CD95L concatemers highlight different stoichiometries of CD95-mediated apoptotic and nonapoptotic pathways.

To better understand the stoichiometry of CD95L required to trigger apoptotic and nonapoptotic signals, we generated several CD95L concatemers from dimer to hexamer conjugated via a flexible link (GGGGS)2 . These ligands reveal that although the hexameric structure is the best stoichiometry to trigger cell death, a dimer is sufficient to induce the apoptotic response in CD95-sensitive Jurkat cells. Interestingly, only trimeric and hexameric forms can implement a potent Ca2+ response, suggesting that while CD95 aggregation controls the implementation of the apoptotic signal, both aggregation and conformation are required to implement the Ca2+ pathway.

High prevalence of infections in non-COVID-19 patients admitted to the Emergency Department with severe lymphopenia.

BACKGROUND: In the Emergency Department (ED), early and accurate recognition of infection is crucial to prompt antibiotic therapy but the initial presentation of patients is variable and poorly characterized. Lymphopenia is commonly associated with bacteraemia and poor outcome in intensive care unit patients. The objective of this retrospective study was to assess the prevalence of community-acquired infection in a cohort of unselected patients admitted to the ED with undifferentiated symptoms and severe lymphopenia. METHODS: This is a retrospective single-center study conducted over a 1 year-period before the COVID-19 pandemic. Consecutive adult patients admitted to the ED with severe lymphopenia (lymphocyte count < 0.5 G/L) were studied. Patients with hematological or oncological diseases, HIV infection, hepato-cellular deficiency, immunosuppression, or patients over 85 years old were excluded. Diagnoses of infection were validated by an independent adjudication committee. The association between various parameters and infection was assessed using a multivariate logistic regression analysis. RESULTS: Of 953 patients admitted to the ED with severe lymphopenia, 245 were studied (148 men; mean age: 63 ± 19 years). Infection was confirmed in 159 patients (65%) (bacterial: 60%, viral: 30%, other: 10%). Only 61 patients (25%) were referred to the ED for a suspected infection. In the univariate analysis, SIRS criteria (OR: 5.39; 95%CI: 3.04-9.70; p < 0.001) and temperature ≥ 38.3 °C (OR: 10.95; 95%CI: 5.39-22.26; p < 0.001) were strongly associate with infection. In the multivariate analysis, only SIRS criteria (OR: 2.4; 95%CI: 1.48-3.9; p < 0.01) and fever (OR: 3.35; 95%CI: 1.26-8.93; p = 0.016) were independently associated with infection. CONCLUSIONS: The prevalence of underlying infection is high in patients admitted to the ED with lymphopenia, irrespective of the reason for admission. Whether lymphopenia could constitute a valuable marker of underlying infection in this clinical setting remains to be confirmed prospectively in larger cohorts. TRIAL REGISTRATION: No registration required as this is a retrospective study.

Locus suicide recombination actively occurs on the functionally rearranged IgH allele in B-cells from inflamed human lymphoid tissues.

B-cell activation yields abundant cell death in parallel to clonal amplification and remodeling of immunoglobulin (Ig) genes by activation-induced deaminase (AID). AID promotes affinity maturation of Ig variable regions and class switch recombination (CSR) in mature B lymphocytes. In the IgH locus, these processes are under control of the 3' regulatory region (3'RR) super-enhancer, a region demonstrated in the mouse to be both transcribed and itself targeted by AID-mediated recombination. Alternatively to CSR, IgH deletions joining Sµ to "like-switch" DNA repeats that flank the 3' super-enhancer can thus accomplish so-called "locus suicide recombination" (LSR) in mouse B-cells. Using an optimized LSR-seq high throughput method, we now show that AID-mediated LSR is evolutionarily conserved and also actively occurs in humans, providing an activation-induced cell death pathway in multiple conditions of B-cell activation. LSR either focuses on the functional IgH allele or is bi-allelic, and its signature is mainly detected when LSR is ongoing while it vanishes from fully differentiated plasma cells or from "resting" blood memory B-cells. Highly diversified breakpoints are distributed either within the upstream (3'RR1) or downstream (3'RR2) copies of the IgH 3' super-enhancer and all conditions activating CSR in vitro also seem to trigger LSR although TLR ligation appeared the most efficient. Molecular analysis of breakpoints and junctions confirms that LSR is AID-dependent and reveals junctional sequences somehow similar to CSR junctions but with increased usage of microhomologies.

