Increased CD4⁺ CD25⁺ T Regulatory Cell Activity in Trauma Patients Depresses Protective T_h1 Immunity

Abstract

Objectives:

We recently reported increased CD4⁺ CD25⁺ T regulatory (Treg) activity after burn injury in mice. This study sought to determine if Tregs mediate the reduction in T_H1-type immunity after serious injury in man and if Treg function is altered by injury.

Methods:

Peripheral blood was withdrawn from 19 consenting adult patients (35.1 ± 16.3 years of age) with Injury Severity Scores (ISS) 36.6 ± 13.9 on days 1 and 7 after trauma and from 5 healthy individuals. CD4⁺ T cells were purified and sorted into Treg (CD25^high) and Treg-depleted populations. After activation of cells with anti-CD3/CD28 antibody, production of the T_H1-type cytokine IFNγ, T_H2-type cytokines (IL-4 and IL-5), and the inhibitory cytokine IL-10 was measured using cytometric bead arrays. Treg activity was measured by in vitro suppression of autologous CD4⁺ T cell proliferation.

Results:

All patients survived, 9 (47%) developed infection postinjury. IFNγ production by patient CD4⁺ T cells was decreased on day 1 and day 7, when compared with healthy controls. However, when Tregs were depleted from the CD4⁺ T cells, the IFNγ production increased to control levels. Tregs were the chief source of IL-4 and IL-5 as well as IL-10. Treg suppression of T cell proliferation increased significantly from day 1 to day 7 after injury.

Conclusions:

We demonstrate for the first time that human Tregs are increased in potency after severe injury. Most significantly, Tregs are important mediators of the suppression of T cell activation and the reduction in T_H1 cytokine production found after injury.

Impaired adaptive immune responses after injury are associated with sespis and organ failure in the host. We demonstrate that CD4⁺ CD25⁺ regulatory T cells (Treg) isolated from injured patients play a major role in mediating adaptive immune suppression and that injury increases this suppressive Treg activity.

Serious injury perturbs the immune system resulting in progressive suppression of adaptive immune responses during the first week posttrauma.^1–4 These impaired adaptive responses are thought to contribute significantly to the development of sepsis and the multiorgan dysfunction syndrome (MODS), which are the leading causes of death in patients who survive the initial injury.¹ Clinical and experimental evidence demonstrates that the increased susceptibility of these patients to infection can, at least in part, be attributed to reduced T-helper-1 (T_H1)-type immune responses and a skewing toward increased T_H2-type or counterinflammatory immune reactivity.^5–12 These observations underlie the idea that major injury triggers the development of a counterinflammatory adaptive immune response that may help control excessive innate immune inflammatory reactivity.^2,4,13

A subpopulation of CD4⁺ T cells that constitutively express the alpha chain of the IL-2 receptor (CD25) has been identified as playing a critical role in controlling autoimmune diseases, transplant tolerance, and infectious and antitumor immune responses.^14–19 These CD4⁺ CD25⁺ cells are therefore designated as T regulatory cells (Tregs). Tregs were first identified in the mouse where they constitute 6% to 10% of lymph node and splenic CD4⁺ T cell populations.¹⁴ Human Tregs are homologous to mouse Tregs but are present in smaller numbers in human peripheral blood than in murine lymph nodes or spleens. They are a less distinct population due to an overlapping spectrum of cells expressing CD25. Only the brightest 1% to 2% (CD25^high) after isolation by fluorescence-activated cell sorting (FACS) demonstrate suppressor activity.^20,21 It is thought that the primary function of this unique CD4⁺ T cell subset is to help control inappropriate T cell responses by actively suppressing CD4⁺ T cell reactivity to self or nonself antigens.²² Tregs can also suppress T_H1 immune responses and promote a shift toward T_H2 counterinflammatory, immunosuppressive responses.²¹ It is this later function of Tregs that suggested to us that they may be involved in the host response to injury.

