Accréditation de la mesure de l'expression des molécules HLA-DR à la surface des monocytes (mHLA-DR) par cytométrie en flux.

INTRODUCTION: Le niveau d'expression des molécules HLA-DR à la surface des monocytes (mHLA-DR) est un marqueur diagnostique utilisé pour évaluer l'immunité des patients en réanimation (choc septique, polytraumatisés, brulures, greffe et plus récemment Covid-19). Il est également utilisé comme un outil de stratification dans les essais cliniques utilisant des thérapies immunostimulantes chez ces patients. L'objectif de cette étude était d'évaluer les performances analytiques d'une méthode de cytométrie en flux pour mesurer mHLA-DR afin de répondre aux exigences de la norme NF EN ISO 15189 dans le cadre de l'accréditation des laboratoires de biologie médicale. Matériels et méthodes. L'évaluation (performances de la technique, étendue de la mesure, comparaison de méthode) a été menée en suivant le SH GTA 04, guide recommandé par le Comité français d'accréditation (COFRAC). En complément, certaines conditions pré analytiques ont été ré-évaluées. Résultats. L'ensemble des coefficients de variation évaluant les performances étaient inférieurs à 10 % (répétabilité, reproductibilité, variabilité interopérateur). Les limites de quantification et de linéarité étaient adaptées à l'utilisation clinique du paramètre. Les résultats étaient identiques quel que soit le type et le fournisseur de cytomètre en flux. Les contraintes de conservation pré-analytiques des échantillons ont été confirmées. CONCLUSION: Les résultats étaient conformes aux exigences de qualité recommandées par le COFRAC. Ils permettent l'accréditation de la mesure de mHLA-DR par cytométrie en flux et son utilisation en soins courants.

Cross-sectional reassessment after 4 years of clinical routine use of AQUIOS CL for absolute T cell quantitation in a university hospital.

BACKGROUND: We had previously reported appropriate performances of automated AQUIOS CL cytometer (Beckman Coulter) for regulatory approval of absolute T cell enumeration. However, after 4 years of routine use, we still observed recurrent histogram anomalies that may affect both absolute values and T cell subset percentages results. The objective of the current study was thus to perform a cross-sectional evaluation of these graphical anomalies within a university hospital context, to assess their influence on results and ultimately to propose a standardized decision tree to circumvent graphical disturbances at the time of results validation. METHODS: Eight hundred and sixty-two blood samples were prospectively analyzed on AQUIOS CL. Results were compared to (i) lymphocyte values from complete blood count; (ii) results from manual staining and analysis on Navios cytometer (Beckman Coulter); (iii) results after washing step and reacquisition on AQUIOS CL. RESULTS: Nearly 75% analyses did not show any graphical anomaly. 20% had single anomaly on "Lymphs (45)" or "Lymphs EV" regions influencing T cells percentages and requiring manual re-gating of "CD3- capture" region. 5% showed concomitant "Lymphs EV" and "Lymphs (45)" anomalies influencing both T cell percentages and absolute counting and requiring additional staining and analysis on Navios. Finally, <1% presented with anomaly on "CD4/CD8" histogram or "CD3+ All" region, influencing both T cell percentages and absolute counting. CONCLUSIONS: Around 25% AQUIOS CL results were flawed due to gating anomalies. In <5% cases, additional back-up procedures should be undertaken to ensure results validity. A simple decision-tree may help in guiding validation process.

Proof of concept study of mass cytometry in septic shock patients reveals novel immune alterations.

Innovative single cell technologies such as mass cytometry (CyTOF) widen possibilities to deeply improve characterisation of immune alterations mechanisms in human diseases. So far, CyTOF has not been used in sepsis - a condition characterized by complex immune disorders. Here, we evaluated feasibility of CyTOF analysis in patients with septic shock. We designed a mass cytometry panel of 25 extracellular markers to study mononuclear cells from 5 septic shock patients and 5 healthy donors. We explored single-cell data with global and specific unsupervised approaches such as heatmaps, SPADE and viSNE. We first validated relevance of our CyTOF results by highlighting established immune hallmarks of sepsis, such as decreased monocyte HLA-DR expression and increased expressions of PD1 and PD-L1 on CD4 T cells and monocytes. We then showed that CyTOF analysis reveals novel aspects of sepsis-induced immune alterations, e.g. B cell shift towards plasma cell differentiation and uniform response of several monocyte markers defining an immune signature in septic patients. This proof of concept study demonstrates CyTOF suitability to analyse immune features of septic patients. Mass cytometry could thus represent a powerful tool to identify novel pathophysiological mechanisms and therapeutic targets for immunotherapy in septic shock patients.

