Fabienne Venet

Chun-Shiang Chung

Hakim Kherouf

Anne Geeraert

Françoise Poitevin

Julien Bohé

Alain Lepape

Alfred Ayala

Guillaume Monneret
email: guillaume.monneret@chu-lyon.fr

Increased circulating regulatory T cells (CD4 + CD25 + CD127 -) contribute to lymphocyte anergy in septic shock patients

Keywords: Septic shock, Anergy, CD4 + CD25 +, CD127, Regulatory T cells, Foxp3

Purpose-Sepsis syndrome represents the leading cause of death in intensive care unit. Patients present features consistent with a decline in immune responsiveness potentially contributing to mortality. We investigated whether CD4 + CD25 + regulatory T cells (Treg) participate in the induction of lymphocyte anergy after sepsis.

Introduction

Sepsis syndrome (i.e. systemic inflammatory response associated with infection) is a common and frequently fatal clinical condition representing a major although largely under-appreciated health care problem worldwide. These are highly lethal conditions, with mortality ranging from 20% in sepsis to over 60% in septic shock [START_REF] Brun-Buisson | EPISEPSIS Study group. EPISEPSIS: a reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units[END_REF][START_REF] Brun-Buisson | The costs of septic syndromes in the intensive care unit and influence of hospital-acquired sepsis[END_REF]. More worrying is a 75% increase in the number of patients diagnosed with severe sepsis observed over the past two decades [START_REF] Brun-Buisson | EPISEPSIS Study group. EPISEPSIS: a reappraisal of the epidemiology and outcome of severe sepsis in French intensive care units[END_REF], which is projected to lead to a significant increase in the incidence of sepsis in the forthcoming years; potentially culminating in over a million cases of severe sepsis in 2020 in the USA alone [START_REF] Angus | Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care[END_REF].

Pathophysiologically, inflammation was initially thought to play a primal role in the patient's response to septic challenge [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF]. This culminated in the testing of many anti-inflammatory agents, unfortunately resulting in the failure of numerous clinical trials and therefore suggesting that we still do not adequately understand the underlying pathological process of sepsis. 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 both innate and adaptive immune responses during sepsis [increased levels of anti-inflammatory cytokines such as interleukin-10 (IL-10), reduced capacity to present antigens associated with the decline of cell surface expression of major histo-compatibility complex (MHC) class II molecules such as HLA-DR and increased evidence of immune cell apoptosis], which in turn, may contribute to death [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF][START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. In particular, the occurrence of a state of lymphocyte anergy has been described after sepsis [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF][START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. This has been known for long and is illustrated by the observation of the loss of delayed-type hypersensitivity reaction to recall skin tests antigens in patients [START_REF] Christou | The delayed hypersensitivity response and host resistance in surgical patients. 20 years later[END_REF][START_REF] Rode | Lymphocyte function in anergic patients[END_REF][START_REF] Heidecke | Selective defects of T lymphocyte function in patients with lethal intraabdominal infection[END_REF][START_REF] Roth | Susceptibility to programmed cell death in T-lymphocytes of septic patients: a mechanism of lymphopenia and Th2 predominance[END_REF]. Moreover, lymphocyte anergy has been shown to be associated with mortality and with the development of secondary infections after sepsis [START_REF] Christou | The delayed hypersensitivity response and host resistance in surgical patients. 20 years later[END_REF][START_REF] Rode | Lymphocyte function in anergic patients[END_REF][START_REF] Heidecke | Selective defects of T lymphocyte function in patients with lethal intraabdominal infection[END_REF][START_REF] Roth | Susceptibility to programmed cell death in T-lymphocytes of septic patients: a mechanism of lymphopenia and Th2 predominance[END_REF]. However, the mechanisms responsible for this phenomenon are still not completely understood.

In this context, the role of a recently described population of naturally occurring regulatory T cells (Treg) has been emphasized [START_REF] Venet | Regulatory T cell populations in sepsis and trauma[END_REF]. In human, these cells are supposed to play a role not only in cancer, autoimmunity, or allergy, but also in infectious diseases [START_REF] Baecher-Allan | Human regulatory T cells and their role in autoimmune disease[END_REF][START_REF] Belkaid | Regulatory T cells and infection: a dangerous necessity[END_REF]. Treg are characterized by the constitutive expression of CD25 (interleukin-2 receptor α) and of intracellular Foxp3 (a transcription factor known to play a fundamental role in Treg's regulatory properties) and possess potent immunosuppressive properties affecting both lymphocyte and antigen presenting cell (APC) responses [START_REF] Baecher-Allan | Human regulatory T cells and their role in autoimmune disease[END_REF]. We have previously demonstrated that human Treg increased Fas/Fas Ligand (FasL)-mediated monocyte apoptosis after stimulation with endotoxin [START_REF] Venet | Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism[END_REF]. They can also induce neutrophil apoptosis after LPS challenge [START_REF] Lewkowicz | Lipopolysaccharide-activated CD4+CD25 + T regulatory cells inhibit neutrophil function and promote their apoptosis and death[END_REF], suggesting that these cells might play a role in the development of innate immune dysfunctions observed after sepsis. However, whether Treg can affect lymphocyte anergy in sepsis is unknown.

To investigate this, the whole blood percentage of Treg (assessed by using a newly described and reproducible gating strategy based on the CD4, CD25, CD127 and FOXP3 flow cytometric measurement [START_REF] Seddiki | Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells[END_REF][START_REF] Liu | CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells[END_REF]) was correlated with the proliferative response of mononuclear cells purified from septic shock patients after mitogenic stimulation. In a second time and because of the difficulty to perform such experiments in patients, a mouse model of polymicrobial septic challenge was used to test whether the downregulation of Treg's specific transcription factor Foxp3 was associated with any restoration of lymphocyte proliferation after sepsis.

Results have been partly presented during the 2008 Experimental Biology Meeting, San Diego, CA [START_REF] Venet | Anergy in septic patients: correlating the increased percentage of circulating CD4+CD25+CD127regulatory T cells with a decline in lymphocyte proliferation[END_REF].

Patients and methods

Septic shock patients admitted to the Intensive Care Units at Lyon-Sud University Hospital were prospectively included. The diagnosis of septic shock was based on criteria of the ACCP/ SCCM [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis[END_REF]. Detailed methods are available in the Electronic Supplementary Material (ESM).

Results

CD4 + CD25 + CD127 -lymphocytes express Foxp3 in healthy individuals and septic shock patients and possess regulatory properties

As cell purification is often not feasible in critically ill patients because of the low blood volume available, we investigated Treg phenotype in whole blood from three septic shock patients and five healthy individuals. As observed previously on purified healthy volunteers' cells (See additional results in the ESM), CD4 + CD25 + lymphocytes did not express CD127 either in healthy individuals or in septic shock patients (Fig. 1, left panel). Moreover, using a 4-color flow cytometry staining including intracellular Foxp3 expression measurement, we observed that the large majority of these cells expressed Foxp3 both in healthy volunteers and in septic patients (Fig. 1, right panel). Therefore, the combined expressions of CD4 and CD25 and the lack of expression of CD127 appeared adequate for the characterization of circulating Treg in whole blood of either healthy volunteers or septic shock patients.

The percentage of CD4 + CD25 + CD127 -lymphocytes is significantly increased in septic patients versus healthy individuals

A total of 30 septic shock patients and 17 healthy individuals were prospectively included in the study. The demographic and clinical characteristics for the septic population are presented in Table 1. Patients were monitored once between 3 and 7 days after the onset of shock. The expression of HLA-DR on their monocytes was significantly reduced in comparison with healthy individuals (60 ± 5 vs. >90%, respectively) in accordance with the occurrence of a state of immunoparalysis in these patients [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF].

We observed a significantly increased percentage of CD4 + CD25 + CD127 -cells in patients in comparison with healthy volunteers (12.0 ± 1.0 vs. 6.8 ± 0.3%, respectively, P < 0.001, Fig. 2a). This was associated with a decreased number of total CD4 + lymphocytes (410 ± 54 vs. 779 ± 63 cells/µl, respectively, P < 0.001; Fig. 2b); as well as of CD4 + CD25 + CD127 -cells (42 ± 5 vs. 52 ± 4 cells/µl, P < 0.05; Fig. 2c) and CD4 + CD25 -CD127 + lymphocytes (368 ± 50 vs. 728 ± 60 cells/µl, P < 0.001; Fig. 2d). However, the extent of the decline in CD4 + CD25 + CD127 -cell's absolute count was significantly less than that of CD4 + CD25 - lymphocyte population (19 vs. 49%, respectively, P < 0.001).

