PeproTech Inc.
Originally published online as doi:10.1189/jlb.0607371 on October 3, 2007

Published online before print October 3, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0607371v1
83/3/523    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Venet, F.
Right arrow Articles by Ayala, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Venet, F.
Right arrow Articles by Ayala, A.
(Journal of Leukocyte Biology. 2008;83:523-535.)
© 2008 by Society for Leukocyte Biology

Regulatory T cell populations in sepsis and trauma

Fabienne Venet*, Chun-Shiang Chung*, Guillaume Monneret{dagger}, Xin Huang*, Brian Horner*, Megan Garber* and Alfred Ayala*,1

* Division of Surgical Research, Rhode Island Hospital/Brown University, Providence, Rhode Island, USA; and
{dagger} Hospices Civils de Lyon, Immunology Laboratory, Hopital Neurologique, Bron, France

1Correspondence: Division of Surgical Research, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903, USA. E-mail: aayala{at}lifespan.org

ABSTRACT

Sepsis syndrome remains the leading cause of mortality in intensive care units. It is now believed that along with the body’s hyperinflammatory response designated to eliminate the underlying pathogen, mechanisms are initiated to control this initial response, which can become deleterious and result in immune dysfunctions and death. A similar state of immune suppression has been described after numerous forms of severe trauma/injury. Although the evidence for immune dysfunctions after sepsis has grown, much remains to be understood about mechanisms underpinning its development and how it acts to increase the morbid state of the critically ill patient. In this context, although the majority of clinical and basic science conducted so far has focused on the roles of myeloid cell populations, the contribution of T lymphocytes and in particular, of regulatory T cells has been somewhat ignored. The studies presented here support the concept that regulatory T lymphocytes (CD4+CD25+ regulatory, {gamma}{delta}, and NK T cells) play a role in the control of immune responses and are affected by injury and sepsis. This may be related to their capacity to interact with components of the innate and adaptive immune responses and to their ability to be activated nonspecifically by bacterial products and/or cytokines and to regulate through direct cell–cell and/or soluble mediators. It is our hope that a better understanding of the mechanism through which those rare lymphocyte subsets exert such a profound effect on the immune response may help in improving our ability not only to diagnose but also to treat the critically ill individual.

Key Words: CD4+CD25+ • NKT cells • {gamma}{delta} T cells • lymphocyte • severe injury

INTRODUCTION

Sepsis syndrome (i.e., systemic inflammatory response associated with infection: sepsis, severe sepsis, and septic shock ranked by increasing severity) is a common and frequently fatal clinical condition. It represents a major, although largely underappreciated, health care problem worldwide. Although variability exists in the reported incidence of severe sepsis, ~750,000 people are affected by this condition each year in the United States [1 ]. In France, the incidence of severe sepsis has been estimated at nearly 60,000 episodes in 2001 [2 ]. These are highly lethal diseases, and mortality ranges from 20% in sepsis to over 60% in septic shock [3 ]. Severe sepsis itself represents the number one cause of mortality in noncoronary European intensive care units (ICUs) [4 ]. In the United States, it is responsible for as many deaths annually as those from myocardial infarctions [1 ]. It is more worrisome that a 75% increase in the number of patients diagnosed with severe sepsis has been observed over the past two decades. This may be explained partly by the improved care of the increasing number of individuals surviving into their 70s, 80s, and 90s and by the associated co-morbidities of the elderly (i.e., cancer and diabetes) [2 ]. Therefore, as the general population continues to age, the incidence of sepsis is projected to increase significantly in the forthcoming years, leading, for example, to over 1 million cases of severe sepsis in 2020 in the United States alone [1 ].

From a pathophysiological perspective, inflammation was thought initially to play a primal role in the patient’s/host’s response to septic challenge [5 ]. Subsequently, several anti-inflammatory agents were tested for the treatment of sepsis, resulting in the failure of numerous clinical trials. Among other reasons, this failure has been attributed to the inability of animal models to correctly mimic the pathophysiological processes leading to sepsis in humans, as well as to the influence of risk factors such as age, nutrition, gender, and various other co-morbidities in patients [6 ]. However, this also suggests that we still do not adequately understand the underlying pathological process of sepsis, which leads to multiple organ dysfunctions.

Accordingly, it is now generally understood that along with the body’s intense hyperinflammatory response designed to eliminate the underlying pathogen, mechanisms are concomitantly initiated to control this initial response. With respect to this latter effect, a number of investigators have suggested that this counter-regulatory process can, if itself not controlled properly, result in dysfunctions of various immunological responses during sepsis, which in turn, may result in death.

In fact, patients with sepsis present features consistent with a decline in their immune responsiveness (immune deficiency). This state is typified by a loss of delayed-type hypersensitivity (DTH) response and an inability to handle secondary infectious challenges, thus predisposing the patient to an increased chance of developing a nosocomial infection. In particular, their immunological status has been delineated frequently by the observation that leukocytes derived from septic patients produce increased levels of anti-inflammatory cytokines such as IL-10 and exhibit T cell anergy sometimes associated with a shift in the Th cell pattern to a predominant Th2 response, reduced capacity to present antigens associated with the reduced cell surface expression of MHC class II molecules such as HLA-DR, and increased evidence of apoptosis of immune cells [5 ].

Moreover, a similar state of immune suppression has been described after numerous forms of severe injuries such as prolonged/invasive surgical procedures, blunt force trauma, severe burn injury, or hemorrhage. This may explain the predisposition of injured patients to the development of not only sepsis but also secondary/nosocomial infections while hospitalized in ICUs, and sepsis and multiple organ failure are the most frequent complications and the most common cause of death in those patients [7 , 8 ].

However, although the evidence for immune suppression/dysfunction in the septic patient/animal has grown, much remains to be understood about what underpins its development and how it acts to increase the morbid state of the critically ill patient, as well as the extent to which aspects of this immunological change can be addressed as valuable therapeutic and/or diagnostic targets. In this particular context, although the vast majority of clinical and basic science conducted during the last three decades examining the septic process has focused mainly on the roles of myeloid cell populations, the contribution of conventional T lymphocytes and the involvement of subpopulations of regulatory T cells (Tregs) have been somewhat overlooked. As a result of their ability to interact not only with cells of the innate immune system (i.e., macrophages, neutrophils, NK cells) but also with other cells of the adaptive immune system, lymphocytes play a central role in the anti-infectious immune response as effectors and regulators of this response. This has been illustrated in humans and in mice by the observation of a correlation between a depressed adaptive immune response and a decreased resistance to infection several days after injury in patients [9 , 10 ] and by the description of an increased mortality, a decreased bacterial clearance, and most important, a dysregulated, proinflammatory immune response after polymicrobial septic challenge in mice lacking T and B cells [11 , 12 ]. Here, we will outline what is understood about the role of subsets of T cells known as Tregs in the control of immune responses after severe trauma and sepsis.

Treg POPULATIONS

In addition to homeostatic mechanisms of regulation of immune responses intrinsic to antigen activation and differentiation [i.e., TCR affinity, apoptosis (activation-induced cell death), regulation through costimulatory molecule (membrane-associated and soluble) expression, and differentiation of CD4+ T cells into subsets expressing different arrays of cytokines], the idea of an extrinsic mechanism of regulation was first developed in the late 1960s with the identification of a subset of CD8+ T cells bearing suppressor functions [13 ]. In the late 1980s, it was found that these suppressor functions could also be mediated by CD4+ T cells independently of CD8+ [14 ]. A decade later, Sakaguchi et al. [15 ] showed for the first time that the suppression mediated by CD4+ T cells appeared to be the function of the small subset of T cells, which expressed CD4+CD25+. With that, the field of CD4+CD25+ Treg research and more generally, of "regulatory T cell" research was initiated. Since then, the field has experienced extensive growth (Fig. 1 ), as tools were developed to define these minor T cell populations, which possess the capacity to regulate not only classic CD4+ and CD8+ T cells but also B cells, macrophages, and other myelocytes.


Figure 1
View larger version (19K):
[in this window]
[in a new window]

  		Figure 1. Number of articles per year referenced on PubMed from 1975 to 2006 for the search "CD4+CD25+", "NKT cell", "{gamma}{delta}", or "regulatory T cell". Nbr, Number.

Regarding their cellular characteristics and mechanism of activation/action, Tregs can be classified as "innate" or "adaptive" Tregs, based largely on the targets on which they were initially observed to act. Innate Tregs comprise {gamma}{delta} T cells and NKT cells, which are not restricted by classic MHC I or II for activation but rather can be stimulated through a nonclassic MHC molecule or nonspecifically activated during inflammatory processes. Adaptive Tregs include CD8+ Tregs, which were described initially 40 years ago and CD4+ Tregs, i.e., the recently described, naturally occurring CD4+CD25+ Tregs.

In this review, we will describe the role of the adaptive and innate Tregs, which have been, thus far, best characterized in the immune response to injury and sepsis.