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.

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.

HHV-6 infection after allogeneic hematopoietic stem cell transplantation: From chromosomal integration to viral co-infections and T-cell reconstitution patterns.

OBJECTIVES: Human herpes virus 6 (HHV-6) can reactivate after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and may be associated with significant clinical manifestations. METHODS: Case control study of HHV-6 infections after allo-HSCT. Chromosomal integration (ciHHV-6) for viral loads ≥ 5.5-log10 copies/mL was investigated. Viral co-infections, T-cell recovery, risk factors and outcome were compared in HHV-6- and non-HHV-6-infected patients. Antiviral treatment strategies were reviewed. RESULTS: Among 366 adult allo-HSCT recipients, 75 HHV-6 infections occurred. Three (4%) recipients were ciHHV-6. HHV-6 infections were associated with CMV (p = 0.05; sdHR 1.73, CI 0.99-3.02) and/or BKV infections (p < 0.0001; sdHR 4.63, CI 2.04-10.53) but not EBV reactivation (p = 0.34). A slower CD8+ T-cells recovery was observed until 6 months after allo-HSCT in the HHV-6-infected group (p < 0.001), independently of acute and/or chronic graft-versus-host disease. The overall probability of survival after allo-HSCT was diminished for active HHV-6-infected patients (p = 0.0326). Cord blood unit recipients had a higher risk of developing HHV-6 infection compared to bone marrow recipients (p = 0.0007; sdHR 3.82, CI 1.76-8.27). Anti-HHV-6 treatment achieved complete response in only 2/3 of the cases. CONCLUSIONS: In this series of allo-HSCT recipients, 4% were ciHHV-6, active HHV-6 infection was likely associated with CMV and BKV co-reactivations, delayed CD8+ T-cell recovery and poorer outcome.

Marked alterations of neutrophil functions during sepsis-induced immunosuppression.

Severe septic syndromes deeply impair innate and adaptive immunity and are responsible for sepsis-induced immunosuppression. Although neutrophils represent the first line of defense against infection, little is known about their phenotype and functions a few days after sepsis, when the immunosuppressive phase is maximal (i.e., between d 3 and 8). The objective of the present study was to perform, for the first time, a global evaluation of neutrophil alterations in immunosuppressed septic patients (at d 3-4 and d 6-8) using phenotypic and functional studies. In addition, the potential association of these parameters and deleterious outcomes was assessed. Peripheral blood was collected from 43 septic shock patients and compared with that of 23 healthy controls. In the septic patients, our results highlight a markedly altered neutrophil chemotaxis (functional and chemokine receptor expressions), oxidative burst, and lactoferrin content and an increased number of circulating immature granulocytes (i.e., CD10(dim)CD16(dim)). These aspects were associated with an increased risk of death after septic shock. In contrast, phagocytosis and activation capacities were conserved. To conclude, circulating neutrophils present with phenotypic, functional, and morphologic alterations a few days after sepsis onset. These dysfunctions might participate in the deleterious role of sepsis-induced immunosuppression. The present results open new perspectives in the mechanisms favoring nosocomial infections after septic shock. They deserve to be further investigated in a larger clinical study and in animal models recapitulating these alterations.

Increased Regulatory T-Cell Percentage Contributes to Poor CD4(+) Lymphocytes Recovery: A 2-Year Prospective Study After Introduction of Antiretroviral Therapy.

Background. The primary aim of this study was to determine the impact of regulatory T cells (Tregs) percentage on immune recovery in human immunodeficiency virus (HIV)-infected patients after antiretroviral therapy introduction. Methods. A 2-year prospective study was conducted in HIV-1 chronically infected naive patients with CD4 count <500 cells/mm(3). Regulatory T cells were identified as CD4(+)CD25(high)CD127(low) cells among CD4(+) lymphocytes. Effect of Treg percentage at inclusion on CD4 evolution overtime was analyzed using a mixed-effect Poisson regression for count data. Results. Fifty-eight patients were included (median CD4 = 293/mm(3), median Treg percentage = 6.1%). Percentage of Treg at baseline and CD4 nadir were independently related to the evolution of CD4 absolute value according to time: (1) at any given nadir CD4 count, 1% increase of initial Treg was associated with a 1.9% lower CD4 absolute value at month 24; (2) at any given Treg percentage at baseline, 10 cell/mm(3) increase of CD4 nadir was associated with a 2.4% increase of CD4 at month 24; and (3) both effects did not attenuate with time. The effect of Treg at baseline on CD4 evolution was as low as the CD4 nadir was high. Conclusions. Regulatory T-cell percentage at baseline is a strong independent prognostic factor of immune recovery, particularly among patients with low CD4 nadir.