The increase in CD4 + CD25 + CD127 -lymphocytes is correlated with a decreased lymphocyte proliferation in septic shock patients

In order to test whether this increased percentage was associated with a functional decline observed in lymphocyte proliferation in patients, the proliferative response of PBMCs purified from a subset of patients and healthy individuals was measured after stimulation with mitogens. This proliferative response was then correlated with the percentage of Treg detected in whole blood using the CD127 gating strategy.

Not surprisingly, we observed that the mitogen-induced proliferative response was severely decreased in patients in comparison with healthy individuals (Fig. 3). Most importantly, we found that the extent of this decrease was closely correlated with the increase in CD4 + CD25 + CD127 -cell's percentage measured in whole blood (Fig. 3, r = -0.943, P < 0.005 for PHA; r = -1.000, P < 0.005 for ConA and r = -0.829 for PWD). This suggests that the increase in Treg percentage may play a role in the decreased proliferative response observed in patients after septic shock.

The decreased Foxp3 expression is associated with a restoration of cell proliferation after polymicrobial sepsis in mice

In order to investigate the involvement of Treg in the decreased cell proliferation observed after sepsis, we tested the effect of reducing Foxp3 expression, a transcription factor known to play a fundamental role in Treg's regulatory properties [START_REF] Campbell | FOXP3 modifies the phenotypic and functional properties of regulatory T cells[END_REF], on lymphocytes' proliferative response purified from the spleen of mice after polymicrobial sepsis (CLP).

We first confirmed that in our model, as observed in septic patients, the percentages of CD4 + CD25 + Foxp3 + (10.1 ± 0.5 vs. 8.4 ± 0.5%, P < 0.05), and CD4 + CD25 + CD127 -cells (11.9 ± 0.5 vs. 9.1 ± 0.6%, P < 0.001) were significantly increased 24 h after CLP in comparison with sham animals. Also, as observed in patients, the proliferative response of splenic lymphocytes harvested from mice after CLP was reduced in comparison with sham animals (Fig. 4b).

Most importantly, in those animals, the transfection of splenic lymphocytes with Foxp3 siRNA, which decreased the percentage of Treg (Fig. 4a), was able to restore the proliferative response of murine lymphocytes to a level not different from sham animals, whereas in this model, transfection with control siRNA had no such effect (Fig. 4b).

Discussion

While numerous of clinical trials aiming at treating septic shock have been completed so far, beyond the modest effect of activated protein C, we have still to observe a significant reduction in the number of sepsis-related deaths [START_REF] Angus | Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care[END_REF]. Meanwhile, there is now growing recognition that, in addition to an overwhelming pro-inflammatory immune response responsible for the shock phase of sepsis, septic patients appear to also rapidly manifest immune dysfunctions consistent with a state of immune suppression [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF][START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. This state is typically characterized by defects in both innate and adaptive immune responses [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF][START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. However, while 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 T lymphocytes, in particular the involvement of sub-populations of regulatory T cells as components of this adaptive immune response, has been somewhat overlooked [START_REF] Venet | Regulatory T cell populations in sepsis and trauma[END_REF].

Here we used a newly described gating strategy to monitor the change in circulating Treg in septic shock patients as compared to healthy individuals [START_REF] Banham | Cell-surface IL-7 receptor expression facilitates the purification of FOXP3(+) regulatory T cells[END_REF]. Moreover, since Treg have been described to be able to decrease T cell's proliferative response [START_REF] Sakaguchi | Foxp3+CD25+CD4+ natural regulatory T cells in dominant self-tolerance and autoimmune disease[END_REF] and since lymphocyte anergy has long been known to occur after sepsis, based on the observation of the loss of delayed-type hypersensitivity reaction to recall skin tests antigens in patients [START_REF] Meakins | Delayed hypersensitivity: indicator of acquired failure of host defenses in sepsis and trauma[END_REF], we investigated the involvement of Treg in the occurrence of lymphocyte anergy after septic shock.

In agreement with our previous reports [START_REF] Monneret | Marked elevation of human circulating CD4+CD25+ regulatory T cells in sepsis-induced immunoparalysis[END_REF][START_REF] Venet | Increased percentage of CD4+CD25+ regulatory T cells during septic shock is due to the decrease of CD4+CD25lymphocytes[END_REF], we observed a significant increase in the percentage of circulating CD4 + CD25 + CD127 -Treg in septic shock patients in comparison with healthy individuals. This was associated with a decrease in the number of CD4 + lymphocytes. Consequently, as previously hypothesized [START_REF] Venet | Increased percentage of CD4+CD25+ regulatory T cells during septic shock is due to the decrease of CD4+CD25lymphocytes[END_REF], we observed that this increase was mainly due to a decrease in CD4 + CD25 -CD127 + T cells number and not so much a rise in the Treg cell count after sepsis (Fig. 2). Once again, this suggested that Treg may be less sensitive to the potentiated apoptotic process that has been reported in patients after septic shock [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF][START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. Although their resistance to Fas/FasL and dexamethasone-induced apoptosis (both known to play a role in injury-induced apoptosis) has been described in vitro [START_REF] Banz | Modulation of Fas-dependent apoptosis: a dynamic process controlling both the persistence and death of regulatory T cells and effector T cells[END_REF][START_REF] Chen | Differential response of murine CD4+CD25+ and CD4+CD25 -T cells to dexamethasone-induced cell death[END_REF], this remains to be demonstrated in vivo in the context of sepsis.

A similar increase in Treg percentage has been observed in trauma patients [START_REF] Macconmara | Increased CD4+CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity[END_REF] and in mice after polymicrobial septic challenge and stroke [START_REF] Scumpia | Increased natural CD4 +CD25+ regulatory T cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis[END_REF][START_REF] Offner | Splenic atrophy in experimental stroke is accompanied by increased regulatory T cells and circulating macrophages[END_REF][START_REF] Chen | Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells[END_REF][START_REF] Wisnoski | The contribution of CD4+CD25+ T-regulatorycells to immune suppression in sepsis[END_REF]. However, despite several observations of an increased regulatory capacity of Treg after trauma/sepsis in patients and in mice (i.e., increased Foxp3 expression and increased suppressive potency after trauma/sepsis in comparison with controls) [START_REF] Macconmara | Increased CD4+CD25+ T regulatory cell activity in trauma patients depresses protective Th1 immunity[END_REF][START_REF] Scumpia | Increased natural CD4 +CD25+ regulatory T cells and their suppressor activity do not contribute to mortality in murine polymicrobial sepsis[END_REF][START_REF] Wisnoski | The contribution of CD4+CD25+ T-regulatorycells to immune suppression in sepsis[END_REF] suggesting that injury primes Treg for enhanced regulatory activity, the actual role of these cells in lymphocyte anergy let alone in mortality/ morbidity after severe injury remains unclear [START_REF] Venet | Regulatory T cell populations in sepsis and trauma[END_REF].

