/ml normal rat IgG to block nonspecific staining, after which PE-labeled anti-CD152 Ab was added for an additional 30-min incubation. The cells were washed twice by centrifugation in permeabilization 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 and labeled with CFSE using the Vybrant Cell Tracer Kit (Molecular Probes) following the manufacturer’s protocol. Briefly, lymph node cells were suspended in prewarmed (37°C) PBS containing CFSE at a 5 µM concentration and then incubated for 15 min at 37°C. Cells were then centrifuged and resuspended in prewarmed (37°C) culture medium, incubated for an additional 30 min to ensure complete modification of the probe, and then rewashed. The CFSE-labeled lymph node cells were added at 2 x 10⁵ cells/well to individual wells of Costar round-bottom 96-well plates. Purified CD4⁺CD25⁺ or CD4⁺CD25^– T cells were then added to the CFSE-labeled lymph node cells at 1:1, 1:2, 1:3, and 1:4 ratios in the absence or presence of 5 µg/ml anti-CD3 Ab. Wells of CFSE-labeled cells without the added CD4⁺CD25⁺ or CD4⁺CD25^– cells served as negative and positive controls. In some experiments, purified CD4⁺CD25^– T cells from sham or burn mice were CFSE-labeled and used in place of CFSE-labeled lymph node 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°C in 5% CO₂ for 72 h, then pelleted by centrifugation, pretreated with Fc Block, and stained with Cy5-labeled anti-CD4 Ab. Flow cytometry was performed by gating on CD4⁺ cells and determining CFSE signal intensity using the FACSCalibur Instrument, and the results were analyzed using CellQuest Pro software.

Transwell CFSE proliferation studies

Lymph node cells from three BALB/cJ mice were labeled with CFSE as described. Cells were cultured in Costar Transwell (0.4-µm pore size) 24-well plates at 1 x 10⁶ cells/well with 5 µg/ml anti-CD3 Ab (soluble) or no additions. Lymph node CD4⁺CD25⁺ and CD4⁺CD25^– cell populations were prepared from six mice at 7 days after sham or burn injury. In addition, T cell-depleted APCs were prepared from the spleens of three BALB/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 x 10⁵) along with T cell-depleted APCs (3 x 10⁵) were added to the well inserts, and the CFSE-labeled lymph node cells were added to the wells. These Transwell cultures were incubated for 3 days at 37°C in 5% CO₂ in the absence or presence of 5 µg/ml anti-CD3 Ab. Control cultures included CFSE-labeled lymph node cells cocultured with CD4⁺CD25⁺ or CD4⁺CD25^– cells in the absence or presence of 5 µg/ml anti-CD3 Ab without well inserts. The levels of cellular 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 prepared from 7-day sham or burn-injured BALB/cJ mice and mixed with CFSE-labeled naive BALB/cJ lymph node cells at a 1:3 ratio in the absence or presence of 5 µg/ml anti-CD3 Ab. Anti-IL-10, anti-TGF-1 Ab, or the appropriate IgG isotype control Abs (rat IgG1 or mouse IgG1, respectively) were added at 0.2, 2, and 20 µg/ml final concentrations. After 3-day incubation at 37°C in 5% CO₂, anti-CD3-induced proliferation was measured by flow cytometry and the results were analyzed as described.

Assessment of the effect of CD25⁺ T cells on Th1-type cytokine production

CD4⁺CD25⁺ and CD4⁺CD25^– T cells were purified from lymph node cell 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 lymph node cells in ratios of 1:1 or 1:3 with or without 5 µg/ml anti-CD3 Ab. After 1-day incubation at 37°C in 5% CO₂, culture supernatants were harvested and IL-2 or IFN- levels were measured by cytokine-specific ELISAs.