Effect of pneumatic tube transport on T lymphocyte subsets analysis.

BACKGROUND: Pneumatic tube system (PTS) for transportation of blood specimens may present with advantages for hospital organization as it provides faster tube transfer from medical wards to routine labs. These characteristics are expected to result in faster sample processing and decreased turnaround time, therefore benefiting the patient particularly in emergency units. However, PTS could affect sample quality and therefore laboratory results. Within the context of routine lab certification, effects of PTS on routine cellular immunology analyses, especially on determination of T lymphocyte subpopulations, need to be evaluated. METHODS: Paired EDTA blood samples were collected from 30 healthy donors. For each pair, one sample was hand-delivered by a courier while the other was transported through a PTS of 2.4 km long (1.6 miles) with two 90-degree turns and one-U turn generating a speed of 5 m/s with a maximal acceleration of 2 g-force. The percentages of CD3+ cells, CD4+ and CD8+ T-cells and their absolute counts were assessed by flow cytometry. RESULTS: For every parameter, results measured by flow cytometry were not significantly different after transport by PTS or hand-delivery. Results comparison revealed an excellent linear correlation between both delivery methods (R ranged from 0.969 to 0.982; P < 0.01). Bland-Altman plots also showed good agreement, indicating that results were not influenced by the transport method. CONCLUSIONS: Our results suggest that, pending validation using clinical samples, PTS does not present with pre-analytical drawbacks and is thus a reliable system for blood samples transportation when performing T cell subset phenotyping.

Flow cytometric evaluation of lymphocyte transformation test based on 5-ethynyl-2'deoxyuridine incorporation as a clinical alternative to tritiated thymidine uptake measurement.

In clinical laboratories, the evaluation of lymphocyte proliferative response (lymphocyte transformation test-LTT) is routinely performed by the measurement of [(3)H]-thymidine uptake after stimulation. In this study we evaluated the performances of a recently developed non-radioactive test based on the detection by flow cytometry of 5-ethynyl-2'deoxyuridine (EdU) incorporation for the measurement of LTT in routine lab conditions. After definition of optimal protocol parameters, EdU incorporation test showed good repeatability and reproducibility. Moreover, this assay was flexible enough to fit important clinical laboratory constraints (delayed stimulation, low number of cells and delayed analysis after staining). Importantly, correlations between results obtained with EdU and [(3)H]-thymidine incorporation assays were excellent both in healthy volunteers and pediatric and septic patients. In particular, the two techniques identified patients presenting with altered LTT. Upon confirmation in a larger cohort of patients, EdU incorporation assay may be a relevant non-radioactive candidate for LLT in clinic.

Comparative dose-responses of recombinant human IL-2 and IL-7 on STAT5 phosphorylation in CD4+FOXP3- cells versus regulatory T cells: a whole blood perspective.

Interleukin(IL)-2 and IL-7 are cytokines with important functions related to CD4(+) lymphocyte proliferation, differentiation and survival. Depending on doses, they theoretically activate regulatory (Treg) and/or effector T cells (Teff) and thus may be indicated with different therapeutic objectives. In this study we assessed ex vivo the differential dose-responses of CD4(+) T cell subsets (Treg versus CD4(+)FOXP3(-) cells) to recombinant human (rh) IL-2 and rhIL-7. Fresh whole blood from healthy donors was stimulated with increasing doses of cytokines. By using a novel flow cytometry procedure of intracellular signaling pathway staining (e.g., detection of STAT5 phosphorylation; a pivotal marker of cytokine-induced activation; in combination with intracellular FOXP3 staining), we were able to specifically measure Treg and CD4(+)FOXP3(-) cell responses in the same tube. Half maximal effective concentrations (EC50) were calculated. We observed a dose-response effect on Treg and CD4(+)FOXP3(-) cells for both cytokines. Interestingly, low doses of hIL-2 preferentially activated Treg (EC50 Treg = 0.15 pg/ml versus CD4(+)FOXP3(-) cells = 750 pg/ml - p < 0.0001) whereas low doses of rhIL-7 preferentially induced CD4(+)FOXP3(-) cell activation (EC50 Treg = 25 pg/ml and CD4(+)FOXP3(-) cells = 2.5 pg/ml - p < 0.0001). To our knowledge, this work is the first to show differential dose-response effects on CD4(+)FOXP3(-) cells versus Treg of rhIL-7 and rhIL-2 in one ex vivo whole blood single tube assay including two intracellular stainings (i.e., pSTAT5 and FOXP3). Beyond the confirmation of the dose-dependent differential effects of IL-2 versus IL-7 on CD4(+)FOXP3(-) cells/Treg, our results illustrate the value of this approach for monitoring drugs' activities by flow cytometry in daily clinical practice.