Anergy is defined as a state of non-responsiveness to antigen. T cells are anergic when they fail to proliferate or secrete cytokines in response to their specific antigens. Lymphocyte anergy after sepsis is illustrated by the observation of the loss of delayed-type hypersensitivity reaction to recall skin tests antigens in patients. This has long been described and is known to be associated with mortality and with the development of secondary infections [START_REF] Christou | The delayed hypersensitivity response and host resistance in surgical patients. 20 years later[END_REF][START_REF] Rode | Lymphocyte function in anergic patients[END_REF][START_REF] Heidecke | Selective defects of T lymphocyte function in patients with lethal intraabdominal infection[END_REF][START_REF] Roth | Susceptibility to programmed cell death in T-lymphocytes of septic patients: a mechanism of lymphopenia and Th2 predominance[END_REF]. In accordance, we observed that the proliferative response of patients' PBMCs after mitogenic stimulation was decreased in comparison with healthy individuals. A similar state of anergy has been described in patients after major surgery [START_REF] Hensler | Distinct mechanisms of immunosuppression as a consequence of major surgery[END_REF], severe burn and trauma [START_REF] Puyana | Both T-helper-1-and T-helper-2type lymphokines are depressed in osttrauma anergy[END_REF][START_REF] Pellegrini | Relationships between T lymphocyte apoptosis and anergy following trauma[END_REF][START_REF] Bandyopadhyay | Negative signaling contributes to T-cell anergy in trauma patients[END_REF]. Anergy is associated with a diminished production of cytokines such as IL-2 and IFN-γ by peripheral blood mononuclear cells [START_REF] Lederer | The effects of injury on the adaptive immune response[END_REF] as well as with a decreased expression of activating co-receptors such as monocyte HLA-DR and CD86 or lymphocyte CD28 and CD3 and an increased expression of inhibitory co-receptors (CTLA-4 or PD-1) [START_REF] Bandyopadhyay | Negative signaling contributes to T-cell anergy in trauma patients[END_REF][START_REF] Manjuck | Decreased response to recall antigens is associated with depressed costimulatory receptor in septic critically ill patients[END_REF]. Therefore, the development of lymphocyte anergy in sepsis is likely to be a multifactorial process involving soluble factors (IL-10), cell-cell interactions (increased expression of inhibitory receptors associated with a decreased expression activating co-receptors on lymphocytes, decreased antigen-presentation by APCs) and increased apoptosis [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. Also, intrinsic defects in intracellular lymphocyte activation pathways are likely to occur after sepsis as shown by Bandyopadhyay et al. in trauma patients [increased levels of inhibitory signal transduction proteins (c-Cbl, and SHP-1) concomitant with depressed phosphorylation of activating signal kinases (Erk, Zap70, and CD3ε)] [START_REF] Bandyopadhyay | Negative signaling contributes to T-cell anergy in trauma patients[END_REF]. T-cell receptor complex phosphorylation and activation of the interleukin-2 pivotal transcriptional complex protein CREB (cAMP response element-binding) were also simultaneously depressed [START_REF] Bandyopadhyay | Negative signaling contributes to T-cell anergy in trauma patients[END_REF]. Most importantly and from a clinical perspective, the severity of this state of anergy has been demonstrated to correlate with poor outcome and the increased occurrence of infectious complications and subsequent multiorgan failure in injured patients [START_REF] De | Induction of global anergy rather than inhibitory Th2 lymphokines mediates posttrauma T cell immunodepression[END_REF].

In this context, we observed a close negative correlation between the increase in the percentage of Treg measured in patients' whole blood and the decline in their proliferative response to mitogens. This suggests that Treg may play a role in the development of lymphocyte anergy after septic shock. In order to investigate this hypothesis, we used the murine model of cecal ligation and puncture (CLP) to induce polymicrobial sepsis in mice. This model is supposed to be the most clinically relevant murine model of sepsis [START_REF] Hubbard | Cecal ligation and puncture[END_REF]. We first recapitulated in septic mice the characteristics observed in patients (i.e. increased percentage of Treg and decreased splenic lymphocytes' proliferative response to mitogens). We then observed that the downregulation of Foxp3 expression in those cells using ex vivo transfection with Foxp3 targeting siRNA was associated with the restoration of their proliferative response to mitogens, therefore confirming the role of Treg in the decreased proliferative response observed after sepsis. These results are concordant with the deleterious role of Treg in the regulation of immune responses described in various animal models of infection (i.e. excessive control of the effector immune response, development of a bystander immunosuppression) [START_REF] Belkaid | Regulatory T cells and infection: a dangerous necessity[END_REF]. Along with our previous study and others demonstrating that Treg increase monocyte and neutrophil apoptosis after LPS stimulation [START_REF] Venet | Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism[END_REF][START_REF] Lewkowicz | Lipopolysaccharide-activated CD4+CD25 + T regulatory cells inhibit neutrophil function and promote their apoptosis and death[END_REF], this indicates that Treg may produce deleterious effects on both arms (innate and adaptive) of the immune response after septic shock and thus may represent central players in the regulation of the developing immune dysfunctions encountered in patients.

From a methodological point of view, the present results demonstrate that the measurement of Treg frequency may represent a simple and valuable surrogate marker of lymphocyte anergy usable on a routine basis for the monitoring of sepsis-induced immune dysfunctions. Indeed, this study shows the validity of the 3-color flow cytometry staining (CD4/CD25/CD127) for the standardized routine monitoring of Treg in patients. While Foxp3 has been described as a specific marker for T cells with regulatory properties [START_REF] Ziegler | FOXP3: of mice and men[END_REF], the intracellular staining procedure required for its determination (4 h for completion) renders this marker unsuitable for a routine analysis of these cells in patients. Thus, until recently, Treg were solely characterized in human whole blood based on the combined expression of CD4 and CD25. Among CD4 + CD25 + cells, only the CD25 high cells are thought to possess regulatory properties in humans [START_REF] Baecher-Allan | MHC class II expression identifies functionally distinct human regulatory T cells[END_REF]. However, since CD25 is not a specific marker for Treg, as it is also over-expressed on activated CD4 + T cells and since no method has been agreed on as a reproducible way to select this CD25 high Treg population, this leads to a high variability in the published percentage of Treg measured in healthy individuals (ranging, for example, from less than 1% to more than 10% of CD4 + T lymphocytes) [START_REF] Baecher-Allan | Human CD4+CD25+ regulatory T cells[END_REF]. This becomes even more problematic in a pathological context, e.g., type 1 diabetes, where it has led to contradictory/-reproducible measured variations of Treg frequency in patients as opposed to controls, thus rendering the determination of their putative involvement in the pathophysiology of the disease hard to reconcile [START_REF] Tree | A mini meta-analysis of studies on CD4+CD25+ T cells in human type 1 diabetes: report of the Immunology of Diabetes Society T Cell Workshop[END_REF]. In this context, the use of the combined expression of CD25 and CD127 (a chain of the IL-7 receptor) has been recently proposed for accurately identify Treg in human whole blood and as a biomarker for human Treg cells in diseases (e.g. type 1 diabetes, organ transplant rejection…) [START_REF] Seddiki | Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells[END_REF][START_REF] Liu | CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells[END_REF][START_REF] Codarri | Expansion and tissue infiltration of an allospecific CD4+CD25+CD45RO +IL-7Ralphahigh cell population in solid organ transplant recipients[END_REF]. CD127 expression is suggested to allow an unambiguous flow-cytometry based distinction of Treg and activated CD4 + T cells [START_REF] Banham | Cell-surface IL-7 receptor expression facilitates the purification of FOXP3(+) regulatory T cells[END_REF]. Here we have confirmed these results since we observed that CD4 + CD25 + CD127 -cells (either purified or in whole blood) expressed a substantially higher percentage of Foxp3 + lymphocytes and possessed regulatory properties. The accuracy of this 3-color staining for the monitoring of Treg in human blood is also further illustrated by the small variation in the percentage of Treg measured in healthy individuals in different studies (6.35% of CD4 + T cells for Seddiki et al. [START_REF] Seddiki | Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells[END_REF], 7-8% for Liu et al. [START_REF] Liu | CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells[END_REF], 6% for Ndhlovu et al. [START_REF] Ndhlovu | FOXP3 expressing CD127lo CD4+ T cells inversely correlates with CD38+CD8+ cell activation levels in primary HIV-1 infection[END_REF], 8.34% for Hoffmann et al. [START_REF] Hoffmann | CD4dimCD25bright Treg cell frequencies above a standardized gating threshold are similar in asthmatics and controls[END_REF] and 6.8% in the present study). Therefore the measurement of CD127 in addition with CD4 and CD25 allows for a more rapid (below 30 min) and standardizable estimation of Treg cell numbers, usable on in multi-centered clinical studies, and should help clarify our understanding of the contribution of these cells to sepsis pathophysiology.