Efficacy of rituximab in thymoma associated minimal change disease: case report.

BACKGROUND: Thymomas have been associated with a broad spectrum of autoimmune diseases. Minimal change disease (MCD) is the most frequent pathological lesion reported. Pathophysiological mechanisms involved in secondary MCD, and linking MCD to thymoma are not yet fully explained, although the hypothesis of T cell dysfunction has been suggested. The fundamental therapeutic principles are steroids and surgical treatment of thymoma, but failures and relapses often require immunosuppressant combinations. CASE PRESENTATION: A 62-year-old female was admitted in our unit for a nephrotic syndrome associated with a thymoma. The diagnosis of thymoma associated MCD was confirmed by kidney biopsy. After surgical resection of the thymoma and steroid therapy, no remission was observed. Immunosuppressive therapy was then intensified with introduction of rituximab. Here, we report a steroid-resistant nephrotic syndrome secondary to MCD associated thymoma, which achieved complete remission after rituximab therapy. To the best of our knowledge, this is the first report of the use and efficacy of rituximab therapy in this pathology. CONCLUSIONS: Our case report suggests that primary and secondary MCD may share similar pathophysiological mechanisms. It does not allow us to draw any conclusions about the mechanism of action of rituximab, but we believe this report argues for the safety and efficacy of rituximab use in thymoma-associated MCD, and therefore constitutes a rationale for future studies.

CBFß-SMMHC creates aberrant megakaryocyte-erythroid progenitors prone to leukemia initiation in mice.

Acute myeloid leukemia (AML) arises through multistep clonal evolution characterized by stepwise accumulation of successive alterations affecting the homeostasis of differentiation, proliferation, self-renewal, and survival programs. The persistence and dynamic clonal evolution of leukemia-initiating cells and preleukemic stem cells during disease progression and treatment are thought to contribute to disease relapse and poor outcome. Inv(16)(p13q22) or t(16;16)(p13.1;q22), one of the most common cytogenetic abnormalities in AML, leads to expression of a fusion protein CBFß-SMMHC (CM) known to disrupt myeloid and lymphoid differentiation. Anemia is often observed in AML but is presumed to be a secondary consequence of leukemic clonal expansion. Here, we show that CM expression induces marked deficiencies in erythroid lineage differentiation and early preleukemic expansion of a phenotypic pre-megakaryocyte/erythrocyte (Pre-Meg/E) progenitor population. Using dual-fluorescence reporter mice in lineage tracking and repopulation assays, we show that CM expression cell autonomously causes expansion of abnormal Pre-Meg/E progenitors with compromised erythroid specification and differentiation capacity. The preleukemic Pre-Meg/Es display dysregulated erythroid and megakaryocytic fate-determining factors including increased Spi-1, Gata2, and Gfi1b and reduced Zfpm1, Pf4, Vwf, and Mpl expression. Furthermore, these abnormal preleukemic Pre-Meg/Es have enhanced stress resistance and are prone to leukemia initiation upon acquiring cooperative signals. This study reveals that the leukemogenic CM fusion protein disrupts adult erythropoiesis and creates stress-resistant preleukemic Pre-Meg/E progenitors predisposed to malignant transformation. Abnormality in Meg/E or erythroid progenitors could potentially be considered an early predictive risk factor for leukemia evolution.

Alcam regulates long-term hematopoietic stem cell engraftment and self-renewal.

