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International audience; Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The immune system plays a key role in sepsis onset and remains dysregulated over time in a heterogeneous manner. Here, we decipher the heterogeneity of the first week evolution of the monocyte HLA-DR (mHLA-DR) surface protein expression in septic patients, a key molecule for adaptive immunity onset. We found and verified four distinctive trajectories endotypes in a discovery (n = 276) and a verification cohort (n = 102). We highlight that 59% of septic patients exhibit low or decreasing mHLA-DR expression while in others mHLA-DR expression increased. This study depicts the first week behavior of mHLA-DR over time after sepsis onset and shows that initial and third day mHLA-DR expression measurements is sufficient for an early risk stratification of sepsis patients. These patients might benefit from immunomodulatory treatment to improve outcomes. Going further, our study introduces a way of deciphering heterogeneity of immune system after sepsis onset which is a first step to reach a more comprehensive landscape of sepsis.

Clostridium difficile infection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. The pathological effects of CDI are primarily attributed to toxins A (TcdA) and B (TcdB). Adequate toxin-specific antibody responses are associated with asymptomatic carriage, whereas insufficient humoral responses are associated with recurrent CDI. While the data supporting the importance of anti-toxin antibodies are substantial, clarity about the toxin domain specificity of these antibodies is more limited. To investigate this matter, combinations of human mAbs targeting multiple domains of TcdB were assessed using toxin neutralization assays. These data revealed that a combination of mAbs specific to all major toxin domains had improved neutralizing potency when compared to equivalent concentrations of a single mAb or a combination of mAbs against one or two domains. The function and toxin domain binding specificity of serum antibodies elicited by immunization of hamsters with a toxoid vaccine candidate was also assessed. Immunization with a toxoid vaccine candidate provoked toxin neutralizing antibodies specific to multiple domains of both TcdA and TcdB. When assessed in a toxin neutralization assay, polyclonal sera displayed greater activity against elevated concentrations of toxins than equivalent concentrations of individual mAbs. These data suggest a potential benefit of any antibody based therapeutic or prophylactic treatment that targets multiple toxin domains.

Physical inactivity is one of the leading contributors to worldwide morbidity and mortality. The elderly are particularly susceptible since the features of physical inactivity overlap with the outcomes of natural aging - including the propensity to develop cardiovascular diseases, cancer, diabetes mellitus, sarcopenia and cognitive impairment. The age-dependent loss of immune function, or immunosenescence, refers to the progressive depletion of primary immune resources and is linked to the development of many of these conditions. Immunosenescence is primarily driven by chronic immune activation and physical activity interventions have demonstrated the potential to reduce the risk of complications in the elderly by modulating inflammation and augmenting the immune system. Since poor vaccination outcome is a hallmark of immunosenescence, the assessment of vaccine efficacy provides a window to study the immunological effects of regular physical activity. Using an accelerator-based study, we demonstrate in a Singaporean Chinese cohort that elderly women (n=56) who walk more after vaccination display greater post-vaccination expansion of monocytes and plasmablasts in peripheral blood. Active elderly female participants also demonstrated lower baseline levels of IP-10 and Eotaxin, and the upregulation of genes associated with monocyte/macrophage phagocytosis. We further describe postive correlations between the monocyte response and the post-vaccination H1N1 HAI titres of participants. Finally, active elderly women reveal a higher induction of antibodies against Flu B in their 18-month second vaccination follow-up. Altogether, our data are consistent with better immunological outcomes in those who are more physically active and highlight the pertinent contribution of monocyte activity.

Clostridium difficile infection (CDI) is a leading cause of nosocomial and antibiotic-associated diarrhea. A vaccine, based on formalin-inactivated toxins A and B purified from anaerobic cultures of C. difficile strain VPI 10463 (toxinotype 0), has been in development for the prevention of symptomatic CDI. We evaluated the breadth of protection conferred by this C. difficile toxoid vaccine in cross-neutralization assessments using sera from vaccinated hamsters against a collection of 165 clinical isolates. Hamster antisera raised against the C. difficile toxoid vaccine neutralized the cytotoxic activity of culture supernatants from several toxinotype 0 strains and heterologous strains from 10 different toxinotypes. Further assessments performed with purified toxins confirmed that vaccine-elicited antibodies can neutralize both A and B toxins from a variety of toxinotypes. In the hamster challenge model, the vaccine conferred significant cross-protection against disease symptoms and death caused by heterologous C. difficile strains from the most common phylogenetic clades, including the most prevalent toxinotypes.