ADAPTIVE Treg POPULATION IN TRAUMA AND SEPSIS: CD4+CD25+ Tregs

Although the role of CD8+ Tregs remains to be clarified in vivo, numerous studies have suggested that CD4+ Tregs play a role in human diseases [16 ]. Several subpopulations of CD4+ Tregs have been described based on their process of activation and their mode of development. They have been theoretically separated into "naturally" (native or resident) occurring Tregs, representing a separate lineage of CD4+ T cells, which develop in the thymus, as opposed to "acquired" (inducible) Tregs [also referred to as type 1 Treg (Tr1) and Th3], which appear in response to a particular condition of antigenic stimulations [17 ]. However, as a result of the lack of specific cell surface markers for each of these cell populations, it is often hard to reconcile, determine, and/or separate the roles of these cells in vitro, let alone in vivo.

Tregs were first characterized by the observation that mice deficient in T cells did not develop autoimmune diseases following adoptive transfers of normal syngenic spleens unless these spleen cells were first depleted of CD4+CD25+ T cells [15 ]. Tregs are typically characterized by constitutive and high surface expression of the IL-2R{alpha} chain (CD25; Table 1 ) and by other surface markers, including CTLA4 and the glucocorticoid-induced TNFR (GITR). However, thus far, no cell surface molecules uniquely distinguish Tregs from conventional, activated CD4+ cells or from Tr1/Th3 cells. The CD25 molecule is, for example, expressed on all peripheral CD4+ T cells after antigenic activation. This is also true for CTLA4 and GITR. However, the recent description of forkhead box p3 (Foxp3), a member of the Forkhead family of DNA-binding transcription factors, in Tregs but not in naïve CD4+CD25– cells, has begun to provide important insight into the development and function of these cells [18 ]. It is important that regarding their function, the level of Foxp3 expression can be correlated with the extent of Treg-suppressive activity [18 ]. In addition, it has been described recently that CD127 expression (IL-7R{alpha}) can also help distinguish Tregs from effector T cells, and CD127 is expressed only on the latter [19 ].


View this table:
[in this window]
[in a new window]

  		Table 1. Treg Properties

Tregs are reported to play an essential role in the control of adaptive and innate immune responses. These cells, typically hyporesponsive to antigenic stimulation in vitro, can proliferate in vivo and inhibit effector responses mediated by CD4+ and CD8+ T cells as well as innate immune cells. Four different mechanisms of action have been described so far. In vitro, their suppressor function appears to be dependent on cell–cell contact supposedly via CTLA4, GITR, or cell-associated TGF-β expression, which in turn, may repress IL-2 transcription in the target cells [20 ]. However, Tregs can also release immunoregulatory cytokines, which may contribute to their suppressor function in vivo. In this respect, their cytokine profile includes a variety of well-known immunoregulatory cytokines, i.e., IL-10, TGF-β, IL-4, and IFN-{gamma}. Another mechanism may involve the regulation of tryptophan production in target cells. Fallarino et al. [21 ] reported that in response to the activation of the indoleamine-2,3-dioxygenase by Tregs, the target cell production of tryptophan was suppressed. Finally, Tregs may also be cytotoxic and induce apoptosis in target cells [22 ].

Tregs have been shown to play a role in autoimmunity, cancer, allergy, and transplantation in animal models and in humans. They may also play a role in infectious diseases. In particular, it has been demonstrated recently that LPS can activate Tregs nonspecifically through their expression of TLRs [23 , 24 ]. These cells could thus be directly responsive to "danger-like signals." However, depending on the type of infection, the timing of activation, and the occurrence of an effective, anti-infectious immune response in the host, the effect of Tregs during infectious processes can be beneficial or deleterious [25 , 26 ]. It is most important that the nature of the Treg’s effect on the immune response after an infection has been shown to be dependent on their relative number versus effector T cells.

Considering their immunosuppressive properties and their role in infectious processes, Tregs may have an impact on the injury-induced immune dysfunctions (Table 2 ). In this respect, Monneret et al. [33 ] were the first to demonstrate, in a small cohort of septic shock patients, the presence of an increased percentage of circulating Tregs in blood. This increase was observed immediately after the diagnosis of sepsis; however, it persisted only in nonsurviving patients. These results were confirmed in a subsequent study by this group, which described further that this relative increase was in fact a result of a decrease of the CD4+CD25– circulating T lymphocyte numbers (Treg counterparts) and not so much a change in the absolute Treg count in patients [34 ]. This group reconfirmed these results by also following the change in intracellular Foxp3 levels in the absence of CD127 cell surface expression (unpublished data). Similar results were also observed in trauma patients [35 ] and in mice after polymicrobial septic challenge and stroke [30 31 32 , 36 ]. It is interesting that Decker et al. [37 ] observed that the percentage of Tregs was increased in the peritoneal drainage fluid but not in the peripheral blood of patients after elective abdominal surgery, suggesting that Tregs were being recruited to the site of inflammation during this process.


View this table:
[in this window]
[in a new window]

  		Table 2. CD4+CD25+ Tregs in Murine Models of Injury

Regarding the functional role of Treg in injury-induced, dysregulated immune response, Heuer et al. [27 ] reported that in a mouse model of septic shock induced by cecal ligation and puncture (CLP), the adoptive transfer of Tregs before or 6 h after CLP had a protective and dose-dependent effect on survival. This was associated with a decreased bacterial count and increased TNF-{alpha} production in the peritoneum. Moreover, recipient host T lymphocytes played an essential role in mediating this effect, as the donor Treg effect was abrogated in lymphopenic mice. This was the first study to demonstrate an improvement in survival after Treg administration in a clinically relevant model of sepsis. Using an adoptive transfer/reconstitution experimental approach, Murphy et al. [28 ] demonstrated that a hyper-reactivity of innate immune cells through TLR4 and TLR2 stimulation was observed in lymphocyte-deficient mice after burn injury and that this could be prevented by the adoptive transfer of Tregs. The authors concluded that Tregs might play a role in the immune response after burn by controlling the inflammatory response for which the immune system is primed after serious injury. Together with the work of Heuer et al. [27 ], this intriguingly suggests that Tregs appear to be important effectors of the innate response to sepsis and injury.

With respect to the role of Treg in trauma, Ni Choileain et al. [29 ] observed an increased regulatory activity of Tregs in lymph nodes 7 days after burn, which coincided with injury-induced immune suppression. Although their phenotype was not modified, this suggests that injury primes Tregs for enhanced immune-suppressive Treg activity. Furthermore, the Treg effect was dependent on cell–cell contact and more specifically, on their membrane-bound TGF-β expression. Finally, using in vivo antibody-mediated depletion, the authors observed that Tregs might play a central role in the suppression of Th1 responses after burn injury.

In humans, MacConmara et al. [35 ] described an increased, suppressive potency of Tregs, also at 7 days after injury, when compared with healthy individuals. This suggests that as observed in mice, injury primes circulating human Tregs for enhanced regulatory activity. They observed that human Tregs exerted a powerful influence on Th1 cytokine production (IFN-{gamma}) and CD4+ T cell proliferation in vitro.

Scumpia et al. [30 ], in a murine model of polymicrobial sepsis, observed an increased percentage of Tregs in the spleen in comparison with sham mice. Here, although the absolute number of Tregs was not modified after sepsis, these cells did express a higher level of Foxp3 mRNA and were found to be more efficient than Tregs from sham mice in altering the proliferative capacity of effector T cells. Nevertheless, in this model, the authors were unable to observe any effect on mortality following the in vivo administration of anti-CD25-depleting antibodies prior to the induction of sepsis. In a similar study, Wisnoski et al. [31 ] observed that polymicrobial sepsis was associated with an increased percentage of Tregs in blood and in spleen and here again, with an increase in Foxp3 expression in spleen. This increase was not present in IL-10-deficient mice, whereas it was enhanced in IL-6-deficient mice, suggesting that instead of naturally occurring Tregs, this increase might be related to the development of Tr1 cells. Last, whereas the in vivo anti-CD25-depleting antibody administration prior to sepsis induction was associated with the restoration of IFN-{gamma} and IL-2 production of CD4+ splenocytes, it also had no effect on overall mortality associated with sepsis. In contrast to these two studies, Chen et al. [32 ] have reported recently that the administration of an anti-CD25 antibody 3 days before CLP reduced the early lethality dramatically following this sublethal septic challenge.

Considering the above, it has been observed consistently in patients and in mice that the number of Tregs (percentage and/or absolute count) is modified after severe injury. This might be explained by a reduction in the sensitivity of Tregs to apoptotic stimuli, which appear to affect other leukocytes markedly after injury [38 , 39 ]. However, although the resistance of Tregs to Fas/Fas ligand (FasL) and dexamethasone-induced apoptosis (both known to play a role in injury-induced apoptosis) has been described in vitro [40 , 41 ], this remains to be demonstrated in vivo in the context of sepsis and/or injury. In addition, it is interesting that the study by Chen at al. [32 ] suggests that TNF-{alpha} might also play a role in this process, as they observed that the increase in Treg percentage after CLP was not present in TNFR2-deficient mice. They indicated that Tregs expressed TNFR2 in mice and that TNF-{alpha}, in combination with IL-2, was able to induce Treg proliferation and to increase their suppressive functions.

