On the Acquisition of an Indefinite Determiner: Evidence for Unselective Binding

This paper investigates the contrast between quantificational and referential properties of the English indefinite determiner during the course of first language acquisition. · Various syntactic, semantic and pragmatic knowledge related to indefinites has previously been probed in the acquisition literature. (See Brown 1973, Maratsos 1976, Warden 1976, Emslie and Stevenson 1980, Zehler and Brewer 1982, Pine and Martindale 1986, Valian 1986, Gerken, Landau and Remez 1990, Philip 1995, Bohnacker 1997, and Burns and Soja 1997, among others.) However, to date, the contrast between referential-type and quantificational/variable-type interpretations of indefinites has not been extensively studied in first language acquisition. In this paper, we investigate the acquisition of knowledge of the indefinite determiner in a context that allows both of these alternative interpretations: the context of VP ellipsis

A Systematic Review of the Effectiveness of the Clubhouse Model

International audienceOBJECTIVE: The aim of the article is to synthesize studies that investigate the effectiveness of clubhouses, to summarize the strength of the evidence for this model, and to discuss methodological issues in the research. METHODS: We collected 216 studies referencing clubhouses in the principal international scientific databases (PsycINFO, Psycarticles, Academic Search Premier, Medline, PubMed, and Science Direct). We then selected 77 studies that used experimental (randomized controlled trial) or quasi-experimental designs (with control group, without randomization and/or pre-post studies). As part of the 77 selected studies, we focused on 15 studies that specifically addressed the effectiveness of clubhouses. RESULTS: There are few studies on the effectiveness of clubhouses, and the existing studies, because of their methodological design, allow us to affirm only moderate (quality of life, employment, rate of hospitalization) or low (symptomatology, social functioning) levels of evidence for the effectiveness of clubhouses. Most studies are limited by a lack of randomization, absence of a comparison group, or absence of a longitudinal design. Furthermore, the diversity of methodologies used makes a comparison of the results difficult. CONCLUSIONS AND IMPLICATIONS FOR PRACTICE: We offer several recommendations for future research to build the evidence base regarding this model and enhance comparability of studies. (PsycINFO Database Recor

BTLA inhibition has a dominant role in the cis-complex of BTLA and HVEM

The engagement of the herpesvirus entry mediator (HVEM, TNFRSF14) by the B and T lymphocyte attenuator (BTLA) represents a unique interaction between an activating receptor of the TNFR-superfamily and an inhibitory receptor of the Ig-superfamily. BTLA and HVEM have both been implicated in the regulation of human T cell responses, but their role is complex and incompletely understood. Here, we have used T cell reporter systems to dissect the complex interplay of HVEM with BTLA and its additional ligands LIGHT and CD160. Co-expression with LIGHT or CD160, but not with BTLA, induced strong constitutive signaling via HVEM. In line with earlier reports, we observed that in cis interaction of BTLA and HVEM prevented HVEM co-stimulation by ligands on surrounding cells. Intriguingly, our data indicate that BTLA mediated inhibition is not impaired in this heterodimeric complex, suggesting a dominant role of BTLA co-inhibition. Stimulation of primary human T cells in presence of HVEM ligands indicated a weak costimulatory capacity of HVEM potentially owed to its in cis engagement by BTLA. Furthermore, experiments with T cell reporter cells and primary T cells demonstrate that HVEM antibodies can augment T cell responses by concomitantly acting as checkpoint inhibitors and co-stimulation agonists

Scientific Reports / Generation of a Jurkat-based fluorescent reporter cell line to evaluate lipid antigen interaction with the human iNKT cell receptor

Invariant natural killer T (iNKT) cells are a specialized subset of T cells contributing to both, the innate and adaptive immune responses. In contrast to conventional T lymphocytes they recognize lipid antigens. The aim of the project is to establish a novel model system, to study iNKT-TCR ligand interaction. An iNKT reporter cell line (JE6-1REP-iNKT) was engineered by introducing the human iNKT-TCR into a human leukemic T cell line carrying an NF-B-driven fluorescent transcriptional reporter construct. Antigen presenting BWSTIM cells expressing human CD1d and CD80 were generated. Reporter induction in JE6-1REP-iNKT cells was assessed by flow cytometry. CRISPR/Cas9 was used for 2M knock out in JE6-1REP-iNKT cells to abrogate CD1d expression and thus excluding antigen self-presentation. Reporter cells were shown to specifically react with iNKT antigens presented via CD1d. Their sensitivity towards -GalCer was comparable to a murine iNKT hybridoma cell line. In conclusion, we created a novel iNKT reporter platform which, compared to traditional iNKT cell assays, is characterized by a shorter turnaround time and lower costs. It thus facilitates the identification of antigenic structures that drive the activation of iNKT cells in health and disease.(VLID)492684