Hematopoietic stem cells (HSCs) reside in a specialized bone marrow (BM) microenvironment that supports the maintenance and functional integrity of long-term (LT)-HSCs throughout postnatal life. The objective of this work is to study the role of activated leukocyte cell adhesion molecule (Alcam) in HSC differentiation and self-renewal using an Alcam-null (Alcam(-/-) ) mouse model. We show here that Alcam is differentially regulated in adult hematopoiesis and is highly expressed in LT-HSCs where its level progressively increases with age. Young adult Alcam(-/-) mice had normal homeostatic hematopoiesis and normal numbers of phenotypic HSCs. However, Alcam(-/-) HSCs had reduced long-term replating capacity in vitro and reduced long-term engraftment potential upon transplantation. We show that Alcam(-/-) BM contain a markedly lower frequency of long-term repopulating cells than wild type. Further, the long-term repopulating potential and engraftment efficiency of Alcam(-/-) LT-HSCs was greatly compromised despite a progressive increase in phenotypic LT-HSC numbers during long-term serial transplantation. In addition, an age-associated increase in phenotypic LT-HSC cellularity was observed in Alcam(-/-) mice. This increase was predominately within the CD150(hi) fraction and was accompanied by significantly reduced leukocyte output. Consistent with an aging-like phenotype, older Alcam(-/-) LT-HSCs display myeloid-biased repopulation activity upon transplantation. Finally, Alcam(-/-) LT-HSCs display premature elevation of age-associated gene expression, including Selp, Clu, Cdc42, and Foxo3. Together, this study indicates that Alcam regulates functional integrity and self-renewal of LT-HSCs.

Phagosomal granulocytic ROS in septic patients induce the bacterial SOS response.

Septic patients with worst clinical prognosis have increased circulating immature granulocytes (IG), displaying limited phagocytosis and reactive oxygen species (ROS) production. Here, we developed an ex vivo model of incubation of human granulocytes, from septic patients or healthy donors, with Escherichia coli. We showed that the ROS production in Sepsis-IG is lower due to decreased activation and protein expression of the NADPH oxidase complex. We also demonstrated that the low level of ROS production and lower phagocytosis of IG in sepsis induce the bacterial SOS response, leading to the expression of the SOS-regulated quinolone resistance gene qnrB2. Without antimicrobial pressure, the sepsis immune response alone may promote antibiotic resistance expression.

Along with PaO2/FiO2 ratio and lymphopenia, low HLA-DR monocytes are the only additional parameter that independently predicts the clinical course of undifferentiated SARS-CoV-2 patients in emergency departments.

Because one-third of patients deteriorate after their admission to the emergency department, assessing the prognosis of COVID-19 patients is of great importance. However, to date, only lymphopenia and the partial pressure of oxygen/fraction of inspired oxygen (PaO2/FiO2) ratio have been reported as partly predictive of COVID-19-related further deterioration, and their association has not been evaluated. We asked whether other key biomarkers of SARS-CoV-2 immunologic defects-increase in circulating immature granulocytes, loss of monocyte HLA-DR (mHLA-DR) expression, and monocyte differentiation blockade-could also predict further COVID-19 deterioration. A series of 284 consecutive COVID-19 patients, with the sole inclusion criterion of being an adult, were prospectively enrolled at emergency department admission (day 0) of 2 different hospitals: 1 for the exploratory cohort (180 patients) and 1 for the confirmatory cohort (104 patients). Deterioration was assessed over the next 7 days. Neither increased immature granulocyte levels nor monocyte differentiation blockade predicted patient worsening. Among more than 30 clinical, biological, and radiological parameters, the value of decreased P/F ratio and lymphopenia for prediction of further COVID-19 deterioration was strongly confirmed, and the loss of mHLA-DR was the only additional independent marker. Combined together in a simple OxyLymphoMono score, the 3 variables perfectly predicted patients who did not worsen and correctly predicted worsening in 59% of cases. By highlighting lymphocyte and monocyte defects as preceding COVID-19 deterioration, these results point on early immunosuppression in COVID-19 deterioration. Combining P/F ratio, lymphopenia, and loss of mHLA-DR together in a simple and robust score could offer a pragmatic method for COVID-19 patient stratification.