Recent evidence from this laboratory demonstrates that murine Treg activity is enhanced after burn injury.²³ Tregs from burn-injured mice at day 7 showed greater suppression of in vitro T cell proliferation and greater suppression of T_H1-type cytokine production than Tregs from sham-injured animals. However, the contribution of Tregs to the altered immune function following injury in humans remains to be determined. In this study, we investigated the function of human Tregs after severe trauma. First, we compared changes in the percentage of circulating CD25^high CD4⁺ T cells (Tregs) among healthy control volunteers and trauma patients. Second, we separated CD4⁺ T cells into subpopulations of Treg and Treg-depleted cells. We then compared CD3/CD28-induced T_H1- and T_H2 cytokine production profiles within these subpopulations in samples from controls and patients to examine injury-associated changes. Finally, we quantified Treg activity by measuring the ability of these cells to suppress autologous naive CD4⁺ T cell proliferation and compared the suppressive potency of these cells at early and late time points after injury and with healthy controls. We demonstrate for the first time that Tregs play a major role in suppressing protective T_H1 cytokine responses after injury. Furthermore, we show that injury induces up-regulation of Tregs potency.

METHODS

Patients

Peripheral blood samples were withdrawn from 19 consenting adult trauma patients with ISS >20 admitted to the Burn/Trauma Intensive Care Unit at the Brigham and Women's Hospital. Patients with major head trauma were excluded along with patients who received immunosuppressive drugs for comorbid conditions. Patients who were unable take adequate oral nourishment received supplemental enteral nutrition to ensure an adequate calorie intake by way of the gastrointestinal tract. All patients were carefully monitored for hyperglycemia, which was rigorously controlled by intravenous insulin infusion. Blood samples in all cases were drawn within the first 24 hours after injury and from the 13 patients who remained in hospital at 7 days after injury. On each occasion, control blood samples were simultaneously drawn from age- and sex-matched healthy volunteers. The study was carried out in compliance with NIH guidelines and with the approval of the Brigham and Women's Hospital Human Research Committee. Clinical data were recorded on patient demographics, trauma etiology, and outcome measures (Table 1). Systemic inflammation (SIRS), sepsis (SIRS with infection), and organ dysfunction were documented using established criteria.^13,24

TABLE 1. Patient Characteristics

Reagents

Culture medium for in vitro studies consisted of RPMI 1640 supplemented with 5% heat-inactivated fetal calf serum, 1 mmol/L glutamine, penicillin/streptomycin/Fungizone, 10 mmol/L HEPES buffer, 100 μmol/L nonessential amino acids, and 2.5 × 10⁻⁵ mol/L 2-mercaptoethanol, all purchased from Gibco Invitrogen Corporation (Grand Island, NY). Carboxy-fluorescein diacetate, succinimidyl ester (CFSE) was obtained from Molecular Probes, Invitrogen (Carlsbad, CA). Magnetic beads coated with anti-CD3 and anti-CD28 Ab for cell culture stimulation were purchased from Dynal, Invitrogen. Magnetic bead sorting kits and columns were purchased from Miltenyi Biotec (Auburn, CA). Human T_H1/T_H2 cytometric bead arrays (CBA) kits were obtained from BD Biosciences (San Diego, CA).

Blood Collection and Processing

Twenty-milliliter blood samples were collected in heparinized Vacutainer CPT tubes (Becton Dickinson, Franklin Lakes, NJ) and centrifuged for 30 minutes at 1500g. The peripheral blood mononuclear cells (PBMC) interface was carefully removed by pipetting, washed once in culture medium, and centrifuged for a further 20 minutes at 100g to remove contaminating platelets. PBMCs were resuspended in culture medium and counted in trypan blue counting solution to determine viability. PBMCs were always >95% viable.

Surface Staining

An aliquot of 2 × 10⁶ PBMC was removed to examine the percentage of CD25⁺CD4⁺ and CD25^highCD4⁺ T cells in the collected samples. PBMCs were incubated for 15 minutes at 4°C with combined rat and mouse immunoglobulin (CalTag Laboratories, Burlingame, CA) to prevent nonspecific binding with Fc receptors. Two-color staining with fluorescein isothiocyanate-labeled anti-CD25 and allophycocyanin (APC)-labeled anti-CD4 was done by incubating for 30 minutes with antibodies along with relevant isotype controls (Pharmingen, San Diego, CA). Samples were washed and fixed for 20 minutes with 100 μL of 0.3% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) at 4°C. Fifty thousand events were collected by flow cytometry, with gating based on forward versus side scatter using a FACScaliber instrument (Becton Dickinson) and CD4⁺ CD25⁺, CD4⁺ CD25high and CD4⁺ CD25⁻ percentages computed using the CELLQuestPro software program.

