Standardized Whole-Blood Transcriptional Profiling Enables the Deconvolution of Complex Induced Immune Responses

SummarySystems approaches for the study of immune signaling pathways have been traditionally based on purified cells or cultured lines. However, in vivo responses involve the coordinated action of multiple cell types, which interact to establish an inflammatory microenvironment. We employed standardized whole-blood stimulation systems to test the hypothesis that responses to Toll-like receptor ligands or whole microbes can be defined by the transcriptional signatures of key cytokines. We found 44 genes, identified using Support Vector Machine learning, that captured the diversity of complex innate immune responses with improved segregation between distinct stimuli. Furthermore, we used donor variability to identify shared inter-cellular pathways and trace cytokine loops involved in gene expression. This provides strategies for dimension reduction of large datasets and deconvolution of innate immune responses applicable for characterizing immunomodulatory molecules. Moreover, we provide an interactive R-Shiny application with healthy donor reference values for induced inflammatory genes

Role of ERM (ezrin-radixin-moesin) proteins in T lymphocyte polarization, immune synapse formation and in T cell receptor-mediated signaling.

International audienceFollowing antigen recognition, T lymphocytes undergo strong actin cytoskeletal rearrangements. These play a crucial role in the molecular reorganization at the contact site between the T lymphocyte and the antigen presenting cell, termed the immunological synapse. Moreover, they are necessary for T cell activation that leads to cytokine secretion, T cell proliferation and effector function. Little is known on how membrane and signaling molecules interact with the actin cytoskeleton during these processes. Here we review the function of the ERM family of membrane-microfilament linkers, making emphasis on the role of these proteins in T lymphocyte physiology. We discuss how ERM proteins are involved in membrane reorganization during T lymphocyte polarization and immune synapse formation, and how these proteins may contribute to T cell receptor-mediated intracellular signaling that leads to T cell activation

Vav proteins, masters of the world of cytoskeleton organization.

International audienceVav proteins are evolutionarily conserved from nematodes to mammals and play a pivotal role in many aspects of cellular signaling, coupling cell surface receptors to various effectors functions. In mammals, there are three family members; Vav1 is specifically expressed in the hematopoietic system, whereas Vav2 and Vav3 are more ubiquitously expressed. Vav proteins contain multiple domains that enable their function in various fashions. The participation of the Vav proteins in several processes that require cytoskeletal reorganization, such as the formation of the immunological synapse (IS), phagocytosis, platelet aggregation, spreading, and transformation will be discussed in this review. We will also cover how the Vav proteins succeed in controlling these processes by their function as guanine nucleotide exchange factors (GEFs) for the Rho/Rac family of GTPases. The contribution of the Vav proteins in a GEF-independent manner to the organization of the cytoskeleton will also be deliberated. The scope of this review is to highlight the numerous roles of the Vav signal transducer proteins in actin organization

Molecular Dynamics at the Immunological Synapse

The immunological synapse (IS) is a specialised cell-cell adhesion that mediates antigen acquisition and regulates the activation of lymphocytes. Initial studies of the IS showed a structure composed of stable supra-molecular activation clusters (SMAC) organised during the interaction of helper T lymphocytes with B lymphocytes, working as antigen presenting cells. A central SMAC of coalesced T cell receptors (TCRs) and a peripheral SMAC for cell-cell adhesion were observed. IS with similar structure was later described during antigen acquisition by B cells and during the interaction of NK cells with target and healthy cells. More recent research developed with microscopy systems that improve the spatial and temporal resolution has showed the complex molecular dynamics at the IS that governs lymphocyte activation. Currently, the IS is seen as a three-dimensional structure where signalling networks for lymphocyte activation and endosomal and cytoskeleton machinery are polarised. A view has emerged in which dynamic microclusters of signalling complexes are composed of molecular components attached to the plasma membrane and other components conveyed on sub-synaptic vesicles transported to the membrane by cytoskeletal fibers and motor proteins. Much information is nonetheless missing about how the dynamics of the endosomal compartment, the cytoskeleton, and signalling complexes are reciprocally regulated to achieve the function of lymphocytes. Experimental evidence also suggests that the environment surrounding lymphocytes exposed to different antigenic challenge regulates IS assembly and functional output, making an even more complex scenario still far from being completely understood. Also, although some signalling molecular components for lymphocyte activation have been identified and thoroughly studied, the function of other molecules has not been yet uncovered or deeply characterised. This research topic aims to provide the reader with the latest information about the molecular dynamics governing lymphocyte activation. These molecular dynamics dictate cell decisions. Thus, we expect that understanding them will provide new avenues for cell manipulation in therapies to treat different immune-related pathologies