Intravenously administered interleukin-7 to reverse lymphopenia in patients with septic shock: a double-blind, randomized, placebo-controlled trial.

BACKGROUND: Profound lymphopenia is an independent predictor of adverse clinical outcomes in sepsis. Interleukin-7 (IL-7) is essential for lymphocyte proliferation and survival. A previous phase II study showed that CYT107, a glycosylated recombinant human IL-7, administered intramuscularly reversed sepsis-induced lymphopenia and improved lymphocyte function. Thepresent study evaluated intravenous administration of CYT107. This prospective, double-blinded, placebo-controlled trial was designed to enroll 40 sepsis patients, randomized 3:1 to CYT107 (10 µg/kg) or placebo, for up to 90 days. RESULTS: Twenty-one patients were enrolled (fifteen CYT107 group, six placebo group) at eight French and two US sites. The study was halted early because three of fifteen patients receiving intravenous CYT107 developed fever and respiratory distress approximately 5-8 h after drug administration. Intravenous administration of CYT107 resulted in a two-threefold increase in absolute lymphocyte counts (including in both CD4+ and CD8+ T cells (all p < 0.05)) compared to placebo. This increase was similar to that seen with intramuscular administration of CYT107, was maintained throughout follow-up, reversed severe lymphopenia and was associated with increase in organ support free days (OSFD). However, intravenous CYT107 produced an approximately 100-fold increase in CYT107 blood concentration compared with intramuscular CYT107. No cytokine storm and no formation of antibodies to CYT107 were observed. CONCLUSION: Intravenous CYT107 reversed sepsis-induced lymphopenia. However, compared to intramuscular CYT107 administration, it was associated with transient respiratory distress without long-term sequelae. Because of equivalent positive laboratory and clinical responses, more favorable pharmacokinetics, and better patient tolerability, intramuscular administration of CYT107 is preferable. TRIAL REGISTRATION: Clinicaltrials.gov, NCT03821038. Registered 29 January 2019, https://clinicaltrials.gov/ct2/show/NCT03821038?term=NCT03821038&draw=2&rank=1 .

Demultiplexing Ig repertoires by parallel mRNA/DNA sequencing shows major differential alterations in severe COVID-19.

To understand the fine differential elements that can lead to or prevent acute respiratory distress syndrome (ARDS) in COVID-19 patients, it is crucial to investigate the immune response architecture. We herein dissected the multiple layers of B cell responses by flow cytometry and Ig repertoire analysis from acute phase to recovery. Flow cytometry with FlowSOM analysis showed major changes associated with COVID-19 inflammation such as an increase of double-negative B-cells and ongoing plasma cell differentiation. This paralleled COVID-19-driven expansion of two disconnected B-cell repertoires. Demultiplexing successive DNA and RNA Ig repertoire patterns characterized an early expansion of IgG1 clonotypes with atypically long and uncharged CDR3, the abundance of this inflammatory repertoire being correlated with ARDS and likely pejorative. A superimposed convergent response included convergent anti-SARS-CoV-2 clonotypes. It featured progressively increasing somatic hypermutation together with normal-length or short CDR3 and it persisted until a quiescent memory B-cell stage after recovery.

Oncogenic activation of the Notch1 gene by deletion of its promoter in Ikaros-deficient T-ALL.