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

Male BALB/cJ mice (n = 18–20/group) were treated with 0.25 mg of purified PC61 Ab or control rat IgG by i.p. injection. This Ab-mediated CD4⁺CD25⁺ T cell depletion protocol depletes CD4⁺CD25⁺ T cells in mice by 3 days postinjection as judged by FACS (see Fig. 9A), as previously described by others (36). At 3 days after PC61 Ab or rat IgG treatment, mice underwent sham or burn injury and were immunized s.c. with 0.1 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 were harvested to prepare serum samples and cell suspensions from individual mice. The cells were stimulated in vitro with an optimal concentration of OVA (0.1 mg/ml), and 48 h later culture supernatants were harvested to measure cytokine production levels. Serum TNP-specific Ig isotype levels were measured by ELISA as described previously (7). In brief, 96-well plates were coated with PBS containing 20 µg/ml TNP-haptenated BSA. Serial dilutions of serum samples prepared from individual mice were added and, after washing specific Ig isotypes, were measured using HRP-conjugated Abs specific for mouse IgM, IgG1, or IgG2a (Caltag Laboratories). ELISA reactions were developed using a NBT substrate buffer, and the plates were read at 450–570 nM using the ELISA plate reader.

View larger version (28K): [in this window] [in a new window]   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 of anti-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 emulsified 1:1 in CFA. Ten days later, mice were sacrificed to harvest blood, spleen, and regional lymph nodes to prepare serum and cells. A, FACS plots showing the 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 indicate the 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 were stimulated 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 nine mice 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-treated burn 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-specific Ab 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.

Statistical analyses were performed by ANOVA using Tukey’s multiple comparisons test or paired t tests. The p values for significance were set at 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Injury augments the regulatory activity of CD4⁺CD25⁺ T cells

It is known that severe injury suppresses T cell-mediated immune responses and that this induced change in immune function coincides with a decreased ability to control opportunistic infections (5). These observations, along with the discovery that a naturally occurring CD4⁺CD25⁺ T cell subset plays a central role in controlling inflammatory responses convinced us to investigate whether the injury response might prime these cell for increased T regulatory activity (31). A well-established mouse burn model was used as a clinically relevant in vivo injury (35). Prior studies using this model have documented that suppressed T cell function is measurable at 7 days, but not 1 day after injury (1). This feature of the injury response prompted us not only to characterize the influence 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 of mice using a two-step magnetic bead cell sorting approach. We consistently obtained purity of >95% for CD4⁺CD25⁺ and CD4⁺CD25^– T cell populations (Fig. 1A). In addition, we wished to characterize further the effects of injury on the cell surface phenotype of CD25⁺ and CD25^–CD4⁺ T cells. We measured by flow cytometry the levels of cell surface markers that are known to be differentially expressed on CD4⁺CD25⁺ Tregs vs conventional CD4⁺ T cells. These included GITR, CD62L, CD45RB, CD69, and TLR4-MD-2. As listed in Table I, we found that the CD4⁺CD25⁺ T cells were predominately CD45RB^low and expressed higher levels of cell surface GITR, CD62L, and TLR4-MD-2 than did CD4⁺CD25^– T cells. The CD4⁺CD25⁺ T cells also expressed significantly lower levels of CD69 than did CD4⁺CD25^– T cells. Interestingly, injury did not markedly alter the cell surface expression levels of any of these molecules on either CD4⁺CD25⁺ or CD4⁺CD25^– T cells at 1 or 7 days after injury. Furthermore, we did not observe a significant change in the overall numbers of CD4⁺CD25⁺ or CD4⁺CD25^– T cells at 1 or 7 days after burn injury (data not shown). We also wished to determine the FoxP3 gene expression levels in CD25^– and CD25⁺ T cells purified from sham vs burn mice. Using a real-time PCR approach, we showed that the purified CD4⁺CD25^– T cells expressed low levels of FoxP3, whereas purified CD4⁺CD25⁺ T cells expressed significantly higher levels of FoxP3 mRNA (Fig. 1B). The results of these FoxP3 studies also showed that burn injury did not significantly alter FoxP3 gene expression levels in either CD25^– or CD25⁺ CD4 T cells. Taken together, these results suggest that the purified CD4⁺CD25⁺ T cells studied here most likely represent natural Tregs rather than induced Tregs as judged by cell surface marker expression and FoxP3 gene expression.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Injury enhances the Treg activity of lymph node CD4+CD25+ T cells. Lymph node CD4+CD25+ and CD4+CD25– T cells purified from six sham or burn-injured BALB/cJ mice were added to 2 x 105 CFSE-labeled BALB/cJ lymph node cells in ratios of 1:1 to 1:4. Cell mixes were stimulated with or without soluble anti-CD3 Ab (5 µg/ml). After 3 days, cells were harvested and proliferation levels were determined by CFSE staining intensity of gated CD4+ T cells. Representative FACS plots from 1-day (A) or 7-day (B) sham and burn mice demonstrate greater inhibition of anti-CD3-stimulated CD4+ T cell proliferation mediated by CD4+CD25+ T cells as compared with CD4+CD25– T cells under all effector to responder ratios tested; p < 0.05 by ANOVA. Seven-day burn CD4+CD24+ T cells show greater Treg potency than do 7-day sham CD4+CD25+ T cells. The marker numbers indicate the percentage of cells demonstrating reduced CFSE staining intensity. C, Plots showing the combined CFSE proliferation assay results measured at 1 and 7 days after injury and representing the mean ± SEM of three independent experiments. *, Differences between groups at all cell ratios tested; p < 0.05 by ANOVA.

