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

Fabienne Venet

Sepsis-Induced Immune Alterations Monitoring by Flow Cytometry as a Promising Tool for Individualized Therapy

Keywords: sepsis, flow cytometry, immunosuppression, HLA-DR, anergy, immune monitoring, sepsis

Septic syndromes remain a major although largely under-recognized health care problem and represent the first cause of mortality in intensive care units. While sepsis has, for long, been solely described as inducing a tremendous systemic inflammatory response, novel findings indicate that sepsis indeed initiates a more complex immunologic response that varies over time, with the concomitant occurrence of both pro-and anti-inflammatory mechanisms. As a resultant, after a short proinflammatory phase, septic patients enter a stage of protracted immunosuppression. This is illustrated in those patients by reactivation of dormant viruses (CMV or HSV) or infections due to pathogens, including fungi, which are normally pathogenic solely in immunocompromised hosts. Although mechanisms are not totally understood, these alterations might be directly responsible for worsening outcome in patients who survived initial resuscitation as nearly all immune functions are deeply compromised. Indeed, the magnitude and persistence over time of these dysfunctions have been associated with increased mortality and health-care associated infection rate. Consequently, new promising therapeutic avenues are currently emerging from those recent findings such as adjunctive immunostimulation (IFN-g, GM-CSF, IL-7, anti-PD1/L1 antibodies) for the most immunosuppressed patients. Nevertheless, as there is no clinical sign of immune dysfunctions, the prerequisite for such therapeutic intervention relies on our capacity in identifying the patients who could benefit from immunostimulation. To date, the most robust biomarkers of sepsis-induced immunosuppression are measured by flow cytometry. Of them, the decreased expression of monocyte HLA-DR appears as a "gold standard." This review reports on the mechanisms sustaining sepsis-induced immunosuppression and its related biomarkers measurable by flow cytometry. The objective is to integrate the most recent facts in an up-to-date account of clinical results, flow cytometry aspects as well as issues in results standardization for multicenter studies. V

INTRODUCTION

Septic syndromes represent a major, although largely under-recognized, healthcare problem worldwide accounting for an unacceptably high number of deaths every year [START_REF] Deutschman | Sepsis: Current dogma and new perspectives[END_REF][START_REF] Angus | Severe sepsis and septic shock[END_REF]. In the USA, they are estimated to represent around 1 million cases and about 200,000 deaths annually. In addition, due to increased longevity and better care of comorbidities such as cancer and diabetes [START_REF] Deutschman | Sepsis: Current dogma and new perspectives[END_REF][START_REF] Angus | Severe sepsis and septic shock[END_REF], sepsis syndrome incidence has dramatically increased by an annual rate of 8-13% over the past decade and this syndrome now claims more lives than bowel and breast cancers combined (Global Sepsis Alliance & World Sepsis Data, www.world-sepsis-day.org). Recently, Adhikari et al. estimated that up to 19 million cases of sepsis occur worldwide annually but the real incidence is likely far higher [START_REF] Adhikari | Critical care and the global burden of critical illness in adults[END_REF]. Meanwhile, mortality remains high, ranging from 20% for sepsis to over 40% for septic shock [START_REF] Deutschman | Sepsis: Current dogma and new perspectives[END_REF][START_REF] Angus | Severe sepsis and septic shock[END_REF]. A recent epidemiological study including 25,000 patients in the USA and in Europe reported that overall hospital mortality from severe sepsis or septic shock is still over 30% [START_REF] Levy | Outcomes of the surviving sepsis campaign in intensive care units in the USA and Europe: A prospective cohort study[END_REF]. This is despite improvement in patients' care guidelines (intravenous fluid resuscitation, source control, and antimicrobial therapy) and the implementation of surviving sepsis campaign and years of various adjunctive therapies aimed at blocking every single inflammatory mediator/mechanism hypothesized to be involved in pathophysiology [START_REF] Dellinger | Surviving sepsis campaign: International guidelines for management of severe sepsis and septic shock, 2012[END_REF]. Therefore, septic syndromes represent the most frequent cause of death in intensive care unit (ICU) patients while, to date, no therapeutic intervention has specifically been approved for the treatment of sepsis. Importantly, according to the Centers for Disease Control and Prevention, in 2008, an estimated $14.6 billion was spent on hospitalizations for sepsis in the USA. Moreover, from 1997 to 2008, the inflation adjusted aggregate costs for the treatment of patients hospitalized for this condition increased on average annually by 12%. Moreover, septic patients are at high risk of developing health-care associated infections (HAI). The latter are associated not only with an increased morbidity, mortality, and length of stay but also with an important augmentation in patient's hospitalization costs. Indeed, HAI-related costs are estimated at 10,000 to 25,000 $ per nosocomial episode [START_REF] Chaix | A model to compute the medical cost of patients in intensive care[END_REF][START_REF] Adrie | Epidemiology and economic evaluation of severe sepsis in France: Age, severity, infection site, and place of acquisition (community, hospital, or intensive care unit) as determinants of workload and cost[END_REF].

Therefore, Deutschman and Tracey, recently qualified sepsis as "the quintessential medical disorder of the 21th century" [START_REF] Deutschman | Sepsis: Current dogma and new perspectives[END_REF].

SEPSIS-INDUCED IMMUNOSUPPRESSION

Historically, (i.e., since 1992) a consensus conference defined sepsis as the host immune response (which is usually called SIRS for systemic inflammatory responses syndromes) to a bacterial, viral, or fungal infection [START_REF] Bone | Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/ SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine[END_REF]. This implies that infection is a trigger and pathophysiology relies on an exacerbated uncontrolled inflammatory process that may lead to organ failures, hypoperfusion, and shock [START_REF] Hutchins | The new normal: immunomodulatory agents against sepsis immune suppression[END_REF]. This consensus definition of sepsis is soon to be changed and would associate a systemic response to infection with the presence of some degree of organ dysfunction [START_REF] Vincent | Sepsis definitions: Time for change[END_REF].

In fact, the inability of various therapies to control the devastating effects of this deadly disease indicates that the initial hypotheses for sepsis pathophysiology may have been inadequately addressed. It is now admitted that the postulate that death after sepsis is solely because of an overwhelming proinflammatory immune response was actually incomplete. Indeed, a large body of evidence now supports that death from septic shock is likely due to the effects of distinct mechanisms rapidly changing over time [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: A new skin for the old ceremony[END_REF][START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF]. Very early in the course of the disease, a massive release of inflammatory mediators (normally designated to trigger immune response against pathogens) is occurring and may be deleterious by inducing organ dysfunctions and hypoperfusion [START_REF] Medzhitov | Disease tolerance as a defense strategy[END_REF]. This "early mortality" (i.e., first 3 days mortality) is regu-larly reported to account for <30% of total mortality [START_REF] Venet | Monitoring the immune response in sepsis: A rational approach to administration of immunoadjuvant therapies[END_REF][START_REF] Otto | The late phase of sepsis is characterized by an increased microbiological burden and death rate[END_REF]. Concomitantly, the body develops compensatory mechanisms to prevent overwhelming inflammation and dampen an overzealous anti-infectious response [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: A new skin for the old ceremony[END_REF][START_REF] Munford | Normal responses to injury prevent systemic inflammation and can be immunosuppressive[END_REF][START_REF] Hotchkiss | Immunosuppression in sepsis: A novel understanding of the disorder and a new therapeutic approach[END_REF]. These negative feedback mechanisms, although having protective effects during the first initial hours, may paradoxically become detrimental as they persist over time leading to immune suppression [START_REF] Hotchkiss | Immunosuppression in sepsis: A novel understanding of the disorder and a new therapeutic approach[END_REF]. Indeed, considerable clinical and experimental facts indicate that patients rapidly present with severely altered immune parameters. The weight of evidence now supports the view that this immune-suppressive phase is the predominant immunologic response in most septic patients [START_REF] Hotchkiss | Immunotherapy for sepsis-a new approach against an ancient foe[END_REF]. Of note, it is also hypothesized that a low grade inflammation may persist over time and thus sustains a chronically compensatory anti-inflammatory that would lead to similar features of immunosuppression [START_REF] Xiao | A genomic storm in critically injured humans[END_REF].

