B-cell immune deficiency in twin sisters expands the phenotype of MOPDI.

Microcephalic osteodysplastic primordial dwarfism type I (MOPDI) is a very rare and severe autosomal recessive disorder characterized by marked intrauterine growth retardation, skeletal dysplasia, microcephaly and brain malformations. MOPDI is caused by biallelic mutations in RNU4ATAC, a non-coding gene involved in U12-type splicing of 1% of the introns in the genome, which are recognized by their specific splicing consensus sequences. Here, we describe a unique observation of immunodeficiency in twin sisters with mild MOPDI, who harbor a novel n.108_126del mutation, encompassing part of the U4atac snRNA 3' stem-loop and Sm protein binding site, and the previously reported n.111G>A mutation. Interestingly, both twin sisters show mild B-cell anomalies, including low naive B-cell counts and increased memory B-cell and plasmablasts counts, suggesting partial and transitory blockage of B-cell maturation and/or excessive activation of naive B-cells. Hence, the localization of a mutation in stem II of U4atac snRNA, as observed in another RNU4ATAC-opathy with immunodeficiency, that is, Roifman syndrome (RFMN), is not required for the occurrence of an immune deficiency. Finally, we emphasize the importance of considering immunodeficiency in MOPDI management to reduce the risk of serious infectious episodes.

Elevated monocyte HLA-DR in pediatric secondary hemophagocytic lymphohistiocytosis: a retrospective study.

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition, and its diagnosis may be challenging. In particular, some cases show close similarities to sepsis (fever, organ failure, and high ferritin), but their treatment, while urgent, differ: prompt broad-spectrum antibiotherapy for sepsis and immunosuppressive treatment for HLH. We questioned whether monocyte human leucocyte antigen (mHLA)-DR could be a diagnostic marker for secondary HLH (sHLH). Methods: We retrospectively reviewed data from patients with a sHLH diagnosis and mHLA-DR quantification. mHLA-DR data from healthy children and children with septic shock, whose HLA-DR expression is reduced, from a previously published study were also included for comparison. Results: Six patients with sHLH had mHLA-DR quantification. The median level of monocyte mHLA-DR expression in patients with sHLH [79,409 antibodies bound per cell (AB/C), interquartile range (IQR) (75,734-86,453)] was significantly higher than that in healthy children and those with septic shock (29,668 AB/C, IQR (24,335-39,199), and 7,493 AB/C, IQR (3,758-14,659), respectively). Each patient with sHLH had a mHLA-DR higher than our laboratory normal values. Four patients had a second mHLA-DR sampling 2 to 4 days after the initial analysis and treatment initiation with high-dose corticosteroids; for all patients, mHLA-DR decreased to within or close to the normal range. One patient with systemic juvenile idiopathic arthritis had repeated mHLA-DR measurements over a 200-day period during which she underwent four HLH episodes. mHLA-DR increased during relapses and normalized after treatment incrementation. Conclusion: In this small series, mHLA-DR was systematically elevated in patients with sHLH. Elevated mHLA-DR could contribute to sHLH diagnosis and help earlier distinction with septic shock.

Assessment of neutrophil subsets and immune checkpoint inhibitor expressions on T lymphocytes in liver transplantation: A preliminary study beyond the neutrophil-lymphocyte ratio.

Background: Advanced stages of cirrhosis are characterized by the occurrence of progressive immune alterations known as CAID (Cirrhosis Associated Immune Dysfunction). In advanced cirrhosis, liver transplantation (LT) remains the only curative treatment. Sepsis, shares many similarities with decompensated cirrhosis in terms of immuno-inflammatory response. In both conditions, the neutrophil-lymphocyte ratio (NLR) is associated with poor outcomes. Based on alterations in sepsis, we hypothesized that we could observe in cirrhotic and LT patients more detailed neutrophil and lymphocyte phenotypes. To this end, along with leukocyte count, we assessed immature neutrophils, LOX-1+ MDSC and PD-1 and TIM-3 lymphocyte expressions in cirrhotic patients before transplantation in association with liver disease severity and during the first month after transplantation. Methods: We conducted a prospective monocentric study including cirrhotic patients registered on LT waiting-list. Blood samples were collected at enrolment before LT and for 1 month post-LT. In addition to NLR, we assessed by whole blood flow cytometry the absolute count of immature neutrophils and LOX-1+ MDSC as well as the expressions of immune checkpoint receptors PD-1 and TIM-3 on T lymphocytes. Results: We included 15 healthy volunteers (HV) and 28 patients. LT was performed for 13 patients. Pre-LT patients presented with a higher NLR compared to HV and NLR was associated with cirrhosis severity. Increased immature neutrophils and LOX-1+ MDSC counts were observed in the most severe patients. These alterations were mainly associated with acute decompensation of cirrhosis. PD-1 and TIM-3 expressions on T lymphocytes were not different between patients and HV. Post-LT immune alterations were dominated by a transitory but tremendous increase of NLR and immature neutrophils during the first days post-LT. Then, immune checkpoint receptors and LOX-1+ MDSC tended to be overexpressed by the second week after surgery. Conclusion: The present study showed that NLR, immature neutrophils and LOX-1+ MDSC counts along with T lymphocyte count and checkpoint inhibitor expression were altered in cirrhotic patients before and after LT. These data illustrate the potential interest of immune monitoring of cirrhotic patients in the context of LT in order to better define risk of sepsis. For this purpose, larger cohorts of patients are now necessary in order to move forward a more personalised care of LT patients.