Conclusion

We observed in the current study that the increased percentage of circulating CD4 + CD25 + CD127 -Treg was strongly and negatively correlated with the decrease in septic patients' proliferative response to mitogens, suggesting that Treg measurement may represent a simple and valuable surrogate biological marker for lymphocyte anergy. In addition, the downregulation of Treg-specific transcription factor Foxp3 in murine splenocytes after sepsis was associated with the restoration of their proliferative response, thus demonstrating for the first time that the relative increase in Treg count might play a role in the state of anergy described after septic shock. Therefore, Treg appear to participate in the defects in both innate and adaptive immune responses observed in septic patients (increased monocyte apoptosis [START_REF] Venet | Human CD4+CD25+ regulatory T lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a Fas/Fas ligand-dependent mechanism[END_REF] and increased lymphocyte anergy), thus suggesting a major role for these cells in sepsisinduced immune dysfunctions. Flow cytometry phenotyping of whole blood lymphocytes from healthy individuals and septic shock patients. EDTA-anticoagulated blood was harvested from healthy individuals and septic shock patients. Cells were stained using 3-color flow cytometry staining (CD4-ECD-CD25-PC5-CD127-PE) or 4-color flow cytometry staining (CD4-ECD-CD25-PC5-CD127-PE-Foxp3-FITC). Red blood cells were lysed using the automated TQ-Prep lysing system or Versalyse reagent. After gating on CD4 + lymphocytes selected based on a SSC/CD4 dot-plot, CD4 + CD25 + CD127 -cells were selected based on a CD25/CD127 2-color dot-plot (left panel). The percentage of Foxp3 expressing cells among this population was subsequently measured (right panel). One representative experiment for healthy individuals (a) and septic shock patients (b) are presented The percentage of CD4 + CD25 + CD127 -cells is increased in whole blood of septic patients in comparison with healthy individuals. EDTA-anticoagulated blood was harvested from 17 healthy individuals (HI, open boxes and circles) and 30 septic shock patients (SS, gray boxes and circles). Cells were stained using 3-color flow cytometry staining (CD4-ECD-CD25-PC5-CD127-PE). Red blood cells were lysed using automated TQ-Prep lysing system. Absolute count measurements were performed using Flow-count fluorospheres™. Results are presented as individual values and box-plots with 5th/95th percentiles. Non-parametric Mann Whitney U test was used for comparison between groups (*P < 0.05, **P < 0.001). a Percentage of CD4 + CD25 + CD127 -lymphocytes measured among CD4 + cells. b Absolute count of peripheral CD4 + T lymphocytes (cells/µl of whole blood). c Absolute count of peripheral CD4 + CD25 + CD127 -T lymphocytes (cells/µl of whole blood). d Absolute count of peripheral CD4 + CD25 -CD127 + T lymphocytes (cells/µl of whole blood) A decreased cell proliferation is associated with an increased circulating percentage of CD4 + CD25 + CD127 -cells in septic shock patients. EDTA-anticoagulated blood was harvested from healthy individuals (n = 2) and septic shock patients (n = 4). Flow cytometry: Whole blood cells were stained using 3-color flow cytometry staining (CD4-ECD-CD25-PC5-CD127-PE). Red blood cells were lysed using automated TQ-Prep lysing system. Results are expressed as percentages of CD4 + CD25 + CD127 -lymphocytes measured among CD4 + cells (y-axis, gray bars: septic patients, open bars: healthy individuals). Cell proliferation: Peripheral blood mononuclear cells (PBMCs) were harvested using Ficoll-Paque Plus gradient centrifugation and cultured in complete RPMI medium (2 × 10 5 cells/200 µl) with phytohaemagglutinin (PHA 10 µg/ml, black circles) or concanavalin A (ConA, 0.1 mg/ml, black triangles) or pokeweed (PWD, 20 µg/ml, black crosses). Proliferation was measured by the increased incorporation of [methyl-3 H]-Thymidine. The assays were carried out in triplicate. Results are expressed as counts per minute (cpm, z-axis). Normal values from the laboratory: PHA and ConA > 15,000 cpm, PWD > 5,000 cpm b After overnight incubation, cells were stained with PKH26 and cultured in 96-well plates in RPMI with or without concanavalin A (0.1 mg/ml, 1 × 10 6 cells/ml) for 5-6 days. Proliferation was assessed by flow cytometric measurement of the decrease in PKH26 (yellow) fluorescence on viable cells selected based on SSC/FCS characteristics. Results are presented as mean ± SEM of proliferation ratio [i.e. (percentage of cells with decreased fluorescence/percentage of highly fluorescent cells) × 100] measured on splenocytes from sham animals (open bar) or from mice after CLP transfected or not (black bar) with Foxp3 (light-gray bar) or control (CTRL) siRNA (medium-gray bar). The Student t test was used for comparison between groups (*P < 0.05) and the Wilcoxon Signed Ranked test was used for comparison between paired values ( # P < 0.05) 682

Fig. 1 .

 1 Fig. 1.

Fig. 2 .

 2 Fig. 2.

Fig. 3 .

 3 Fig. 3.

Fig. 4 .

 4 Fig. 4. Transfection of CD4 + CD25 + Foxp3 + CD127 -regulatory T cells with Foxp3 siRNA is associated with recovery of splenocytes proliferative capacity in septic mice. Murine splenocytes were harvested 24 h after the induction of a polymicrobial septic challenge (cecal ligation and puncture model: CLP, n = 17) or from sham/control animals (Sh, n = 17). Immediately after harvesting, 2 × 10 6 cells were transfected with 2 µM Foxp3 siRNA or control siRNA or left untouched. a Cells were stained using 3-color flow cytometry staining (CD4-PECy7-CD25-PE-Foxp3-APC) 16 h after transfection or not with siRNA. Results are presented as mean ± SEM [black bar: septic mice, light gray bar: septic mice after transfection with Foxp3 siRNA, dark gray bar: septic mice after transfection with control (CTRL) siRNA].

Intensive Care Med. Author manuscript; available in PMC 2010 April 1.

Acknowledgments

Aspects of the work presented here were supported by funds from the Hospices Civils de Lyon (to G.M.) and a grant from the NIH R01 GM46354 (to A.A.). The study was conducted thanks to the logistic support of Centre d'Investigation Clinique (CIC 201) of INSERM and Hospices Civils de Lyon.

Supplementary Material

Refer to Web version on PubMed Central for supplementary material.


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Sylvain Clair

Self-assembly of s-indacene-tetrone on Cu(111): molecular trapping and patterning of Cu adatoms

The supramolecular self-assembly of s-indacene-1,3,5,7(2H,6H)-tetrone on the Cu(111) surface was investigated in ultrahigh vacuum by room-temperature scanning tunneling microscopy supported by theoretical modelling based on the density functional theory. In total, six different phases were found, driven by hydrogen bonding, metal ligand coordination or covalent coupling. Host-guest interactions allowed for the accommodation of molecular or metal clusters inside the open nanoporous patterns. In one phase, molecular trapping was stochastically observed inside the large periodic nanopores created inside the supramolecular network. The three metal-organic networks observed resulted in the creation of different kinds of regular arrays of isolated metal adatoms or adatom clusters with a lattice period larger than 1 nm.