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Compartmentalization of the effects of injury on Treg activity: injury fails to augment the Treg activity of spleen CD4+CD25+ T cells. Purified CD4+CD25+ and CD4+CD25– T cells purified from the spleens of sham and burn BALB/cJ mice at 7 days post-injury were added in ratios of 1:1 to 1:4 to CFSE-labeled lymph node cells. Cells were stimulated with soluble anti-CD3 Ab (5 µg/ml). After 3 days in culture, cells were harvested and proliferation levels were determined by CFSE staining intensity of gated CD4+ T cells by FACS. Results shown represent the mean ± SEM of three independent experiments; n = 6 mice/group. There was no significant difference between sham and burn Treg activity at all cell ratios examined.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. CD4+CD25+ T cells can directly inhibit CD4+CD25– T cell proliferation. Lymph node CD4+CD25+ and CD4+CD25– T cells were harvested and purified from sham and burn BALB/cJ mice at 7 days after injury. The pure CD4+CD25– T cells were labeled with CFSE and mixed with pure CD4+CD25+ T cells at the indicated ratios. Cells were stimulated with plate-bound anti-CD3 Ab. After 3-day incubation, cells were harvested and assessed for proliferation by FACS analysis. Results shown represent the mean ± SEM of three independent experiments; n = 3 mice/group. *, p < 0.05 by ANOVA for differences between burn CD25+ and sham CD25+ at all ratios tested.

Severe injury skews polyclonal or Ag-specific CD4⁺ T cell cytokine responses toward a Th2 phenotype (35, 37). This influence of injury on the peripheral T cell pool does not occur early, but it is easily detected 7–10 days later. The temporal overlap between injury-enhanced Treg activity displayed by lymph node CD4⁺CD25⁺ T cells and the injury-biased Th2-type cytokine response suggested to us that there might be a link between these two injury-induced changes in host immune responses. To address this possibility, we directly compared Th1, Th2, and Treg-type cytokine production profiles by anti-CD3-stimulated purified populations of CD4⁺CD25⁺ and CD4⁺CD25^– T cells prepared from the lymph nodes or spleens of mice at 1 and 7 days after sham or burn injury. The results of these studies revealed some significant differences 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 produced significantly higher levels of IFN- than did CD4⁺CD25⁺ T cells, whereas CD4⁺CD25⁺ T cells released low levels of IL-2 and IFN- and higher levels of IL-10 and TGF- (Fig. 5). Burn injury did not significantly alter IL-2, IFN-, or TGF- production levels by either CD4⁺ T cell subset at 1 or 7 days post-injury; however, at 7 days after burn injury, we observed a significant increase in IL-10 production by both lymph node and spleen CD4⁺CD25⁺ T cells (Fig. 5).

View larger version (25K): [in this window] [in a new window]   FIGURE 5. CD4+CD25+ T cells produce increased IL-10 at 7 days after injury. Purified CD4+CD25+ or CD4+CD25– T cells (2 x 105) from the lymph nodes (A) or spleens (B) of day 1 or day 7 sham and burn mice were stimulated with plate-bound anti-CD3 Ab. At 1 day, culture supernatants were harvested and tested for IL-2, IFN-, IL-10, and TGF- levels by cytokine-specific ELISA. The results shown represent the mean ± SEM of triplicate wells and represent three independent experiments involving six mice/group. *, p < 0.05 for sham vs burn groups by ANOVA.