The Notch pathway is frequently activated in T-cell acute lymphoblastic leukemias (T-ALLs). Of the Notch receptors, Notch1 is a recurrent target of gain-of-function mutations and Notch3 is expressed in all T-ALLs, but it is currently unclear how these receptors contribute to T-cell transformation in vivo. We investigated the role of Notch1 and Notch3 in T-ALL progression by a genetic approach, in mice bearing a knockdown mutation in the Ikaros gene that spontaneously develop Notch-dependent T-ALL. While deletion of Notch3 has little effect, T cell-specific deletion of floxed Notch1 promoter/exon 1 sequences significantly accelerates leukemogenesis. Notch1-deleted tumors lack surface Notch1 but express γ-secretase-cleaved intracellular Notch1 proteins. In addition, these tumors accumulate high levels of truncated Notch1 transcripts that are caused by aberrant transcription from cryptic initiation sites in the 3' part of the gene. Deletion of the floxed sequences directly reprograms the Notch1 locus to begin transcription from these 3' promoters and is accompanied by an epigenetic reorganization of the Notch1 locus that is consistent with transcriptional activation. Further, spontaneous deletion of 5' Notch1 sequences occurs in approximately 75% of Ikaros-deficient T-ALLs. These results reveal a novel mechanism for the oncogenic activation of the Notch1 gene after deletion of its main promoter.

Bicentric evaluation of stabilizing sampling tubes for assessment of monocyte HLA-DR expression in clinical samples.

BACKGROUND: Diminished expression of human leukocyte antigen DR on circulating monocytes (mHLA-DR), measured by standardized flow cytometry procedure, is a reliable indicator of immunosuppression in severely injured intensive care unit patients. As such, it is used as stratification criteria in clinical trials evaluating novel immunostimulating therapies. Preanalytical constraints relative to the short delay between blood sampling and flow cytometry staining have nevertheless limited its use in multicentric studies. The objective of the present work was to compare mHLA-DR expression between whole blood samples simultaneously drawn in EDTA or Cyto-Chex BCT tubes. METHODS: In two university hospitals, mHLA-DR was assessed in fresh whole blood from septic patients (n = 12) and healthy donors (n = 6) simultaneously sampled on EDTA and Cyto-Chex BCT tubes. Staining was performed immediately after sampling and after blood storage at room temperature. RESULTS: We confirmed that samples collected in Cyto-Chex tube had substantially enhanced stability for mHLA-DR results (48-72 h) over those collected in EDTA. On baseline values, despite good correlation between tubes (r = 0.98, p < 0.001), mHLA-DR expression was systematically lower with Cyto-Chex BCT. CONCLUSION: The present reports confirms the potential of Cyto-Chex BCT tubes to stabilize mHLA-DR expression before staining and extends the work of Quadrini et al. [Cytometry B 2021;100:103-114]. In centers without rapid access to flow cytometry facilities, it enables to tolerate delays in mHLA-DR staining. However, a 30% gap exists between results obtained with EDTA and Cyto-Chex BCT tubes. As current thresholds for clinical decisions were obtained with EDTA samples, further studies are needed to confirm clinical thresholds with Cyto-Chex BCT tubes.

De novo natural anti-M alloantibody emergence in severe Coronavirus Disease 2019.

The immune response is a key player in the course of SARS-CoV-2 infection, and is often seriously dysfunctional in severe Coronavirus Disease 2019. The hyperinflammatory status has been described to be accompanied by the appearance of autoantibodies. In a lethal COVID-19 infection, we observed the emergence of a de novo natural alloantibody which targeted the M antigen from the MNS blood group on red blood cells (RBC) without evidence of any cross-reaction with SARS-CoV-2 antigens. This IgM lambda alloantibody was unmutated and unswitched. Here, we describe for the first time the emergence of a bystander de novo natural alloantibody against RBCs in a severe COVID-19 patient, highlighting the extra-follicular humoral response reported in these cases.

Immature granulocytes can help the diagnosis of pulmonary bacterial infections in patients with severe COVID-19 pneumonia.