CD4⁺ T Cell Purification and Subpopulation Sorting

Magnetic bead sorting (Miltenyi human CD4⁺ isolation kit) was used to prepare CD4⁺ cells by negative selection according to manufacturer's guidelines. The CD4⁺ cells were always >95% pure when assessed by flow cytometry using APC-labeled anti-CD4 staining. The CD4⁺ cells were then stained for surface CD25 using phycoerythrin (PE)-conjugated anti-CD25 antibody (BD Pharmingen) and incubated with anti-PE conjugated magnetic beads. CD4⁺ cells were sorted by positive selection according to affinity. The strongly positive CD25 cells (CD25^high) were removed by passage through a low-affinity column followed by a second high-affinity column to remove all CD25 expressing cells. Four CD4⁺ T cell populations were collected: CD25^high (Tregs), Treg-depleted, CD25⁻, and unsorted CD4⁺ cells.

In Vitro Cytokine Production Assays

Each CD4⁺ T cell population (unsorted CD4⁺, Treg, Treg-depleted, and CD25⁻) was counted using Trypan Blue and 1 × 10⁵ cells in 200 μL volume were incubated in round-bottom 96-well plates (Costar, Corning, NY) in the presence of beads coated with anti-CD3/CD28 antibodies (2 × 10⁵ beads/well, Dynabeads) or culture medium alone at 37°C in 5% CO₂ for 48 hours. The supernatant was removed and cytokine bead array technology used to determine levels of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), interleukin (IL)-10, IL-6, IL-4, and IL-2 in 20 μL samples (BD CBA Human Th Kit).

T Cell Suppression Assays

CD4⁺ CD25⁻ cells isolated using the magnetic bead sorting technique were labeled with CFSE. This was done by incubating the cells with 2.5 μmol/L CFSE/10⁷cells in 1 mL of warm PBS for 10 minutes before washing and further incubation in culture medium for 30 minutes. These CFSE-labeled autologous cells were added at a concentration of 5 × 10⁴ cells/well and incubated along with 5 × 10⁴ cells from each CD4⁺ T cell population (unsorted CD4⁺, Treg, Treg-depleted and CD25⁻) in round-bottom 96-well plates in the presence beads coated with anti-CD3/CD28 antibodies or of culture medium alone at 37°C in 5% CO₂ for 72 hours. Samples were washed and fixed for 20 minutes with 100 μL of 0.3% paraformaldehyde in PBS (pH 7.4) at 4°C. Twenty-five thousand events were collected by flow cytometry, with gating based on APC-labeled CD4⁺ cells and the intensity of CFSE measured within each cell. The pattern of progressive reduction in CFSE intensity that follows the redistribution of the CFSE among daughter cells after cell division was analyzed using the CELLQuestPro software program to determine the percentage of proliferation among labeled CD4⁺ CD25⁻ T cells.

Statistics

Observations on controls and patients at days 1 and 7 were compared by ANOVA. The χ² test was used to evaluate differences in the incidence of complications between patient groups. The Prism 3.0 software (GraphPad, San Diego, CA) was used for all calculations. P < 0.05 was considered significant.

RESULTS

Circulating CD25⁺ and CD25^high CD4⁺ T cells

The absolute numbers of CD4⁺ T cells fell in patients early after injury and remained low on day 7 (Table 2). Within the CD4⁺ T cell population, the percentage of cells expressing CD25 early after injury was not increased above the level seen in healthy controls. However, by day 7, a significant increase in CD25⁺ CD4⁺ T cell percentage was observed (Fig. 1B). The percentage of CD25^high CD4⁺ T cells, which represent the human Treg population, was also unchanged relative to controls at 1 day postinjury but was significantly increased by day 7 after injury (Fig. 1B). These findings indicate that injury induces a significant increase in the percentage of CD25-expressing CD4⁺ T cells by 7, but not 1 day after injury. Moreover, we demonstrate that there is an injury-induced increase in the percentage of CD25^high Tregs.

TABLE 2. Hematology Values (10³/μL)

FIGURE 1. Changes in circulating CD25⁺ CD4⁺ T cells after injury. A, Representative FACS plots showing CD25 staining on circulating human CD4⁺ T cells. CD25⁺ cells were measured by comparison with the isotype Ab (negative control) staining and represent a heterogenous group of cells. CD25^high cells were determined by gating on only the brightly stained cells. B, The percentage of CD25⁺ and CD25^high staining found in CD4⁺ T cells from patients at days 1 and 7 postinjury as well as healthy controls. There were similar percentages of CD25⁺ and CD25^high cells on day 1 after injury compared with controls; however, both populations showed significant increases by day 7 (P < 0.01 and P < 0.05, respectively).