The increased IL-10 production by highly purified CD4⁺CD25⁺ T cells detected at day 7 after injury led us to question whether IL-10 might mediate, at least in part, the injury-induced increase in Treg activity. We also wished to learn whether CD4⁺CD25⁺ T cells acted through a cell-contact-dependent mechanism as previously described (26). To address these questions, we first used a conventional Transwell approach to test whether the measured Treg activity was mediated by a soluble factor. We added purified CD4⁺CD25^– or CD4⁺CD25⁺ lymph node T cells from 7-day sham or burn mice along with T cell-depleted splenic APCs to one side of the semipermeable membrane and CFSE-labeled lymph node responder cells to the other side at a 1:3 purified T cell to lymph node responder cell ratio. After 3-day incubation in the presence of soluble anti-CD3 Ab, proliferation levels were assessed by FACS analysis. As shown in Fig. 6A, preventing cell contact by separating the CFSE-labeled lymph node responder cells from CD4⁺CD25⁺ T cells eliminated the Treg activity normally displayed by these cells. The purified CD4⁺CD25^– T cells from sham or burn mice also did not alter the proliferation of the CFSE-labeled lymph node responder cells under identical experimental conditions. As a positive control, purified CD4⁺CD25⁺ lymph node T cells from 7-day sham or burn mice cultured in the same well with CFSE-labeled lymph node responder cells at a 1:3 ratio again showed a marked suppression of anti-CD3-induced T cell proliferation. These findings indicate that cell contact is required for CD4⁺CD25⁺ T cells to mediate their Treg activity and that IL-10 or other soluble mediators are not likely to contribute directly to the observed injury-dependent increase in Treg function.

View larger version (23K): [in this window] [in a new window]   FIGURE 6. CD4+CD25+ T cell-mediated inhibition of CD4+ T cell proliferation is cell contact and TGF- dependent, but does not involve IL-10. For the results shown in A, CD4+CD25+ and CD4+CD25– T cells purified from the lymph nodes of sham or burn mice were cultured with 1 x 106 CFSE-labeled BALB/cJ lymph node cells at a 1:3 ratio. The CFSE-labeled lymph node cells in the bottom well were separated from the purified cells and T cell-depleted spleen cells (APCs) in the Transwell insert by a semipermeable membrane. All cell mixtures were stimulated with anti-CD3 Ab (5 µg/ml). Controls included sham or burn CD4+CD25+ T cells added to CFSE-labeled lymph node cells at a 1:3 ratio, but without separation. After 3-day incubation, the cells were harvested and tested for anti-CD3-induced proliferation by FACS. The results shown represent the mean ± SEM of three experiments with six mice/group. To address the role of IL-10 (B) and TGF- (C) in mediating the CD4+CD25+ Treg activity, anti-IL-10 or anti-TGF-1 Abs, along with the appropriate IgG isotype control Abs, were tested at the indicated concentrations for their ability to block CD4+CD25+-mediated Treg activity. For all these experiments, CD4+CD25+ and CD4+CD25– T cells purified from the lymph nodes of sham or burn mice were added to CFSE-labeled BALB/cJ lymph node cells at a 1:3 ratio and were stimulated with anti-CD3 Ab (5 µg/ml). The proliferation of gated-CFSE-labeled CD4+ T cells was assessed by FACS. The results represent the mean ± SEM of three independent experiments using four mice per group; *, p < 0.05 by ANOVA for differences between burn CD25+ and all other groups at the highest Ab concentration tested.

The enhanced CD4⁺CD25⁺ Treg activity after injury is associated 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 membrane TGF- might be responsible for CD4⁺CD25⁺ T cell-mediated Treg function detected in our studies. Using an approach similar to the anti-IL-10 experiments, we tested the effects of adding a monoclonal anti-TGF-1 Ab or its isotype control at a range of concentrations to cultures of purified CD4⁺CD25⁺ or CD4⁺CD25^– lymph node T cells harvested from 7-day sham or burn-injured mice mixed at a 1:3 ratio with CFSE-labeled lymph node responder cells. As shown in Fig. 6C, we observed a significant dose-dependent effect of the anti-TGF-1 Ab on CD4⁺CD25⁺ T cell-mediated Treg activity. At the highest dose tested, 20 µg/ml, we observed a complete inhibition of Treg function by burn CD4⁺CD25⁺ T cells and a significant 2-fold reduction in Treg function mediated by sham CD4⁺CD25⁺ T cells. Taken together, these results indicate that the counterproliferative activity displayed by the CD4⁺CD25⁺ T cells in our experiments is mediated in part by TGF-1.