HLA-DR expression on monocytes and outcome of anti-CD19 CAR T-cell therapy for large B-cell lymphoma.

Despite their unprecedented success in relapsed/refractory (R/R) large B-cell lymphoma (LBCL), anti-CD19 CAR T cells are associated with significant toxicity, and more than half of patients relapse. As monocytes emerged as key players in CAR therapy, we sought to evaluate the evolution of HLA-DR expression on monocytes (mHLA-DR) before and after commercial anti-CD19 CAR T-cell infusion in a large cohort (n = 103) of patients with R/R LBCL and its association with adverse events and treatment response. Cy-Flu-based lymphodepletion (LD) upregulated mHLA-DR in 79% of the cases, whereas in 2l% of cases (15 patients), the mHLA-DR level decreased after LD, and this decrease was associated with poorer outcome. Low mHLA-DR at day minus 7 (D-7) (<13 500 antibodies per cell) before CAR T-cell infusion correlated with older age, poorer performance status, higher tumor burden, and elevated inflammatory markers. With a median follow-up of 7.4 months, patients with low mHLA-DR D-7 exhibited a poorer duration of response and survival than the higher mHLA-DR D-7 group. For toxicity management, tocilizumab was more frequently used in the low-mHLA-DR D-7 group. These data suggest that monocyte dysregulation before LD, characterized by the downregulation of mHLA-DR, correlates with an inflammatory and immunosuppressive tumor environment and is associated with failure of anti-CD19 CAR T cells in patients with R/R LBCL. Modulation of these myeloid cells represents a promising field for improving CAR therapy.

Emergence of immunosuppressive LOX-1+ PMN-MDSC in septic shock and severe COVID-19 patients with acute respiratory distress syndrome.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with immunosuppressive properties. In cancer patients, the expression of lectin-type oxidized LDL receptor 1 (LOX-1) on granulocytic MDSC identifies a subset of MDSC that retains the most potent immunosuppressive properties. The main objective of the present work was to explore the presence of LOX-1+ MDSC in bacterial and viral sepsis. To this end, whole blood LOX-1+ cells were phenotypically, morphologically, and functionally characterized. They were monitored in 39 coronavirus disease-19 (COVID-19, viral sepsis) and 48 septic shock (bacterial sepsis) patients longitudinally sampled five times over a 3 wk period in intensive care units (ICUs). The phenotype, morphology, and immunosuppressive functions of LOX-1+ cells demonstrated that they were polymorphonuclear MDSC. In patients, we observed the significant emergence of LOX-1+ MDSC in both groups. The peak of LOX-1+ MDSC was 1 wk delayed with respect to ICU admission. In COVID-19, their elevation was more pronounced in patients with acute respiratory distress syndrome. The persistence of these cells may contribute to long lasting immunosuppression leaving the patient unable to efficiently resolve infections.

[Study of monocytic myeloid-derived suppressor cells and CD4 lymphopenia in septic shock-induced immunosuppression]. / Étude des cellules myéloïdes immunosuppressives monocytaires au cours de l'immunodépression associée au choc septique.