During COVID-19, immature granulocyte (IG) concentration is heterogeneous with higher concentrations than those found in bacterial sepsis. We investigated the relationship between IG levels at ICU admission and on days 7 (± 2) and 15 (± 2) and associated pulmonary bacterial infections in intensive care unit (ICU) patients hospitalized for an acute respiratory distress syndrome (ARDS) related to SARS-CoV-2. Patients with associated pulmonary bacterial infection had a peak of IGs. IG thresholds of 18% or 2 G/L allowed discriminating patients with ventilator associated pneumonia with 100% sensitivity and specificity. Our study supports that IGs could help identifying pulmonary bacterial infections in this population.

Comparative immune profiling of acute respiratory distress syndrome patients with or without SARS-CoV-2 infection.

Acute respiratory distress syndrome (ARDS) is the main complication of coronavirus disease 2019 (COVID-19), requiring admission to the intensive care unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS differs from other causes of ARDS remains unknown. To address this question, here, we build 3 cohorts of patients categorized in COVID-19-ARDS+, COVID-19+ARDS+, and COVID-19+ARDS-, and compare, by high-dimensional mass cytometry, their immune landscape. A cell signature associating S100A9/calprotectin-producing CD169+ monocytes, plasmablasts, and Th1 cells is found in COVID-19+ARDS+, unlike COVID-19-ARDS+ patients. Moreover, this signature is essentially shared with COVID-19+ARDS- patients, suggesting that severe COVID-19 patients, whether or not they experience ARDS, display similar immune profiles. We show an increase in CD14+HLA-DRlow and CD14lowCD16+ monocytes correlating to the occurrence of adverse events during the ICU stay. We demonstrate that COVID-19-associated ARDS displays a specific immune profile and may benefit from personalized therapy in addition to standard ARDS management.

Phenotypical and functional alteration of unconventional T cells in severe COVID-19 patients.

COVID-19 includes lung infection ranging from mild pneumonia to life-threatening acute respiratory distress syndrome (ARDS). Dysregulated host immune response in the lung is a key feature in ARDS pathophysiology. However, cellular actors involved in COVID-19-driven ARDS are poorly understood. Here, in blood and airways of severe COVID-19 patients, we serially analyzed unconventional T cells, a heterogeneous class of T lymphocytes (MAIT, γδT, and iNKT cells) with potent antimicrobial and regulatory functions. Circulating unconventional T cells of COVID-19 patients presented with a profound and persistent phenotypic alteration. In the airways, highly activated unconventional T cells were detected, suggesting a potential contribution in the regulation of local inflammation. Finally, expression of the CD69 activation marker on blood iNKT and MAIT cells of COVID-19 patients on admission was predictive of clinical course and disease severity. Thus, COVID-19 patients present with an altered unconventional T cell biology, and further investigations will be required to precisely assess their functions during SARS-CoV-2-driven ARDS.

Notch activation is an early and critical event during T-Cell leukemogenesis in Ikaros-deficient mice.

The Ikaros transcription factor is both a key regulator of lymphocyte differentiation and a tumor suppressor in T lymphocytes. Mice carrying a hypomorphic mutation (Ik(L/L)) in the Ikaros gene all develop thymic lymphomas. Ik(L/L) tumors always exhibit strong activation of the Notch pathway, which is required for tumor cell proliferation in vitro. Notch activation occurs early in tumorigenesis and may precede transformation, as ectopic expression of the Notch targets Hes-1 and Deltex-1 is detected in thymocytes from young Ik(L/L) mice with no overt signs of transformation. Notch activation is further amplified by secondary mutations that lead to C-terminal truncations of Notch 1. Strikingly, restoration of Ikaros activity in tumor cells leads to a rapid and specific downregulation of Notch target gene expression and proliferation arrest. Furthermore, Ikaros binds to the Notch-responsive element in the Hes-1 promoter and represses Notch-dependent transcription from this promoter. Thus, Ikaros-mediated repression of Notch target gene expression may play a critical role in defining the tumor suppressor function of this factor.