Elevated plasmatic level of soluble IL-7 receptor is associated with increased mortality in septic shock patients.

PURPOSE: Adjunctive immunoadjuvant therapies are now proposed in the treatment of septic patients that develop immune dysfunctions. However, a prerequisite is to identify patients at high risk of death that would benefit from such therapy. Knowing that rhIL-7 is a putative candidate for septic shock treatment, we evaluated the association between increased plasmatic level of soluble CD127 (sCD127, IL-7 receptor alpha chain) and mortality after septic shock. METHODS: sCD127 plasmatic level was measured in 70 septic shock patients sampled at day 1-2 (D1) and day 3-4 (D3) after the onset of shock and 41 healthy volunteers. RESULTS: Compared with survivors, non-survivors presented with significantly higher sCD127 concentrations at D1 and D3 (p < 0.001 and p = 0.002). At D1, the area under the receiver operating characteristic curve for sCD127 level association with mortality was 0.846 (p < 0.0001). Kaplan-Meier survival curves illustrated that mortality was significantly different after stratification based on D1 sCD127 level (log rank test, hazard ratio 9.10, p < 0.0001). This association was preserved in multivariate logistic regression analysis including clinical confounders (age, SAPS II and SOFA scores, odds ratio 12.71, p = 0.003). Importantly, patient stratification on both D1 sCD127 value and SAPS II score improved this predictive capacity (log rank test, p = 0.0001). CONCLUSIONS: Increased sCD127 plasmatic level enables the identification of a group of septic shock patients at high risk of death. After confirmation in a larger cohort, this biomarker may be of interest for patient stratification in future clinical trials.

Assessment of a novel flow cytometry technique of one-step intracellular staining: example of FOXP3 in clinical samples.

BACKGROUND: By measuring multiple parameters on a single-cell basis, flow cytometry is a potent tool to dissect the phenotypes and functions of cell subsets. However, because this technique may be time-consuming, particularly for intracellular staining, it could be problematic for its use in daily routine or in large cohorts. Recently, a novel reagent has been developed to perform intracellular staining in one step. The objective of our study was thus to assess this new method in comparison with the reference technique by focusing on FOXP3 staining in clinical samples. METHODS: Peripheral blood was collected from 15 HIV-1-infected patients, 5 critically ill patients, and 5 healthy volunteers and stained using the two different methods. Different subsets of FOXP3 positive cells were investigated by flow cytometry. RESULTS: When comparing results obtained with the two techniques, no statistical differences between the percentages of CD4+FOXP3+, CD4+CD25+FOXP3+, and CD4+CD25+CD127-FOXP3+ cells were observed. Besides, a strong correlation between percentages of CD4+FOXP3+CD25+CD127- lymphocytes measured with both techniques was found in patients (r: 0.843, P < 0.001, intra-class correlation coefficient: 0.820, P < 0.001). Importantly, flow cytometry stainings obtained with the one-step method were very robust with an excellent intra-assay precision, a better discriminative power and correct stability and reproducibility of the staining even after blood storage. CONCLUSIONS: With a strong correlation between the percentages of FOXP3+ Tregs when compared with the reference method, a better staining quality, a shorter realization time and no need of isotype control, this one step procedure may represent an important improvement for a daily routine use of intracellular staining.

Decreased T-cell repertoire diversity in sepsis: a preliminary study.