Introduction The host response to septic shock is dynamic and complex. A sepsis-induced immunosuppression phase has recently been acknowledged and linked to bad outcomes and increased healthcare costs. Moreover, a marked suppression of the immune response has also been partially described in patients hospitalized in intensive care unit (ICU) for severe trauma or burns. It has been hypothesized that immune monitoring could enable identification of patients who might most benefit from novel, adjunctive immune-stimulating therapies. However, there is currently neither a clear definition for such injury-induced immunosuppression nor a stratification biomarker compatible with clinical constraints. Methods and analysis We set up a prospective, longitudinal single-centre clinical study to determine the incidence, severity and persistency of innate and adaptive immune alterations in ICU patients. We optimized a workflow to describe and follow the immunoinflammatory status of 550 patients (septic shock, severe trauma/burn and major surgery) during the first 2 months after their initial injury. On each time point, two immune functional tests will be performed to determine whole-blood TNF-α production in response to ex vivo lipopolysaccharide stimulation and the T lymphocyte proliferation in response to phytohaemagglutinin. In addition, a complete immunophenotyping using flow cytometry including monocyte HLA-DR expression and lymphocyte subsets will be obtained. New markers (ie, levels of expression of host mRNA and viral reactivation) will be also evaluated. Reference intervals will be determined from a cohort of 150 age-matched healthy volunteers. This clinical study will provide, for the first time, data describing the immune status of severe ICU patients over time. Ethics and dissemination Ethical approval has been obtained from the institutional review board (no 69HCL15_0379) and the French National Security agency for drugs and health-related products. Results will be disseminated through presentations at scientific meetings and publications in peer-reviewed journals. Trial registration number Clinicaltrials.gov Registration number: NCT02638779. Pre-results.

Clostridiumdifficile (C.difficile)is a significant human pathogen. C.difficile infection (CDI) causesclinical symptoms ranging from diarrhea to life-threatening fulminant pseudo membranous colitis. The pathogenesis of C. difficile is mediated by two large exotoxins, toxins A and B. These two toxins are highly homologous, single chain proteins consisting of four functional domains: N-terminal glucosyl transferase domain (GTD), cysteine protease domain (CPD), translocation domain (TLD) and a C-terminal receptor-binding domain (RBD). The important role played by anti-toxin sera and antibodies in the prevention of primary and recurrent CDI has been described and demonstrated in both clinical and pre-clinical studies; however, work focused on the impact of the toxin domain specificity of these antibodies is limited. To address this deficit, sera from a C. difficile vaccine immunized hamsters and toxin-specific human monoclonal antibodies (mAbs) were used to assess the impact of toxin domain specificity on antibody mediated inhibition of cytotoxicity in a Vero cell-based functional assay. Results from toxin domain immunoabsorption assays using hamster anti-toxinsera indicated that no single domain fragment from either toxin A or toxin B could inhibit neutralizing activities. Anti-toxin A activity was prevented with a combination of GTD and CTD fragments while anti-toxin B activity required the GTD, CTD and CPD fragments to block activity. Assays with human mAbs demonstrated that combining mAbs that target multiple toxin domains greatly improves neutralizing potency when compared to equivalent concentrations of either a single mAb or a combination of mAbs against a single domain.

The anti-proliferative effect of Bcl-2 acts mainly at the level of the G0/G1 phase of the cell cycle. Deletions and point mutations in the bcl-2 gene show that the anti-proliferative activity of Bcl-2, can in some cases, be dissociated from its anti-apoptotic function. This indicates that the effect of Bcl-2 on cell cycle progression can be a direct effect and not only a consequence of its anti-apoptotic activity. Bcl-2 appears to mediate its anti-proliferative effect by acting on both signal transduction pathways (NFAT, ERK) and on specific cell cycle regulators (p27, p130).