Clinically, this is illustrated by patients' difficulty to overcome the primary bacterial infection. For example, a recent postmortem study showed that 80% septic patients had unresolved septic foci at time of death [START_REF] Torgersen | Macroscopic postmortem findings in 235 surgical intensive care patients with sepsis[END_REF]. In addition, reactivation of dormant viruses (CMV, TTV, and HSV) or infections due to pathogens, including fungi, which are normally pathogenic solely in immunocompromised hosts (Acinetobacter, Candida. . .) are observed in septic patients [START_REF] Landelle | Nosocomial infection after septic shock among intensive care unit patients[END_REF][START_REF] Monneret | ICU-acquired immunosuppression and the risk for secondary fungal infections[END_REF][START_REF] Walton | Reactivation of multiple viruses in patients with sepsis[END_REF]. These alterations and subsequent infectious complications might be directly responsible for worsening outcome in patients who survived initial resuscitation and for increasing length of stay in ICU (Fig. 1).

Biologically, both arms of immunity (innate and adaptive) are markedly suppressed and nearly all immune responses are deeply compromised or shifted to antiinflammatory functions after sepsis [START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF]. Although not exhaustively understood, some mechanisms at play are now described. In particular, epigenetic regulation, energetic failure, central and endocrine regulations, increased apoptosis, release of anti-inflammatory mediators (IL-10, Prostaglandin E2; TGF-b. . .) or endotoxin tolerance have been shown to be playing a role both in clinical observations and animal models of sepsis [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: A new skin for the old ceremony[END_REF][START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF].

As our capacity to treat patients during the very first hours of shock has improved (early and aggressive initial supportive therapy), many patients now survive this critical step but eventually die later in a state of immunosuppression [START_REF] Otto | The late phase of sepsis is characterized by an increased microbiological burden and death rate[END_REF]. This second step of septic shock is hypothesized to be associated with >70% of total mortality [START_REF] Venet | Monitoring the immune response in sepsis: A rational approach to administration of immunoadjuvant therapies[END_REF]. Importantly, although this aspect is not well documented yet, it is speculated that immune dysfunctions may persist over years after sepsis and might contribute to increased delayed mortality. For example, it has been recently demonstrated that total lymphocyte count <600 cells/lL at Day 4 after septic shock is associated with significantly increased mortality after one year [START_REF] Drewry | Persistent lymphopenia after diagnosis of sepsis predicts mortality[END_REF]. Long-term outcome studies after sepsis have shown its association with increased mortality for several years after the initial infectious episode (up to 10 years). Similarly, an increased risk of recurrent infections in the year following ICU discharge has also been observed for septic patients [START_REF] Yende | Long-term outcomes from sepsis[END_REF][START_REF] Wang | Long-term mortality after community-acquired sepsis: A longitudinal population-based cohort study[END_REF][START_REF] Linder | Longterm (10-year) mortality of younger previously healthy patients with severe sepsis/septic shock is worse than that of patients with nonseptic critical illness and of the general population[END_REF]. These data support the hypothesis that septic patients retain persistent immune defects that may render them more vulnerable to subsequent infections or less able to totally eradicate their indolent primary infections.

Thus, in addition to the timely antibiotic administration and development of clinical guidelines that avoid infections, focus should shift to therapies that may boost host immunity in patients that present with sepsisinduced immune alterations. A second important implication may be that antibiotics alone are unlikely to improve sepsis mortality because the major underlying defects leading to delayed mortality may be patient's impaired immunity [START_REF] Hotchkiss | Immunosuppression in sepsis: A novel understanding of the disorder and a new therapeutic approach[END_REF]. This may also concern patients with severe immune defects from other origins, e.g., bone marrow transplant patients facing fungal secondary infections [START_REF] Monneret | ICU-acquired immunosuppression and the risk for secondary fungal infections[END_REF].

Consequently, from those recent findings, new promising therapeutic strategies are currently emerging in the treatment of septic patients such as adjunctive immunostimulation for the most immunodepressed patients or those who present with persistent immune defects. The most evident candidates are IFN-g, GM-CSF, IL-7, anti-PD1/L1 antibodies [see further details in recent reviews, [START_REF] Hutchins | The new normal: immunomodulatory agents against sepsis immune suppression[END_REF][START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF][START_REF] Hotchkiss | Immunosuppression in sepsis: A novel understanding of the disorder and a new therapeutic approach[END_REF]] whereas thymolisin-a or toll-like receptors agonists are also under investigations [START_REF] Davis | TLR3 agonist improves survival to secondary pneumonia in a double injury model[END_REF][START_REF] Wu | The efficacy of thymosin alpha 1 for severe sepsis (ETASS): A multicenter, single-blind, randomized and controlled trial[END_REF]. In any cases, as there is no clinical sign of immune dysfunctions, the prerequisite for immunostimulation administration will rely on our capacity in identifying the patients who could benefit from it. Therefore, it is crucial to identify the best biological tools for patients' stratification according to their immune status (a missing step in most of previous clinical trials]. This would define the right action (i.e., timulating innate immunity and/or adaptive immunity) at the right time (early or delayed treatment) in the right patient (individualized/ tailored therapy). Hopefully, this should help in partly decrease heterogeneity of patients enrolled in clinical trials and better take into account the rapidly changing pro-/anti-inflammatory response after sepsis.

In this article, we will show that flow cytometry is, for now, the most appropriate tool to monitor septic patients' immune response, to define targeted and individualized adjunctive therapy and also to control drug efficacy. As this review is focused on sepsis-induced immunosuppression, the topic of sepsis diagnosis will not be addressed here. This article will review the novel/recently described flow cytometry biomarkers in regard with the mechanisms sustaining sepsis-induced immunosuppression (Table 1).

NEUTROPHILS

Neutrophils represent the first line of defense against infection. They play a central role in the control of pathogens and initiation of effective immune responses.

As such, their monitoring might be of great interest in infected patients.

Most data on neutrophils during sepsis have been obtained during the first hours of the syndrome and showed inconsistent modifications (increased or altered functions) such as impaired bacterial phagocytosis, increased radical oxygen species production, loss of chemotaxis activities-every aspect measurable by flow cytometry [START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF]. However, none of these alterations has been clinically validated in large cohorts of patients and finally retained in any immuno-monitoring protocol.

With that said, the more informative neutrophil parameter regarding its association with deleterious outcomes appears to be one described for decades in sepsis. Indeed, it has been regularly described that septic patients present with markedly increased number of circulating immature neutrophils. For example, in 2014, several articles published by different groups indicated that increased numbers of immature granulocytes ("band cells" identified by automatic hematology analyzer, ADVIA 2120 or Sysmex XE-5000) were associated with mortality [START_REF] Park | Sepsis affects most routine and cell population data (CPD) obtained using the Sysmex XN-2000 blood cell analyzer: Neutrophil-related CPD NE-SFL and NE-WY provide useful information for detecting sepsis[END_REF][START_REF] Ha | Fraction of immature granulocytes reflects severity but not mortality in sepsis[END_REF][START_REF] Nahm | Delta neutrophil index in automated immature granulocyte counts for assessing disease severity of patients with sepsis[END_REF]. In addition, immature neutrophils phenotypically identified according to lower CD10/CD16 expressions, were also reported to be associated with increased early mortality after sepsis [START_REF] Guerin | Circulating immature granulocytes with T-cell killing functions predict sepsis deterioration*[END_REF] and to present with immunosuppressive functions. In line, Pillay and colleagues demonstrated in a human model of endotoxemia (administration of low dose LPS) the existence of a neutrophil subset (CD16 bright and CD62L dim ) able to suppress T lymphocyte proliferation [START_REF] Pillay | A subset of neutrophils in human systemic inflammation inhibits T cell responses through Mac-1[END_REF]. These recent results aggregate with the expending description of myeloid-derived suppressor cells (MDSC). In experimental models of sepsis, those cells have been shown to be able to block T lymphocyte function [START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF] and, as such, they could participate in sepsisinduced immunosuppression. However, to date, no relevant clinical studies investigated MDSC in septic patients have been published. This might be due to the complexity of their immunophenotyping by flow cytometry associated with the absence of any consensually accepted phenotype in humans [START_REF] Dumitru | Neutrophils and granulocytic myeloid-derived suppressor cells: Immunophenotyping, cell biology and clinical relevance in human oncology[END_REF].