It is also clear from these studies that the suppressive capacity of Tregs is amplified following sepsis or injury. This suggests that injury primes Tregs for enhanced regulatory capacity. It is therefore surprising that their resultant effect (beneficial or deleterious) on mortality and their actual role in injury-induced immune dysfunction remain unresolved. Indeed, the observed effect of Tregs on injury-induced mortality appears to be highly dependent on the methodology used for its study (Table 2) . Adoptive transfer experiments tend to illustrate a more protective effect of Tregs on injury-induced mortality, whereas anti-CD25 antibody-mediated depletion studies failed in several cases to demonstrate any effect. This might be explained by the inability of anti-CD25 antibodies to actually deplete Tregs or even decrease their suppressive properties [42 , 43 ]. Moreover, as CD25 is not a specific marker for Tregs, these antibodies may also affect other lymphocyte subpopulations, which might be immune-enhancing or valuable to the host, such as activated CD4+CD25– lymphocytes, therefore balancing the effect of Treg depletion. The development of more specific targeting methods, such as small interfering RNA to Foxp3, or transgenic mice selectively deficient for Tregs, such as diphtheria toxin receptor-Foxp3 mice, should provide more reliable tools to study Treg involvement in injury-induced immune dysfunctions going forward. Finally, the fundamental difference between the expansion of this cell population after therapeutic administration of ex vivo-activated Tregs versus the depletion of the endogenously, somewhat expanded, native Tregs in mice can also explain the discrepancy between those two types of studies. In other words, the relative increase of Tregs after severe injury might not be sufficient to allow these cells to show any protective effect during the early response after injury, whereas massive infusion of Treg numbers might un-naturally amplify their effect and thus, render it measurable. Also, ex vivo conditions used to activate Tregs might be different/stronger than those encountered in vivo by Tregs after injury, therefore leading to different regulatory capacities for these cells.

Another explanation might be related to the timing of Treg depletion after injury. Considering the potent immunosuppressive properties of Tregs, these cells may more likely play a role in the late compensatory, anti-inflammatory immune response after injury rather than during the initial injury-induced, proinflammatory immune response. Therefore, the study of the time-selective depletion of Tregs as a post-treatment after the induction of injury in mice would be interesting from a pathologic and potential therapeutic perspective.

Nonetheless, as Treg have been shown to affect adaptive and innate immune responses, the relationship between changes in their numbers or potency and patient outcome after trauma/sepsis is likely to be complex. Based on their known properties and on the results of these various studies in mice, one could postulate a role for Tregs, not only in the decreased Th1 immune responsiveness and T cell anergy observed but also in the suppression of proinflammatory cytokine production described secondarily after severe injury (Fig. 2 ). However, the precise mechanisms (IL-10/TGF-β production, CTLA4 interaction, apoptosis) involved in this process remain to be established. An increased understanding of the alterations in human Treg function in critically ill patients, together with novel animal studies addressing mechanisms underlying this phenomenon, will help to clarify the role of these cells.


Figure 2
View larger version (46K):
[in this window]
[in a new window]

  		Figure 2. Hypothetical mechanism by which Treg, {gamma}{delta}-T, and NKT cell activation, in response to the diverse signal(s) derived from tissue injury and infectious microbial challenge, leads to (A) clearance of the septic challenge (infection) and wound healing, (B) overzealous activation of innate immune via overt NKT and/or Treg cell activation, and (C) immune suppression of classic CD4 Th1 cell via anergic/chronic stimulation of Treg and/or NKT cell effect or loss of {gamma}{delta}-T cells. Solid <-, Potentiates immune response; dotted <-, regulates immune response; Ags, antigens; M{phi}, monocytes/macrophages; DC, dendritic cells; PMN, polymorphonuclear leukocytes.

INNATE Treg POPULATIONS IN TRAUMA AND SEPSIS: {gamma}{delta} AND NKT CELLS

{gamma}{delta} T cells and CD1-restricted NKT cells are usually classified as innate T lymphocytes, in part, as these cells are highly concentrated in tissue beds of various organs as opposed to lymphoid organs, at times comprising up to 50–100% of the T cell population. They are, therefore, often considered to be a component of the first line of defense against infection (Table 1) .

These innate T lymphocytes express germ line-encoded TCR with limited diversity in comparison with the highly diverse adaptive peripheral {alpha}β T lymphocyte TCRs. Therefore, {gamma}{delta} and NKT cells are ready to respond rapidly and expansively to antigenic stimulation through these conserved TCRs. A variety of effector functions has been attributed to these cells in vitro, and developing evidence supports an effector and a regulatory role for {gamma}{delta} T cells and NKT lymphocytes in biological responses to a variety of pathological challenges in vivo [44 , 45 ].

{gamma}{delta} T LYMPHOCYTES

A subpopulation of T cells expressing a TCR composed of {gamma} and {delta} chains associated with the CD3 molecule was discovered more than 20 years ago ({gamma}{delta} T cells) [45 ] (Fig. 1) [46 ]. However, as {gamma}{delta} T cells constitute a minor T cell subpopulation in the peripheral lymphoid organs and in peripheral blood, their physiological functions remained enigmatic for a long time [46 ].

{gamma}{delta} T cells are preferentially localized in mucosal organs containing epithelia such as the skin, lungs, intestines, and genito-urinary tracts. Alternatively, they represent less than 5% of total cells in the mouse spleen and only 1–10% of circulating lymphocytes in humans (Table 1) .

Although the genes coding for {gamma}{delta} TCR possess a great potential for recombination, the actual diversity of the {gamma}{delta} TCR is typically lower than that of the {alpha}β TCR [47 ]. Moreover, the expression of the V{gamma} and V{delta} chains appears to be tissue-specific. For example, in mice, the majority of {gamma}{delta} T cells expressing V{gamma}3/V{gamma}1 chains is found in the epidermis, whereas the reproductive epithelium contains a preponderance of V{gamma}4/V{gamma}1 T cells [48 ].

Similar to NKT cells, {gamma}{delta} T cells recognize antigens presented by MHC class I-like molecules such as CD1. They can also recognize antigens independent of the need for cellular antigen presentation/processing and can be activated nonspecifically/antigen-independently by pathogen-associated molecular patterns (PAMPs), LPS, TNF-{alpha}, or superantigens [47 , 49 , 50 ]. For this reason, {gamma}{delta} T cells are considered to be part of the innate immune system.

Multiple functions have been ascribed to these cells, including immune surveillance, regulation of wound repair, the potentiation of inflammation, and protection from malignancy [51 ]. It is most important that their preferential epithelial localization allows {gamma}{delta} T cells to play a major role as the first line of defense against invading pathogens. They are supposed to play a role in innate anti-infectious immune response through two major properties: cytokine production (IFN-{gamma} and TNF-{alpha}) and cytotoxic capacity (through the perforin/granzyme pathway) [45 , 52 ]. For example, {gamma}{delta} T cells have been observed to have the ability to lyse cells infected by Mycobacterium tuberculosis or Toxoplasma gondii in vitro in humans and in mice [47 ]. In vivo, an increased number of {gamma}{delta} T cells have been observed at the site of infection, in peripheral blood, and in secondary lymphoid organs in humans and in mice after exposure to various pathogens (Herpes viridae, HIV, cytomegalovirus, Salmonella spp., Listeria monocytogenes, M. tuberculosis or Mycobacterium leprae, Plasmodium falciparum, T. gondii) [45 ].

In addition to their capacity to produce various cytokines and act cytotoxic, {gamma}{delta} T cells have been reported to possess immune and nonimmune cell regulatory properties [53 ]. {gamma}{delta} T cells can, for example, regulate macrophage activity through their production of anti-inflammatory cytokines such as IL-10 [54 ]. {gamma}{delta} T cells also appear to play a potentiating role in mediating the production of TNF-{alpha} by monocytes in response to LPS [55 ]. These regulatory properties are also illustrated by the exaggerated {alpha}β T cell-mediated immunopathology observed in the absence of {gamma}{delta} T cells in various murine models of infection, suggesting that {gamma}{delta} T cells also possess immunoregulatory properties directed at {alpha}β T cells [56 ].

Therefore, their role during the infectious process could be different depending on the stage of the immune response where they are activated and depending on the nature of the cellular environment in a given tissue bed [45 ]. It has been postulated that {gamma}{delta} T cells located at mucosal sites are ideally situated to contribute to the initial response to infection [45 ]. They, thus, may act as a link between innate and adaptive immune responses, favoring the development of an {alpha}β T cell-mediated immune response. However, {gamma}{delta} T cells, which are recruited to the site of infection in response to subsequent stimulation over time, may play more of an anti-inflammatory role, taking part in the resolution processes of the immune response, again through their capacity to regulate proinflammatory macrophage functions [45 ].

Considering their preferential tissue/skin localization and their involvement in tissue repair, numerous studies have emphasized a role for {gamma}{delta} T cells in immune dysfunctions occurring after thermal injury (Table 3 ). Major thermal injury is associated, not only with a marked inflammatory component contributing to the development of a systemic inflammatory response and subsequent multiple organ failure but also with immune dysfunctions contributing to the subsequent susceptibility to the development of sepsis in these patients [68 ].


View this table:
[in this window]
[in a new window]

  		Table 3. {gamma}{delta} T Cells in Animal Models of Injury

Using a full-thickness model of burn injury on {gamma}{delta} T cell-deficient mice, Schwacha et al. [57 ] demonstrated that {gamma}{delta} T cells played an important role in post-burn survival, which is associated with a reduced production of proinflammatory cytokines, suggesting that {gamma}{delta} T cells, through the modulation of macrophage activity, may have contributed to the immune dysfunctions observed after thermal injury.