Because we did not detect increased TGF- production by anti-CD3-stimulated CD4⁺CD25⁺ T cells purified from 7-day burn mice, we reasoned that changes in cell surface TGF- expression levels might be responsible for the augmented Treg activity detected at 7 days after burn injury. To test this possibility, we performed three-color FACS studies using a PE-labeled anti-TGF-1 Ab to detect cell surface expression of TGF-1 on lymph node and spleen CD4⁺CD25⁺ and CD4⁺CD25^– T cells prepared from 1- or 7-day sham or burn mice. We found a significant increase in TGF-1 expression on 7-day burn lymph node-derived CD4⁺CD25⁺ T cells (Fig. 7). A similar injury-induced increase in cell surface TGF-1 was not detected on CD4⁺CD25⁺ T cells purified from the spleens of burn-injured mice, and we did not detect any cell surface TGF-1 on the CD4⁺CD25^– T cell subsets. The fact that expression of cell surface TGF-1 was restricted to the CD4⁺CD25⁺ T cell subset in mice fully agrees with several published reports (26, 38, 39). However, the observed up-regulation of cell surface TGF-1 expression on CD4⁺CD25⁺ T cells after injury is novel. Moreover, the association between the tissue-compartmentalized influence of injury on CD4⁺CD25⁺ Treg function and changes in TGF-1 expression suggests that the enhanced Treg function expressed by lymph node CD4⁺CD25⁺ T cells at 7 days after burn injury is likely mediated by increased cell surface TGF-1 expression.

View larger version (46K): [in this window] [in a new window]   FIGURE 7. Injury increases cell surface TGF-1 expression on lymph node, but not spleen CD4+CD25+ T cells at 7 days after injury. Spleen and lymph node cells harvested from sham and burn mice at 1 or 7 days after injury were stained with Cy5-conjugated anti-CD4 Ab, FITC-conjugated anti-CD25 Ab and PE-conjugated anti-TGF-1 Ab, or PE-conjugated IgG1 isotype control Abs. The representative FACS plots of TGF-1 staining on gated CD4+CD25+ (A) and CD4+CD25– (B) T cells illustrate that TGF-1 is selectively expressed on CD4+CD25+ T cells. As shown in C, injury caused a significant increase in cell surface TGF-1 expression on lymph node CD4+CD25+ T cells. *, p < 0.001 by ANOVA. No significant injury-induced changes in cell surface TGF-1 expression were observed on day 1 post-injury lymph node or spleen CD4+CD25+ T cells or on day 7 spleen-derived CD4+CD25+ T cells. Results shown represent the mean ± SEM of six individual mice.

The results of our proliferation studies, which showed enhanced Treg function by CD4⁺CD25⁺ T cells at 7 days after injury, suggested that these cells might also be able to suppress Th1-type cytokine production. To explore this possibility, we tested the effects of CD4⁺CD25⁺ or CD4⁺CD25^– T cells on anti-CD3 Ab-induced IL-2 and IFN- production by naive lymph node cells. For these experiments, purified CD4⁺CD25⁺ or CD4⁺CD25^– lymph node T cells from 7-day sham or burn mice were added at 1:3 ratios to lymph node cells, which were then stimulated with anti-CD3 Ab. As shown in Fig. 8, we found that the addition of CD4⁺CD25⁺ T cells caused a significant reduction in CD3-stimulated IL-2 and IFN- production, whereas the addition of CD4⁺CD25^– T cells did not suppress the production of either cytokine. Moreover, CD4⁺CD25⁺ T cells from 7-day burn mice were more effective at suppressing IL-2 and, in particular, IFN- production than were sham CD4⁺CD25⁺ T cells. Similar results were observed when experiments were performed using a 1:1 purified T cell to lymph node cell ratio, except that the CD4⁺CD25⁺ T cell-mediated suppression of Th1-type cytokine production was even more profound (data not shown).