To note, in a currently submitted study in septic shock patients presenting with immune alterations, we observed marked alterations of neutrophils functions after sepsis and an association between CD10 dim / CD16 dim immature neutrophil number and mortality. However, in this study, the potentially immunosuppressive neutrophil subset (CD16 bright /CD62L dim ) described by Pillay et al. was not consistently observed in septic shock patients except for very few cases (personal data, under submission).

Collectively, these data indicate that there is a rational to reinvestigate the number and the role of immature neutrophils (and their putative link with MDSC) in sepsis, especially during the immunosuppressive phase on the disease. In particular, to date, studies with appropriate numbers of patients allowing the study of biological parameters' associations with deleterious outcomes (mortality, HAI rate, viral reactivation) are missing. The most convincing results were obtained in a study including 177 septic patients in which immature granulocytes number was only assessed as a predictor of early mortality [START_REF] Guerin | Circulating immature granulocytes with T-cell killing functions predict sepsis deterioration*[END_REF]. Therefore, the deleterious aspect of the persistence overtime of this CD10 dim /CD16 dim neutrophil subset in patients deserves additional investigations.

DENDRITIC CELLS (DC)

Decreased DCs number (spleen, blood) and functions have been largely reported in septic mice in association with global alterations of immune functions [START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF]. Indeed, it has been shown that DCs are particularly vulnerable to sepsis-induced apoptosis [START_REF] Hotchkiss | Depletion of dendritic cells, but not macrophages, in patients with sepsis[END_REF]. Importantly, as key players in orchestrating immune responses, DCs likely play a pivotal role in the development of sepsisinduced immunosuppression [START_REF] Huang | Changes in dendritic cell function in the immune response to sepsis. Cell-& tissue-based therapy[END_REF]. In a postmortem study, Hotchkiss et al. initially observed a dramatic reduction in splenic DCs number after septic shock [START_REF] Hotchkiss | Depletion of dendritic cells, but not macrophages, in patients with sepsis[END_REF]. Similarly, reduced circulating DCs count has been reported in septic patients [START_REF] Guisset | Decrease in circulating dendritic cells predicts fatal outcome in septic shock[END_REF][START_REF] Grimaldi | Profound and persistent decrease of circulating dendritic cells is associated with ICUacquired infection in patients with septic shock[END_REF][START_REF] Riccardi | Flow cytometric analysis of peripheral blood dendritic cells in patients with severe sepsis[END_REF]. This loss was more important in septic patients who died than in survivors [START_REF] Guisset | Decrease in circulating dendritic cells predicts fatal outcome in septic shock[END_REF]. Moreover, a profound and persistent decrease was associated with the secondary occurrence of HAI [START_REF] Grimaldi | Profound and persistent decrease of circulating dendritic cells is associated with ICUacquired infection in patients with septic shock[END_REF]. In addition, in septic patients, this diminished DCs count was found to be associated with phenotypic alterations (decreased MHC class II molecules, CD80 and CD86 expressions) [START_REF] Poehlmann | Phenotype changes and impaired function of dendritic cell subsets in patients with sepsis: A prospective observational analysis[END_REF]. Importantly, these alterations lasted several weeks after patients' admission as it necessitates weeks after sepsis to recover normal DCs number of functions [START_REF] Poehlmann | Phenotype changes and impaired function of dendritic cell subsets in patients with sepsis: A prospective observational analysis[END_REF]. In total, although circulating DCs count might appear as a useful biological parameter to be monitored after sepsis, we still lack data obtained in large cohorts of patients to definitely include this parameter within a monitoring panel of sepsis-induced immune dysfunctions. Furthermore, as DCs identification and absolute counting in whole blood requires a multicolor flow cytometric protocol and calibrated beads, the cost of this parameter might be elevated.

MONOCYTES Functional Testing

Since it directly measures ex vivo the capacity of a cell population to respond to an immune challenge, functional testing ideally represents the best method to establish the occurrence of sepsis-induced immunosuppression. Regarding monocytes, many groups have described the altered capacity of septic patients' cells to release pro-inflammatory cytokines in response to stimulation with LPS, various TLR agonists or whole bacteria in vitro [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: A new skin for the old ceremony[END_REF][START_REF] Cavaillon | Bench-to-bedside review: Endotoxin tolerance as a model of leukocyte reprogramming in sepsis[END_REF][START_REF] Cavaillon | Determining the degree of immunodysregulation in sepsis[END_REF]. In contrast, their capacity to release anti-inflammatory molecules was conserved (e.g., IL-10). These tests, based on cytokine measurements in cell culture supernatant, represent reliable methods to assess the phenomenon of endotoxin tolerance, i.e., refractory state to subsequent endotoxin challenge [START_REF] Biswas | Endotoxin tolerance: New mechanisms, molecules and clinical significance[END_REF]. Although not really suitable to routine monitoring as they are time consuming (incubation time 1 cytokine assay), one of them (i.e., whole blood TNF release) has been used to stratify patients in a small clinical trial evaluating GM-CSF as an immuno-adjuvant therapy in sepsis [START_REF] Hall | Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome[END_REF]. To date, intracellular cytokine staining protocols are mainly used in experimental or observational clinical studies investigating monocytes functionalities. To our knowledge, nothing has been conducted in significant clinical cohorts of patients whereas preliminary results provided convincing results. Indeed, Fumeaux et al. nicely observed that septic monocytes were characterized by a predominant anti-inflammatory phenotype: increased IL-10/TNF intracellular ratio [START_REF] Fumeaux | Immune monitoring of patients with septic shock by measurement of intraleukocyte cytokines[END_REF]. Nevertheless, on the basis of improved protocols for intracellular stainings and functional testing (as described below for phospho STAT5 measurement), we may expect in a near future some easily standardizable whole blood protocols for rapid detection of intracellular cytokine by flow cytometry. This may constitute a major step in the immune monitoring of various clinical conditions, including sepsis.

Cell Surface Expression

The expressions of several membrane receptors have been shown to be altered on monocytes after sepsis. Decreased expressions of CX3CR1, CD80/86, or increased PD-L1 have been reported [START_REF] Pachot | Decreased expression of the fractalkine receptor CX3CR1 on circulating monocytes as new feature of sepsis-induced immunosuppression[END_REF][START_REF] Guignant | Programmed death-1 levels correlate with increased mortality, nosocomial infection and immune dysfunctions in septic shock patients[END_REF][START_REF] Manjuck | Decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients[END_REF]. Nevertheless, in this topic, the decreased major histocompatibility complex class II (MHC II) molecules expression on monocyte surface has become an unquestionable marker of injury-induced monocyte impairment. Indeed, there is an established consensus for considering low monocyte HLA-DR (mHLA-DR) as a good surrogate marker of monocyte anergy as it correlates with lower TNF-a and IL-1 releases in response to bacterial challenges (50) and reduced ability for antigen presentation [START_REF] Wolk | Impaired antigen presentation by human monocytes during endotoxin tolerance[END_REF]. This biomarker has been popularized by the group of Prof. HD. Volk (Charit e, Berlin) and has become the most published parameter in the field. In clinics, the magnitude and the persistent overtime of this altered phenotype is demonstrated to be associated with increased mortality and HAI in ICU patients [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF][START_REF] Landelle | Low monocyte human leukocyte antigen-DR is independently associated with nosocomial infections after septic shock[END_REF]. This aspect has been extensively reviewed elsewhere [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: A new skin for the old ceremony[END_REF][START_REF] Venet | Monitoring the immune response in sepsis: A rational approach to administration of immunoadjuvant therapies[END_REF]. It is noteworthy that, after adjustment for usual clinical confounders in multivariate analysis, decreased mHLA-DR remains an independent predictor of mortality/occurrence of HAI after sepsis [START_REF] Monneret | Persisting low monocyte human leukocyte antigen-DR expression predicts mortality in septic shock[END_REF][START_REF] Landelle | Low monocyte human leukocyte antigen-DR is independently associated with nosocomial infections after septic shock[END_REF]. This illustrated that monocyte anergy, and immunosuppression by extension, may contribute by itself to increased risk of adverse events in sepsis. This consequently reinforces the idea that immune recovery is a key parameter in favorable patients' outcome. Importantly, low mHLA-DR was also observed in various organs in post-mortem biopsies after septic shock [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF] and on BAL fluids macrophages of trauma patients in association with secondary pneumonia [START_REF] Nakos | Immunoparalysis in patients with severe trauma and the effect of inhaled interferon-gamma[END_REF][START_REF] Muehlstedt | Increased IL-10 production and HLA-DR suppression in the lungs of injured patients precede the development of nosocomial pneumonia[END_REF][START_REF] Chalk | Dysfunction of alveolar macrophages after cardiac surgery and postoperative pneumonia?-An observational study[END_REF]. This indicates that sepsis-induced immunosuppression is not restricted to the circulating compartment but must be considered as a systemic process.