In a similar model, Toth et al. [58 ] subsequently demonstrated that {gamma}{delta} T cells also play a central role in the initiation of neutrophil-mediated tissue damage in the lung and small intestine through their production of chemokines. The authors observed that the number of activated {gamma}{delta} T cells was increased in the circulation early after burn injury, suggesting a causative relationship among {gamma}{delta} T cell activation, chemokine production, and the post-burn inflammatory response. Similarly, Wu et al. [62 ] observed that the apoptosis and proliferation index in the gut as well as TNF-{alpha} production were diminished significantly in {gamma}{delta} T cell-deficient mice after burn injury. However, gut mucosal atrophy associated with burn could not be modified in this model. Finally, using sponges implanted beneath the burn wound site, Daniel et al. [60 ] have shown that {gamma}{delta} T cells are essential to the induction of immune cell infiltration into the s.c. wound sponges and to the development of an inflammatory response at that site. These results, along with the observation that in WT mice, only a small percentage of infiltrating cells are T lymphocytes, are consistent with the concept that resident {gamma}{delta} T cells are important in the orchestration of the infiltrating, inflammatory response but are not the actual infiltrating, immune cells.

{gamma}{delta} T cells might also play a key role in the wound-healing process after severe burn. Jameson et al. [61 ] observed that {gamma}{delta} T cell deficiency was associated with a reduced wound closure and with a decreased keratinocyte proliferation in comparison with WT mice during wound-healing studies. Moreover, the authors demonstrated that the recognition of antigens presented by neighboring epithelial cells following injury was necessary for this process to occur. Furthermore, this process was mediated through the stimulation of the production of keratinocyte growth factors and chemokines by the activated {gamma}{delta} T cells. Moreover, this group demonstrated recently that {gamma}{delta} T cells play a role in macrophage infiltration into the wound site by inducing glycosaminoglycan production by keratinocytes [69 ].

Similarly, in a murine model of burn injury, the absence of {gamma}{delta} T cells was associated with a profound decrease in inflammation and immune cell infiltration at the burn site [59 ]. It is most important that a profound decrease in growth factors such as G-CSF, fibroblast growth factor, and platelet-derived growth factor levels was also observed, suggesting that {gamma}{delta} T cells, through their production and/or regulation of growth factor release, are critical to the healing process after burn injury.

In total, these studies suggest that {gamma}{delta} T cells, located at the burn site and activated after injury, may play a central role, not only in inflammation but also in wound healing after burn. It is most important that the {gamma}{delta} T cells appear to be critical to overall survival following burn injury.

Several studies have also emphasized the degree to which {gamma}{delta} T cells have a role in the immune response after sepsis (Table 3) . In a rat model of polymicrobial sepsis, Berguer and Ferrick [63 ] observed that in the spleen, {gamma}{delta} T cells were vigorously activated early on after CLP, as measured by their overexpression of the IL-2R and their increased IFN-{gamma} production. Similarly, in an ovine model of multiple injuries, the percentage of T cells expressing CD8 and {gamma}{delta} TCRs in the lymphatic compartment was decreased after trauma, whereas it was unaffected in blood [64 ]. The authors proposed that this decrease might be caused by the activation and sequestration of these cells at the site of injury.

Using a murine model of sepsis-induced lung injury, Hirsh et al. [66 ] observed an increased number of {gamma}{delta} T cells in the lung after CLP. However, those cells were dysfunctional, as they exhibited a decreased cytolytic activity and a modified cytokine profile in comparison with {gamma}{delta} T cells purified from sham mice. The authors proposed that {gamma}{delta} T cells recruited to the lung during a septic process might be unable to effectively control the inflammatory process occurring in this organ after sepsis, therefore leading to acute lung injury. Chung et al. [65 ] demonstrated that polymicrobial sepsis was associated with an increased percentage of gut {gamma}{delta} T cells in mice. In this study, early mortality after sepsis was increased in {gamma}{delta} T cell-deficient mice, and peripheral proinflammatory cytokine production was decreased in comparison with WT animals. This suggests that {gamma}{delta} T cells play a critical role in the development of a competent innate/proinflammatory response and most importantly, in survival after sepsis.

However, divergent results have been observed in another study using a similar model of polymicrobial sepsis in {gamma}{delta} T cell-deficient mice. In this study, Enoh et al. [67 ] observed an up-regulation of the marker CD69 on splenic {gamma}{delta} T cells after CLP, suggesting the activation of this T cell subset. However, as opposed to {alpha}β T cell-deficient mice, no difference in mortality, bacterial clearance in the blood, and peritoneal fluid or in proinflammatory cytokine responses in plasma and various tissues could be observed in {gamma}{delta} T cell-deficient mice when compared with WT mice after CLP. The authors concluded that {gamma}{delta} T cells do not play a crucial role in facilitating acute CLP-induced systemic inflammation and injury. One likely explanation for the discrepancy between these two studies might be related to the difference in the acuity of injury between the CLP models. In the first study, using a model of chronic sepsis, mortality after CLP in WT mice was 40% after 4 days, whereas in the second study, a model of acute septic shock, the mortality was 100% after 48 h, suggesting that the role of those cells after sepsis might be different in the earlier versus later phases of the immune response and might also be dependent on the strength of the injury.

In humans, Matsushima et al. [70 ] evaluated the percentage of {gamma}{delta} T cells in peripheral blood of patients with systemic inflammatory response syndrome (SIRS), examining their role in acute phase after severe trauma and sepsis. They observed a decrease in the absolute number of {gamma}{delta} T cells in the peripheral blood of SIRS patients when compared with healthy individuals, but their frequency was only decreased in septic patients. CD69 and HLA-DR expressions (as markers of lymphocytic activation) were increased on {gamma}{delta} T cells in SIRS patients, again suggesting that these cells are activated in the peripheral blood of these patients. This would be in keeping with the concept that {gamma}{delta} T cells may represent early responders to an acute inflammatory insult in humans. These results have been in part confirmed by a second clinical study in septic shock patients. In those patients, the percentage of {gamma}{delta} T cells in peripheral blood was decreased significantly in comparison with healthy individuals [71 ]. However, this was associated with a reduced CD3 expression on their surface, suggesting that circulating {gamma}{delta} T cells might be dysfunctional after septic shock.

Overall, it appears that {gamma}{delta} T cells are activated and may be recruited to the site of inflammation after severe injury or infection. However, this last point remains to be demonstrated in humans. Once localized in tissue (gut, lung, burn wound), {gamma}{delta} T cells (resident and/or recruited) appear to play a major role, not only in the development of a regional tissue immune response through the induction of a local proinflammatory response but also via the recruitment of other immune cells such as neutrophils. Moreover, these cells may play an essential role in mediating tissue repair after burn. However, the functional importance of {gamma}{delta} T cells in contributing to systemic inflammation and global immune responses, as well as their role in more chronic models of injury remain controversial. The observation that {gamma}{delta} T cell deficiency can have divergent effects on mortality in studies of polymicrobial sepsis in mice suggests that the contribution of these cells in the immune response after sepsis may be different in the earlier versus later phases of this response and may also be dependent on the strength of the injury. Therefore, it is unclear at this point how the balance of immunoprotective versus immunopathogenic effects of {gamma}{delta} T cells after injury relates to eventual clinical outcomes and whether these cells can eventually become a potential clinical target in the future.

NKT LYMPHOCYTES

NKT cells were first described in 1987 as murine thymocytes expressing a restricted TCR repertoire in combination with NK cell markers [72 ] (Fig. 1) . Therefore, NKT cells are characterized by the concomitant expression of an {alpha}β TCR associated with the CD3 molecule-like conventional T cells and of NK cell markers such as CD56 and NK1.1 (CD161).

In mice, NKT cells comprise 1–2% of lymphocytes in the spleen, lymph nodes, and peripheral circulation (Table 1) . However, they make up the vast majority of lymphocytes found within the liver [73 ]. As of yet, little information is available regarding their tissue distribution in humans. They may constitute anywhere from 0.02% to 0.2% of the peripheral blood T cells [72 ]. In contrast with conventional effector T cells, the majority of NKT cells express invariant TCR{alpha} chains associated with a variety of β chains, and the most widely studied is the V{alpha}14/J{alpha}281 subset in mice and V{alpha}24/J{alpha}Q in humans [74 ]. As such, these cells are often referred to as "invariant NKT cells". Although the majority of NKT cells express CD4, most of the remaining cells express neither CD4 nor CD8. Furthermore, humans contain a subset of CD8+ NKT cells, which are not present in mice [74 ].