The Staphylococcus aureus Enterotoxin B Superantigen Induces Specific T Cell Receptor Down-regulation by Increasing Its Internalization

Superantigens are able to stimulate T lymphocyte populations expressing T cell antigen receptors (TCR) belonging to particular V beta families. Moreover, the presence of these superantigens may induce long term unresponsiveness (anergy) of these sensitive cells. Some bacterial toxins are potent superantigens. We have analyzed in vitro the capacity of some Staphylococcus aureus enterotoxin superantigens to modulate T cell antigen receptor expression and the cellular mechanisms involved. Staphylococcus enterotoxin B (SEB) induced rapid down-regulation of surface T cell antigen receptors in V beta 3-expressing T lymphocytes, as assessed by flow cytometry. This phenomenon was a consequence of the direct interaction between the toxin and the TCR since it was observed in the absence of cells expressing major histocompatibility complex class II molecules. The cellular mechanism involved in SEB-induced down-regulation of TCR was further investigated. Immunofluorescence and confocal microscopy experiments showed that toxin B induced intracellular accumulation of TCR.CD3 in endocytic vesicles. Moreover, SEB induced an increase in T cell receptor endocytosis as measured using radiolabeled Fab fragments of an anti-CD3 monoclonal antibody. Taken together, our observations indicate that Staphylococcus enterotoxin B superantigen induced changes in the dynamics of surface T cell receptors, which resulted in the fast reduction of membrane receptor numbers

Analysis of hematopoietic lineage cells in the spleen of control and SLP76-S376A mice.

<p>Splenocytes isolated from wild type (open bars) or SLP76-S376A (filled bars) were stained for detection of main T and NK cell (<b>A</b>), B cell (<b>B</b>) or myeloid cell (<b>C</b>) sub-populations, then analyzed by flow cytometry. Histograms represent the mean frequencies of each population in total splenocytes (A,C) or within B cells (B, right panel). Error bars represent SD (n = 6). Plasm.: plasma cells, Ag-exp.: antigen-experienced B cells. Mem.: memory B cells; MZ: marginal zone B cells. Gr: granulocytes; Eos: eosinophils; Mono: monocytes; Mac: macrophages; cDCs: conventional dendritic cells; pDCs: plasmacytoid dendritic cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170396#pone.0170396.s004" target="_blank">S2 Table</a> for marker definition of each cell type.</p

Ser376 mutation impairs 14-3-3 binding to SLP76 and affects activation of several T cell signalling pathways.

<p><b>A.</b> Total lymph node cells from wild type (CTRL) or mutant (S376A) mice were isolated and either left unstimulated (medium) stimulated by anti-CD3 antibody crosslinking (CD3) or treated with 50 μM calyculin A (calyc.A) for 10 min at +37°C. Cells were then lysed and soluble protein extracts analysed by immunoblotting with the indicated antibodies. <b>B.</b> CD4⁺ T cells purified from lymph nodes of wild type and SLP76-S376A mice were left unstimulated (-) or stimulated by anti-CD3 plus anti-CD28 antibody crosslinking for the indicated time points. Cells were subsequently lysed and analysed by immunoblotting using the indicated antibody. Images were acquired with an Odyssey infrared scanner (LI-COR) and quantified. Data are representative of two independent experiments. <b>C.</b> Quantification of signaling protein phosphorylation. For each phosphoprotein, band intensities were quantified as described in B and normalized by the β-tubulin relative amount in the same lane. Normalized intensities were then divided by the mean normalized intensity of the same experiment. Each data point represents mean±SEM from three independent experiments. Statistical analysis was performed by two-way ANOVA. The p-value indicating the significance of the difference between the two curves (CTRL vs S376A) is indicated in each panel. A.u.: arbitrary units.</p

Cytokine secretion by <i>in vitro</i> differentiated and restimulated SLP76-S376A T cells.

<p>Naïve CD4+ T cells from control (empty bars) or SLP76-S376A mice (filled bars) were stimulated by anti-CD3 and anti-CD28 antibodies <i>in vitro</i> for 5 days in non-polarizing (Th0) or Th1 or Th2 polarizing conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170396#sec002" target="_blank">Materials and Methods</a>). Cells were then washed and restimulated with antibodies for 24 h. Secretion of IFNγ (top panel) and IL-4 (bottom) was assessed in culture supernatants by ELISA. Data represents mean+SEM from five independent experiments, each run at least in duplicate. Statistical significance was assessed by a Mann-Withney test.</p