Christophe Malcus

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.

Protein kinase Cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation

OBJECTIVE Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE. METHODS We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. RESULTS We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. CONCLUSION Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.

Lack of recovery in monocyte human leukocyte antigen-DR expression is independently associated with the development of sepsis after major trauma

IntroductionMajor trauma is characterized by an overwhelming pro-inflammatory response and an accompanying anti-inflammatory response that lead to a state of immunosuppression, as observed after septic shock. Diminished monocyte Human Leukocyte Antigen DR (mHLA-DR) is a reliable marker of monocyte dysfunction and immunosuppression. The main objective of this study was to determine the relation between mHLA-DR expression in severe trauma patients and the development of sepsis.MethodsWe conducted a prospective observational study over 23 months in a trauma intensive care unit at a university hospital. Patients with an Injury Severity Score (ISS) over 25 and age over 18 were included. mHLA-DR was assessed by flow cytometry protocol according to standardized protocol. Mann-Whitney U-test for continuous non-parametric variables, independent paired t test for continuous parametric variables and chi-square test for categorical data were used.ResultsmHLA-DR was measured three times a week during the first 14 days. One hundred five consecutive severely injured patients were monitored (ISS 38 ± 17, SAPS II 37 ± 16). Thirty-seven patients (35%) developed sepsis over the 14 days post-trauma. At days 1-2, mHLA-DR was diminished in the whole patient population, with no difference with the development of sepsis. At days 3-4, a highly significant difference appeared between septic and non-septic patients. Non- septic patients showed an increase in mHLA-DR levels, whereas septic patients did not (13,723 ± 7,766 versus 9,271 ± 6,029 antibodies per cell, p = .004). Most importantly, multivariate logistic regression analysis, after adjustment for usual clinical confounders (adjusted OR 5.41, 95% CI 1.42-20.52), revealed that a slope of mHLA-DR expression between days1-2 and days 3-4 below 1.2 remained associated with the development of sepsis.ConclusionsMajor trauma induced an immunosuppression, characterized by a decrease in mHLA-DR expression. Importantly, after multivariate regression logistic analysis, persistent decreased expression was assessed to be in relation with the development of sepsis. This is the first study in trauma patients showing a link between the lack of immune recovery and the development of sepsis on the basis of the standardized protocol. Monitoring immune function by mHLA-DR measurement could be useful to identify trauma patients at a high risk of infection.

Assessment of pro-vasopressin and pro-adrenomedullin as predictors of 28-day mortality in septic shock patients.

PurposeImprovements in survival after septic shock will most likely rely on our capacity to manage individualized therapies based on the measurement of rapidly accessible biomarkers. As the early phase of septic shock is dominated by severe alterations of the cardiovascular system, the predictive value for mortality of pro-vasopressin (pro-AVP) and pro-adrenomedullin (pro-ADM), two vasoactive pro-hormones, was assessed.MethodsIn 99 consecutive patients, pro-hormone concentrations were measured (immunoluminometric assay) three times within the first week after the onset of septic shock.ResultsPro-AVP and pro-ADM concentrations were significantly increased in non-survivors in comparison with survivors and were significantly associated with mortality after both univariate and multivariate analysis. Importantly, when assessed as a pair, pro-ADM and pro-AVP were even more informative.ConclusionsBoth Pro-ADM and pro-AVP appear to be good biomarkers for the prediction of 28-day mortality after septic shock. However, their association in a single variable tends to improve their predictive capacity.