TLR4/CD14/MD2 Revealed as the Limited Toll-like Receptor Complex for <i>Chlamydia trachomatis</i>-Induced NF-κB Signaling

Chlamydia trachomatis (Ct) is the most common cause of genital tract infections as well as preventable blindness worldwide. Pattern recognition receptors such as toll-like receptors (TLRs) represent the initial step in recognizing pathogenic microorganisms and are crucial for the initiation of an appropriate immune response. However, our understanding of TLR-signaling in Chlamydia-infected immune cells is incomplete. For a better comprehension of pathological inflammatory responses, robust models for interrogating TLR-signaling upon chlamydial infections are needed. To analyze the TLR response, we developed and utilized a highly sensitive and selective fluorescent transcriptional cellular reporter system to measure the activity of the transcription factor NF-κB. Upon incubation of the reporter cells with different preparations of Ct, we were able to pinpoint which components of TLRs are involved in the recognition of Ct. We identified CD14 associated with unique characteristics of different serovars as the crucial factor of the TLR4/CD14/MD2 complex for Ct-mediated activation of the NF-κB pathway. Furthermore, we found the TLR4/CD14/MD2 complex to be decisive for the uptake of Ct-derived lipopolysaccharides but not for infection and replication of Ct. Imaging flow cytometry provided information about inclusion formation in myeloid- as well as lymphocytic cells and was highest for Ct L2 with at least 25% of inclusion forming cells. Ct E inclusion formation was eminent in Jurkat cells without CD14 expression (11.1%). Thus, our model enables to determine Ct uptake and signal induction by pinpointing individual components of the recognition and signaling pathways to better understand the immune response towards infectious pathogens

PD-1 Blockade Promotes Emerging Checkpoint Inhibitors in Enhancing T Cell Responses to Allogeneic Dendritic Cells

Immune checkpoint inhibitors, which target coinhibitory T cell molecules to promote anticancer immune responses, are on the rise to become a new pillar of cancer therapy. However, current immune checkpoint-based therapies are successful only in a subset of patients and acquired resistances pose additional challenges. Finding new targets and combining checkpoint inhibitors might help to overcome these limitations. In this study, human T cells stimulated with allogeneic dendritic cells (DCs) were used to compare immune checkpoint inhibitors targeting TIM-3, BTLA, LAG-3, CTLA-4, and TIGIT alone or in combination with a PD-1 antibody. We found that PD-1 blockade bears a unique potency to enhance T cell proliferation and cytokine production. Other checkpoint inhibitors failed to significantly augment T cell responses when used alone. However, antibodies to TIM-3, BTLA, LAG-3, and CTLA-4 enhanced T cell proliferation in presence of a PD-1 antibody. Upregulation of coinhibitory T cell receptors upon PD-1 blockade was identified as a potential mechanism for synergistic effects between checkpoint inhibitors. Donor-specific variation in response to immune checkpoint inhibitors was attributed to the T cells rather than DCs. Additionally, we analyzed the regulation of checkpoint molecules and their ligands on T cells and allogeneic DCs in coculture, which suggested a PD-1 blockade-dependent crosstalk between T cells and APC. Our results indicate that several immune checkpoint inhibitors have the capacity to enhance T cell responses when combined with PD-1 blockade. Additional in vitro studies on human T cells will be useful to identify antibody combinations with the potential to augment T cell responses in cancer patients

A human monocytic NF-κB fluorescent reporter cell line for detection of microbial contaminants in biological samples