View larger version (15K): [in this window] [in a new window]   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 x 105) from sham or burn mice (n = 6/group) were added to BALB/cJ lymph node cells at a 1:3 ratio. Controls included BALB/cJ lymph node cells without purified cell additions. These cell mixes were stimulated overnight with anti-CD3 Ab (5 µg/ml), after which supernatants were harvested and tested for IL-2 and IFN- levels by ELISA. Both sham and burn CD4+CD25+ T cells caused a significant reduction in IL-2 and IFN- production by anti-CD3-stimulated lymph node cells. *, p < 0.001 by ANOVA; **, significant difference in IL-2 and IFN- production mediated by CD4+CD25+ T cells from burn as compared with sham mice (p < 0.05 by ANOVA).

To further investigate the influence of injury on CD4⁺CD25⁺ T cell phenotype, we measured injury effects on several T cell costimulatory and immune regulatory receptors (CD28, CD152, ICOS, and PD-1) that are known to be associated with CD4⁺CD25⁺ T cell activation and function (Table II) (40). At 1 day after injury, we detected increased expression of CD28 and ICOS on lymph node and spleen CD4⁺CD25⁺ T cells but not CD4⁺CD25^– T cells. By 7 days after injury, we found a significant increase in intracellular CD152 expression by lymph node and spleen CD4⁺CD25⁺ T cells, whereas ICOS was significantly increased only on lymph node CD4⁺CD25⁺ T cells. Collectively, these observations suggest that injury provides an early CD4⁺ T cell-activating signal that may cause a sustained increase in the expression of CD152 and ICOS, two regulatory receptors associated with inhibiting proinflammatory immune responses.

To directly measure the functional influence of Treg cells on the development of Ag-specific T cell responses in vivo, we examined whether sham and burn mice immunized with the T cell-dependent Ag TNP-OVA respond differently after in vivo Treg depletion. In these studies, mice were given 10 mg/kg anti-CD25 Ab or control IgG at the same concentration by i.p. injection. As illustrated in Fig. 9A, this single i.p. injection of anti-CD25 Ab reduces the level of 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 injury and were immunized s.c with 0.1 mg of TNP-OVA emulsified 1:1 in CFA. After 10 days, blood, spleen, and regional lymph nodes were harvested to measure TNP-specific Ab isotype levels and OVA-induced IFN- production. As shown in Fig. 9B, Ag-stimulated IFN- production by lymph node cells was markedly increased in sham or burn mice that were Treg-deficient at the time of injury. A similar, though less dramatic, effect of Treg depletion on OVA-induced IFN- production was observed in spleen cell cultures. These findings indicate that Tregs control the development of Ag-specific IFN- responses in vivo and that Treg depletion restores Ag-driven IFN- responses in burn-injured mice to normal levels.

We next measured the influence of Treg depletion on Ag-specific Ab isotype formation by measuring the levels of TNP-specific IgM, IgG1, and IgG2a in serum prepared from TNP-OVA-immunized burn and sham mice. We found that Treg depletion markedly and significantly increased titers of the Th1-dependent Ab isotype IgG2a in both sham and burn mice, whereas Treg depletion did not significantly alter IgM or IgG1 Ab isotype levels (Fig. 9C). The effect of Treg depletion on Ag-specific IgG2a Ab production was most dramatic in the sham group, which showed a supernormal IgG2a response. Nonetheless, we found that Treg-depleted burn mice displayed a significantly higher IgG2a Ab response than did control Ig-treated sham or burn mice, suggesting that Tregs contribute to suppressed Th1-type Ab responses after injury. Taken together with the observation that Treg depletion boosted Ag-specific IFN- production (Fig. 9B), these findings support the idea that Tregs play a central role in suppressing Th1 responses after burn injury.

	Discussion

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The stress and tissue damage associated with severe injury appear to initiate a programmed immune response that involves both innate and adaptive immune cell types and mediators (5, 9). Investigations into how the immune system compensates for the initial inflammatory response to injury have led us and other investigators (9, 12) to conclude that T cells play a central role. For example, we recently reported evidence that Rag1^–/– mice, which lack an adaptive immune system, acquire a more proinflammatory phenotype after injury than do wild-type mice, suggesting that the adaptive immune system must play an active role in modulating innate immune reactivity after injury (41). Further investigations into the adaptive immune cell types that mediate this regulatory response showed that CD4⁺CD25⁺ T cells were the primary cell population involved in controlling the inflammatory reactivity of innate immune cells after injury (34). Other studies have shown that injury-induced alterations in T cell function include a deviation toward Th2-type reactivity and suppressed Th1-type immune function (7, 35). We report here a potential mechanism involved in the development of suppressed adaptive immunity after severe injury. Specifically, we show that injury primes CD4⁺CD25⁺ T cells for enhanced T regulatory activity. It is noteworthy that this enhanced Treg function is detected at a time coincident with the expression of injury-induced immune suppression, as judged by reduced resistance to bacterial or viral infections in mice and man (3, 42, 43). Another interesting aspect of these findings is the relative restriction of the enhanced Treg activity to the lymph nodes draining the injury site. This compartmentalized effect of injury on Treg cell function suggests that tissue injury-associated Ags or lymph node-specific APC types might play an important role in initiating or amplifying the injury-associated increase in Treg activity.