Importantly, it was initially shown, in a small nonrandomized clinical trial that administration of GM-CSF to septic patients induced a sustained mHLA-DR recovery, which was accompanied by a restoration of ex vivo TNF-a production capacity [START_REF] Nierhaus | Reversal of immunoparalysis by recombinant human granulocytemacrophage colony-stimulating factor in patients with severe sepsis[END_REF]. These results were then confirmed in a randomized clinical trial enrolling 38 septic patients [START_REF] Meisel | Granu-locyte-macrophage colony-stimulating factor to reverse sepsisassociated immunosuppression: A double-blind, randomized, placebo-controlled multicenter trial[END_REF]. Of major importance, in this last study, patients were included on the basis of a low mHLA-DR (i.e., below 8,000 AB/C) on two consecutive days. This biomarker-guided GM-CSF therapy has been demonstrated to be safe and effective in restoring monocyte functionality and shortening the time of mechanical ventilation and hospital/ICU stay [START_REF] Meisel | Granu-locyte-macrophage colony-stimulating factor to reverse sepsisassociated immunosuppression: A double-blind, randomized, placebo-controlled multicenter trial[END_REF]. The next step would be to conduct multicentric studies to confirm these convincing GM-CSF effects on larger cohorts of patients. However, this necessitates a standardized flow cytometric protocol for mHLA-DR measurement so as to obtain reproducible results between laboratories certifying comparable patients' stratification between sites. Indeed, mHLA-DR measurement protocol by flow cytometry has evolved over years. Results were historically provided either as % of positive cells (including different protocols for setting up positivity threshold) or means of fluorescence intensities (MFI). None of them being acceptable in terms of strict standardization, a group of experts [START_REF] Docke | Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: A multicenter standardized study[END_REF] proposed a consensual protocol based on the use of calibrated beads (with known amounts of fluorochrome) to convert MFI in numbers of antibodies bound per cell (AB/C) (Fig. 2). In addition, mHLA-DR measurement requires strict preanalytical conditions: use of EDTA blood, staining within 1.5 h after blood collection or 4 h if storage at 148C, a washing step after staining [START_REF] Docke | Monitoring temporary immunodepression by flow cytometric measurement of monocytic HLA-DR expression: A multicenter standardized study[END_REF][START_REF] Monneret | Analytical requirements for measuring monocytic human lymphocyte antigen DR by flow cytometry: Application to the monitoring of patients with septic shock[END_REF]. Such strict protocol provided excellent correlations between mHLA-DR results (Fig. 3) [START_REF] Demaret | Inter-laboratory assessment of flow cytometric monocyte HLA-DR expression in clinical samples[END_REF]. Importantly, this observation was valid between different flow cytometers from different manufacturers (i.e., Beckman-Coulter and Becton-Dickinson) demonstrating that mHLA-DR is ready for prime time as a sepsisinduced immune alterations biomarker. Of note, due to preanalytical requirements and as flow cytometers are usually not available 24/7 in hospitals, alternative meth-ods have been developed for the measurement of HLA-DR, as for example by qRT-PCR in whole blood [START_REF] Cazalis | Decreased HLA-DR antigen-associated invariant chain (CD74) mRNA expression predicts mortality after septic shock[END_REF][START_REF] Cajander | Preliminary results in quantitation of HLA-DRA by realtime PCR: A promising approach to identify immunosuppression in sepsis[END_REF]. Although the putative development of automated systems for qRT-PCR may represent a possible perspective, for now, host response mRNA measurement remains based on homemade protocols. In addition, no data are available regarding results standardization (interlaboratory assessment, choice of housekeeping genes), analytical specifications (reproducibility, coefficient of variation) and validation in large cohorts of various ICU patients. Thus, for now, flow cytometry remains the most appropriate and validated tool for ICU patients immunomonitoring [START_REF] Monneret | Monocyte HLA-DR in sepsis: Shall we stop following the flow?[END_REF]. Furthermore, recent fully automated flow cytometer systems offer dedicated applications (CD41 lymphocyte absolute counts, CD64 expression on neutrophils) that may be extended to mHLA-DR measurements.

LYMPHOCYTES Lymphopenia

A markedly decreased number of circulating lymphocytes has been repeatedly observed in patients after sepsis [START_REF] Guignant | Programmed death-1 levels correlate with increased mortality, nosocomial infection and immune dysfunctions in septic shock patients[END_REF][START_REF] Tulzo | Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome[END_REF][START_REF] Roth | Susceptibility to programmed cell death in T-lymphocytes from septic patients: a mechanism for lymphopenia and Th2 predominance[END_REF][START_REF] Spolarics | Depressed interleukin-12-producing activity by monocytes correlates with adverse clinical course and a shift toward Th2type lymphocyte pattern in severely injured male trauma patients[END_REF][START_REF] Felmet | Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure[END_REF][START_REF] Hotchkiss | Prevention of lymphocyte apoptosis-a potential treatment of sepsis?[END_REF][START_REF] Venet | Early assessment of leukocyte alterations at diagnosis of septic shock[END_REF][START_REF] Guignant | Increased MerTK expression in circulating innate immune cells of patients with septic shock[END_REF][START_REF] Venet | Decreased T cell repertoire diversity in sepsis: A preliminary study[END_REF][START_REF] Inoue | Reduction of immunocompetent T cells followed by prolonged lymphopenia in severe sepsis in the elderly*[END_REF]. Every lymphocyte subpopulation was affected (except regulatory T cells, see below). Most importantly, the extent of this lymphopenia has been shown to correlate with the development of HAI and/or death at 28 days [START_REF] Drewry | Persistent lymphopenia after diagnosis of sepsis predicts mortality[END_REF][START_REF] Tulzo | Early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome[END_REF][START_REF] Felmet | Prolonged lymphopenia, lymphoid depletion, and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure[END_REF][START_REF] Inoue | Reduction of immunocompetent T cells followed by prolonged lymphopenia in severe sepsis in the elderly*[END_REF][START_REF] Heffernan | Failure to normalize lymphopenia following trauma is associated with increased mortality, independent of the leukocytosis pattern[END_REF][START_REF] Chung | Severe lymphopenia is associated with elevated plasma interleukin-15 levels and increased mortality during severe sepsis[END_REF]. Importantly, this lymphopenia may last for several weeks after the onset of sepsis and this lack of recovery was associated with delayed infectious complications [START_REF] Inoue | Reduction of immunocompetent T cells followed by prolonged lymphopenia in severe sepsis in the elderly*[END_REF].

NK Cells, cD Lymphocytes, Mucosal-Associated Invariant T Lymphocytes

The preferential tissue locations of these lymphocyte subpopulations along with their low percentages in peripheral blood render their study in clinic difficult. This may explain why those cell subsets have been somewhat ignored during years in sepsis pathophysiology. Recent works investigated NK cells in sepsis and showed altered functionalities [START_REF] Chiche | The role of natural killer cells in sepsis[END_REF][START_REF] Souza-Fonseca-Guimaraes | Natural killer (NK) cells in antibacterial innate immunity: Angels or devils?[END_REF][START_REF] Souza-Fonseca-Guimaraes | NK cell tolerance to TLR agonists mediated by regulatory T cells after polymicrobial sepsis[END_REF]. More specifically, regarding flow cytometry results, the number of circulating NK cells was markedly decreased after injury in patients [START_REF] Venet | Early assessment of leukocyte alterations at diagnosis of septic shock[END_REF][START_REF] Forel | Phenotype and functions of natural killer cells in critically-ill septic patients[END_REF]. This decrease may persist for one Week [START_REF] Holub | Lymphocyte subset numbers depend on the bacterial origin of sepsis[END_REF] and was found to be associated with increased mortality [START_REF] Tsaganos | Early changes of CD4-positive lymphocytes and NK cells in patients with severe Gram-negative sepsis[END_REF]. However, to date, no data describes that a specific fall in NK cells (out of other lymphocyte subsets) might be by itself responsible for such deleterious outcome. With that said, due to their central role in antiviral defense, one may speculate that such decreased NK lymphocyte number may favor viral reactivations frequently described in ICU patients [START_REF] Walton | Reactivation of multiple viruses in patients with sepsis[END_REF][START_REF] Luyt | Viral infections in the ICU[END_REF][START_REF] Cook | Cytomegalovirus reactivation in critically ill immunocompetent hosts: A decade of progress and remaining challenges[END_REF]. Again, this deserves to be demonstrated.