Unlike classic CD4+ or CD8+ {alpha}β TCR effector T cells, which recognize peptide antigens, NKT cells recognize and kill target cells expressing lipid antigens presented by the MHC I-like molecule designated as CD1 [16 ]. NKT cells, thus, recognize antigens usually ignored by conventional effector T cells. The CD1 molecule, like MHC class I molecules, associates with β2 microglobulin to form a complex on the surface of the APCs [73 ]. Humans possess five distinct CD1 genes (a–e), whereas mice only express CD1d. Because of a remarkable degree of evolutionary conservation, mouse CD1d-restricted NKT cells recognize human CD1d and vice versa [73 ]. NKT cells recognize self and foreign glycolipid antigens in the context of CD1d. The nature of the glycolipid recognized by NKT cells was described initially by the observation that {alpha}-galactosylceramide ({alpha}-GalCer), which had been isolated from a marine sponge, could serve to stimulate NKT cells to release large amounts of cytokines (IFN-{gamma}, IL-4, IL-10, IL-13, and TGF-β) rapidly [16 ]. Subsequently, homologues for {alpha}-GalCer, which might serve more realistically as nascent natural antigens such as bacterial-derived antigens, have also been found to stimulate mouse and human NKT cells in vitro. However, the identity of the ligand recognized by NKT cells in vivo remains unclear [75 ]. NKT cells can also be activated by IL-12 or IL-18, even in the absence of TCR stimulation by endogenous antigens presented by CD1 [74 ]. This, along with their expression of various TLRs [76 , 77 ], allows NKT cells to make a rapid response to a wide range of infectious agents, similar to the other cells that participate in the innate immune response.

However, the type of NKT cell response after an antigenic stimulation varies depending on the nature of the antigen and the cytokine background in which it is presented. NKT cells acquire an IL-4- or IFN-{gamma}-producing phenotype depending on their environment. Moreover, the secretion of IFN-{gamma} or IL-4 by the NKT cells during the acute phase of the immune response further influences the progression of Th1 versus Th2 differentiation of effector T lymphocytes, respectively. Besides this, NKT cells, like Tregs and {gamma}{delta} T cells, also possess effector functions, which involve IL-12-mediated perforin and FasL cytotoxic mechanisms. These could be relevant in immunity against intracellular microorganisms and tumors [75 ]. Therefore, NKT cells are able to regulate innate and adaptive immune responses.

NKT cells have been shown to play a role in various clinical conditions, including autoimmunity, allergy, and cancer [16 , 72 ]. Many studies have suggested that an important function for NKT cells might be in serving as a protective brake (particularly in vital organs such as the liver) on the damaging effects of the local inflammatory immune response [44 ]. Alternatively, because of their ability to undergo activation rapidly after antigenic exposure, NKT cells may also play a major role in the first line of defense against invading pathogens [74 , 75 ]. Indeed, a role in pathogen clearance has been described for NKT cells in bacterial, viral, fungal, as well as parasitic infections [74 ]. NKT cells can also inhibit the synthesis of proinflammatory cytokines during an infectious process, thereby preventing inflammation and decreasing immunopathology in some cases [74 ]. However, as shown in malaria or in Chlamydia infections, NKT cells may have detrimental effects, which contribute to infection-induced pathogenesis and injury. In summary, despite being a minority T cell population, there is abundant evidence that NKT cells are important in mice for host response against various bacteria, viruses, and parasites [74 ]. There are also data, especially for viral infections [78 ], indicating that this population could be important in humans as well. What then do we know about the role of these cells in the response to sepsis and injury?

Although the contribution of NKT cells to immune dysfunctions associated with injury has not been studied extensively (Table 4 ), a role for NKT cells has been described recently in the post-burn immune response in a murine dorsal scald-injury model. Faunce et al. [79 ] found that in burned mice pretreated with anti-CD1d-blocking antibodies, NKT cells isolated from the spleen 24 h post-burn increased their production of IL-4 and decreased their production of IFN-{gamma}. This was correlated with the development of injury-induced suppression of T cell immunity. As in other models of injury and sepsis, increased IL-4 appears to direct the immune response in a Th2 direction and actively suppresses the effector functions of lymphocytes and APCs [79 ]. This suggests that suppression of T cell-mediated immunity after burn occurred in part from an active suppression of T cell function mediated by CD1d-restricted, invariant NKT.


View this table:
[in this window]
[in a new window]

  		Table 4. NKT in Murine Models of Injury

Subsequent studies by Palmer et al. [80 ] revealed that NKT cell suppression of T cell immunity is antigen-specific and requires APCs, which express CD1d. Adoptive transfer of purified, CD1d-positive but not CD1d-negative APCs from burn mice was sufficient to suppress a primed T cell response in an injured recipient. CD1d-positive APCs from burn-injured mice were not, however, able to induce immune suppression in the unburned NKT cell-deficient recipient. This data not only indicate that CD1d-expressing APCs and CD1d-restricted NKT cells are required for the development of immune suppression after burn injury but also that direct interaction between these cells leads to the production of immune-suppressive cytokines by NKT cells.

Finally, a recently published study by this group confirmed that the NKT cell subset responsible for T cell suppression after burn was the CD1d-restricted invariant NKT cell population. This study suggested further that NKT cells may suppress peripheral CD4 T cell immunity after burn injury, not only by the direct suppression of CD4 T cell function but also by acting on APCs through the negative regulation of MHC and costimulatory molecule expression [81 ].

A small number of studies have examined the involvement of NKT cells in immune dysfunctions occurring after sepsis. Rhee et al. [82 ] studied the role of NKT cells in immune dysfunctions developing after a polymicrobial septic challenge in mice pretreated with anti-CD1d-blocking antibodies. The percentage of NKT cells in the spleen was higher after CLP compared with control mice, and this increase was blocked by antibody administration. Mice treatment with anti-CD1d-blocking antibodies had a significant increase in survival after CLP in comparison with nonspecific, IgG-treated animals. Moreover, the ablation of NKT cell activation by these antibodies led to the suppression of the increased IL-10 and IL-6 production typically seen after sepsis. This suggests that NKT cells may play a role in the initial phase of immune response occurring after sepsis, thereby decreasing sepsis-induced mortality. Similarly, Tsujimoto et al. [77 ] emphasized a role for NKT cells in the acute proinflammatory immune response to bacterial DNA during polymicrobial sepsis. They observed that TLR9 (receptor for bacterial DNA) expression was increased on liver NKT cells but not NK cells after CLP in mice. The depletion of NK and NKT cells reduced the IFN-{gamma} level after TLR9 stimulation and was associated with a reduced mortality after CLP or TLR9 stimulation in vivo. The authors concluded that in cases of overwhelming and long-term bacterial infection, bacterial DNA might induce a toxic, proinflammatory response in liver NKT cells, thereby causing a detrimental effect on survival after polymicrobial sepsis. In a comparable model, Scott et al. [83 ] observed an increased CD69 expression on NKT cells in blood, spleen, and peritoneal lavage in mice after CLP, suggesting that these cells were activated after sepsis.

Li et al. [84 ] considered the role of NKT cells in renal IRI, which led to an increase in the number of NKT cells in the kidney associated with increased IFN-{gamma} production. This was associated with an augmented neutrophil recruitment. Treatment with anti-CD1d antibodies suppressed the increase in NKT cells and neutrophil recruitment and also protected the kidney from IRI. Similar results were observed in NKT cell-depleted mice and in NKT cell-deficient mice, demonstrating that activation of NKT cells and their production of IFN-{gamma} mediate neutrophil infiltration and renal tissue injury following IRI in mice [84 ].

Overall, the potential of NKT cells to produce pro- and anti-inflammatory cytokines provides them with the capacity to promote or inhibit immune responses following severe injury or sepsis. The results of the few studies published in animal models so far suggest that NKT cells appear to play a deleterious role following injury and might participate in the induction of immune dysfunctions. It is interesting that this is mediated in some instances by their polarization toward IL-4 production, therefore playing a role in the Th2 shift of immune responses observed after injury; and in other cases, the data suggest that NKT cells might also play a role in organ injury as a result of their role in the recruitment of neutrophils. Clearly, further experiments will be necessary to confirm these initial results and determine the extent to which they apply in the critically ill patient.

DISCUSSION/CONCLUSION

The idea that considerably rare populations of regulatory lymphocytes have such profound effects on immunity after severe injury is a relatively new concept in the study of immune consequences of trauma and systemic inflammation. The studies presented here support the concept that regulatory T lymphocytes (including Tregs, {gamma}{delta} T cells, and NKT cells) indeed appear to play a role in the control of immune responses to but are also affected by injury and sepsis (Fig. 2) . This may be explained by their unique capacity to interact with components of the innate and the adaptive immune responses, their ability to be activated nonspecifically by bacterial products and/or cytokines, as well as their capacity to regulate through direct cell–cell and/or soluble mediators. Indeed, it is of significant interest that Treg activation can be affected by molecules released during injury/infection processes, such as PAMPs, or damage-associated molecular patterns, such as heat shock proteins [50 , 85 ]. This can result in a positive or a negative regulation, depending on the patterns of TLRs expressed on a given Treg population and on the cytokine environment encountered at the time of activation. It is most important that this allows Tregs, not only to sense their environment and thus, modify their behavior in response to stress/infection but also to play different roles (i.e., activator and/or inhibitor), depending on the conditions in which they are activated (Fig. 2) .

Moreover, considering that their unique properties are used frequently in a tissue-restricted setting, each Treg population appears to possess a distinctive and nonredundant role in the immune response after injury. Yet, the characterization of this precise role deserves further experimentation, as significant issues exist about their local versus systemic and early versus late responses to injury or sepsis and/or their response to acute versus chronic injury. Last but not least, much work remains to be done in defining if their particular role will be beneficial or detrimental to the host.