We analyzed regulation of the prosurvival Bcl-2 homologue A1, following T-cell receptor (TCR) or cytokine receptor engagement. Activation of CD4(+) or CD8(+) T cells by antigenic peptides induced an early but transient IL-2-independent expression of A1 and Bcl-xl mRNA and proteins, whereas expression of Bcl-2 was delayed and required IL-2. Cytokines such as IL-2, IL-4, IL-7 or IL-15 prevented apoptosis of activated T cells that effect being associated with the maintenance of Bcl-2, but not of A1 expression. However, restimulation of activated or posteffector T cells with antigenic peptide strongly upregulated A1 mRNA and maintained A1 protein expression. IL-4, IL-7 or IL-15 also prevented cell death of naive T cells. In those cells, cytokines upregulated Bcl-2, but not A1 expression. Therefore, in naive, activated and posteffector T cells, expression of A1 is dependent on TCR but not on cytokine receptor engagement, indicating that A1 is differently regulated from Bcl-xl and Bcl-2.

Exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane is a key feature of apoptosis. As the signals underlying these phenomena are unknown, it is generally assumed that PS exposure is a consequence of caspase activation, another hallmark of apoptosis. In this study we investigated the role of caspases in PS externalization during apoptosis of activated PBL triggered by drugs (etoposide, staurosporine), CD95 engagement, or IL-2 withdrawal. Anti-CD95 mAb induces a rapid activation of caspases, followed by PS exposure and mitochondrial transmembrane potential (ΔΨm) disruption. In contrast, etoposide (ETO), staurosporine (STS), or IL-2 withdrawal triggers concomitant caspase activation, PS exposure, and ΔΨm disruption. Such kinetics suggest that PS exposure could be independent of caspase activation. As expected, in activated PBL treated by anti-CD95 mAb, the pan-caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethylketone and the caspase-8 inhibitor Cbz-Leu-Glu-Thr-Asp(OMe)-fluoromethylketone, but not the caspase-9 inhibitor Cbz-Leu-Glu-His-Asp(OMe)-fluoromethylketone, inhibit PS externalization and ΔΨm disruption. Surprisingly, during apoptosis induced by ETO, STS, or IL-2 withdrawal, none of those caspase inhibitors prevents PS externalization or ΔΨm disruption, whereas they all inhibit DNA fragmentation as well as the morphological features of nuclear apoptosis. In Jurkat and H9 T cell lines, as opposed to activated PBL, PS exposure is inhibited by Cbz-Val-Ala-Asp(OMe)-fluoromethylketone during apoptosis induced by CD95 engagement, ETO, or STS. Thus, caspase-independent PS exposure occurs in primary T cells during apoptosis induced by stimuli that do not trigger death receptors.

Mycophenolic acid (MPA), the active metabolite of the immunosuppressive drug mycophenolate mofetil, is a selective inhibitor of inosine 5′-monophosphate dehydrogenase type II, a de novo purine nucleotide synthesis enzyme expressed in T and B lymphocytes and up-regulated upon cell activation. In this study, we report that the blockade of guanosine nucleotide synthesis by MPA inhibits mitogen-induced proliferation of PBL, an effect fully reversed by addition of guanosine and shared with mizoribine, another inhibitor of inosine 5′-monophosphate dehydrogenase. Because MPA does not inhibit early TCR-mediated activation events, such as CD25 expression and IL-2 synthesis, we investigated how it interferes with cytokine-dependent proliferation and survival. In activated lymphoblasts that are dependent on IL-2 or IL-15 for their proliferation, MPA does not impair signaling events such as of the extracellular signal-regulated kinase 2 and Stat5 phosphorylation, but inhibits down-regulation of the cyclin-dependent kinase inhibitor p27Kip1. Therefore, in activated lymphoblasts, MPA specifically interferes with cytokine-dependent signals that control cell cycle and blocks activated T cells in the mid-G1 phase of the cell cycle. Although it blocks IL-2-mediated proliferation, MPA does not inhibit cell survival and Bcl-xL up-regulation by IL-2 or other cytokines whose receptors share the common γ-chain (CD132). Finally, MPA does not interfere with IL-2-dependent acquisition of susceptibility to CD95-mediated apoptosis and degradation of cellular FLIP. Therefore, MPA has unique functional properties not shared by other immunosuppressive drugs interfering with IL-2R signaling events such as rapamycin and CD25 mAbs.