Introduction

Supramolecular self-assembly of small planar aromatic molecules on well-defined surfaces is driven by the subtle balance between intermolecular bonding and molecule-substrate interactions. [START_REF] Dong | Self-assembly of metal-organic coordination structures on surfaces[END_REF][START_REF] Clair | Controlling a Chemical Coupling Reaction on a Surface: Tools and Strategies for On-Surface Synthesis[END_REF][START_REF] Brown | Structural Polymorphism as the Result of Kinetically Controlled Self-Assembly[END_REF][START_REF] Barth | Molecular Architectonic on Metal Surfaces[END_REF][START_REF] Kudernac | Two-dimensional supramolecular self-assembly: nanoporous networks on surfaces[END_REF][START_REF] Bouju | Bicomponent Supramolecular Architectures at the Vacuum-Solid Interface[END_REF][START_REF] Clair | STM Study of Terephthalic Acid Self-Assembly on Au(111): Hydrogen-Bonded Sheets on an Inhomogeneous Substrate[END_REF] While hydrogen bonds remain ubiquitous and largely dominating in many systems, [START_REF] Brown | Structural Polymorphism as the Result of Kinetically Controlled Self-Assembly[END_REF][START_REF] Barth | Molecular Architectonic on Metal Surfaces[END_REF][START_REF] Kudernac | Two-dimensional supramolecular self-assembly: nanoporous networks on surfaces[END_REF][START_REF] Bouju | Bicomponent Supramolecular Architectures at the Vacuum-Solid Interface[END_REF][START_REF] Clair | STM Study of Terephthalic Acid Self-Assembly on Au(111): Hydrogen-Bonded Sheets on an Inhomogeneous Substrate[END_REF][START_REF] Yu | Self-assembly of hydrogen-bonded chains of molecular landers[END_REF] the formation of surfacesupported metal-organic frameworks through complexation with metal adatoms [START_REF] Dong | Self-assembly of metal-organic coordination structures on surfaces[END_REF][START_REF] Barth | Fresh Perspectives for Surface Coordination Chemistry[END_REF][START_REF] Lin | In Templates in Chemistry Iii[END_REF][START_REF] Liu | On-Surface Synthesis: A New Route Realizing Single-Layer Conjugated Metal-Organic Structures[END_REF][START_REF] Ashworth | Metal-organic framework nanosheets (MONs): a new dimension in materials chemistry[END_REF] is particularly interesting because it represents an efficient approach for patterning a surface with a regular tessellation of isolated metal atoms with preserved properties for catalysis [START_REF] Grumelli | Bio-inspired nanocatalysts for the oxygen reduction reaction[END_REF] or magnetism. [START_REF] Gambardella | Supramolecular Control of the Magnetic Anisotropy in Two-Dimensional High-Spin Fe Arrays at a Metal Interface[END_REF][START_REF] Giovanelli | Magnetic Coupling and Single-Ion Anisotropy in Surface-Supported Mn-Based Metal-Organic Networks[END_REF][START_REF] Xie | Switching the Spin on a Ni Trimer within a Metal-Organic Motif by Controlling the On-Top Bromine Atom[END_REF] A wide range of different patterns, among the 17 wallpaper groups, [START_REF] Liu | Wallpaper group kirigami[END_REF] can be achieved by adequate control on the design of the organic precursor and on the growth conditions, [START_REF] Stepanow | Steering Molecular Organization and Host-Guest Interactions using Tailor-Made Two-Dimensional Nanoporous Coordination Systems[END_REF] as revealed at the single molecule level by scanning probe microscopy. [START_REF] Dong | Self-assembly of metal-organic coordination structures on surfaces[END_REF][START_REF] Clair | Controlling a Chemical Coupling Reaction on a Surface: Tools and Strategies for On-Surface Synthesis[END_REF][START_REF] Brown | Structural Polymorphism as the Result of Kinetically Controlled Self-Assembly[END_REF][START_REF] Barth | Molecular Architectonic on Metal Surfaces[END_REF][START_REF] Kudernac | Two-dimensional supramolecular self-assembly: nanoporous networks on surfaces[END_REF] When supramolecular open networks are created on a surface, the 2D nanoporosity [START_REF] Kudernac | Two-dimensional supramolecular self-assembly: nanoporous networks on surfaces[END_REF][START_REF] Jasper-Tonnies | Coverage-Controlled Superstructures of C-3-Symmetric Molecules: Honeycomb versus Hexagonal Tiling[END_REF][START_REF] Zhang | Self-assembled monolayers of new dendron-thiols: manipulation of the patterned surface and wetting properties[END_REF] can lead to particular confinement effects [START_REF] Muller | Confinement properties of 2D porous molecular networks on metal surfaces[END_REF][START_REF] Kumar | Two-Dimensional Band Structure in Honeycomb Metal Organic Frameworks[END_REF] such as the formation of quantum dots [START_REF] Piquero-Zulaica | Precise engineering of quantum dot array coupling through their barrier widths[END_REF] , host-guest molecular complexes, [START_REF] Khan | Supramolecular Chemistry: Host-Guest Molecular Complexes[END_REF][START_REF] Cui | Solution and Air Stable Host/Guest Architectures from a Single Layer Covalent Organic Framework[END_REF][START_REF] Stöhr | Lateral manipulation for the positioning of molecular guests within the confinements of a highly stable self-assembled organic surface network[END_REF][START_REF] Kalashnyk | Self-Accommodating Honeycomb Networks from Supramolecular Self-Assembly of s-Indacene-tetrone on Silver Surfaces[END_REF] or adatom trapping. [START_REF] Schiffrin | Self-aligning atomic strings in surface-supported biomolecular gratings[END_REF][START_REF] Zhang | Complex Supramolecular Interfacial Tessellation through Convergent Multi-Step Reaction of a Dissymmetric Simple Organic Precursor[END_REF][START_REF] Pivetta | Formation of Fe Cluster Superlattice in a Metal-Organic Quantum-Box Network[END_REF][START_REF] Zhang | Stepwise Synthesis of N-Ag-N and C-Ag-C Organometallic Structures on a Ag(111) Surface[END_REF] In this article we show the formation of a variety of supramolecular networks based on hydrogen bonding or metal-organic complexation and exhibiting remarkable properties of molecular and adatom trapping.

We have previously investigated extensively the molecule s-indacene-1,3,5,7(2H,6H)-tetrone (INDO4, see inset in Fig. 1a) [START_REF] Krief | s-indacene-1,3,5,7(2H,6H)-tetraone ('Janus dione') and 1,3-dioxo-5,6-indane-dicarboxylic acid: Old and new 1,3indandione derivatives[END_REF][START_REF] Sprick | Acid catalyzed synthesis of carbonyl-functionalized microporous ladder polymers with high surface area[END_REF] on various silver surfaces. [START_REF] Kalashnyk | Self-Accommodating Honeycomb Networks from Supramolecular Self-Assembly of s-Indacene-tetrone on Silver Surfaces[END_REF][START_REF] Kalashnyk | The Orientation of Silver Surfaces Drives the Reactivity and the Selectivity in Homo-Coupling Reactions[END_REF][START_REF] Kalashnyk | Molecular adaptation in supramolecular self-assembly: brickwall-type phases of indacene-tetrone on silver surfaces[END_REF][START_REF] Kalashnyk | On-Surface Synthesis of Aligned Functional Nanoribbons Monitored by Scanning Tunneling Microscopy and Vibrational Spectroscopy[END_REF] This compound is formed of a small indacene backbone surrounded by four oxygen atoms with D2h symmetry and presents a remarkable ability to establish various intermolecular in-plane hydrogen bonds. [START_REF] Sigalov | O bonding in the anions of 1,3-indandione derivatives[END_REF] A variety of ordered phases can be obtained upon deposition of INDO4 on silver surfaces, depending on the crystallographic orientation of the surface and on the growth conditions. [START_REF] Kalashnyk | Self-Accommodating Honeycomb Networks from Supramolecular Self-Assembly of s-Indacene-tetrone on Silver Surfaces[END_REF][START_REF] Kalashnyk | The Orientation of Silver Surfaces Drives the Reactivity and the Selectivity in Homo-Coupling Reactions[END_REF][START_REF] Kalashnyk | Molecular adaptation in supramolecular self-assembly: brickwall-type phases of indacene-tetrone on silver surfaces[END_REF][START_REF] Kalashnyk | On-Surface Synthesis of Aligned Functional Nanoribbons Monitored by Scanning Tunneling Microscopy and Vibrational Spectroscopy[END_REF] Motivated by the exceptional versatility of this precursor, we wanted to investigate its potential for creating metal-organic frameworks. The Cu(111) surface was chosen because it exhibits a large density of diffusing adatoms at room temperature and it represents a model substrate for creating coordination networks. [START_REF] Lin | Real-time single-molecule imaging of the formation and dynamics of coordination compounds[END_REF][START_REF] Faraggi | Role of Deprotonation and Cu Adatom Migration in Determining the Reaction Pathways of Oxalic Acid Adsorption on Cu(111)[END_REF][START_REF] Kunkel | Rhodizonic Acid on Noble Metals: Surface Reactivity and Coordination Chemistry[END_REF][START_REF] Bebensee | A Surface Coordination Network Based on Copper Adatom Trimers[END_REF] Similar to the case of silver surfaces, we observed the activation of covalent homocoupling at high annealing temperatures, but here exclusively based on the Knoevenagel reaction. The molecular precursors were deposited in ultrahigh vacuum (UHV) conditions and analyzed by room-temperature scanning tunneling microscopy (STM). Theoretical modeling is often necessary to interpret experimental results. Recently, a series of coarse-grained Monte Carlo simulations have been developed to interpret various two-dimensional molecular patterns, [START_REF] Nieckarz | Understanding Pattern Formation in 2D Metal-Organic Coordination Systems on Solid Surfaces[END_REF][START_REF] Gdula | On-Surface Self-Assembly of Metal-Organic Architectures: Insights from Computer Simulations[END_REF] especially for tetratomic molecules. [START_REF] Bischoff | Tailoring Large Pores of Porphyrin Networks on Ag(111) by MetalOrganic Coordination[END_REF][START_REF] Nieckarz | On-surface self-assembly of tetratopic molecular building blocks[END_REF][START_REF] Nieckarz | Surface-Confined Self-Assembly of Asymmetric Tetratopic Molecular Building Blocks[END_REF] Here, numerical calculations based on the density functional theory (DFT) were performed to evidence the stability of the various coordination structures.