Sepsis is one of the leading causes of in-hospital mortality. In some patients, sepsis-induced immunosuppression is associated with increased risk of death and secondary infections. In oncology, myeloid-derived suppressor cells (MDSCs) have been described to inhibit various immune functions. Monocytic MDSCs (M-MDSCs) represent a subtype of MDSCs. The objectives of the present study was to determine by flow cytometry the % M-MDSCs (among total monocyte population) in a cohort of septic shock patients and to assess its association with deleterious outcomes: 28-day mortality and occurrence of nosocomial infections. The cohort included 301 patients. They presented with immune alterations usually found 3-4 days after the onset of shock: lymphopenia (median T CD4: 362/µL, quartiles: 235-591/µL) and low monocytic HLA-DR expression (median: 4,944 AB/C, quartiles: 3,104-8,266 AB/C). From admission until the end of the first week, % M-MDSCs was significantly increased in patients compared with healthy donors (p < 0.01). In early samples, no association with deleterious outcomes was identified. However, after one week, patients who were going to die or to develop nosocomial infections presented with significantly higher % M-MDSCs than non-survivors and non-infected patients (p < 0.01). These associations remained significant in multivariate analyses, odds ratio of 4.4 (p = 0.001) regarding 28-day mortality and 2.4 (p = 0.013) regarding occurrence of nosocomial infections. In conclusion, % M-MDSCs was markedly increased after septic shock. One week-persistence of an increased proportion of M-MDSCs was associated with unfavorable outcomes.

Characterization of Circulating IL-10-Producing Cells in Septic Shock Patients: A Proof of Concept Study.

Sepsis is a worldwide health priority characterized by the occurrence of severe immunosuppression associated with increased risk of death and secondary infections. Interleukin 10 (IL-10) is a potent immunosuppressive cytokine which plasma concentration is increased in septic patients in association with deleterious outcomes. Despite studies evaluating IL-10 production in specific subpopulations of purified cells, the concomitant description of IL-10 production in monocytes and lymphocytes in septic patients' whole blood has never been performed. In this pilot study, we characterized IL-10 producing leukocytes in septic shock patients through whole blood intracellular staining by flow cytometry. Twelve adult septic shock patients and 9 healthy volunteers were included. Intracellular tumor necrosis factor-α (TNFα) and IL-10 productions after lipopolysaccharide stimulation by monocytes and IL-10 production after PMA/Ionomycine stimulation by lymphocytes were evaluated. Standard immunomonitoring (HLA-DR expression on monocytes, CD4+ T lymphocyte count) of patients was also performed. TNFα expression by stimulated monocytes was reduced in patients compared with controls while IL-10 production was increased. This was correlated with a reduced monocyte HLA-DR expression. B cells, CD4+, and CD4- T lymphocytes were the three circulating IL-10 producing lymphocyte subsets in both patients and controls. No difference in IL-10 production between patients and controls was observed for B and CD4- T cells. However, IL-10 production by CD4+ T lymphocytes significantly increased in patients in parallel with reduced CD4+ T cells number. Parameters reflecting altered monocyte (increased IL-10 production, decreased HLA-DR expression and decreased TNFα synthesis) and CD4+ T lymphocyte (increased IL-10 production, decreased circulating number) responses were correlated. Using a novel technique for intracellular cytokine measurement in whole blood, our results identify monocytes and CD4+ T cells as the main IL-10 producers in septic patients' whole blood and illustrate the development of a global immunosuppressive profile in septic shock. Overall, these preliminary results add to our understanding of the global increase in IL-10 production induced by septic shock. Further research is mandatory to determine the pathophysiological mechanisms leading to such increased IL-10 production in monocytes and CD4+ T cells.

Occurrence of marked sepsis-induced immunosuppression in pediatric septic shock: a pilot study.

BACKGROUND: While the process of sepsis-induced immunosuppression is now well described in adults, very little information is available on immune functions in pediatric sepsis. The current study investigated this in children with septic shock by performing immunomonitoring, including both innate (monocyte human leukocyte antigen-DR, mHLA-DR, expression) and adaptive immunity (lymphocyte subsets count), as well as cytokine concentrations (IL-6, IL-8, IL-10, IL-1Ra, TNF-α, IFN-γ). Subsequent objectives were to assess the associations between inflammatory response, potential immunosuppression and secondary acquired infection occurrence. METHODS: Single-center prospective observational study, including children aged between 1 month and 18 years admitted to pediatric intensive care unit (PICU) for septic shock. Age-matched controls were children hospitalized for elective surgery without any infectious criteria. Blood was sampled at day 1-2, 3-5, and 7-9 after sepsis onset. mHLA-DR and lymphocyte subsets count were measured by flow cytometry and cytokine concentrations by Luminex technology. RESULTS: A total of 26 children and 30 controls were included. Patients had lymphopenia, and mHLA-DR levels were significantly lower than controls at each time point (p < 0.0001). All cytokines peaked at day 1-2. Children with secondary acquired infection had lower day 3-5 mHLA-DR and higher pro-inflammatory cytokine concentrations (IL-6, IL-8 and TNF-α) at day 1-2 compared to children without secondary acquired infection. CONCLUSIONS: The higher initial inflammatory cytokine production was, the more innate immunity was altered, while evaluated by low mHLA-DR expression. Children with decreased mHLA-DR expression developed more secondary acquired infections. Upon confirmation in multicenter cohorts, these results pave the way for immunostimulation for the most immunosuppressed children in order to prevent nosocomial infections in PICU. Trial registration PedIRIS study NCT02848144. Retrospectively registered 28 July 2016.