Tregs Suppress T_H1-Type Cytokine Production After Injury

Injury led to a significant depression in the ability of CD4⁺ T cells to produce T_H1-type cytokines as demonstrated by the dramatic reduction in anti-CD3/CD28 Ab stimulated IFNγ production at 1 and 7 days after injury. However, removal of Tregs permitted the remaining CD4⁺ T cells to produce IFNγ at levels comparable to those produced by similarly prepared CD4⁺ T cells from healthy control volunteers (Fig. 2). Purified Tregs from controls or injured patients produced little IFNγ but were found to be the primary source of the T_H2-type cytokines, IL-4 and IL-5. Following injury, IL-10 production by CD4⁺ T cells was lower than similarly prepared control CD4⁺ T cells. However, when subpopulations were examined, it appeared that injury reduced IL-10 production by Treg-depleted cells but not by Tregs. Indeed, Tregs showed a trend toward increased IL-10 production and were the dominant source of IL-10 within the CD4⁺ cell population in patients 7 days posttrauma. Finally, we found that injury induced significant increases in IL-4 production by Tregs, while having no effect on CD4⁺ T cells depleted of Tregs. Taken together, these results suggest that injury causes significant changes in stimulated cytokine production profiles and, in particular, Tregs acquire increased ability to suppress IFNγ production and to produce higher levels of T_H2-type cytokines.

FIGURE 2. T_H1- and T_H2-type cytokine production by CD4⁺ T cell populations. IFNγ production by CD4⁺ T cells after anti-CD3/CD28 Ab stimulation was reduced after injury; however, when Tregs were removed, normal levels of this T_H1-type cytokine were produced by the Treg-depleted CD4⁺ T cells. Tregs produced most IL-4 and IL-5, and the IL-4 production by day 7 Tregs was significantly greater than controls. IL-10 production in response to this stimulus was significantly reduced in patient CD4⁺ cells; however, Tregs were insensitive to this shutdown and showed increased production by day 7. #P < 0.01, control CD4⁺ versus patient CD4⁺. *P < 0.05, patient CD4⁺ day 7 versus patient Treg-depleted cells. **P < 0.01, control Tregs versus patient day 7 Tregs (all ANOVA).

Treg Suppression of CD4⁺ T Cell Proliferation

To test whether injury might alter the antiproliferative function of human Tregs (CD25^high CD4 T cells), Tregs were purified from peripheral blood samples taken from patient and healthy control volunteers and tested for their ability to suppress the proliferation of autologous CD25⁻ CD4⁺ T cells. To accomplish this, we adapted a mouse Treg assay approach that measures the dilution of CFSE staining intensity upon cell division. A representative FACS plot illustrates that CFSE-stained human CD25⁻ CD4⁺ T cells proliferate well in response to anti-CD3/CD28 Ab stimulation with 79% of the cells undergoing one or more rounds of cell division (Fig. 3). We also show in Figure 3 that the addition of CD25^high CD4⁺ T cells reduced the level of proliferating cells to 22%, while the addition of Treg-depleted CD4⁺ T cells failed to suppress CD25⁻ CD4⁺ T cell proliferation with 86% of the cells showing one or more rounds of proliferation. Having established our ability to accurately measure human Treg activity, we performed Treg assays on circulating CD25^high CD4⁺ T cells from healthy volunteers and trauma patients at 1 and 7 days after injury. As shown in Figure 4, we found that injury induced a significant increase in the potency of Tregs at day 7 after injury (P < 0.05) above the level observed in similarly prepared Tregs from healthy controls or from day 1 trauma patients. To our knowledge, these findings represent the first demonstration that injury promotes a progressive increase in Treg activity in trauma patients.

FIGURE 3. In vitro suppression of CD4⁺ T cell proliferation by CD25^high CD4⁺ T cells (Tregs). Representative CFSE FACS plots showing in vitro proliferation. The autologous CD25− CD4⁺ T cells were first labeled with the fluorescent dye, CFSE. The CFSE then distributed evenly among daughter cells, giving peaks on the histogram as the signal decayed with each round of proliferation. Each plot shows events gated on labeled cells only. The control plots indicate proliferation of unstimulated and stimulated naive cells without any additional T cells. Tregs caused a significant suppression of labeled cell proliferation (from 79% to 22%); however, the addition of Treg-depleted cells failed to suppress anti-CD3/CD28-induced cell proliferation (86% proliferating cells).