Results and discussion

Similarly to the case of INDO4 deposited on Ag surfaces, [START_REF] Kalashnyk | Self-Accommodating Honeycomb Networks from Supramolecular Self-Assembly of s-Indacene-tetrone on Silver Surfaces[END_REF][START_REF] Kalashnyk | The Orientation of Silver Surfaces Drives the Reactivity and the Selectivity in Homo-Coupling Reactions[END_REF][START_REF] Kalashnyk | Molecular adaptation in supramolecular self-assembly: brickwall-type phases of indacene-tetrone on silver surfaces[END_REF][START_REF] Kalashnyk | On-Surface Synthesis of Aligned Functional Nanoribbons Monitored by Scanning Tunneling Microscopy and Vibrational Spectroscopy[END_REF] it is possible to form a large range of supramolecular networks upon adsorption of these molecules on Cu(111). Depending on the annealing temperature, we could identify six different phases, as reported in Table 1. At mild annealing temperature, the hydrogen-bonded phases KG1 and KG2 are formed while at higher annealing temperatures metalorganic self-assembly takes place in P3, P4 and P5 phases. At 470 K, a homo-coupling reaction is activated leading to the formation of a disordered covalent network (P6 phase). All STM experiments were performed at room temperature after the corresponding annealing procedures. A noisy background is systematically observed around the different molecular domains, thus revealing the presence of an additional molecular gas phase with high mobility.

Growth of H-bonded molecular networks

Upon deposition of INDO4 derivative on the Cu(111) surface held at room temperature, we observe the growth of small ordered molecular islands (see Fig. 1a,b). A zoom-in STM image discloses the formation of the Kagome lattice KG1 composed of bright elongated protrusions whose dimensions match the size of the INDO4 molecule. This structure is characterized by a large hexagonal network with a unit cell size of ~6.6 Å and comprising three molecules. A ( 6 1 -1 7

) superlattice unit cell can be determined for this molecular assembly, and evidences a purely epitaxial growth of the indacenes on the surface. The density of indacenes within this phase is thus reckoned at 1.23 molecules per nm 2 . This phase exhibits chiral asymmetry and the opposite chirality was also observed (see Fig. S8). superlattice unit cell can be determined for this phase, with a density of 1.17 molecules per nm 2 .

Remarkably, in both KG1 and KG2 phases, the orientation of the long axis of indacene with respect to the [11 ̅ 0]-close packed directions of the Cu(111) substrate remains the same (i.e. ±25°, depending on the chiral orientation of the domain). These patterns are actually based on a single hydrogen-bonded unit, a molecular trimer arranged in triangular shape, that is organized either in a hexagonal lattice (in KG1) or Although KG1 and KG2 structures have similar molecular densities (~1.2 nm -2 ), the latter exhibits clearly large pores and can be regarded effectively as an open nanoporous network. By looking in closer details at the STM images of KG2 in Fig. 1c it is possible to observe an important number of defects appearing in the center of the pores of the network, as reported in Fig. 2. In fact, the pores are large enough to accommodate guest molecules. Because of the high symmetry of the pores, the guest may adopt different equivalent positions. As a result, the apparent STM contrast corresponds to the average occupation position, similar to other studies displaying dynamical systems. [START_REF] Kalashnyk | Self-Accommodating Honeycomb Networks from Supramolecular Self-Assembly of s-Indacene-tetrone on Silver Surfaces[END_REF][START_REF] Palma | Topological Dynamics in Supramolecular Rotors[END_REF][START_REF] Palma | Visualization and thermodynamic encoding of single-molecule partition function projections[END_REF][START_REF] Karamzadeh | Sequential nested assembly at the liquid/solid interface[END_REF] The molecular trapping inside a pore has been investigated by calculating the intermolecular interactions between the inside molecule and the six surrounding molecules forming a ring structure (Fig. 2d,e and Fig. S7). A more preferable configuration (see Table S2) is found for the inside molecule located in lateral position (Fig. 2d) closer to the molecules of the ring, indicating stronger attractive intermolecular interactions by hydrogen bonding as compared to the central position where van der Waals interactions are favored (Fig. 2e). Further increase of the annealing temperature to 390 K yields to the formation of a new chiral supramolecular phase with a hexagonal lattice (P3, see Fig. 3). A careful inspection of the high resolution STM images evidences the presence of not only elongated protrusions ascribed to the molecules but also of round shaped point-like features. The latter can be identified as Cu adatoms involved in a metalorganic phase. The high annealing temperature delivered an increased amount of intrinsic Cu adatoms that get coordinated by the organic precursors. The superstructure is characterized by a large hexagonal network with a unit cell size of 9.6 Å and comprising six molecules. A ( 4 7 -7 11

Formation of metal-organic frameworks

) superlattice unit cell can be determined for this phase, with a density of 1.14 molecules per nm 2 . This phase again exhibits chiral asymmetry and the opposite chirality was also observed (see Fig. S9).

Similar to the previous KG1 and KG2 cases, a peculiar trimer motif can be identified as the basic bonding unit in P3 that is further organized in a honeycomb lattice. The intrinsic trimer here is of metal-organic nature and its stability and structure could be well reproduced by DFT calculations (see Fig. 3c and Fig. S3 for details). The copper adatoms are naturally located above hollow sites forming a triangle of (6.71 ± 0.05) Å, and are positioned below the mean plane generated by the molecular skeletons. The intermolecular interactions in the presence of the three adatoms generate the trimer with a Cu-O bond equal to (2.00 ± 0.04) Å. Actually, there are two sets of distance, (1.96 ± 0.01) Å and (2.04 ± 0.01) Å, and the main distances between the surface plane and the six oxygen atoms close to the three Cu adatoms are (3.26 ± 0.01) Å and (2.90 ± 0.03) Å, respectively. This has to be compared with the z-position of the six other oxygen atoms, which is (2.27 ± 0.13) Å, indicating that the molecules do not stay flat above the surface in that case. The main molecular axis forms an angle with the Cu-Cu adatoms of (100.30 ± 0.05)° and of (29 ± 2)° with respect to [11 ̅ 0]-direction of the surface. Moreover, the three adatoms are positively charged (around -0.5 e -, with e -= -|e|= -1.6 × 10 -19 C) due to the proximity of the oxygen atoms (~ +1.1 e -) of the molecules. These results suggest that the structure is in rather strong interaction with the substrate. Indeed, the metal-organic interaction can be estimated as one order of magnitude larger than the hydrogen-bonding in this trimer structure (Fig. S5).

Furthermore, we could simulate a free-standing sheet of the metal-organic P3 network (Fig. 3d) based on the previous information. It should be noted here that, because the intrinsic adatoms can be sometimes difficult to evidence in STM images, [START_REF] Blowey | Re-evaluating how charge transfer modifies the conformation of adsorbed molecules[END_REF][START_REF] Ryan | Thermodynamic Driving Forces for Substrate Atom Extraction by Adsorption of Strong Electron Acceptor Molecules[END_REF] the proposed model that we have simulated may differ from the real network in terms of number and position of the metal adatoms. Nevertheless, our calculation shows that the reciprocal interaction between the molecules and the metal adatoms in this complex network is well equilibrated and can produce a robust and well-ordered organization. We could propose a model structure for the P4 (Fig. 4c) and P5 (Fig. 5c) phases in a free-standing configuration. For the P5 structure (Fig. 5c), we propose that adatom trimers are trapped inside the pores because the dimension of the latter is large enough to accommodate more than a single adatom (see also Fig. S6). However, because it is often impossible to distinguish between an isolated metal adatom and a small cluster in the STM images, [START_REF] Bebensee | A Surface Coordination Network Based on Copper Adatom Trimers[END_REF][START_REF] Rochefort | Role of the Structure and Reactivity of Cu and Ag Surfaces in the Formation of a 2D Metal-Hexahydroxytriphenylene Network[END_REF][START_REF] Zhang | Atomic-Scale Visualization of the Stepwise Metal-Mediated Dehalogenative Cycloaddition Reaction Pathways: Competition between Radicals and Organometallic Intermediates[END_REF] we cannot conclude unambiguously whether the center metal structure is composed of a single adatom or an adatom trimer. In the P5 phase, the distances between the trapped metal adatoms and the ligand are remarkably large (the mean distance between the trimer barycenter and the six surrounding hydrogen atoms of the molecules is (5.8 ± 1.0) Å). Similar structures have been also previously observed, where adatoms were included inside quite large nanocavities formed by a supramolecular network. [START_REF] Schiffrin | Self-aligning atomic strings in surface-supported biomolecular gratings[END_REF][START_REF] Zhang | Complex Supramolecular Interfacial Tessellation through Convergent Multi-Step Reaction of a Dissymmetric Simple Organic Precursor[END_REF][START_REF] Pivetta | Formation of Fe Cluster Superlattice in a Metal-Organic Quantum-Box Network[END_REF][START_REF] Zhang | Stepwise Synthesis of N-Ag-N and C-Ag-C Organometallic Structures on a Ag(111) Surface[END_REF] In the present case, it has to be noted that the trapping of the adatoms is made possible thanks to a relatively important decrease of the molecular density of the network (see Table 1).