Septic patients also present with decreased numbers of circulating mucosal-associated invariant T (MAIT) lymphocytes [START_REF] Grimaldi | Specific MAIT cell behaviour among innate-like T lymphocytes in critically ill patients with severe infections[END_REF]. The incidence of ICU-acquired infections was higher in patients with persistent MAIT cell depletion [START_REF] Grimaldi | Specific MAIT cell behaviour among innate-like T lymphocytes in critically ill patients with severe infections[END_REF]. Similarly, a reduced percentage of gd lymphocytes has been reported [START_REF] Venet | Both percentage of gammadelta T lymphocytes and CD3 expression are reduced during septic shock[END_REF]. And these cells, appear to no longer be able to proliferate after sepsis [START_REF] Galley | Characterisation of gamma delta (gammadelta) T cell populations in patients with sepsis[END_REF].

Collectively, investigations in clinic on the specific roles of these lymphocyte subsets would be of great interest after sepsis. However, clinical data demonstrating the putative interest of their monitoring are still missing. Additional observational studies are necessary before proposing inclusion of these parameters in a panel of markers for the monitoring of septic patients.

T Cell Exhaustion

Many alterations have been reported regarding T lymphocytes during sepsis-induced immunosuppression: functional impairments (decreased proliferation, cytokine production IL-2/IFNg, STAT-5 phosphorylation upon TCR activation), decreased TCR related molecule expression (decreased CD3, CD28), increased pro-apoptotic molecules expression, decreased repertoire, alterations of all T helper subsets (objectivized by decreased transcription factors) [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: A new skin for the old ceremony[END_REF][START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF][START_REF] Venet | Monitoring the immune response in sepsis: A rational approach to administration of immunoadjuvant therapies[END_REF][START_REF] Cabrera-Perez | Impact of sepsis on CD4 T cell immunity[END_REF].

Functional testing

Lymphocyte anergy (i.e., lack of proliferation) has been historically assessed by 3 H-thymidine uptake (that presents with radioactivity drawbacks). More recently, by flow cytometry, fluorescent probes (e.g., edU) have been validated in comparison with this reference method and provided convincing alternative for clinical use [START_REF] Poujol | Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement[END_REF]. However, as proliferation test require long incubation time (2-3 days for mitogens), they are not suitable for clinical decision-making and thus are not performed on a routine basis. Thus, a functional surrogate marker of lymphocyte proliferation capacity, easily measurable by flow, remains highly desirable. Some alternatives exist thanks to the development of standardized shortened protocol for intracellular staining. For example, a decreased phosphorylation of STAT-5 in response to cytokine activation was recently shown to identify a group of nonsurviving septic shock patients [START_REF] Demaret | STAT5 phosphorylation in T cell subsets from septic patients in response to recombinant human interleukin-7: A pilot study[END_REF]. As the protocol takes less than 90 min to be completed in whole blood, upon clinical validation in large cohort of patients, it may provide a smart approach to circumvent drawbacks of lymphocyte proliferation testing.

Phenotypic alteration

One recent and important feature of lymphocyte anergy in sepsis is the description of various increased co-inhibitory receptors expressions: PD-1 (CD279), LAG-3 (CD223), CTLA-4 (CD152), TIM-3, BTLA (CD272) [START_REF] Guignant | Programmed death-1 levels correlate with increased mortality, nosocomial infection and immune dysfunctions in septic shock patients[END_REF][START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF][START_REF] Boomer | A prospective analysis of lymphocyte phenotype and function over the course of acute sepsis[END_REF][START_REF] Monneret | Marked elevation of human circulating CD41CD251 regulatory T cells in sepsis-induced immunoparalysis[END_REF][START_REF] Hou | Tim-3 negatively mediates natural killer cell function in LPS-induced endotoxic shock[END_REF][START_REF] Wang | Up-regulation of Programmed cell death 1 ligand 1 on neutrophils may be involved in sepsis-induced immunosuppression: An animal study and a prospective case-control study[END_REF][START_REF] Zhao | Blockade of the T cell immunoglobulin and mucin domain protein 3 pathway exacerbates sepsis-induced immune deviation and immunosuppression[END_REF][START_REF] Shubin | BTLA expression contributes to septic morbidity and mortality by inducing innate inflammatory cell dysfunction[END_REF][START_REF] Shubin | B and T lymphocyte attenuator expression on CD41 T-cells associates with sepsis and subsequent infections in ICU patients[END_REF] contributing to the definition of T cell exhaustion as that described in chronic viral infections. In septic patients, although only low levels of expressions were observed at the onset of syndrome; over the first week, these expressions were progressively upregulated on lymphocytes [START_REF] Boomer | A prospective analysis of lymphocyte phenotype and function over the course of acute sepsis[END_REF]. Regarding PD-1, its overexpression (as well as that of its ligand, PD-L1) has been found to be associated with mortality and the occurrence of HAI in septic shock patients and was inversely correlated with lymphocyte proliferation capacity [START_REF] Guignant | Programmed death-1 levels correlate with increased mortality, nosocomial infection and immune dysfunctions in septic shock patients[END_REF]. These results observed in circulating cells complement the work on postmortem biopsies in which the authors showed that lymphocyte exhaustion is a severe and profound mechanism occurring not only systematically but also locally in organs after septic shock [START_REF] Boomer | Immunosuppression in patients who die of sepsis and multiple organ failure[END_REF]. Indeed, the authors demonstrated that, in deceased septic shock patients, both lung and spleen lymphocytes were markedly phenotypically and functionally affected (decreased cell counts, altered functional response to stimulation and increased coinhibitory receptor expressions). This increased coinhibitory receptors expressions is likely of great interest as we are to treat septic patients with blocking antibodies (anti-PD-L1 and anti-CTLA-4) targeting these immune check-points. The measurement of these target molecules by flow might be a suitable tool to identify the patients who could mostly benefit from such innovative therapies. Although a large panel of coinhibitory receptor phenotyping has been investigated, the low number of patients included in each study precludes any robust conclusion, especially regarding correlations with deleterious clinical outcomes. The true potential of these receptors (in terms of monitoring) still needs prospective observational studies in larger cohorts of patients.

Regulatory T cells

One major feature of T lymphocyte dysfunctions after sepsis is the increased proportion, among circulating patients' lymphocytes, of a cell subpopulation with known regulatory properties. This was initially described in septic shock patients [START_REF] Monneret | Marked elevation of human circulating CD41CD251 regulatory T cells in sepsis-induced immunoparalysis[END_REF]. This rise was observed immediately after the onset of shock but persisted only in non-surviving patients. These results were further extended by showing that this relative increase was due to a decrease of the effector T lymphocyte number and not so much to a change in the absolute Treg count [START_REF] Venet | Increased percentage of CD41CD251 regulatory T cells during septic shock is due to the decrease of CD41CD25-lymphocytes[END_REF]. Since these pioneer works, many groups have confirmed those results in experimental and clinical sepsisincreased percentages being observed either in blood or in spleens (septic mice) and in other clinical contexts [START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF]. It seems likely that increased Treg play a deleterious role in sepsis-induced immune alterations as described in various animal models [START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF]. In septic shock patients, it was observed that augmented Treg percentage significantly and inversely correlated with decreased lymphocyte proliferation [START_REF] Venet | Increased circulating regula-tory T cells (CD4(1)CD25 (1)CD127 (-)) contribute to lymphocyte anergy in septic shock patients[END_REF]. This phenomenon, recapitulated in CLP-mice, was totally abrogated by using FOXP3 siRNA inhibiting Treg functions [START_REF] Venet | Increased circulating regula-tory T cells (CD4(1)CD25 (1)CD127 (-)) contribute to lymphocyte anergy in septic shock patients[END_REF]. Recent clinical works aligned with initial descriptions (100). Of note, Treg overexpression of CD39 in septic patients has been reported to be significantly associated with mortality (101). CD39 is an ectoenzyme that hydrolyses ATP into ADP, known to be possessing immunosuppressive properties. Interestingly, in HIV-patients, IL-7 negatively regulates Treg by decreasing CD39 activity (102). Thus, CD39 monitoring in Treg deserves further investigations in sepsis. Regarding results standardization in clinic, Treg phenotyping by flow cytometry remains difficult as various cut-off values and protocols are used between laboratories (i.e., CD41CD251, CD41CD251CD127-, CD41FOXP31. . .). A multicentric study, assessing Treg values in sepsis according to a common definition would be desirable.