FIGURE 4. Injury induces increased Treg-mediated suppression of CD4⁺ T cell proliferation. Suppression was defined as the difference between proliferation of naive CFSE-labeled T cells in the presence of CD25⁻ T cells and of Tregs. There was a significant injury-associated increase in the potency of Treg suppression on day 7 after injury (P < 0.05, ANOVA).

Correlation of Clinical Outcome With Circulating T Cell Numbers and Cytokine Expression

Nine of the 19 patients with severe trauma developed sepsis (as diagnosed by the development of SIRS with infection) with most infections originating in the respiratory system (78%). The development of sepsis was associated with the severity of injury. Six of the 8 patients with ISS scores >35 developed sepsis, whereas only 3 of 11 with ISS scores ≤35 had a septic episode (P = 0.04, χ² test). There was no association between initial white blood cells, lymphocyte, or CD4⁺ T cell numbers and sepsis. A lower percentage of CD4⁺ CD25⁺ T cells on day 1 was found to be associated with later sepsis (P = 0.025); however, the patients destined to develop sepsis did not show any percentage difference of CD25^high T cells on day 1.

There was no association between sepsis and IFNγ production by CD4⁺ T cells on day 1. Interestingly, there was an association between both IL-10 and IL-4 production by Tregs on day 1 and later sepsis (P = 0.01 and P = 0.03, respectively, χ² test).

DISCUSSION

Trauma and its associated tissue injury perturb both innate and adaptive immunity. Following the initial innate immune-driven inflammatory response to injury, a compensatory anti-inflammatory response develops associated with suppression of adaptive immunity.¹³ This sequence of events is thought to have very important clinical consequences since sepsis and MODS are the most frequent causes of death in patients who survive the first 24 hours.² We report here a mechanism that appears to play a major role in the development of suppressed adaptive immunity after severe traumatic injury. We demonstrate, for the first time, that Tregs inhibit T cell proliferation after injury in trauma patients and disrupt protective T_H1-type cytokine production. We also provide the first evidence that injury primes circulating human Tregs for enhanced regulatory activity.

In mouse studies, naive CD4⁺ T cells expressing the alpha chain of the IL-2 receptor (CD25) represent a distinct population of potent regulatory cells with immunosuppressive function. However, in humans, prior T cell exposure to environmental or self antigens results in a higher percentage of activated CD25-expressing CD4⁺ T cells. This complicates the identification and purification of Tregs. However, there is good evidence that the fraction of circulating CD4⁺ T cells that express the highest level of cell-surface CD25 represent the human Treg population.^20,21,25 These CD25^high cells constitute approximately 1% to 2% of the total CD4⁺ T cell population. Furthermore, they express a transcription factor called forkhead box P3 (FOXP3) that has been shown to be critical for the development and function of these regulatory cells in humans and in mice.²⁶ The CD25^high CD4⁺ T cell population display Treg function, while the low intensity staining CD25⁺ population does not mediate Treg activity.^19,21 In agreement with these observations, we found that there is a continuous spectrum from low to high cell-surface CD25 staining intensity in circulating CD4 T cells and that the shown Treg activity was found only in the CD25^high subpopulation (Figs. 1A, 4). Moreover, we found by a real-time PCR assay that the CD25^high CD4⁺ T cell population expresses significantly higher levels of FOXP3 mRNA than the CD25^low CD4⁺ T cells (data not shown).

A primary objective of this study was to determine if traumatic injury alters the percentage of both CD25⁺ and CD25^high CD4⁺ T cells in peripheral blood. We found a significant increase in the percentage of CD25⁺ CD4⁺ T cells on day 7 but not on day 1 after injury. This finding is consistent with a previous report that was published prior to the identification of Tregs.²⁷ In that study, the authors demonstrated that cell-surface CD25 expression on CD4⁺ T cells increased by 5 days after injury and remained elevated for at least 2 weeks. At the time, it was presumed that CD25 expression was increased due to injury-induced CD4⁺ T cell activation. However, we demonstrate here that the increased percentage of circulating CD25⁺ CD4⁺ T cells in our trauma patients was attributable to both T cell activation and a significant increase in the percentage of CD25^high CD4⁺ cells. This increase in circulating CD4⁺ CD25^high T cells may represent Treg expansion or possibly migration of these cells from immunologically active sites influenced by the injury. These are important mechanistic issues that can be studied in more detail using animal models of injury.