The effect of etoposide and camptothecin, two topoisomerase inhibitors directed against topoisomerases II and I, respectively, was evaluated on human peripheral blood lymphocytes. Etoposide and camptothecin induced apoptosis of mitogen-activated but not resting CD4+ and CD8+ T lymphocytes. Cell sensitivity to these agents required G1 to S-phase transition of the cell cycle. Conversely, daunorubicin, an intercalating agent and topoisomerase II inhibitor, induced apoptosis of both resting and activated lymphocytes. Although etoposide and camptothecin induced CD95-ligand mRNA expression, drug-induced apoptosis of activated human lymphocytes was not inhibited by CD95 antagonists. Drug-induced cell death was also not inhibited by p55 TNFR-Ig fusion protein. Activation of the caspases cascade was suggested by the partial inhibitory effect of the tripeptide zVAD-fmk and documented by activation of caspase 3. Finally etoposide and camptothecin induced a rapid production of ceramide in activated but not resting peripheral blood lymphocytes, suggesting that ceramide might initiate the signaling apoptotic cascade in sensitive cells.

We recently demonstrated that the engagement of HLA class I alpha1 domain induced Fas-independent apoptosis in human T and B lymphocytes. We analyzed the signaling pathway involved in HLA class I-mediated apoptosis in comparison with Fas (APO-1, CD95)-dependent apoptosis. The mouse mAb90 or the rat YTH862 monoclonal antibodies which bind the human HLA class I alpha1 domain induced the production of ceramide which was blocked by addition of the phosphatidylcholine-dependent phospholipase C inhibitor, D609. Furthermore, HLA class I-mediated apoptosis involved at least two different caspases, an interleukin-1 converting enzyme-like protease and another protease inhibited by the CPP32-like protease inhibitor Ac-DEVD-CHO. Despite similarity between Fas and HLA class I signaling pathways, we failed to demonstrate any physical association between these two molecules. We also report that the pan-caspase inhibitory peptide zVAD-fmk, but not Ac-DEVD-CHO and Ac-YVAD-CHO, inhibited decrease of mitochondrial transmembrane potential and generation of ceramide induced by anti-HLA class I and anti-Fas monoclonal antibodies, whereas all three peptides efficiently inhibited apoptosis. Altogether these results suggest that signaling through Fas and HLA class I involve caspase(s), targeted by zVAD-fmk, which act upstream of ceramide generation and mitochondrial events, whereas interleukin-1 converting enzyme-like and CPP32-like proteases act downstream of the mitochondria.

T1 and T2 transitional B cells are precursors for marginal zone B cells (MZB), which surround splenic follicles. MZB are essential for marginal zone formation, are central to the innate immune response, and contribute to adaptive immunity. Differentiation, migration, and homing of MZB and their precursors remain to be fully understood. We show that SWAP-70, a RhoGTPase-interacting and F-actin-binding protein with functions in cell polarization, migration, and adhesion regulates MZB development and marginal zone formation. The percentage of MZB in spleen of Swap70(-/-) mice was reduced to about one-third of that found in WT mice. Swap70(-/-) T1 cells accumulated in integrin ligand(high) regions of the splenic red pulp and failed to efficiently develop into T2 cells. Adoptive transfer and mixed BM chimera experiments demonstrated this to be a B-cell intrinsic phenotype. T-cell-independent antibody production was not impaired, however, and thus suggests that this process does not require correct homing of MZB precursors. B-cell adhesion through α(L)β(2) and α(4)β(1) integrins was hyper-activated in vitro and on tissue sections, and S1P-stimulated chemokinesis of MZB was reduced in the absence of SWAP-70. Thus, SWAP-70 acts as a regulator of the adhesion process, particularly important for differentiation control of B-cell precursors and their contribution to splenic tissue formation.

Germinal centers (GCs) are lymphoid tissue structures central to the generation of long-lived, high-affinity, antibody-forming B cells. However, induction, maintenance, and regulation of GCs are not sufficiently understood. The F-actin–binding, Rac-interacting protein SWAP-70 is strongly expressed in activated B cells like those in B follicles. Recent work suggests that SWAP-70 is involved in B-cell activation, migration, and homing. Therefore, we investigated the role of SWAP-70 in the T-dependent immune response, in GC formation, and in differentiation into plasma and memory B cells. Compared with wt, sheep red blood cell (SRBC)–, or NP-KLH–immunized SWAP-70−/− mice have strongly reduced numbers of GCs and GC-specific B cells. However, SWAP-70−/− NP-specific B cells accumulate outside of the B follicles, and SWAP-70−/− mice show more plasma cells in the red pulp and in the bone marrow, and increased NP-specific Ig and antibody-forming B cells. Yet the memory response is impaired. Thus, SWAP-70 deficiency uncouples GC formation from T-dependent antibody and long-lived plasma cell production and causes extrafollicular generation of high-affinity plasma cells, but does not adequately support the memory response.