OBJECTIVE: Septic syndromes are the leading causes of mortality in intensive care units. In patients, the occurrence of sepsis-induced immune suppression is associated with delayed mortality, although the exact role of lymphocyte dysfunctions is not well established. The objective of this study was to investigate T-cell receptor diversity, an important feature of T-cell response, in patients with septic shock. DESIGN: Preliminary prospective observational study. SETTING: Adult intensive care units in a university hospital. SUBJECTS: Patients with septic shock (n = 41) sampled twice after the onset of shock (early after inclusion [day 1] and at the end of the first week [day 7]). MEASUREMENTS AND MAIN RESULTS: Using a novel molecular biology technique, the combinatorial diversity of human T-cell receptor ß-chain (TRB locus) was measured in peripheral blood. Patients with septic shock presented with a marked decreased T-cell receptor diversity after the onset of shock in comparison with normal values. Importantly, in paired samples, a very steep recovery slope of T-cell receptor diversity, never described in other clinical situations, was observed between day 1 and day 7 (p < 0.0001, Wilcoxon's paired test). Decreased T-cell receptor diversity was associated with mortality (log-rank test, p = 0.0058; hazard ratio = 4.48; 95% confidence interval 1.96-53.32), and the development of nosocomial infections (p < 0.05, Mann-Whitney U test). CONCLUSION: Our results show for the first time that septic patients present with a marked decreased T-cell receptor diversity that returned rapidly toward normal values over time. This opens novel cognitive research perspectives that deserve to be investigated in experimental models of sepsis. After confirmation in larger cohorts of these preliminary results, T-cell receptor diversity measurements may become a crucial tool to monitor immune functions in ICU patients.

CD4+CD25+CD127- assessment as a surrogate phenotype for FOXP3+ regulatory T cells in HIV-1 infected viremic and aviremic subjects.

BACKGROUND: Although likely pivotal, the role of regulatory T cells (Tregs) in HIV pathogenesis remains elusive. This can be partly explained by analytical issues regarding their phenotypic identification in clinical studies. Instead of intracellular FOXP3 staining, CD4+CD25+CD127- phenotype has been proposed as an alternative to identify Tregs in clinical samples. However, its use remains controversial in viremic patients. Therefore, the objective of the present study was to assess the correlation between frequencies of CD4+CD25+CD127- and CD4+CD25+FOXP3+ lymphocytes in viremic and matched aviremic HIV-infected patients. METHODS: Peripheral blood was collected from HIV-1 infected patients. Eleven viremic patients (Viral Load > 40 copies/mL) were matched (age, sex, CD4+ cell number) with 8 aviremic patients under highly active antiretroviral therapy (HAART). Fresh whole blood was immediately stained to analyze by flow cytometry the correlation between CD4+CD25+CD127- and the reference phenotype CD4+CD25+FOXP3+ lymphocytes in the same tube (four color staining CD4/CD25/CD127/FOXP3 for concomitant analysis of cell surface and intracellular markers). RESULTS: In both groups, no significant differences were observed when comparing CD4+CD25+CD127- and CD4+CD25+FOXP3+ cell frequencies. In line, a strong correlation between CD4+CD25+CD127- and CD4+CD25+FOXP3+ lymphocyte percentages was observed in the whole patient population (r: 0.948, P < 0.001) or each group separately: aviremic (r: 0.968, P < 0.001), viremic (r: 0.9, P < 0.001). Finally, we found that most CD4+FOXP3+ cells were indeed CD25+CD127-, both in viremic and aviremic groups (88.5% and 90.9%, respectively). CONCLUSIONS: We observed that CD4+CD25+CD127- phenotype is a good and easy-to-perform surrogate identification strategy for FOXP3+ regulatory T cells in both viremic and aviremic HIV-1-infected subjects. Thus, it represents a useful tool for monitoring Tregs in clinical research studies based on large cohorts of patients prospectively monitored, including HIV-infected subjects.

IL-7 restores lymphocyte functions in septic patients.

Septic syndrome is the leading cause of mortality for critically ill patients worldwide. Patients develop lymphocyte dysfunctions associated with increased risk of death and nosocomial infections. In this study, we performed preclinical experiments testing the potential of recombinant human IL-7 (rhIL-7) as a lymphostimulating therapy in sepsis. Circulating IL-7 and soluble IL-7 receptor α-chain (soluble CD127) concentrations were measured in plasma, whereas cellular CD127 expression was evaluated on circulating CD4(+) and CD8(+) lymphocytes from septic shock patients and healthy volunteers. Lymphocyte proliferation, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction were measured ex vivo in response to T cell stimulation in the presence or not of rhIL-7. We show that IL-7 pathway (plasmatic IL-7 concentration and cellular and soluble CD127 expressions) is not overtly altered and remains activable in septic patients. Most importantly ex vivo treatment of patients' cells with rhIL-7 significantly improves lymphocyte functionality (CD4(+) and CD8(+) lymphocyte proliferations, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction after stimulation). To our knowledge, this constitutes the first report of rhIL-7 ability to restore normal lymphocyte functions in septic patients. These results support the rational for initiating a clinical trial testing rhIL-7 in septic shock.