There are numerous reports indicating that T cell-mediated immune function is impaired after injury.^5,7,28 It is thought that suppressed or altered CD4⁺ T cell responses contribute to the development of postinjury immune suppression. Some of these injury-induced changes in T cell activity include reduced proliferation in response to polyclonal stimulation and a shift toward increased T_H2-type cytokine production with an accompanying loss of T_H1-type responses.⁸ In this report, we examined the effect of Tregs on CD4⁺ T cell responses and found they exert a powerful influence on both T_H1-type cytokine production and CD4⁺ T cell proliferation. Our data demonstrate that, when Tregs are removed from the CD4⁺ T cell population from controls or patients, the remaining CD4⁺ T cells produce higher levels of the signature T_H1-type cytokine, IFNγ (Fig. 3). Of note, CD4⁺ T cell cultures prepared from trauma patients, which contained Tregs, showed significantly lower level IFNγ production than controls, but when Treg cells were depleted, IFNγ production levels increased above those observed in Treg-depleted CD4⁺ T cells from controls (Fig. 3). These observations suggest that Tregs from trauma patients can more potently suppress T_H1-type cytokine production than those from controls and further supports the hypothesis that injury enhances Treg potency.

Purified CD4⁺ T cells, Treg-depleted CD4⁺ T cells, and Tregs from healthy control volunteers and trauma patients were stimulated with anti-CD3/CD28 antibody-coated-beads to profile T_H1- and T_H2-type cytokine production. We chose this T cell stimulation approach because it allows for high-level T cell activation in an antigen presenting cell-independent fashion. Increased T_H2 cytokine production by CD4⁺ T cells from trauma or burn patients has been reported previously, but to our knowledge, this is the first study to examine injury-induced changes in cytokine production by isolated Tregs and CD4⁺ T cells prepared from trauma patients. We found that Tregs were the primary source of the T_H2-type cytokines, IL-4 and IL-5. Under the same experimental conditions, activated human Tregs failed to produce significant levels of IFNγ. Most importantly, we observed that Tregs prepared from trauma patients produced significantly higher levels of T_H2-type cytokines than similarly prepared Tregs from healthy controls. An additional finding was that Tregs from day 7 trauma patients produced significantly higher levels of IL-10. Since IL-10 has been shown to suppress inflammatory responses, this result suggests that human Tregs may also help control the development of SIRS following major trauma. Higher IL-10 production by human Tregs may also contribute to the heightened Treg activity seen at 7 days after injury reported here (Fig. 4) and in prior studies performed in a mouse burn injury model.²³

Perhaps the most important observation reported in this study is that Treg-suppressive activity was significantly higher at 7 days after injury when compared with the activity of Tregs from healthy controls or patients at day 1 after injury. We have recently reported a similar injury-induced increase in functional Treg potency by mouse CD25⁺ CD4⁺ T cells.²³ These murine Tregs were contact dependent and required TGFβ₁ for in vitro suppression. TGFβ₁ surface expression was increased after injury, suggesting a potential pathway used during injury to increase Treg potency. Interestingly, the murine Treg changes were observed principally in the lymph nodes draining the site of injury, suggesting that the influence of injury on Treg activity is compartmentalized. We have not yet examined CD25⁺ CD4⁺ T cells from mouse blood to test whether circulating mouse Tregs also demonstrate increased Treg activity.

The clinical consequences of altered Treg activity in critically injured patients are at present unclear. We did not see a relationship between Treg percentage or activity and sepsis or MODS. However, this study lacks sufficient power, at present, to determine with an acceptable level of confidence the relationship between Treg activity and these later complications. Larger numbers of patients are needed to overcome the many confounding variables that exist given the heterogeneity of trauma patients. We are also investigating the relationship between injury-induced changes in Treg activity and later sepsis using a technique of in vivo depletion of Tregs in our mouse burn-injury model.