Formation of covalent structures

When the temperature is raised above 470 K the homocoupling reaction is taking place leading to a disordered covalent network (phase P6, Fig. 6). Remarkably here, only the Knoevenagel coupling is allowed and no oxidative coupling was observed, in opposition to the case of silver surfaces. [START_REF] Kalashnyk | The Orientation of Silver Surfaces Drives the Reactivity and the Selectivity in Homo-Coupling Reactions[END_REF][START_REF] Kalashnyk | On-Surface Synthesis of Aligned Functional Nanoribbons Monitored by Scanning Tunneling Microscopy and Vibrational Spectroscopy[END_REF][START_REF] Hussein | On-surface homocoupling reactivity of a chiral bifunctional bromoindanone molecule on Cu(111)[END_REF] This is directly related to the fact that the Knoevenagel reaction is a less energy-demanding reaction than the oxidative coupling. Indeed, oxidative coupling can only be carried out in the presence of a metal catalyst in order to lower the energy barrier of the reaction. [START_REF] Funes-Ardoiz | Oxidative Coupling Mechanisms: Current State of Understanding[END_REF] In absence of metal catalyst, an insurmountable energy barrier exists so that the coupling reaction is unable to proceed. This is not the case for Knoevenagel reactions that can be carried out in metal-catalyst free conditions. [START_REF] Dalessandro | Mechanism and free energy profile of basecatalyzed Knoevenagel condensation reaction[END_REF] Typically, Knoevenagel reactions are catalyzed with a base and in this field, piperidine is a popular base. By using this base, theoretical calculations and experimental results revealed the free energy barrier of the reaction to be low, around 20.0 kcal/mol. [START_REF] Dalessandro | Mechanism of the Piperidine-Catalyzed Knoevenagel Condensation Reaction in Methanol: The Role of Iminium and Enolate Ions[END_REF] Additionally, based on the supramolecular phases P4/P5 obtained at lower temperatures, the orientations of the molecules are favorable so that the activated methylene groups of INDO4 can directly react with the adjacent ketone functions. Indeed, the Knoevenagel reaction consists in a dehydrative coupling between a carbonyl group and an activated methylene group, what is achieved in INDO4 with the presence of two electron-withdrawing ketone groups standing on both sides of the methylene group.

Conclusions

For the system INDO4/Cu(111) at room temperature, we have described the formation of six phases based on hydrogen bonding, metal ligand coordination or covalent coupling. Two Kagome networks were found, one of them with an important nanoporosity providing the ability to self-accommodate guest molecules. [START_REF] Kalashnyk | Self-Accommodating Honeycomb Networks from Supramolecular Self-Assembly of s-Indacene-tetrone on Silver Surfaces[END_REF] Kagome supramolecular lattices are particularly attractive due to their specific topological electronic structure. [START_REF] Hua | Highly Degenerate Ground States in a Frustrated Antiferromagnetic Kagome Lattice in a Two-Dimensional Metal-Organic Framework[END_REF][START_REF] Hernandez-Lopez | Searching for kagome multi-bands and edge states in a predicted organic topological insulator[END_REF][START_REF] Anindya | Controlling the magnetic properties of two-dimensional carbon-based Kagome polymers[END_REF][START_REF] Pawlak | On-Surface Synthesis of Nitrogen-Doped Kagome Graphene[END_REF][START_REF] Kumar | Manifestation of Strongly Correlated Electrons in a 2D Kagome Metal-Organic Framework[END_REF] The different metal-organic phases P3, P4 and P5 represent well-defined robust self-organized patterns of metal adatoms, either directly involved in metal-organic bonds, or isolated and well-separated from the neighboring molecules. The complexity of the unit cells involving a large number of molecules and their relatively large sizes (up to 1 nm) can provide potentially interesting properties to such systems with regard to catalysis or magnetism.

Finally, for higher annealing temperature we observed the homocoupling of the precursor from the Knoevenagel reaction. The latter has only been scarcely reported on surfaces [START_REF] Kalashnyk | The Orientation of Silver Surfaces Drives the Reactivity and the Selectivity in Homo-Coupling Reactions[END_REF][START_REF] Au-Yeung | On-Surface Formation of Cyano-Vinylene Linked Chains by Knoevenagel Condensation[END_REF][START_REF] Geng | Formation of C=C Bond via Knoevenagel Reaction between Aromatic Aldehyde and Barbituric Acid at Liquid/HOPG and Vapor/HOPG Interfaces[END_REF] and represents a promising approach for the formation of C=C bonds.

Methods

The DFT calculations were performed via the Vienna ab initio simulation package (VASP), [START_REF] Kresse | From ultrasoft pseudopotentials to the projector augmented-wave method[END_REF] where the projector augmented wave (PAW) potential [START_REF] Blöchl | Projector augmented-wave method[END_REF] was employed, while the exchange-correlation functional along with the generalized gradient approximation (GGA) by Perdew, Burke, and Ernzerhof (PBE) [START_REF] Perdew | Generalized gradient approximation made simple[END_REF] was used. The 400 eV cut-off energy with the convergence criteria of 10 -4 eV were set for the minimization.

Only a single k-point, namely the Г-point, was used for the calculations. The structure were relaxed until the residual forces were smaller than 0.02 eV/Å. Moreover, the van der Waals dispersion correction term of DFT-D3 method proposed by Grimme [START_REF] Grimme | A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu[END_REF] was also adopted.

Figure 1 .

 1 Figure 1. STM images of the hydrogen-bonded phases obtained upon deposition of INDO4 (inset in (a)) on Cu(111) (a,b) at room temperature (KG1 phase) and (c,d) after annealing at 350 K (KG2 phase). Both phases are based on the same trimer motif modelled by DFT in (e) (see also Fig. S2). Scale bars: (a,c) 10 nm; (b,d) 2 nm.

  in a honeycomb lattice (in KG2). The intrinsic stability and structure of the hydrogen-bonded trimer was investigated by DFT calculations (see Fig.1eand SI Fig. S2 for details). The triangle formed by the three inner oxygen atoms is almost isosceles (3.20 Å, 3.21 Å, 3.01 Å) whereas the three C-H⋯O hydrogenbonds are characterized by (2.33 ± 0.09) Å and (151 ± 2)°. The partial Bader charges on each atom of the molecule do not differ from the case of a single adsorbed molecule (Fig. S1). In particular, the molecular axis is rotated by (36.8 ± 0.2)° with respect to the [11 ̅ 0]-direction.

Figure 2 .

 2 Figure 2. Zoomed-in STM images inside KG2 phase showing its nanoporosity (a) that is occasionally filled with a molecule in two different configurations (b,c). Scale bars: 1 nm. (d,e) DFT modelling of a single nanopore with a trapped molecule in lateral (d) or central position (e).

Figure 3 .

 3 Figure 3. (a,b) STM images of the metal-organic phase P3 obtained upon deposition of INDO4 on Cu(111) and annealing at 390 K. This phase is based on a trimer motif modelled by DFT in (c) (see also Fig. S3). (d) Configuration obtained from gas-phase DFT calculations. Scale bars: (a) 10 nm; (b) 2 nm.

Figure 4 .

 4 Figure 4. (a,b) STM images of the metal-organic phase P4 obtained upon deposition of INDO4 on Cu(111) and annealing at 430 K and (c) corresponding model obtained from gas-phase DFT calculations. Scale bars: (a) 10 nm; (b) 2 nm.

Figure 5 .