EMERGING FIELDS

Importantly, the mechanisms sustaining such immunosuppression are not specific to sepsis as they are induced each time the body develops an exacerbated inflammatory response. Therefore we propose the use of a more generic term of "injury-induced immunosuppression". Indeed, decreased mHLA-DR expression has been observed in patients with trauma, burns, pancreatitis, acute cirrhosis, surgery, extra-corporal circulation in association with increased mortality and/or HAI occurrence [START_REF] Venet | Monitoring the immune response in sepsis: A rational approach to administration of immunoadjuvant therapies[END_REF]. This is also the case for decreased DCs number, lymphocyte anergy, increased inhibitory receptors, increased Treg. . . [START_REF] Monneret | Monitoring immune dysfunctions in the septic patient: A new skin for the old ceremony[END_REF][START_REF] Hotchkiss | Sepsis-induced immunosuppression: From cellular dysfunctions to immunotherapy[END_REF](103)(104)(105)(106)(107)(108)(109)(110). As these injured patients all present with an increased risk of HAI; an immunomonitoring tool available in routine clinical setting may help in identifying those with marked immune alterations for whom one may envisage to initiate preemptive actions to decrease this secondary infectious risk. This may open new angle in battling and preventing nosocomial infections in hospital.

First reports in neonatology/pediatric sepsis indicate interesting results as similar immune alterations seem to be associated with poor outcome and/or HAI risk (decreased mHLA-DR, decreased lymphocyte cytokine production, lymphopenia) [START_REF] Hall | Immunoparalysis and nosocomial infection in children with multiple organ dysfunction syndrome[END_REF](111)(112)(113)(114). As sepsis remains a major cause of death in pediatrics, further investigations in immunological monitoring appear of major importance.

As previously mentioned, long term mortality (i.e., up to 10 years) is known to be increased after severe sepsis [START_REF] Yende | Long-term outcomes from sepsis[END_REF][START_REF] Wang | Long-term mortality after community-acquired sepsis: A longitudinal population-based cohort study[END_REF][START_REF] Linder | Longterm (10-year) mortality of younger previously healthy patients with severe sepsis/septic shock is worse than that of patients with nonseptic critical illness and of the general population[END_REF]115). A significant percentage of this delayed mortality (and of hospital readmission) is attributable to infectious complications reinforcing the hypothesis that immune alterations may last for years after sepsis. Thus, another major field of investigation would be the monitoring over years of immune functions in patients who survived sepsis.

In addition, mHLA-DR starts to be reported in non-ICU, noninfectious contexts. In lymphoma, lowest mHLA-DR values are observed in patients with relapses (116). Surprisingly, loss of mHLA-DR expression has been reported as early as 1988 in cancer patients but has remained somewhat ignored (117). In solid organ (118) and hematopoietic stem cell transplantations (119), decreased values are observed in patients who developed secondary infections (reflecting a too strong effect of immunosuppressant drugs as hypothesized by the authors). Although we do not know if results could be interpreted similarly to those of septic patients (i.e., similar thresholds to define immunosuppression associated-risks), these fields deserve to be further explored.

On a more general perspective, it has been recently demonstrated that variation in the human immune response is largely driven by nonheritable influences (120). Among various components, comorbidities (including infectious exposure through life) and age are of major importance. As septic patients are aged (median around 65 years) and present with various comorbidities, this reinforce the idea that an individual monitoring of immune parameters is of crucial interest as, depending on its own history, no patient will immunologically respond like another one.

Collectively, these data indicate that mHLA-DR determination has an interest in many clinical conditions for which level of immunocompetence needs to be assessed. Given its clinical interest and considering its relative simplicity (two color flow cytometry staining), moderate price (<15 e), and possible standardized expression of results, mHLA-DR might become a blockbuster tool in immunomonitoring.

CONCLUSION

In sepsis, innovative immuno-monitoring strategies that characterize the host immune response and thus permit personalized medicine approaches appear as a reasonable perspective. This would allow to decrease heterogeneity of septic population and to better take into account the rapidly changing immune response overtime after initial infection, from exacerbated inflammation to severe immunosuppression. For such purpose, the most validated tools are based on flow cytometry (Table 1). To date, a panel including neutrophil CD10/ CD16 expressions measurements, total lymphocyte and CD4 counts (along with Treg) and monitoring of HLA-DR expression on monocyte would provide, for a reasonable cost, useful information on the main circulating type of cells (i.e., lymphocytes, monocytes, neutrophils). As immunostimulation will not be started immediately at septic shock patients' admission in order to avoid any potentially uncontrolled reactivation of the inflammatory cascade, this monitoring may start 2-3 days after the onset of sepsis and then twice a week. The exact timing of this monitoring will depend on first results from clinical trials based on IL-7 and GM-CSF that should be starting before the end of 2015. Obviously and ideally, a large multicenter study investigating various and more detailed aspects of immune response after sepsis would be of major interest to perfectly identify and compare the most appropriate immuno-monitoring markers after sepsis. Beyond sepsis, in other medical conditions, such simple and robust tools are desirable to assess the degree of immune competence of a given patient. For this purpose, flow cytometry remains the most appropriate technology so far.

FIG. 1 .

 1 FIG. 1. Main features of sepsis-induced immunosuppression and related consequences. Abbreviations: DC, dendritic cell; Ag, antigen; NK, natural killer cells; T CD4, CD4 lymphocyte; T CD8, CD8 lymphocyte; Treg, regulatory T cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

FIG. 2 .

 2 FIG. 2. (A) Illustrative examples of monocyte gating and typical monoparametric HLA-DR histograms (gated on CD14-positive monocyte population) in septic patients and healthy controls. Results are provided as MFI (median of fluorescence intensity) and AB/C (antibody bound per cell). (B) Based on medians of fluorescence intensity measured on each bead subpopulation and on the number of phycoerythrin (PE) molecules per bead provided by the manufacturer, a calibration curve can be generated for the calculation of AB/C. From this curve, MFI are transformed in AB/C and become standardized between centers. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Table 1

 1 Immune Parameters/Alterations Measurable by Flow Cytometry in Patients Presenting with Sepsis-Induced Immunosuppression

		Variation observed
	Parameter	in patients	Link with outcome	Putative therapy
	% CD10 Dim CD16 Dim (among neutrophils)	"	Yes-death	?
	PD-L1 expression (on monocytes)	"	Yes-HAI	Anti PD-L1 Abs
	HLA-DR expression (on monocytes)	#	Yes-mortality and HAI	GM-CSF, Interferon-g
	intracellular TNF (in monocytes)	#	Not observed	GM-CSF, Interferon-g
	Total lymphocytes (absolute count)	#	Yes-death	IL-7
	Total CD3 or CD4 (absolute count)	#	Yes-death	IL-7
	Apoptosis (in lymphocytes)	"	Yes-death	-
	pSTAT5 expression (in lymphocytes)	#	Yes-death	IL-7
	% regulatory T cells (among CD4)	"	Yes-death	IL-7
	CD39 expression (on regulatory T cells)	"	Yes-HAI	IL-7
	PD-1 expression (on lymphocytes)	"	Yes-death	Anti PD-1/PD-L1 Abs
	BTLA, CTLA-4, TIM-3 expressions	"	Not observed	Monoclonals?
	(on lymphocytes)
	Innate lymphocytes (absolute count	#	Yes-HAI	-
	or among total Ly)
	HAI, Health-care associated infection; Abs, antibodies.

Cytometry Part B: Clinical Cytometry 15524957, 2016, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/cyto.b.21270 by Cochrane France, Wiley Online Library on [06/04/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License

ACKNOWLEDGMENTS

This invited review is based on a presentation (Monneret G, Cell surface markers for sepsis) given in Seattle, WA for the 29th International Clinical Cytometry Meeting (October 2014). The authors declare they have no conflict of interest.