Septic Shock Shapes B Cell Response toward an Exhausted-like/Immunoregulatory Profile in Patients.

Septic shock is accompanied by the development of immune dysfunctions whose intensity and duration are associated with increased risk of secondary infections and mortality. Although B lymphocytes play a pivotal role in the immune response to infections, no comprehensive exploration of circulating B cell status has been performed during the immunosuppressive phase of septic shock. Thus, our aim was to extensively characterize the phenotype and function of B cells in septic shock, including IL-10 production. Circulating B lymphocyte phenotype and function were evaluated by flow cytometry on fresh whole blood and after ex vivo stimulation in adult septic shock patients sampled at day 1, 3, and 6 after the onset of shock. The circulating B cell number was reduced in septic shock patients, whereas the B cell proportion among total lymphocytes was increased. The remaining circulating B lymphocytes presented with decreased MHC class II expression and increased CD21low CD95high exhausted-like phenotype but showed no change in maturation status. Circulating B cell functions were markedly altered after sepsis with reduced ex vivo activation and proliferation capacities. Finally, B cell response after septic shock was characterized by a clear plasmacytosis and an increased IL-10 production in remaining B cells from patients after ex vivo stimulation. During the sepsis-induced immunosuppression phase, B cell response is altered and is oriented toward an exhausted-like/immunoregulatory profile. Further studies are now needed to confirm the immunoregulatory properties of B lymphocytes and evaluate their role in sepsis-induced immunosuppression.

Decreased intra-lymphocyte cytokines measurement in septic shock patients: A proof of concept study in whole blood.

Functional testing protocols are thought to be the gold standard for the exploration of the immune system. However, in terms of routine analysis, they present numerous drawbacks and consequently their use is mainly limited to research applications. In the clinical context of septic shock, characterized by marked lymphocyte alterations, a new approach for lymphocyte intracellular cytokine measurement in whole blood upon was evaluated in a proof-of-concept study. Following lymphocyte activation, simultaneous intracellular labeling of Interferon-γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α), and Interleukin-2 (IL-2) was performed in CD4+ and CD8+ T cells (identified by surface marking). The analysis was carried out by flow cytometry (6 colors). Results obtained in septic patients (n=22) were compared to those of healthy volunteers (n=8). Independently of lymphopenia, there were significant differences between groups. In particular there was significant decrease in the production of IL-2 and TNF-α in septic patients, while the production of IFN-γ was not significantly altered. Polyfunctional results showed that patients presented with increased percentages of triple negative lymphocytes. In contrast, volunteers had higher proportions of triple positive cells. The approach could be performed in a robust and consistent way, taking 4.5h to complete. Moreover, clear differences could be observed between clinical groups with this modified method. These characteristics illustrate the potential of this novel whole blood protocol for clinical applications. However, further research is required to determine the applicability compared to alternative test and to evaluate clinical performances in larger cohorts of patients.

IL-7 and Its Beneficial Role in Sepsis-Induced T Lymphocyte Dysfunction.

Sepsis, defined as life-threatening organ dysfunction caused by dysregulated host response to infection, has recently been acknowledged as a worldwide health priority. Sepsis remains the leading cause of mortality in intensive care units and accounts for 6 million deaths every year. Few therapeutic options targeting host immune response in sepsis have demonstrated their efficacy so far. Increasing evidence suggests that a profound immune suppression develops following sepsis, affecting innate and adaptive immune response, of which intensity and duration is associated with increased risk of death and nosocomial infection. Immunostimulant treatments are thus now evaluated in sepsis, and recombinant human IL-7 (rhIL-7) represents a promising candidate. rhIL-7 has been evaluated in several clinical trials in patients with altered lymphocytic responses (HIV infection, hematopoietic stem cell transplantation, and cancer). Recent studies in animal models and in patients' samples ex vivo demonstrated its efficacy in improving sepsis-induced T cell alterations. Finally, the first clinical trial evaluating rhIL-7 in septic shock patients has just been published. This review will discuss the use of rhIL-7 to treat sepsis-induced T cell dysfunction by introducing the pathophysiology of sepsis and sepsis-related lymphocyte alterations before focusing on rhIL-7 and its potential use of as a therapeutic intervention in patients.