Nucleotide synthesis inhibitors are currently used in neoplastic diseases or as immunosuppressive agents for the prevention of acute rejection in organ transplantation and the treatment of autoimmune disorders. We have previously described that these inhibitors interfere with proliferation and survival of primary T cells in vitro. However, the precise effects of nucleotide restriction on effector and memory functions have not been elucidated. In this study, we investigated the impact of nucleotide synthesis inhibition on CD8 T cell differentiation by using TCR transgenic mice (F5) specific for the influenza virus nucleoprotein 68 peptide presented on the H-2Db molecule. Our results show that methotrexate and 5-fluorouracil prevent the acquisition of effector functions, such as IFN-γ, granzyme B expression, and cytotoxic function following antigenic stimulation of naive cells. Surprisingly, in the presence of mycophenolate mofetil, activated F5 cells are still able to produce granzyme B and to kill target cells but to a lesser extent compared with control. All three inhibitors interfere with the differentiation of naive cells into memory CD8 T cells. In contrast, the drugs are unable to inhibit the development of improved cytotoxic functions displayed by memory CD8 T cells.

Purine and pyrimidine nucleotides play critical roles in DNA and RNA synthesis as well as in membrane lipid biosynthesis and protein glycosylation. They are necessary for the development and survival of mature T lymphocytes. Activation of T lymphocytes is associated with an increase of purine and pyrimidine pools. However, the question of how purine vs pyrimidine nucleotides regulate proliferation, cell cycle, and survival of primary T lymphocytes following activation has not yet been specifically addressed. This was investigated in the present study by using well-known purine (mycophenolic acid, 6-mercaptopurine) and pyrimidine (methotrexate, 5-fluorouracil) inhibitors, which are used in neoplastic diseases or as immunosuppressive agents. The effect of these inhibitors was analyzed according to their time of addition with respect to the initiation of mitogenic activation. We showed that synthesis of both purine and pyrimidine nucleotides is required for T cell proliferation. However, purine and pyrimidine nucleotides differentially regulate the cell cycle since purines control both G1 to S phase transition and progression through the S phase, whereas pyrimidines only control progression from early to intermediate S phase. Furthermore, inhibition of pyrimidine synthesis induces apoptosis whatever the time of inhibitor addition whereas inhibition of purine nucleotides induces apoptosis only when applied to already cycling T cells, suggesting that both purine and pyrimidine nucleotides are required for survival of cells committed into S phase. These findings reveal a hitherto unknown role of purine and pyrimidine de novo synthesis in regulating cell cycle progression and maintaining survival of activated T lymphocytes.

The anthracyclines daunorubicin and doxorubicin were shown to induce apoptosis of hematopoietic cell lines. Here we report that they induce apoptosis of both nonactivated and phytohemagglutinin-activated human peripheral blood lymphocytes. Apoptosis demonstrated by surface expression of phosphatidylserine and typical nuclear alterations reached a maximum after 48 h of incubation with these agents. In contrast to topoisomerase inhibitors (etoposide and camptothecin) and antimetabolites (methotrexate and 5-fluorouracil) that induced apoptosis of activated cells only, daunorubicin and doxorubicin triggered apoptosis of cells in the G0-G1 phases of the cell cycle. In agreement with in vitro data, a single i.p. injection of daunorubicin or doxorubicin in BALB/c mice induced T- and B-cell depletion in spleen, lymph nodes, and to a lesser extent in the thymus. Soluble Fas-Fc, CD95 antagonistic antibodies, as well as the p55 tumor necrosis factor receptor-immunoglobulin fusion protein, did not inhibit drug-induced apoptosis. The level of reactive oxygen species was significantly increased in the presence of daunorubicin or doxorubicin only in nonactivated lymphocytes. However, antioxidants such as N-acetyl-L-cysteine or glutathione did not prevent apoptosis. Activation of caspase-3 after daunorubicin or doxorubicin treatment of either nonactivated or activated lymphocytes was demonstrated by the cleavage of poly(ADP-ribose) polymerase, which was, as apoptosis, inhibited by the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Finally, daunorubicin and doxorubicin induced a rapid production of ceramides. These data indicate that anthracyclines may induce major peripheral T-cell deletion, a property not shared by many cytotoxic agents.