We demonstrate that the Treg activity measured in this study is cell-contact dependent and mediated in part by TGF-1. This conclusion is supported by the observations that anti-TGF-1 Ab abrogates the ability of purified CD4⁺CD25⁺ T cells to inhibit anti-CD3-induced CD4⁺ T cell proliferation. However, the finding that CD4⁺CD25⁺ T cells from sham and burn-injured mice secrete equivalent amounts of TGF- and the fact that injury significantly up-regulates cell surface TGF-1 expression on regional lymph node but not splenic CD4⁺CD25⁺ T cells leads us to conclude that the enhanced regulatory function of the CD4⁺CD25⁺ T cell is not attributable to TGF- release, but rather to an injury-induced increase in cell surface TGF-1 expression. Yet, some reports conclude that TGF- activity is not responsible for CD4⁺CD25⁺ Treg function 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 active TGF-, and anti-TGF- Ab levels may need to be high enough to rapidly block TGF- activity during this conversion process (32). The fact that we observed significant abrogation of Treg activity at the highest anti-TGF-1 concentration tested (20 µg/ml) but not at the lower concentrations (0.2 or 2 µg/ml) supports this possibility. An additional report suggests that TGF-1 cannot be solely responsible for CD4⁺CD25⁺ Treg activity because Treg activity persists in TGF--deficient mice (44). Although the results obtained using these mice are convincing, it is unclear what compensatory 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 by TGF- alone or whether other factors such as IL-10 may also play a contributing role. We verified in this study that CD4⁺CD25⁺ T cells produced higher levels of IL-10 than did CD4⁺CD25^– T cells. Moreover, we found that anti-CD3 Ab-stimulated CD4⁺CD25⁺ T cells from 7-day burn mice produced significantly higher levels of IL-10 than did CD4⁺CD25⁺ T cells from sham mice. Nevertheless, we were unable to link Treg activity in vitro to IL-10 as judged by anti-IL-10 Ab blocking studies. Despite these findings, it is still possible that IL-10 contributes to CD4⁺CD25⁺ T cell-mediated activities in vivo. For example, it was shown that the transfer of CD4⁺CD45RB^high T cells into SCID mice led to the development of colitis, but the transfer of these cells along with CD4⁺CD45RB^low T cells prevented colitis, whereas anti-IL-10 or anti-IL-10R treatment blocked the CD4⁺CD45RB^low Treg effect (45). The results of that study suggest that IL-10 may be an important factor in mediating in vivo Treg activity. Therefore, it is possible that IL-10 may play a significant in vivo role in the increase in Treg activity observed in the present report. Other studies addressing the function of IL-10 during the injury response have indicated that blocking its activity early after injury promoted Th1-type immunity and prevented the development of postinjury immune suppression, suggesting that IL-10 does indeed influence the host immune response to injury (7, 11).

This report does not conclusively demonstrate whether burn injury enhances the regulatory function of natural Tregs or induces a naive population of Treg cells that are responsible for the enhanced Treg activity at 7 days after injury. However, detailed examination of the cell surface phenotype of CD25⁺ vs CD25^– CD4 T cells suggests that injury did not significantly alter those markers that identify CD25⁺ CD4 T cells as a natural Treg cell population (Table I). Moreover, we did not detect a significant change in FoxP3 gene expression by CD25⁺ CD4 T cells from burn as compared with sham mice. Taken together, these findings suggest that the purified CD25⁺ CD4 T cells examined in this study are not a mixture of induced and natural Tregs. This evidence leads us to favor the idea that injury enhances the regulatory activity of natural Treg cells rather than inducing CD4 T cells with Treg cell properties. However, a clearer picture of whether or not injury induces a unique population of Treg cells will emerge from the results of future studies addressing the contribution of factors such as IL-10, TGF-, or LPS on Treg function after burn injury (28, 36, 46, 47).