Evaluation of a novel automated volumetric flow cytometer for absolute CD4+ T lymphocyte quantitation.

BACKGROUND: Bead-based single platform cytometry technology (SPT) is the gold standard when performing CD4 absolute counting. However, it presents drawbacks as precision depends on various critical steps (for example, pipetting methodology, overtime stability of beads, stability of fluidics, regular recalibration…) and thus requires skilled operators. The fully automated volumetric SPT AQUIOS CL (Beckman Coulter) has recently emerged as an alternative with no need of beads. It may help improving results standardisation and fulfilling requirements for certification (ISO 15189). In this study, we assessed SPT AQUIOS CL performances in accordance to requirements for ISO 15189 accreditation. METHODS: We evaluated repeatability and reproducibility (precision), bias (trueness), uncertainty (total error), range limits (linearity, quantification, detection limits), and inter-reagent/inter-sample contaminations in enumerating CD4+ T-cells. Concomitantly, we compared AQUIOS CL CD4+ T-cell values with the results obtained with our routine bead-based SPT (that is, FC500 Beckman Coulter, bead-based SPT), on blood samples from 148 patients representative of clinical laboratory routine workload. RESULTS: Every result (repeatability, reproducibility, trueness, total error) was below the acceptable thresholds proposed in international recommendations. Contamination results and range limits (linearity, quantification, and detection limits) were all found perfectly suitable to routine analysis. The comparison between AQUIOS CL and FC500 exhibited excellent correlation and agreement (Pearson R = 0.99, P < 0.001; Lin's concordance correlation coefficient: Lin ρc = 0.991, Cb = 0.999), and Bland-Altman analysis did not reveal any systematic error. CONCLUSIONS: Our results demonstrate that, upon subsequent validation in more routine conditions, the AQUIOS CL could be a suitable tool for clinical flow cytometry laboratories facing accreditation process. © 2016 International Clinical Cytometry Society.

Novel Approach in Monocyte Intracellular TNF Measurement: Application to Sepsis-Induced Immune Alterations.

OBJECTIVE: The monitoring of septic shock induced immunosuppression has been proposed to identify patients who could benefit from specific immunoadjuvant therapies. Among potential biomarkers to monitor immunological status, functional testing remains the gold standard because it directly measures the capacity of a cell population to respond to an immune challenge. We investigated a new approach in intracellular staining for flow cytometry to measure tumor necrosis factor (iTNF) produced in vitro by monocytes in response to lipopolysaccharide. DESIGN, SETTING, SUBJECTS, AND INTERVENTIONS: Observational study in intensive care unit and immunology laboratory of a university medical center. Sixteen septic shock patients and eight control subjects were included. MAIN RESULTS: Monocyte iTNF was determined by flow cytometry in whole blood and completed in 2.5 h according to a no-wash, no centrifuge procedure. Lipopolysaccharide challenge induced a tremendous expression of iTNF that was statistically more pronounced in controls than in patients. This was observed when results were expressed as medians of fluorescence intensity (median: 16.1 [IQR: 14.5-19.1] vs. 5 [4.0-8.0], P = 0.0001) or as percentages of positive cells (99.7 [99.6-99.8] vs. 85 [74-97], P = 0.0001). iTNF expression was correlated to monocyte HLA-DR expression in patients and controls. CONCLUSIONS: These preliminary results illustrate the feasibility of immune functional testing on a routine manner in septic shock patients. They now deserve to be widely assessed and validated in various intensive care unit conditions. This could be a major step to characterize the rapidly changing immune response overtime and thus permit personalized medicine.

Sepsis in PD-1 light.

Increasing evidence suggests that after the first pro-inflammatory hours, sepsis is characterized by the occurrence of severe immunosuppression. Several mechanisms have been reported to participate in sepsis-induced immune alterations affecting both innate and adaptive immunity. Of these, the concept of 'cell exhaustion' has gained a lot of interest because some parallels can be drawn with the cancer field in which immunostimulation approaches through blocking immune checkpoints currently obtain remarkable success. Herein, perspectives regarding co-inhibitory receptors' contribution to lymphocyte exhaustion in sepsis will be discussed in the context of a recently published study investigating the potential of PD-1 molecule expression (i.e. PD-1 on lymphocytes, PD-L1 on monocytes) to predict mortality in septic shock patients.