Interleukin-7 restores lymphocytes in septic shock: the IRIS-7 randomized clinical trial.

BACKGROUND: A defining pathophysiologic feature of sepsis is profound apoptosis-induced death and depletion of CD4+ and CD8+ T cells. Interleukin-7 (IL-7) is an antiapoptotic common γ-chain cytokine that is essential for lymphocyte proliferation and survival. Clinical trials of IL-7 in over 390 oncologic and lymphopenic patients showed that IL-7 was safe, invariably increased CD4+ and CD8+ lymphocyte counts, and improved immunity. METHODS: We conducted a prospective, randomized, double-blind, placebo-controlled trial of recombinant human IL-7 (CYT107) in patients with septic shock and severe lymphopenia. Twenty-seven patients at academic sites in France and the United States received CYT107 or placebo for 4 weeks. Primary aims were to determine the safety of CYT107 in sepsis and its ability to reverse lymphopenia. RESULTS: CYT107 was well tolerated without evidence of inducing cytokine storm or worsening inflammation or organ dysfunction. CYT107 caused a 3- to 4-fold increase in absolute lymphocyte counts and in circulating CD4+ and CD8+ T cells that persisted for weeks after drug administration. CYT107 also increased T cell proliferation and activation. CONCLUSIONS: This is the first trial of an immunoadjuvant therapy targeting defects in adaptive immunity in patients with sepsis. CYT107 reversed the marked loss of CD4+ and CD8+ immune effector cells, a hallmark of sepsis and a likely key mechanism in its morbidity and mortality. CYT107 represents a potential new way forward in the treatment of patients with sepsis by restoring adaptive immunity. Such immune-based therapy should be broadly protective against diverse pathogens including multidrug resistant bacteria that preferentially target patients with impaired immunity. TRIAL REGISTRATION: Trials registered at clinicaltrials.gov: NCT02640807 and NCT02797431. FUNDING: Revimmune, NIH National Institute of General Medical Sciences GM44118.

Multicentric Standardized Flow Cytometry Routine Assessment of Patients With Sepsis to Predict Clinical Worsening.

BACKGROUND: In this study, we primarily sought to assess the ability of flow cytometry to predict early clinical deterioration and overall survival in patients with sepsis admitted in the ED and ICU. METHODS: Patients admitted for community-acquired acute sepsis from 11 hospital centers were eligible. Early (day 7) and late (day 28) deaths were notified. Levels of CD64pos granulocytes, CD16pos monocytes, CD16dim immature granulocytes (IGs), and T and B lymphocytes were assessed by flow cytometry using an identical, cross-validated, robust, and simple consensus standardized protocol in each center. RESULTS: Among 1,062 patients screened, 781 patients with confirmed sepsis were studied (age, 67 ± 48 years; Simplified Acute Physiology Score II, 36 ± 17; Sequential Organ Failure Assessment, 5 ± 4). Patients were divided into three groups (sepsis, severe sepsis, and septic shock) on day 0 and on day 2. On day 0, patients with sepsis exhibited increased levels of CD64pos granulocytes, CD16pos monocytes, and IGs with T-cell lymphopenia. Clinical severity was associated with higher percentages of IGs and deeper T-cell lymphopenia. IG percentages tended to be higher in patients whose clinical status worsened on day 2 (35.1 ± 35.6 vs 43.5 ± 35.2, P = .07). Increased IG percentages were also related to occurrence of new organ failures on day 2. Increased IG percentages, especially when associated with T-cell lymphopenia, were independently associated with early (P < .01) and late (P < .01) death. CONCLUSIONS: Increased circulating IGs at the acute phase of sepsis are linked to clinical worsening, especially when associated with T-cell lymphopenia. Early flow cytometry could help clinicians to target patients at high risk of clinical deterioration. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT01995448; URL: www.clinicaltrials.gov.