M.D Xavier Carcopino

Ph.D Maggie Cruickshank

PhD Simon Leeson

MD Charles Redman

MD Pekka Nieminen

MD Maggie Cruickshank

PANDEMIC ON SCREENING PROGRAMMES FOR CERVICAL CANCER PREVENTION ACROSS EUROPE

Keywords: screening, COVID-19, cervical cancer, consequences screening, COVID-19, cervical cancer, consequences

Objective

To estimate the impact of COVID-19 pandemic on the practice of cervical cancer screening in European countries.

Methods

A 3 rounds e-survey was conducted among the 31 European Federation for Colposcopy (EFC) member countries during 2020. Each representative was asked to answer to each questionnaire for their own country. Questionnaires were not anonymous. The first questionnaire was sent in April 2020 and second and third in June and December 2020, respectively. Results 25 of the 31 European countries solicited responded. A total of 19 (70.4%) countries reported that screening for cervical cancer was suspended at least once during the 3 rounds of questionnaires. Additionally, 11 countries reported stopping colposcopy and treatments for cervical precancerous lesions at least once during the 3 rounds of questionnaires. These situations evolved with time, with the highest rate of countries recommending suspension of screening, colposcopy and treatments during the second round of the survey. At round 3, no country recommended screening, colposcopy and treatment, and 12 (57.5%) countries reported normal screening was fully implemented.

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Conclusion

Our results suggest massive disruption in cervical cancer screening programmes across Europe resulting from COVID-19 pandemic. Increase in the incidence of cervical cancer is to be expected.

Introduction

Cervical cancer remains one of the most common cancers and causes of cancer-related deaths in women worldwide. In Europe, it accounted for 33000 cases and 15000 deaths in 2018. [START_REF] Arbyn | Estimates of incidence and mortality of cervical cancer in 2018: a worldwide analysis[END_REF] With the possibility for screening and human papillomavirus (HPV) vaccination, cervical cancer is one of the few preventable cancers. In August 2020, the WHO adopted the global strategy for cervical cancer elimination with the recommendation to achieve 90% HPV vaccination coverage, 70% screening coverage and 90% access to treatment of screen-detected lesions. [START_REF]WHO's Global strategy to accelerate the elimination of cervical cancer as a public health problem[END_REF] Screening is key to the prevention and early detection of the disease. In Europe, implemented screening programmes have shown to be extremely efficient, resulting in massive reduction in cervical cancer mortality. [START_REF] Jansen | Effect of organised cervical cancer screening on cervical cancer mortality in Europe: a systematic review[END_REF] Reduction in the incidence rate is estimated to be 50-60%. [START_REF] Vaccarella | Preventable fractions of cervical cancer via effective screening in six Baltic, central, and eastern European countries 2017-40: a population-based study[END_REF] In the first half of 2020, the COVID-19 pandemic has massively impacted population worldwide. Hospitals in most impacted areas across Europe have been overcrowded, with the need to devote a large part of the medical resources to the management of COVID patients, reinforcement of intensive care units' resources and opening of dedicated wards. The suspension or postponement of non-urgent procedures were among the first adopted strategies.

With health care services overrun, measures to avoid non-urgent medical procedures were endorsed, resulting in disruption of many medical activities. Several medical societies have recommended cancelling, postponing, or adapting non-urgent procedures of cancer patients. [START_REF] Hanna | Cancer, COVID-19 and the precautionary principle: prioritizing treatment during a global pandemic[END_REF][START_REF] Burki | Cancer guidelines during the COVID-19 pandemic[END_REF][START_REF] Akladios | Recommendations for the surgical management of gynecological cancers during the COVID-19 pandemic -FRANCOGYN group for the CNGOF[END_REF][START_REF] Colombo | ESMO management and treatment adapted recommendations in the COVID-19 era: gynaecological malignancies[END_REF] Additionally, the implementation of social distancing and extensive lockdown periods, restriction of mobility and patients' fear of getting infected with coronavirus have severely jeopardized elective medical care and existing preventions programmes.

Recent data has already shown a significant reduction in breast, colorectal and cervical cancer screening with several countries reporting a massive drop in screening, diagnosis and treatment because of COVID-19. [START_REF] Vecchio Blanco | The impact of COVID-19 pandemic in the colorectal cancer prevention[END_REF][START_REF] Basu | Cancer Screening in the Coronavirus Pandemic Era: Adjusting to a New Situation[END_REF][START_REF] Fagundes | Dealing with cancer screening in the COVID-19 era[END_REF] Thus, a 28 to 100 % decrease in colorectal cancer screening depending on countries and different times after the onset of the COVID-19 has been recently reported. [START_REF] Mazidimoradi | Impact of the COVID-19 Pandemic on Colorectal Cancer Screening: a Systematic Review[END_REF] This unprecedent worldwide situation will possibly result in delay in diagnosis with more advance stage at diagnosis possibly resulting in an increased mortality from preventable cancers. Using a national population-based modelling study based upon the English National Health Service (NHS) cancer registration and hospital dataset, a recent publication estimated a 15.3-16.6% increase in the 5-years mortality due to colorectal cancer and 7.9-9.6% increase in the 5-years mortality due to breast cancer. [START_REF] Maringe | The impact of the COVID-19 pandemic on cancer deaths due to delays in diagnosis in England, UK: a national, population-based, modelling study[END_REF] To date, very few data about the impact of COVID-19 pandemic on cervical cancer screening have been published. Few authors have already reported major concerns about future consequences of the repeated blows of COVID-19 on cervical cancer screening programmes. [START_REF] Arbyn | Tackling cervical cancer in Europe amidst the COVID-19 pandemic[END_REF][START_REF] Gourd | COVID-19 pandemic causes cervical cancer screening crisis[END_REF] Investigating the long-term consequences of COVID-19 outbreak on current cervical cancer screening programmes and the impact of different decisions taken across Europe is therefore a major public health matter.

The aim of this survey was to estimate the impact of COVID-19 pandemic on the practice of cervical cancer screening in European countries.

Materials and methods

This survey was endorsed by the European Federation of Colposcopy (EFC). A 3 round survey was conducted using online questionnaires that were sent to the 31 EFC member countries during 2020. All questionnaires were sent using the google forms web platform: https://www.google.fr/intl/fr/forms/about/. At each round of the survey, a link to each online questionnaire was sent by email to the representative of each national society of colposcopy.

Each representative was asked to answer to each questionnaire for his own country.

Questionnaires were not anonymous. IRB approval was not necessary as this study does not rely on research on human subjects.

The first questionnaire was sent the 12 th of April 2020, shortly after the first wave of the pandemic in Europe. It included 16 questions with 13 structured multiple-choice questions (MCQs) and three open questions. Questions focussed on the COVID situation in each country at the time of questionnaire, with specific evaluation of the specific impact on screening, practice of colposcopy and of treatment of precancerous lesions of the cervix. Finally, respondents were asked if specific national recommendations had been issued in their country regarding how screening, the practice of colposcopy and treatment should be adapted to the current situation. The second and third questionnaires were sent on the first of June 2021 and on the14 th of December 2020, respectively. They consisted of 17 ( 14MCQs and three open questions) and 14 (12 MCQs and two open) questions respectively. Questions were mostly a repetition the ones from first questionnaire, but additional questions about the negative impact on screening and cervical cancer prevention were added. For each questionnaire, a one-month period was offered for all representatives to respond. During each round, two reminder emails were sent to representatives.

Answers were collected and analysed using excel v16 spreadsheets (Microsoft).

Results

25 of the 31 European countries solicited returned a questionnaire. Nineteen answered all three rounds of questionnaires, four answered two and two countries provided answers to only one of the three questionnaires. There were 23 respondents in each of the first two rounds of the survey and 21 in the last one.

Among the participating countries, 12 (52.2%) reported using HPV testing as the primary test for cervical cancer screening. A lockdown with the national population confined at home except for vital needs was reported by all responding countries during round one, with progressive reduction in this rate over the subsequent two other rounds of the survey (Table 1). A total of 19 (70.4%) countries reported they had been advised to stop screening for cervical cancer at least during one of the 3 rounds of questionnaires (Figure 1). Additionally, 11 countries reported that they had to stop performing colposcopy and treatments for cervical precancerous lesions in at least at one of the three rounds of questionnaires. These situations evolved with time, with the highest cessation rates of screening, colposcopy and treatments being reported during the second round of the survey (Table 1). In round three, no country reported that there were recommendations to stop screening, colposcopy or treatment anymore, and 12 (57.5 %) countries reported they had gone back fully to normal screening.