 5 Figure 5. (a,b) STM images of the metal-organic phase P5 obtained upon deposition of INDO4 on Cu(111) and annealing at 430 K and (c) the corresponding model obtained from gas-phase DFT calculations. Scale bars: (a) 10 nm; (b) 2 nm.

Figure 6 .

 6 Figure 6. (a,b) STM images of the disordered covalent network P6 obtained upon deposition of INDO4 on Cu(111) and annealing at 470 K. Scale bars: (a) 10 nm; (b) 3 nm. (c) High resolution STM images of the different molecular features observed inside the network and corresponding molecular models based on the Knoevenagel reaction.

  The substrate is simulated by a 5-layers Cu(111) surface onto which we add the Cu-ITO molecules. The separation distance between the slabs, (top vacuum) is set to 20 Å.The experiments were performed in ultra-high vacuum (UHV, base pressure in the low 10 -10 mbar range). The single-crystal Cu(111) surface was cleaned by several cycles of Ar + bombardment followed by annealing. INDO4 molecules were provided by Sigma-Aldrich as rare and unique chemical. The precursors were thoroughly degassed prior to deposition onto the atomically clean substrate held at room temperature (RT). The molecular powder was thermally sublimated from an evaporator heated to 150 °C for typical dosing time of 30 min providing coverage of ~0.5 monolayer. STM measurements were performed with a commercial Omicron VT-STM system operated at room temperature. The STM images were acquired in constant current mode with typical tunneling current IT≈0.3 nA and sample bias VT ≈ -(1-1.5) V. All images were subsequently calibrated using atomically resolved images of the pristine surfaces. Images were partly treated with the free softwares WSxM[START_REF] Horcas | A software for scanning probe microscopy and a tool for nanotechnology[END_REF] and LMAPper.70 An error bar of typically ± 1 Å is found for each distance measurement in the STM images. The proposed superstructure matrices are derived from the measured unit cell sizes and the symmetry considerations of each phase corresponding to the observation of the different symmetryequivalent domains.

Acknowledgments

HD and XB mention that this study has been supported through the EUR grant NanoX n°ANR-17-EURE-0009 in the framework of the "Programme des Investissements d'Avenir". This work was granted access to the HPC resources of CALMIP supercomputing center under the allocation 2021-P0832.


Guillaume Monneret
email: guillaume.monneret@chu-lyon.fr

Fabienne Venet

A rapidly progressing lymphocyte exhaustion after severe sepsis

e work of Boomer and colleagues [START_REF] Boomer | A prospective analysis of lymphocyte phenotype and function over the course of acute sepsis[END_REF] in the previous issue of Critical Care provides further insights into the concept of sepsis-induced immunosuppression [START_REF] Hotchkiss | The pathophysiology and treatment of sepsis[END_REF][START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: a new skin for the old ceremony[END_REF]. Clinical observations have shown that many patients with severe sepsis survive the first critical hours of the syndrome but eventually die in a state of immuno suppression.

is immune failure is illustrated by patients' difficulty in fighting the primary bacterial infection, decreased resistance to secondary nosocomial infections, and reactivation of viral infections that normally are solely pathogenic in immunocompromised hosts [START_REF] Monneret | ICU-acquired immunosuppression and the risk for secondary fungal infections[END_REF][START_REF] Kalil | Prevalence and mortality associated with cytomegalovirus infection in nonimmunosuppressed patients in the intensive care unit[END_REF]. Both innate and adaptive immune responses are affected after sepsis. However, in the last five to 10 years, much attention has been given to the study of the innate side of the immune response, in particular via monitoring of the decreased expression of monocyte HLA-DR (a monocyte anergy biomarker whose decreased levels are associated with mortality and nosocomial infections in critically ill patients). Meanwhile, sepsis-induced adaptive immune failure has been somewhat under-appreciated.

Sepsis-induced lymphocyte alterations include severe lymphopenia due to increased apoptosis, decreased Tcell proliferation and cytokine production after stimulation, and increased percentage of circulating regulatory T (Treg) cells. Very recently, the loss of T-cell receptor repertoire diversity (assessed at the molecular level) was shown to be associated with increased mortality and risk for nosocomial infections in patients with septic shock, independently of lymphopenia [START_REF] Venet | Decreased T cell repertoire diversity in sepsis: a preliminary study[END_REF].

e study by Boomer and colleagues reinforces these observations and complements the outstanding work on post-term biopsies recently published by the same group [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF]. Importantly, the latter study showed that sepsisinduced immunosuppression is a severe and profound mechanism occurring not only systematically but also locally in organs. Indeed, the authors demonstrated that, in deceased patients with septic shock, both lung and spleen lymphocytes were markedly immunodepressed (decreased cell counts, altered functional response to stimulation, and increased co-inhibitory receptor expressions).

Here, the investigators extended these observations by prospectively monitoring circulating lymphocytes in patients with sepsis. More specifically, a panel of coinhibitory receptor expressions (PD-1, BTLA, CTLA-4, TIM-3, and LAG-3) along with CD127 (receptor for interleukin 7 (IL-7), a key cytokine in lymphocyte function) were determined in parallel with Treg cell per centage and lymphocyte functional response. In regard to coinhibitory receptor expressions, the main result of the present study is to show that, although only low levels of expressions were observed at the onset of sepsis, these expressions over the first week were progressively upregulated on lymphocytes of patients with sepsis. Along with the decreased CD127 expression (a feature of exhausted lymphocytes) and the increased percentage of circulating Treg cells (the major immunosuppressive cells in the body), this upregulation suggests that a full picture of lymphocyte exhaustion occurs within a few days in patients with sepsis [START_REF] Wherry | T cell exhaustion[END_REF]. Interestingly, this observation is very similar to clinical observations made in patients Abstract Septic syndromes induce immune alterations that have long been considered solely an overwhelming pro-in ammatory response. Increasing evidence now suggests that, after the rst pro-in ammatory hours, sepsis is accompanied by the occurrence of a systemic immune failure. Here, novel perspectives regarding sepsis-induced lymphocyte alterations will be discussed in the context of a recently published study investigating overtime evolution of co-inhibitory lymphocyte receptor expressions in patients with severe sepsis.

with chronic viral infections such as HIV. Importantly, these results support the concept of 'delayed immunosuppression' after sepsis.

Another interesting observation is that these increased co-inhibitory receptor expressions were associated with altered functional responses (that is, decreased inter ferongamma production after stimulation). Most importantly, the authors suggest that impaired lymphocyte functions after sepsis could be reversible. Here, ex vivo incubation of patients' cells with fresh media improved responses. This improvement likely indicates that it remains possible to rejuvenate lymphocytes after sepsis and thus this opens novel therapeutic strategies in battling sepsisinduced immunosuppression. This is of the utmost importance since sepsis-induced immune altera tions are known to be associated with increased mortality and nosocomial infection rates in patients. Given the central role of lymphocytes (mainly, CD4 + T cells) in orches tra ting immune responses, one may expect that restoring their function can generate a positive global effect, mediated by both lymphocytes and innate immune cells, on the overall immune response.

Two therapeutic strategies are conceivable: blocking negative/co-inhibitory pathways (for example, with anti-PD-1 antibodies) or boosting lymphocyte functions. In the latter case, recombinant human IL-7 would represent a major candidate. Of note, these two approaches are already used and assessed in cancer and chronic viral infections [START_REF] Topalian | Safety, activity, and immune correlates of anti-PD-1 antibody in cancer[END_REF][START_REF] Morre | Interleukin-7 and immune reconstitution in cancer patients: a new paradigm for dramatically increasing overall survival[END_REF], which are clinical conditions that share many similarities with lymphocyte dysfunctions described in sepsis.

However, one limitation, duly acknowledged by the authors, needs to be reiterated. Although a large battery of co-inhibitory receptor phenotyping was performed, the low number of patients included precludes any robust conclusion, especially in regard to correlations with clinical outcomes. Despite this limitation, this study reinforces the idea that, after severe sepsis, patients develop a phenotype of lymphocyte exhaustion. This agrees with previous results postulating that immunostimulating therapies aimed at battling lymphocyte anergy and exhaustion could restore immune function in immunodepressed patients [START_REF] Hotchkiss | Immunotherapy for sepsis -a new approach against an ancient foe[END_REF] and thus help in decreasing mortality after sepsis.

Abbreviations

IL, interleukin; Treg, regulatory T.

Competing interests

The authors declare that they have no competing interests.