Since Tregs have been shown to control both adaptive immunity and innate inflammatory responses, the relationship between changes in their numbers or potency and patient outcome after trauma is likely to be complex.^23,29 However, the findings reported here demonstrate that Tregs play a significant role in the development of suppressed adaptive immune function after major injury. Removal of Tregs was sufficient to permit the remaining T cells to function normally, as measured by normal levels of IFNγ production and normal levels of proliferation. An increased understanding of the alterations in human Treg function in injured patients, together with controlled animal studies addressing mechanisms underlying this phenomenon, will help clarify the role of these cells in patient outcome after serious injury.

Discussions

Dr. Ronald V. Maier (Seattle, Washington): This current study is a logical extension of the previous work that has been done in Dr. Mannick's lab over many years by he and his colleagues elucidating the cellular molecular mechanisms underlying immunosuppression following severe injury. This study is unique in that it is the first time the impact of T regulatory T cells on the immune dysfunction has been studied in the human system following injury.

As was mentioned, the T regulatory cell is the past forgotten T suppressor cell that could not be isolated and therefore fell into disrepute until the new biomarkers were identified, the CD4⁺CD25⁺ cell markers, that allowed it to be isolated and interrogated specifically to identify its effects. This T cell has been shown to prevent autoimmunity in animal models. It infiltrates tumors and facilitates tumor growth escaping the normal immune system. It is being tested to prevent GVHD and transplant rejection. And now in this current study it has been shown to explain in large part the immune suppression seen following severe injury. This study demonstrates a decrease in gamma interferon that is protective and the concomitant increase in the anti-inflammatory IL-4, IL-5 and IL-10 producing the concomitant immune dysfunction.

I have several questions that may help elaborate their findings. First, what causes the upregulation of these T regulatory cells? Is it the reactive oxygen intermediates that are generated during reperfusion after severe illness and total body ischemia? Or is it the massive release of tissue antigens from injured tissue recognized as a potential autoimmune stimulus and upregulation of the T regulatory cells prevents a destructive autoimmune response?

Secondly, expression of the transcription factor of Fox-P-3 messenger RNA was identified by the authors, as a marker for this T regulatory cell. Do they believe that this is a predetermined subpopulation of T cells from the time they are created, or whether they are a phenotypic overexpression of a subpopulation of the overall T cell population? Does the excessive stress of severe injury drive a portion of the cells to this level of activity, or are they a select subpopulation that are unique and separate from the rest of the T cells?

How long does the response last? In the animal models it peaks at about 7 to 10 days. Is the time course similar in humans, does it peak at 7 days and then resolve at later time points regarding the T regulatory upregulation?

Others have shown that a major component of the T regulatory cell effect is cell-cell contact dependent and is presumably due to surface expression of TGF beta and the activation of the SMAD pathways. Is the same effect found in vitro using supernatants from the T regulatory cell populations or is cell-cell contact necessary to express the optimal inhibitory effect? Does selective antibody inhibition of the various immune components, such as IL-4, IL-5, IL-10 or TGF beta, identify which is most important for the effect they saw.

Lastly, a philosophic issue. While the data support what we all believe is the current and correct paradigm for immunosuppression following severe injury, the question really remains as to whether this is merely an epiphenomena. Is the increase in T regulatory cells merely a biomarker or surrogate for severity of injury and disregulation of the immune system? As the authors point out in their manuscript, there really was no direct correlation between the level of the T regulatory cell activation state and the risk for subsequent secondary infections in the patients. Is this really a causal mechanism or just a correlative process that occurs after severe injury?

Dr. Malcolm P. MacConmara (Boston, Massachusetts): If I may start with your final question first, regarding the importance of Tregs in post-injury sequelae. This study is based upon 19 patients and lacks sufficient power, at present, to determine with an acceptable level of confidence the relationship between Treg activity and later complications. Many potentially confounding variables exist given the heterogeneity of trauma patients. We will continue to increase the numbers in order to determine the full importance of Treg function on the outcome. We are also investigating how Tregs affect outcome using model of Treg depletion in a mouse injury model.

As regards the method of injury-induced upregulation of Tregs, we are limited by size of sample permitted. In this study we collect 20 mls of blood which yields approximately 300,000 Tregs after purification. The patient assays carried out thus far have not included assays to interrogate the mechism of upregulation, however we are working with our animal model to determine potential pathways. In the mouse we find that these Tregs are contact dependent. Furthermore, murine Treg TGFβ₁ surface expression is increased after injury and antibodies to TGFβ₁ can, at least in vitro, reverse the enhanced suppression of T cell proliferation. Currently, we are investigating the effect that blocking TGFβ₁ in vivo in the mouse has on enhanced Treg function after injury. We are also planning additional patient studies to determine whether cell contact and surface TGFβ₁ are equally important in the activity of circulating human Tregs.