Mutations in CECR1 associated with a neutrophil signature in peripheral blood

BackgroundA reduction of ADA2 activity due to autosomal recessive loss of function mutations in CECR1 results in a newly described vasculopathic phenotype reminiscent of polyarteritis nodosa, with manifestations ranging from fatal systemic vasculitis with multiple strokes in children to limited cutaneous disease in middle-aged individuals. Evidence indicates that ADA2 is essential for the endothelial integrity of small vessels. However, CECR1 is not expressed, nor is the ADA2 protein detectable, in cultured human endothelial cells, thus implicating additional cell types or circulating factors in disease pathogenesis.MethodsConsidering the phenotypic overlap of ADA2 deficiency with the type I interferonopathy Aicardi-Goutières syndrome due to mutations in SAMHD1, we looked for the presence of an interferon signature in the peripheral blood of two newly ascertained ADA2-deficient patients.ResultsWe identified biallelic CECR1 mutations in two patients consistent with ADA2 deficiency. Both patients demonstrated an upregulation of interferon stimulated gene transcripts in peripheral blood. More strikingly however, genome-wide analysis revealed a marked overexpression of neutrophil-derived genes, suggesting that the vasculitis seen in ADA2 deficiency may be an indirect effect resulting from chronic and marked activity of neutrophils.ConclusionsWe hypothesise that ADA2 may act as a regulator of neutrophil activation, and that a reduction of ADA2 activity results in significant endothelial damage via a neutrophil-driven process.

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 &bgr;-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.

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.

Preferential increase in memory and regulatory subsets during T-lymphocyte immune reconstitution after Thymoglobulin induction therapy with maintenance sirolimus vs cyclosporine.

BACKGROUND Sirolimus maintenance therapy with Thymoglobulin induction is a promising regimen that may preserve renal function. Data are lacking, however, about the immunologic effects of combined Thymoglobulin-sirolimus. METHODS In a 12-month, prospective, randomised, open-label, single-centre pilot study, de novo deceased-donor kidney transplant patients were randomised to receive cyclosporine or sirolimus, with Thymoglobulin induction, mycophenolate mofetil and corticosteroids. Flow cytometry analysis of peripheral blood was used to evaluate immune reconstitution. RESULTS Nineteen patients were recruited (sirolimus 9, cyclosporine 10). Reconstitution of the CD4(+) T-lymphocyte subset was significantly lower with sirolimus versus cyclosporine over year 1, but CD8(+) reconstitution did not differ significantly between groups. The proportion of naïve CD4(+) T-lymphocytes showed an initial decrease with sirolimus versus cyclosporine. Naïve CD8(+) T-lymphocytes increased versus baseline in the cyclosporine cohort at months 1 and 3, but remained unchanged with sirolimus. Memory CD4(+) T-lymphocytes occurred more frequently in sirolimus- versus cyclosporine-treated patients during year 1. The proportion of memory CD8(+) T-lymphocytes decreased at months 1 and 3 compared to baseline in the CsA arm, but did not change in the sirolimus cohort. By month 12, the proportion of both naïve and memory CD4(+) and CD8(+) T-lymphocytes had become similar with sirolimus or cyclosporine. There were fewer naïve B-lymphocytes in the sirolimus cohort and more CD19(-)IgD(+/-)CD27(+) memory B-lymphocytes. CONCLUSIONS In this small population, homeostatic reconstitution after Thymoglobulin induction showed disproportionately high recovery of memory T-lymphocyte subsets during sirolimus therapy, which may explain the higher rejection rate seen with sirolimus versus cyclosporine following kidney transplantation.

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., CD10dimCD16dim). 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.

B-cell subpopulations in children: National reference values

Peripheral B‐lymphocytes undergo a series of changes during the first few years of life. Encounters with foreign antigens lead to maturation and differentiation. Several primary antibody deficiencies (PADs) affecting B‐cell development are associated with abnormalities in the composition and/or differentiation of B‐cell compartments. The most recent international classifications of primary immunodeficiencies (PIDs) and common variable immunodeficiencies (CVID) have highlighted the importance of B‐cell immunophenotyping and age‐specific reference intervals for diagnostic purposes. We established national reference values for memory B‐cell subpopulations, on the basis of CD27 and surface IgD expression in the peripheral blood of 242 healthy children. We report here the absolute counts and percentages of naive, switched and non‐switched memory B‐cells for seven age groups, from neonates to adults. We found that the naive B‐cells percentage declined between the ages of 6 months and 8 years, after which it remained stable at about 70–80%. Memory B‐cells are already present at birth and their numbers increase throughout childhood, stabilizing between the ages of 12 and 18 years. The definition of reference intervals for pediatric B‐cell levels should facilitate the screening and diagnosis of various B‐cell immunodeficiencies. This multicenter study, providing national reference values, should thus facilitate immunological diagnosis in children.