Also, thus far, the discussion of Treg populations has largely been held in the absence of the consideration of possible cross-talk between the populations. For example, activated NKT cells are able to modulate Treg functions quantitatively and qualitatively through IL-12-dependent mechanisms, whereas Tregs have been reported to suppress the proliferation, cytokine release, and cytotoxic activity of NKT cells by a cell contact-dependent mechanism [86 , 87 ]. It is important that Tregs and NKT cells share crucial signaling pathways, which could be responsible for their concerted responses [86 ].

Finally, as mentioned in the beginning of our discussion of Treg populations, with the continuing advances in our ability to define these cell populations, we will need to continue to consider the involvement of other novel Treg subpopulations in the development of injury/sepsis-induced immune dysfunctions. One example of this is the need for further information about the role for the recently described Th17 Tregs in immune dysfunctions occurring after polymicrobial sepsis in mice [88 ]. They represent a separate lineage of proinflammatory lymphocytes and may play a role in early host defense against pathogens through the recruitment of neutrophils. It is interesting that they are regulated in an inversely coordinate manner and have opposite functions with Tregs, which are known to be increased after severe injury [89 , 90 ].

In conclusion, although considerably more research is needed to understand how rare (low frequency/number) lymphocyte subsets exert such profound regulation over the immune response, the studies reviewed here have begun to allow us to develop the framework for their immunopathologic contributions in trauma and infection. It is our hope that by better understanding the role of these sentinels of innate and adaptive immunity, we would be better able to diagnose, if not treat, the critically ill.

ACKNOWLEDGEMENTS

Aspects of the work presented here were supported by a grant from the National Institutes of Health, R01 GM46354 (to A. A.), and funds from the Hospices Civils de Lyon (to G. M.). The authors thank Mary Sun for critical reading of the manuscript.

Received June 9, 2007; revised August 24, 2007; accepted September 8, 2007.