To explore the functional significance to up-regulated Treg activity after injury, we examined whether Tregs might contribute to the injury-induced suppression of Ag-specific Th1-type immune reactivity because this is an established feature of the mammalian injury response. Because prior studies have shown that mice immunized at the time of injury with a T cell-dependent Ag displayed a significant decrease in Th1-type Ab isotype formation, we used this observation to determine how Treg cell depletion might influence the development of Th1 and Th2 responses (7). We found that mice treated with a Treg-depleting dose of anti-CD25 Ab showed a marked increase in Th1-type reactivity. Most importantly, burn mice that were depleted of CD4⁺CD25⁺ T cells at the time of injury did not show suppressed Th1 Ab responses, indicating that injury in the absence of Tregs did not result in suppression of Th1 immunity. However, the most dramatic effect of Treg depletion on Th1 Ab production was observed in TNP-OVA-immunized sham mice, which displayed extremely high levels of serum TNP-specific IgG2a. Another recent report shows a similar consequence of Treg depletion on Ag-specific Th1-type Ab responses (48). Interestingly, burn-injured mice that were rendered Treg deficient did not show this abnormally high Th1-type reactivity, but did show higher Th1-type Ab production than did sham or burn mice that were not CD4⁺CD25⁺ T cell depleted. This finding indicates that Tregs do play a role in suppressing Th1-type reactivity after injury but that other mechanisms may prevent the development of the supranormal Ag-driven Th1 response seen in sham mice. The idea that Tregs control the level of Th1 reactivity in vivo is further supported by our finding that lymph node and spleen cells from immunized, Treg-depleted mice produced significantly higher levels of Ag-specific IFN- than did cells from control Ig-treated mice. The latter finding also suggests that injury induces a significant increase in Treg activity in the spleen even though such an increase was not observed in our in vitro studies of antiproliferative function.

Although we demonstrate that mice given anti-CD25 Ab to deplete CD25⁺ T cells are able to respond to immunization with a T cell-dependent Ag, there was concern that residual anti-CD25 Ab may influence the emergence of activated T cells after injury and immunization. Therefore, we performed an additional immunization study in sham and burn mice using a concentration of anti-CD25 Ab (1 mg/kg) that was found to be the minimally effect dose needed to deplete mice of CD25⁺ CD4 T cells but that allows for the repopulation or emergence of CD25-expressing CD4 T cells by 14 days after anti-CD25 Ab treatment. In this immunization study, we found that burn mice showed enhanced Ag-specific Th1-type Ab formation as was reported in Fig. 9C, suggesting that transient depletion of Treg cells also restores Th1 responses in mice after burn injury (data not shown).

The detection of augmented regional lymph node, but not spleen, CD4⁺CD25⁺ Treg activity suggests that the exposure of lymph node cells to endogenous Ag(s) released at the injury site may be responsible for this phenomenon. Other factors that are known to enhance CD4⁺CD25⁺ Treg activity include the C3b product of complement activation (which is a ligand for CD46 expressed on CD4⁺ T cells), TGF-, and bacterial LPS or potential endogenous agonists activating TLR4 uniquely expressed on CD4⁺CD25⁺ T cells (28, 49, 50, 51). Other endogenous activating agents may include uric acid or a TLR2-activating Ag that is released by cells after necrotic cell death (52, 53).

However, further investigations will be required to identify the contribution of these and other potential factors to the increase in Treg activity after injury. The identification of such factors or Ags would be of great interest to the immunological community and, since loss of Th1 immune function after injury is associated with diminished resistance to infection, a more complete understanding of injury-induced alterations in Treg function may be of major significance for developing therapeutic strategies for the benefit of injured patients in the future.

	Acknowledgments

 
We thank Drs. Peter Sayles and Mohammed Sayegh for their critical reading of this manuscript.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by funding from National Institutes of Health Grants GM57664 and GM35633. Additional support was provided by the Julian and Eunice Cohen and Brook Family Funds for Surgical Research.

² Address correspondence and reprint requests to Dr. James A. Lederer, Department of Surgery, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail address: jlederer{at}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.

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

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