Another potential mechanism which we are currently investigating is the lipopolysaccharide (LPS) receptor, TLR4. Tregs are the only CD4⁺ T cells known to express TLR4 on their surface and their activity has been shown to increase after LPS treatment in vitro. In support of a potential role in the injury-induced Treg changes we have found the surface expression of TLR4 increases after injury.

Our time course was limited to 1 and 7 days, although again we plan to follow patients out further, where possible. You may have noted that in spite of an average ISS of 36, 6 of our 19 patients were discharged by day 7 and following these patients beyond one week will present logistic challenges as well as potentially introducing bias.

The question of natural versus induced regulatory cells is a difficult one to answer. FOXP3 messenger RNA was found predominantly in the CD25^high group. We would like to use a novel FOXP3 reporter mouse in order to see if the injury actually induces cells to become FOXP3 positive and regulatory in nature or whether it is natural Tregs that proliferate in response to the injury.

Dr. David N. Herndon (Galveston, Texas): I would like to congratulate, as Dr. Maier did, Dr. MacConmara and Dr. Mannick and their associates for the first time demonstrating in humans that T regulatory cells are increased after injury and are important mediators of the suppression of T cell activation and the reduction of Th1 cytokine production.

The authors imply that reduction of interferon gamma represents a suppression of protective immunity, but since correlation with outcome, as just discussed, is as yet elusive, do you predict that increased T regulatory cell activity is good or bad? Have you sought to modulate this response in your mouse studies, or do you intend to?

As you mentioned, 9 of the 19 patients developed sepsis. A lower percent of CD4, CD25⁺ cells on day one was associated with later development of sepsis. IL-10 and IL-4 production was also modulated. But was it predicted? And in what way? Can you speculate any causative relationship between these activities?

Finally, I would like to comment—it is most interesting that the CD25 high cells constitute only 1% to 2% of the total CD4⁺ T cells. The low intensity CD25 cell population does not mediate, it appears, T regulatory cell activity. Is the increase in CD4⁺ CD25 high cells from expansion, activation, or migration from immunologically privileged areas in those populations?

Dr. Malcolm P. MacConmara (Boston, Massachusetts): The relationship between Tregs and immune dysfunction after injury is complex, and consequently so too is the ultimate outcome of the patient. We tried to determine whether or not Tregs are good for you using a depletion model in the mouse. One of the difficulties encountered has been the dual effect of Tregs. So, while they appear deleterious by suppressing the adaptive system, they also seem to benefit the host after injury by suppressing excessive innate inflammatory responses. It seems that simple removal of Tregs does not benefit the host and instead selectively modifying the Treg suppression of adaptive responses may be required to provide benefit.

As far a predictive role for IL-10 levels and Treg numbers in patients who later develop sepsis, again with the small numbers we can't demonstrate a comment accurately on causal relationships at present. With greater patient numbers we hope perform multivariate analysis. The circulating Tregs in trauma patients appear thus far to be the natural Tregs described in the literature. We are unable to determine whether the increased numbers of CD4⁺CD25^high cells represent altered migration or Treg proliferation.

Dr. David B. Hoyt (San Diego, California): Very nice study. Congratulations on finding the suppressor cell. My question, though, goes back to Dr. Maier, what the etiology of this might be. Can you take injured patients, serum or plasma, or injured mouse lymph exudate, mix that with normal T cells and engender expression of these Tregs? If you can, can you then take it out of that environment and culture it normally and lose that ability or can you do that with osmotic stress? What is the origin of these cells? What is the stimulation for them to come out?

Dr. Malcolm P. MacConmara (Boston, Massachusetts): The culture of injured plasma with normal T cells is an interesting approach. As yet, we haven't carried out these experiments using either human or mouse tissue.

The enhanced Treg function is cell contact dependent in the mouse and not related to humoral factors. However, enhanced Treg activity was most prominent in lymph nodes draining the site of injury and therefore injured tissue would appear to play an important role in upregulating Treg function. We would like to examine circulating mouse Tregs to determine if similarity exists with our finding in patients.