REFERENCES

    1
  1. Angus, D. C., Linde-Zwirble, W. T., Lidicker, J., Clermont, G., Carcillo, J., Pinsky, M. R. (2001) Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care Crit. Care Med. 29,1303-1310[CrossRef][Medline]
    2. 2
  2. Brun-Buisson, C., Meshaka, P., Pinton, P., Vallet, B., . EPISEPSIS Study Group (2004) EPISEPSIS: a reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units Intensive Care Med. 30,580-588[CrossRef][Medline]
    3. 3
  3. Brun-Buisson, C., Roudot-Thoraval, F., Girou, E., Grenier-Sennelier, C., Durand-Zaleski, I. (2003) The costs of septic syndromes in the intensive care unit and influence of hospital-acquired sepsis Intensive Care Med. 29,1464-1471[CrossRef][Medline]
    4. 4
  4. Vincent, J. L., Sakr, Y., Sprung, C. L., Ranieri, V. M., Reinhart, K., Gerlach, H., Moreno, R., Carlet, J., Le Gall, J. R., Payen, D., . Sepsis Occurrence in Acutely Ill Patients Investigators (2006) Sepsis in European intensive care units: results of the SOAP study Crit. Care Med. 34,344-353[CrossRef][Medline]
    5. 5
  5. Hotchkiss, R. S., Karl, I. E. (2003) The pathophysiology and treatment of sepsis N. Engl. J. Med. 348,138-150[Free Full Text]
    6. 6
  6. Vincent, J. L., Sun, Q., Dubois, M. J. (2002) Clinical trials of immunomodulatory therapies in severe sepsis and septic shock Clin. Infect. Dis. 34,1084-1093[CrossRef][Medline]
    7. 7
  7. Moore, F. A., Sauaia, A., Moore, E. E., Haenel, J. B., Burch, J. M., Lezotte, D. C. (1996) Postinjury multiple organ failure: a bimodal phenomenon J. Trauma 40,501-510[Medline]
    8. 8
  8. Mannick, J. A., Rodrick, M. L., Lederer, J. A. (2001) The immunologic response to injury J. Am. Coll. Surg. 193,237-244[CrossRef][Medline]
    9. 9
  9. Faist, E., Kupper, T. S., Baker, C. C., Chaudry, I. H., Dwyer, J., Baue, A. E. (1986) Depression of cellular immunity after major injury: its association with post traumatic complications and its restoration with immunomodulatory agents Arch. Surg. 121,1000-1005[Abstract/Free Full Text]
    10. 10
  10. Wood, J. J., Rodrick, M. L., O'Mahony, J. B., Palder, S. B., Saporoschetz, I., D'Eon, P., Mannick, J. A. (1984) Inadequate interleukin 2 production: a fundamental immunological deficiency in patients with major burns Ann. Surg. 200,311-320[Medline]
    11. 11
  11. Shelley, O., Murphy, T., Paterson, H., Mannick, J. A., Lederer, J. (2003) Interaction between the innate and adaptive immune systems is required to survive sepsis and control inflammation after injury Shock 20,123-129[Medline]
    12. 12
  12. Hotchkiss, R. S., Chang, K. C., Swanson, P. E., Tinsley, K. W., Hui, J. J., Klender, P., Xanthoudakis, S., Roy, S., Black, C., Grimm, E., et al (2000) Caspase inhibitors improve survival in sepsis: a critical role of the lymphocyte Nat. Immunol. 1,496-501[CrossRef][Medline]
    13. 13
  13. Gershon, R. K., Kondo, K. (1971) Infectious immunological tolerance Immunology 21,903-914[Medline]
    14. 14
  14. Sakaguchi, S., Fukuma, K., Kuribayashi, K., Masuda, T. (1985) Organ-specific autoimmune diseases induced in mice by elimination of T cell subset. I. Evidence for the active participation of T cells in natural self-tolerance; deficit of a T cell subset as a possible cause of autoimmune disease J. Exp. Med. 161,72-87[Abstract/Free Full Text]
    15. 15
  15. Sakaguchi, S., Sakaguchi, N., Asano, M., Itoh, M., Toda, M. (1995) Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor {alpha}-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases J. Immunol. 155,1151-1164[Abstract]
    16. 16
  16. Jiang, H., Chess, L. (2004) An integrated view of suppressor T cell subsets in immunoregulation J. Clin. Invest. 114,1198-1208[CrossRef][Medline]
    17. 17
  17. Jonuleit, H., Schmitt, E. (2003) The regulatory T cell family: distinct subsets and their interrelations J. Immunol. 171,6323-6327[Free Full Text]
    18. 18
  18. Campbell, D. J., Ziegler, S. F. (2007) FOXP3 modifies the phenotypic and functional properties of regulatory T cells Nat. Rev. Immunol. 7,305-310[CrossRef][Medline]
    19. 19
  19. Banham, A. H. (2006) Cell-surface IL-7 receptor expression facilitates the purification of FOXP3(+) regulatory T cells Trends Immunol. 27,541-544[CrossRef][Medline]
    20. 20
  20. von Boehmer, H. (2005) Mechanisms of suppression by suppressor T cells Nat. Immunol. 6,338-344[CrossRef][Medline]
    21. 21
  21. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bianchi, R., Belladonna, M. L., Fioretti, M. C., Alegre, M. L., Puccetti, P. (2003) Modulation of tryptophan catabolism by regulatory T cells Nat. Immunol. 4,1206-1212[CrossRef][Medline]
    22. 22
  22. Venet, F., Pachot, A., Debard, A. L., Bohe, J., Bienvenu, J., Lepape, A., Powell, W. S., Monneret, G. (2006) Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism J. Immunol. 177,6540-6547[Abstract/Free Full Text]
    23. 23
  23. Sakaguchi, S. (2003) Control of immune responses by naturally arising CD4+ regulatory T cells that express Toll-like receptors J. Exp. Med. 197,397-401[Free Full Text]
    24. 24
  24. Caramalho, I., Lopes-Carvalho, T., Ostler, D., Zelenay, S., Haury, M., Demengeot, J. (2003) Regulatory T cells selectively express Toll-like receptors and are activated by lipopolysaccharide J. Exp. Med. 197,403-411[Abstract/Free Full Text]
    25. 25
  25. Kretschmer, U., Bonhagen, K., Debes, G. F., Mittrucker, H-W., Erb, K. J., Liesenfeld, O., Zaiss, D., Kamradt, T., Syrbe, U., Hamann, A. (2004) Expression of selectin ligands on murine effector and IL-10-producing CD4+ T cells from non-infected and infected tissues Eur. J. Immunol. 34,3070-3081[CrossRef][Medline]
    26. 26
  26. Belkaid, Y., Rouse, B. T. (2005) Natural regulatory T cells in infectious disease Nat. Immunol. 6,353-360[CrossRef][Medline]
    27. 27
  27. Heuer, J. G., Zhang, T., Zhao, J., Ding, C., Cramer, M., Justen, K. L., Vonderfecht, S. L., Na, S. (2005) Adoptive transfer of in vitro-stimulated CD4+CD25+ regulatory T cells increases bacterial clearance and improves survival in polymicrobial sepsis J. Immunol. 174,7141-7146[Abstract/Free Full Text]
    28. 28
  28. Murphy, T. J., Ni Choileain, N., Zang, Y., Mannick, J. A., Lederer, J. A. (2005) CD4+CD25+ regulatory T cells control innate immune reactivity after injury J. Immunol. 174,2957-2963[Abstract/Free Full Text]
    29. 29
  29. Ni Choileain, N., MacConmara, M., Zang, Y., Murphy, T. J., Mannick, J. A., Lederer, J. A. (2006) Enhanced regulatory T cell activity is an element of the host response to injury J. Immunol. 176,225-236[Abstract/Free Full Text]
    30. 30
  30. Scumpia, P. O., Delano, M. J., Kelly, K. M., O'Malley, K. A., Efron, P. A., McAuliffe, P. F., Brusko, T., Ungaro, R., Barker, T., Wynn, J. L., et al (2006) Increased natural CD4+CD25+ regulatory T cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis J. Immunol. 177,7943-7949[Abstract/Free Full Text]
    31. 31
  31. Wisnoski, N., Chung, C. S., Chen, Y., Huang, X., Ayala, A. (2007) The contribution of CD4+ CD25+ T-regulatory-cells to immune suppression in sepsis Shock 27,251-257[CrossRef][Medline]
    32. 32
  32. Chen, X., Bäumel, M., Männel, D. N., Howard, O. M., Oppenheim, J. J. (2007) Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells J. Immunol. 179,154-161[Abstract/Free Full Text]
    33. 33
  33. Monneret, G., Debard, A. L., Venet, F., Bohe, J., Hequet, O., Bienvenu, J., Lepape, A. (2003) Marked elevation of human circulating CD4+CD25+ regulatory T cells in sepsis-induced immunoparalysis Crit. Care Med. 31,2068-2071[CrossRef][Medline]
    34. 34
  34. Venet, F., Pachot, A., Debard, A. L., Bohe, J., Bienvenu, J., Lepape, A., Monneret, G. (2004) Increased percentage of CD4+CD25+ regulatory T cells during septic shock is due to the decrease of CD4+CD25– lymphocytes Crit. Care Med. 32,2329-2331[Medline]
    35. 35
  35. MacConmara, M. P., Maung, A. A., Fujimi, S., McKenna, A. M., Delisle, A., Lapchak, P. H., Rogers, S., Lederer, J. A., Mannick, J. A. (2006) Increased CD4+ CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity Ann. Surg. 244,514-523[Medline]
    36. 36
  36. Offner, H., Subramanian, S., Parker, S. M., Wang, C., Afentoulis, M. E., Lewis, A., Vandenbark, A. A., Hurn, P. D. (2006) Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages J. Immunol. 176,6523-6531[Abstract/Free Full Text]
    37. 37
  37. Decker, D., Tolba, R., Springer, W., Lauschke, H., Hirner, A., von Ruecker, A. (2005) Abdominal surgical interventions: local and systemic consequences for the immune system—a prospective study on elective gastrointestinal surgery J. Surg. Res. 126,12-18[CrossRef][Medline]
    38. 38
  38. Wesche, D. E., Lomas-Neira, J. L., Perl, M., Chung, C. S., Ayala, A. (2005) Leukocyte apoptosis and its significance in sepsis and shock J. Leukoc. Biol. 78,325-337[Abstract/Free Full Text]
    39. 39
  39. Oberholzer, C., Oberholzer, A., Clare-Salzler, M., Moldawer, L. L. (2001) Apoptosis in sepsis: a new target for therapeutic exploration FASEB J. 15,879-892[Abstract/Free Full Text]
    40. 40
  40. Banz, A., Pontoux, C., Papiernik, M. (2002) Modulation of Fas-dependent apoptosis: a dynamic process controlling both the persistence and death of regulatory T cells and effector T cells J. Immunol. 169,750-757[Abstract/Free Full Text]
    41. 41
  41. Chen, X., Murakami, T., Oppenheim, J. J. (2004) Differential response of murine CD4+CD25+ and CD4+CD25– T cells to dexamethasone-induced cell death Eur. J. Immunol. 34,859-869[CrossRef][Medline]
    42. 42
  42. Couper, K. N., Blount, D. G., de Souza, J. B., Suffia, I., Belkaid, Y., Riley, E. M. (2007) Incomplete depletion and rapid regeneration of Foxp3+ regulatory T cells following anti-CD25 treatment in malaria-infected mice J. Immunol. 178,4136-4146[Abstract/Free Full Text]
    43. 43
  43. Kohm, A. P., McMahon, J. S., Podojil, J. R., Begolka, W. S., DeGutes, M., Kasprowicz, D. J., Ziegler, S. F., Miller, S. D. (2006) Cutting edge: Anti-CD25 monoclonal antibody injection results in the functional inactivation, not depletion, of CD4+CD25+ T regulatory cells J. Immunol. 176,3301-3305[Abstract/Free Full Text]
    44. 44
  44. Godfrey, D. I., Hammond, K. J. L., Poulton, L. D., Smyth, M. J., Baxter, A. G. (2000) NKT cells: facts, functions and fallacies Immunol. Today 21,573-582[CrossRef][Medline]
    45. 45
  45. Carding, S. R., Egan, P. J. (2002) {gamma}{delta} T cells: functional plasticity and heterogeneity Nat. Rev. Immunol. 2,336-345[CrossRef][Medline]
    46. 46
  46. Nanno, M., Shiohara, T., Yamamoto, H., Kawakami, K., Ishikawa, H. (2007) {gamma}{delta} T cells: firefighters or fire boosters in the front lines of inflammatory responses Immunol. Rev. 215,103-113[CrossRef][Medline]