<div><p>Sensing of pathogens by innate immune cells is essential for the initiation of appropriate immune responses. Toll-like receptors (TLRs), which are highly sensitive for various structurally and evolutionary conserved molecules derived from microbes have a prominent role in this process. TLR engagement results in the activation of the transcription factor NF-κB, which induces the expression of cytokines and other inflammatory mediators. The exquisite sensitivity of TLR signalling can be exploited for the detection of bacteria and microbial contaminants in tissue cultures and in protein preparations. Here we describe a cellular reporter system for the detection of TLR ligands in biological samples. The well-characterized human monocytic THP-1 cell line was chosen as host for an NF-ᴋB-inducible enhanced green fluorescent protein reporter gene. We studied the sensitivity of the resultant reporter cells for a variety of microbial components and observed a strong reactivity towards TLR1/2 and TLR2/6 ligands. Mycoplasma lipoproteins are potent TLR2/6 agonists and we demonstrate that our reporter cells can be used as reliable and robust detection system for mycoplasma contaminations in cell cultures. In addition, a TLR4-sensitive subline of our reporters was engineered, and probed with recombinant proteins expressed in different host systems. Bacterially expressed but not mammalian expressed proteins induced strong reporter activity. We also tested proteins expressed in an <i>E</i>. <i>coli</i> strain engineered to lack TLR4 agonists. Such preparations also induced reporter activation in THP-1 cells highlighting the importance of testing recombinant protein preparations for microbial contaminations beyond endotoxins. Our results demonstrate the usefulness of monocytic reporter cells for high-throughput screening for microbial contaminations in diverse biological samples, including tissue culture supernatants and recombinant protein preparations. Fluorescent reporter assays can be measured on standard flow cytometers and in contrast to established detection methods, like luciferase-based systems or Limulus Amebocyte Lysate tests, they do not require costly reagents.</p></div

BTLA-derived peptides as inhibitors of BTLA/HVEM complex formation – design, synthesis and biological evaluation

Immune checkpoints can be divided into co-stimulatory and co-inhibitory molecules that regulate the activation and effector functions of T cells. The co-inhibitory pathways mediated by ICPs are used by cancer cells to escape from immune surveillance, and therefore the blockade of these receptor/ligand interactions is one of the strategies used in the treatment of cancer. The two main pathways currently under investigation are CTLA-4/CD80/CD86 and PD-1/PD-L1, and the monoclonal Abs targeting them have shown potent immunomodulatory effects and activity in clinical environments. Another interesting target in cancer treatment is the BTLA/HVEM complex. Binding of BTLA protein on T cells to HVEM on cancer cells leads to inhibition of T cell proliferation and cytokine production. In the presented work, we focused on blocking the HVEM protein using BTLA-derived peptides. Based on the crystal structure of the BTLA/HVEM complex and MM/GBSA analysis performed here, we designed and synthesized peptides, specifically fragments of BTLA protein. We subsequently checked the inhibitory capacities of these compounds using ELISA and a cellular reporter platform. Two of these peptides, namely BTLA(35−43) and BTLA(33−64)C58Abu displayed the most promising properties, and we therefore performed further studies to evaluate their affinity to HVEM protein, their stability in plasma and their effect on viability of human PBMCs. In addition, the 3D structure for the peptide BTLA(33−64)C58Abu was determined using NMR. Obtained data confirmed that the BTLA-derived peptides could be the basis for future drugs and their immunomodulatory potential merits further examination

THP-1 NF-κB-eGFP reporter cells show a selective sensitivity towards TLR ligands.

<p>(A) THP-1 NF-κB-eGFP cells were incubated with Pam3CSK4 (TLR1/2; 100 nM), FSL-1 (TLR2/6; 100 nM), MALP-2 (TLR2/6; 46,8 nM), Poly I:C (TLR3; 10 μg/ml), standard LPS (TLR2/4; 300 ng/ml), LPS ultrapure (UP) (TLR4; 300ng/ml), flagellin (TLR5; 100 ng/ml), imidazoquinoline (TLR7/8; 10 μg/ml) and CpG ODN 2006 (TLR9; 50 μg/ml). After 24 h, eGFP expression was assessed by flow cytometry. Bar graphs show geometric mean of fluorescence intensity (gMFI). Mean and SE were calculated from triplicates of five independently performed experiments (n = 5). (B) Representative flow cytometry histograms of reporter gene expression in THP-1 NF-κB-eGFP cells as described in A. Open histograms: TLR-activated reporter cells; filled histograms: unstimulated reporter cells. Numbers show gMFI. (C) Fluorescent microscopy images of reporter cells activated with standard LPS (3 μg/ml) for 24 h (right panel). Unstimulated cells served as negative control (left panel). Bright field images are shown for comparison (top row). Scale bar: 10 μm. (D) Immature human monocyte-derived DCs were incubated with various TLR ligands (used at the same concentrations as in A) for 24 h or were left untreated. Expression of maturation markers CD83 and CD86 was assessed by flow cytometry. Bar graphs show total percentage of gMFI normalized to standard LPS.</p

Frontiers in Immunology / Neuropilin-1 Acts as a Receptor for Complement Split Products

Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a KD value of 0.71 M. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.(VLID)490490