Assessment of plasmatic immunoglobulin G, A and M levels in septic shock patients.

Polyvalent immunoglobulin (Ig) therapy has been tested as adjunctive treatment in sepsis and septic shock, but its efficacy is still a matter of debate. This has been explained because clinical trials were mostly performed on small numbers of patients. Moreover, the endogenous level of circulating Ig in patients was never taken into account. In this study, plasmatic Ig classes and protein concentrations were measured at Days (D) 1-2, D3-4 and D5-7 in 62 septic shock patients. At D1-2 as well as at D3-4, patients presented with a significant reduction of plasmatic IgG concentrations. Indeed, at D1-2, 61% of the patients had IgG level below the lowest limit of our age-matched reference values. Plasmatic IgM levels were decreased as well in comparison with reference values from the lab whereas IgA concentrations were not modified. Circulating IgG and IgM concentrations tends to increase overtime. Indeed, at D5-7, most patients (61%) had IgG and IgM levels within the range of normal values. These alterations did not appear to be associated with increased mortality, morbidity or severity after septic shock. However, at D1-2 and D3-4, decreased circulating Ig level was significantly correlated with reduced plasmatic protein concentrations. Overall, our results suggest that an apparent hypogammaglobulinemia is present at D1-2 and D3-4 in septic shock patients, which seems to be related with reduced circulating protein concentration after septic shock. These results need to be confirmed in a larger cohort of patients.

Lymphocyte subset reconstitution after unrelated cord blood or bone marrow transplantation in children.

We report the post-transplant lymphocyte subset recovery of 226 children treated with Unrelated Cord Blood transplant (UCBT) (n = 112) or Unrelated Bone Marrow Transplant (UBMT) (n = 114) for malignant or non-malignant diseases. Absolute numbers of natural killer (NK), B and T cells were monitored by flow cytometry up to 5 years post-transplant. Immunological endpoints were: time to achieve a CD3(+) cell count > 0·5 and 1·5 × 109/l, CD4(+) > 0·2 and 0·5 × 109/l, CD8(+) > 0·25 ×109/l, CD19(+) > 0·2 × 109/l, NK > 0·1 × 109/l. These endpoints were analysed through the use of cumulative incidence curves in the context of competing risks. CD8(+) T cell recovery was delayed after UCBT with a median time to reach CD8(+) T cells > 0·25 × 109/l of 7·7 months whereas it was 2·8 months in UBMT (P < 0·001). B cell recovery was better in UCBT, with a median time to reach CD19(+) cells > 0·2 × 109/l of 3·2 months in UCBT and 6·4 months in UBMT (P = 0·03). Median time for CD4(+) T cell and NK cell recovery was similar in UCBT and UBMT. CD4(+) T cells recovery was negatively correlated to age (better reconstitution in younger patients, P = 0·002). CD8(+) T cells recovery was shorter in recipients with a positive cytomegalovirus serology (P =0·001).

Early assessment of leukocyte alterations at diagnosis of septic shock.

A dramatic decrease in circulating lymphocyte number is regularly described after septic shock. However, it is unknown how early this alteration develops after diagnosis of shock and if it remains stable over time. Twenty-one septic shock patients with no comorbidities were included within 2 h after the beginning of vasopressive treatment. Flow cytometry phenotyping of circulating leukocyte subpopulations and quantitative real-time polymerase chain reaction of T-bet, GATA-3, FOXP3, and RORγ mRNA were performed in patients from the diagnosis of shock and every 6 h during the subsequent 48 h. From their admission in the intensive care unit, patients present with major alterations of circulating leukocyte count (leukocytosis, neutrophilia, and major lymphopenia). The numbers of every lymphocyte subpopulations (T, B, and natural killer cells) were diminished. Gene expression analysis of transcription factors specific for TH1, TH2, CD4CD25 regulatory, and TH17 lymphocytes showed a severe decrease in comparison with healthy individuals' values. These alterations remain stable during the first 48 h after inclusion in the protocol despite early and aggressive resuscitation and antibiotherapy administered in patients. At the time of diagnosis of shock and admission in the intensive care unit, septic patients already present with severe lymphopenia involving every lymphocyte subsets including CD4 T-cell subpopulations. No significant variation could be detected within the first 48 h. This should be taken into account in the forthcoming clinical trials testing immunomodulating therapies in septic shock patients.