    47. 47
  47. Allison, T. J., Winter, C. C., Fournié, J. J., Bonneville, M., Garboczi, D. N. (2001) Structure of a human {gamma}{delta} T-cell antigen receptor Nature 411,820-824[CrossRef][Medline]
    48. 48
  48. Haas, W., Pereira, P., Tonegawa, S. (1993) {gamma}/{delta} Cells Annu. Rev. Immunol. 11,637-685[Medline]
    49. 49
  49. Lahn, M., Kalataradi, H., Mittelstadt, P., Pflum, E., Vollmer, M., Cady, C., Mukasa, A., Vella, A. T., Ikle, D., Harbeck, R., O'Brien, R., Born, W. (1998) Early preferential stimulation of {gamma} {delta} T cells by TNF-{alpha} J. Immunol. 160,5221-5230[Abstract/Free Full Text]
    50. 50
  50. Hedges, J. F., Lubick, K. J., Jutila, M. A. (2005) {gamma} {delta} T cells respond directly to pathogen-associated molecular patterns J. Immunol. 174,6045-6053[Abstract/Free Full Text]
    51. 51
  51. Komano, H., Fujiura, Y., Kawaguchi, M., Matsumoto, S., Hashimoto, Y., Obana, S., Mombaerts, P., Tonegawa, S., Yamamoto, H., Itohara, S., et al (1995) Homeostatic regulation of intestinal epithelia by intraepithelial {gamma} {delta} T cells Proc. Natl. Acad. Sci. USA 92,6147-6151[Abstract/Free Full Text]
    52. 52
  52. Jameson, J., Witherden, D., Havran, W. L. (2003) T-cell effector mechanisms: {gamma} {delta} and CD1d restricted subsets Curr. Opin. Immunol. 15,349-353[CrossRef][Medline]
    53. 53
  53. Bluestone, J. A., Abbas, A. K. (2003) Natural versus adaptive regulatory T cells Nat. Rev. Immunol. 3,253-257[CrossRef][Medline]
    54. 54
  54. Andrew, E. M., Carding, S. R. (2005) Murine {gamma}{delta} T cells in infections: beneficial or deleterious? Microbes Infect. 7,529-536[CrossRef][Medline]
    55. 55
  55. Nishimura, H., Emoto, M., Hiromatsu, K., Yamamoto, S., Matsuura, K., Gomi, H., Ikeda, T., Itohara, S., Yoshikai, Y. (1995) The role of {gamma} {delta} T cells in priming macrophages to produce tumor necrosis factor-{alpha} Eur. J. Immunol. 25,1465-1468[Medline]
    56. 56
  56. Hayday, A., Tigelaar, R. (2003) Immunoregulation in the tissues by {gamma}{delta} T cells Nat. Rev. Immunol. 3,233-242[CrossRef][Medline]
    57. 57
  57. Schwacha, M. G., Ayala, A., Chaudry, I. H. (2000) Insights into the role of {gamma}{delta} T lymphocytes in the immunopathogenic response to thermal injury J. Leukoc. Biol. 67,644-650[Abstract]
    58. 58
  58. Toth, B., Alexander, M., Daniel, T., Chaudry, I. H., Hubbard, W. J., Schwacha, M. G. (2004) The role of {gamma}{delta} T cells in the regulation of neutrophil-mediated tissue damage after thermal injury J. Leukoc. Biol. 76,545-552[Abstract/Free Full Text]
    59. 59
  59. Alexander, M., Daniel, T., Chaudry, I. H., Choudhry, M. A., Schwacha, M. G. (2007) T cells of the {gamma}{delta} T-cell receptor lineage play an important role in the postburn wound healing process J Burn Care Res. 27,18-25
    60. 60
  60. Daniel, T., Thobe, B. M., Chaudry, I. H., Choudhry, M. A., Hubbard, W. J., Schwacha, M. G. (2007) Regulation of the postburn wound inflammatory response by {gamma}{delta} T cells Shock 28,278-283[CrossRef][Medline]
    61. 61
  61. Jameson, J., Ugarte, K., Chen, N., Yachi, P., Fuchs, E., Boismenu, R., Havran, W. L. (2002) A role for skin [{gamma}][{delta}] T cells in wound repair Science 296,747-749[Abstract/Free Full Text]
    62. 62
  62. Wu, X., Woodside, K. J., Song, J., Wolf, S. E. (2004) Burn-induced gut mucosal homeostasis in TCR {delta} receptor-deficient mice Shock 21,52-57[CrossRef][Medline]
    63. 63
  63. Berguer, R., Ferrick, D. A. (1995) Differential production of intracellular {gamma} interferon in {alpha} β and {gamma} {delta} T-cell subpopulations in response to peritonitis Infect. Immun. 63,4957-4958[Abstract]
    64. 64
  64. Aguilar, M. M., Battistella, F. D., Owings, J. T., Olson, S. A., MacColl, K. (1998) Posttraumatic lymphocyte response: a comparison between peripheral blood T cells and tissue T cells J. Trauma 45,14-18[Medline]
    65. 65
  65. Chung, C. S., Watkins, L., Funches, A., Lomas-Neira, J. L., Cioffi, W. G., Ayala, A. (2006) Deficiency of {gamma}{delta} T-lymphocytes contributes to mortality and immunosuppression in sepsis Am. J. Physiol. Regul. Integr. Comp. Physiol. 291,R1338-R1343[Abstract/Free Full Text]
    66. 66
  66. Hirsh, M., Dyugovskaya, L., Kaplan, V., Krausz, M. M. (2004) Response of lung {gamma}{delta} T cells to experimental sepsis in mice Immunology 112,153-160[CrossRef][Medline]
    67. 67
  67. Enoh, V. T., Lin, S. H., Lin, C. Y., Toliver-Kinsky, T., Murphey, E. D., Varma, T. K., Sherwood, E. R. (2007) Mice depleted of {alpha}β but not {gamma}{delta} T cells are resistant to mortality caused by cecal ligation and puncture Shock 27,507-519[CrossRef][Medline]
    68. 68
  68. Schwacha, M. G. (2003) Macrophages and post-burn immune dysfunction Burns 29,1-14[CrossRef][Medline]
    69. 69
  69. Jameson, J., Havran, W. L. (2007) Skin {gamma}{delta} T-cell functions in homeostasis and wound healing Immunol. Rev. 215,114-122[CrossRef][Medline]
    70. 70
  70. Matsushima, A., Ogura, H., Fujita, K., Koh, T., Tanaka, H., Sumi, Y., Yoshiya, K., Hosotsubo, H., Kuwagata, Y., Shimazu, T. (2004) Early activation of {gamma}{delta} T lymphocytes in patients with severe systemic inflammatory response syndrome Shock 22,11-15[CrossRef][Medline]
    71. 71
  71. Venet, F., Bohe, J., Debard, A. L., Bienvenu, J., Lepape, A., Monneret, G. (2005) Both percentage of {gamma}{delta} T lymphocytes and CD3 expression are reduced during septic shock Crit. Care Med. 33,2836-2840[CrossRef][Medline]
    72. 72
  72. Van der Vliet, H. J., Molling, J. W., von Blomberg, B. M., Nishi, N., Kölgen, W., van den Eertwegh, A. J., Pinedo, H. M., Giaccone, G., Scheper, R. J. (2004) The immunoregulatory role of CD1d-restricted natural killer T cells in disease Clin. Immunol. 112,8-23[CrossRef][Medline]
    73. 73
  73. Schneider, D. F., Glenn, C. H., Faunce, D. E. (2007) Innate lymphocyte subsets and their immunoregulatory roles in burn injury and sepsis J. Burn Care Res. 28,365-379[Medline]
    74. 74
  74. Tupin, E., Kinjo, Y., Kronenberg, M. (2007) The unique role of natural killer T cells in the response to microorganisms Nat. Rev. Microbiol. 5,405-417[CrossRef][Medline]
    75. 75
  75. Hansen, D. S., Schofield, L. (2004) Regulation of immunity and pathogenesis in infectious diseases by CD1d-restricted NKT cells Int. J. Parasitol. 34,15-25[CrossRef][Medline]
    76. 76
  76. Emoto, M., Miyamoto, M., Yoshizawa, I., Emoto, Y., Schaible, U. E., Kita, E., Kaufmann, S. H. (2002) Critical role of NK cells rather than V {alpha} 14(+)NKT cells in lipopolysaccharide-induced lethal shock in mice J. Immunol. 169,1426-1432[Abstract/Free Full Text]
    77. 77
  77. Tsujimoto, H., Ono, S., Matsumoto, A., Kawabata, T., Kinoshita, M., Majima, T., Hiraki, S., Seki, S., Moldawer, L. L., Mochizuki, H. (2006) A critical role of CpG motifs in a murine peritonitis model by their binding to highly expressed Toll-like receptor-9 on liver NKT cells J. Hepatol. 45,836-843[CrossRef][Medline]
    78. 78
  78. Biron, C. A., Brossay, L. (2001) NK cells and NKT cells in innate defense against viral infections Curr. Opin. Immunol. 13,458-464[CrossRef][Medline]
    79. 79
  79. Faunce, D. E., Gamelli, R. L., Choudry, M. A., Kovacs, E. J. (2003) A role for CD1d-restricted NKT cells in injury-associated T cell suppression J. Leukoc. Biol. 73,747-755[Abstract/Free Full Text]
    80. 80
  80. Palmer, J. L., Tulley, J. M., Kovacs, E. J., Gamelli, R. L., Taniguchi, M., Faunce, D. E. (2006) Injury-induced suppression of effector T cell immunity requires CD1d-positive APCs and CD1d-restricted NKT cells J. Immunol. 177,92-99[Abstract/Free Full Text]
    81. 81
  81. Tulley, J. M., Palmer, J. L., Gamelli, R. L., Faunce, D. E. (2007) Prevention of injury-induced suppression of T-cell immunity by the CD1D/NKT cell-specific ligand {alpha}-galactosylceramide Shock Epub ahead of print
    82. 82
  82. Rhee, R. J., Carlton, S., Chung, C. S., Lomas, J. L., Cioffi, W. G., Ayala, A. (2003) Inhibition of CD1d activation suppresses septic mortality: a role for NK-T cells in septic immune dysfunction J. Surg. Res. 115,74-81[CrossRef][Medline]
    83. 83
  83. Scott, M. J., Hoth, J. J., Turina, M., Woods, D. R., Cheadle, W. G. (2006) Interleukin-10 suppresses natural killer cell but not natural killer T cell activation during bacterial infection Cytokine 33,79-86[CrossRef][Medline]
    84. 84
  84. Li, L., Huang, L., Sung, S. S., Lobo, P. I., Brown, M. G., Gregg, R. K., Engelhard, V. H., Okusa, M. D. (2007) NKT cell activation mediates neutrophil IFN-{gamma} production and renal ischemia-reperfusion injury J. Immunol. 178,5899-5911[Abstract/Free Full Text]
    85. 85
  85. Sutmuller, R. P., Morgan, M. E., Netea, M. G., Grauer, O., Adema, G. J. (2006) Toll-like receptors on regulatory T cells: expanding immune regulation Trends Immunol. 27,387-393[CrossRef][Medline]
    86. 86
  86. La Cava, A., Van Kaer, L., Shi, F. D. (2006) CD4+CD25+ Tregs and NKT cells: regulators regulating regulators Trends Immunol. 27,322-327[CrossRef][Medline]
    87. 87
  87. Ghiringhelli, F., Ménard, C., Martin, F., Zitvogel, L. (2006) The role of regulatory T cells in the control of natural killer cells: relevance during tumor progression Immunol. Rev. 214,229-238[CrossRef][Medline]
    88. 88
  88. Gao, H., Neff, L. M., Hoesel, L. M., Flierl, M. A., Guo, R-F., Sarma, V., Zetoune, F. S., Ward, P. A. (2007) Protective effects of IL-17 blockade in sepsis Shock 27,A38
    89. 89
  89. McKenzie, B. S., Kastelein, R. A., Cua, D. J. (2006) Understanding the IL-23-IL-17 immune pathway Trends Immunol. 27,17-23[CrossRef][Medline]
    90. 90
  90. Stockinger, B., Veldhoen, M. (2007) Differentiation and function of Th17 T cells Curr. Opin. Immunol. 19,281-286[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Innate ImmunityHome page
S. B. Flohe, S. Flohe, and F. U. Schade
Invited review: Deterioration of the immune system after trauma: signals and cellular mechanisms
Innate Immunity, December 1, 2008; 14(6): 333 - 344.
[Abstract] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
M. M. Wurfel, A. C. Gordon, T. D. Holden, F. Radella, J. Strout, O. Kajikawa, J. T. Ruzinski, G. Rona, R. A. Black, S. Stratton, et al.
Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis
Am. J. Respir. Crit. Care Med., October 1, 2008; 178(7): 710 - 720.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0607371v1
83/3/523    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Venet, F.
Right arrow Articles by Ayala, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Venet, F.
Right arrow Articles by Ayala, A.