In round one and two, 10 (43.5%) and 8 (34.8%) respondents expressed concerns about actual risk to women's health, respectively. Finally, in round three, 19 (90.5 %) reported that the COVID pandemic had had a negative impact on cervical cancer screening and prevention.

Discussion

Our survey provides a crude overview of the short-term consequences of the COVID-19 pandemic on cervical cancer screening in Europe. With 70.4% of participating countries reporting having stopped cervical cancer screening during at least one of the three rounds of the survey, our results show massive disruption in cervical cancer screening programmes across Europe. Additionally, it is noticeable that diagnosis and treatment activities have also massively suffered from the COVID-19 pandemic, with colposcopy and treatments being temporarily interrupted in almost half of European participating countries. The question remains whether such decisions will result in an increased cervical cancer incidence and related mortality in Europe. With all respondents reporting in round three a withdrawal of recommendations to limit screening, colposcopy and treatment, our findings suggest that the disruption lasted only for a limited period, possibly up to six months. This resumption was probably related to the fall in the incidence of the COVID cases at the time of round two, as previously modelized. [START_REF] Basu | Cancer Screening in the Coronavirus Pandemic Era: Adjusting to a New Situation[END_REF] Initially, when information on the virus and mode of transmission was limited, there were concerns if colposcopy treatments could be aerosol-generating procedures. Although most treatments can be done as out-patient procedures under local anaesthetic, there were more restrictions on general anaesthetic due to covid risks. It is however notable that the suspension of the recommendation to stop screening, colposcopy and treatment did not necessarily meant a fully return to normal services, as only 57.5% of respondents reported so at round three. Therefore, a long-term effect of the COVID-19 pandemic on global cervical cancer prevention activities across Europe cannot be ruled out.

Although most respondents expected that the COVID-19 pandemic had a negative impact on cervical cancer prevention, the genuine impact of cervical cancer screening and prevention remains unknown. Only modelled data regarding breast, colorectal, lung and oesophageal cancers related mortality are currently available with the anticipation of substantial increases in the incidence and mortality of these avoidable cancers. [START_REF] Maringe | The impact of the COVID-19 pandemic on cancer deaths due to delays in diagnosis in England, UK: a national, population-based, modelling study[END_REF] Although data on the COVID-19 impact on cervical cancer incidence and related mortality are not available yet, our results suggest that the same is to be expected for cervical cancer. Monitoring of cervical cancer incidence within next years in Europe will therefore be paramount.

There is definitely an urge to carefully manage the resumption of suspended cervical cancer prevention activities. [START_REF] Ciavattini | European Federation for Colposcopy (EFC) and European Society of Gynaecological Oncology (ESGO) joint considerations about human papillomavirus (HPV) vaccination, screening programs, colposcopy, and surgery during and after the COVID-19 pandemic[END_REF] Patients who should have been screened at the time of the suspension period should be offered an appropriate catch-up screening test. It is also crucial to ensure that all patients with positive screening test who have missed proper colposcopic diagnosis evaluation should be recalled. However, there is no guarantee that such catch-up procedure will always be applicable. Cervical cancer screening in Europe is not homogeneous, as some countries have central funded organized screening programmes whilst others are opportunistic with much practice done by independent practitioners. There is no ascertainment of the capacity to catch-up with back logs of cytology or colposcopy and triaging referrals to colposcopy. One can therefore expect this strategy to dramatically vary across different health systems. With the ongoing persistence of the COVID-19 pandemic, there is also a need to adapt cervical cancer screening guidelines. Several societies, including the EFC have provided guidelines for the triage of patients needing a prompt colposcopic evaluation and for those whose care could be safely postponed, management of screen-positive women, preinvasive lesions of the lower genital tract and post-treatment follow up test of cure. [START_REF] Ciavattini | European Federation for Colposcopy (EFC) and European Society of Gynaecological Oncology (ESGO) joint considerations about human papillomavirus (HPV) vaccination, screening programs, colposcopy, and surgery during and after the COVID-19 pandemic[END_REF][START_REF] Ciavattini | Expert consensus from the Italian Society for Colposcopy and Cervico-Vaginal Pathology (SICPCV) for colposcopy and outpatient surgery of the lower genital tract during the COVID-19 pandemic[END_REF][START_REF] Asccp | ASCCP Interim Guidance for Timing of Diagnostic and Treatment Procedures for Patients with Abnormal Cervical Screening Tests[END_REF][START_REF] Bsccp | Colposcopy guidance during COVID 19 pandemic[END_REF] This unprecedented situation also forces us to consider alternative prevention methods and to promote more cost-efficient evidence-based protocols. With only half of respondents reporting having an HPV testing-based screening, there is a need for the implementation of more efficient prevention protocols across Europe. HPV screening was demonstrated to offer greater protection against cervical cancer than cytological screening and to offer the possibility for longer intervals between tests. [START_REF] Koliopoulos | Cytology versus HPV testing for cervical cancer screening in the general population[END_REF][START_REF] Ronco | Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials[END_REF] Additionally, the COVID-19 pandemic generates opportunities for considering self-sampling HPV testing-based screening programmes. This method offers the possibility of at-home sample collection by the women themselves and was shown to be efficient with reliable results. [START_REF] Arbyn | Detecting cervical precancer and reaching underscreened women by using HPV testing on self samples: updated meta-analyses[END_REF] It also avoids patients having to travel if test kits are posted to them and the exposure within clinics, therefore reducing both the risks of contamination for medical staff and patients.

Although our study provides a overview of COVID-19 impact on cervical cancer screening and prevention across Europe, it suffers from limitations that should be carefully considered when interpreting our results. As we only collected answers from representatives of national societies and did not access to crude screening data, we cannot assess the genuine impact of the pandemic on the screening participation rate. Therefore, we are unable to assess how and for how long the individuals recommendations to stop screening, colposcopic examinations and treatment have been applied in routine practice amongst European countries. With only half of respondents acknowledging a return to normal screening at round three, despite restrictions to screening having been lifted in all countries, it is possible that consequences might be even worse than what our data suggest. For all these reasons, we believe that increase in the incidence of cervical cancer is to be expected.

Conclusion

The COVID-19 pandemic has resulted in the disruption cervical cancer prevention activities in most European countries. The consequences were not limited to cervical cancer screening activities but also impacted both diagnostic and treatment activities. There is an absolute need to carefully manage the resumption of suspended cervical cancer prevention activities. All European countries that interrupted their screening programmes should ensure that an. appropriate catch-up screening test is offered as soon as possible for those to all women whose screening was interrupted. It is also crucial to ensure that all women with positive screening tests receive appropriate colposcopic diagnosis. Without these measures, increases in the incidence and the mortality of cervical cancer is to be expected in Europe as collateral damage of the COVID-19 pandemic. Declaration of Interests: All authors have no conflict of interest to declare.

Table 1 .

 1 Impact of COVID outbreak on cervical cancer screening and prevention in European countries during the 3 rounds of the survey.

		Round 1	Round 2	Round 3
		(n=23)	(n=23)	(n=21)
	Population confined at home	23 (100)	15 (65.2)	10 (47.6)
	Advised to
	Stop screening	17 (73.9)	19 (82.6)	0 (0)
	Stop performing colposcopy	7 (30.4)	10 (43.5)	0 (0)
	Stop performing treatment	11 (47.8)	10 (43.5)	0 (0)

Table

  Click here to access/download;Table;Table 1 .docx Figure Figure 1. Representation of all participating European countries who reported they were recommended to stop cervical cancer screening at least in one of the 3 rounds of questionnaires.

Acknowledgements:

We are particularly grateful to all the participating European national societies for colposcopy and their representatives: Austria, Belgium, Cyprus, Croatia, England, Estonia, Finland, France, Georgia, Germany, Greece, Hungary, Ireland, Italy, Lithuania, Macedonia, Moldavia, Netherlands, Norway, Poland, Portugal, Romania, Spain, Switzerland and Wales list of Abbreviations and Acronyms used in the manuscript: EFC: European Federation for Colposcopy HPV: Human Papillomavirus MCQs: Multiple-Choice Questions WHO: World Health Organization