Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation

Serine/threonine phosphorylation of the T cell adaptor proteins SLP76 and GADS by HPK1 induces their release from signaling microclusters and subsequent termination of the T cell response

High Incidence of Scrapie Induced by Repeated Injections of Subinfectious Prion Doses

To clarify the mechanisms leading to the development of Creutzfeldt-Jakob disease in some recipients of pituitary-derived human growth hormone (hGH), we investigated the effects of repeated injections of low prion doses in mice. The injections were performed, as in hGH-treated children, by a peripheral route at short intervals and for an extended period. Twelve groups of 24 mice were intraperitoneally inoculated one, two, or five times per week for 200 days with 2 × 10(−5) to 2 × 10(−8) dilutions of brain homogenate containing the mouse-adapted C506M3 scrapie strain. Sixteen control mice were injected once a week for 200 days with a 2 × 10(−4) dilution of normal brain homogenate. Of mice injected in a single challenge with a scrapie inoculum of a 2 × 10(−4), 2 × 10(−5), or 2 × 10(−6) dilution, 2/10, 1/10, and 0/10 animals developed scrapie, respectively. Control mice remained healthy. One hundred thirty-five of 135 mice injected with repeated prion doses of a 2 × 10(−5) or 2 × 10(−6) dilution succumbed to scrapie. Of mice injected with repeated scrapie doses of a 2 × 10(−7) or 2 × 10(−8) dilution, 52/59 and 38/67 animals died of scrapie, respectively. A high incidence of scrapie was observed in mice receiving repeated doses at low infectivity, whereas there was no disease in mice that were injected once with the same doses. Repeated injections of low prion doses thus constitute a risk for development of prion disease even if the same total dose inoculated in a single challenge does not induce the disease

Chapter 1 - Imaging polarized granule release at the cytotoxic T cell immunological synapse using TIRF microscopy: Control by polarity regulators

International audienceImmunological synapse formation results from a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, and the intracellular traffic of several vesicular organelles. T cell polarization is key for both T cell activation lead- ing to T cell proliferation and differentiation, and for T cell effector functions such as polarized secretion of cytokines by helper T cells, or polarized delivery of lytic granules by cytotoxic T cells. Efficient targeting of lytic granules by cytotoxic T cells is a crucial event for the con- trol and elimination of infected or tumor cells. Understanding how lytic granule delivery is regulated and quantifying its efficiency under physiological and pathological conditions may help to improve immune responses against infection and cancer

Chapter 1 - Imaging polarized granule release at the cytotoxic T cell immunological synapse using TIRF microscopy: Control by polarity regulators

International audienceImmunological synapse formation results from a profound T cell polarization process that involves the coordinated action of the actin and microtubule cytoskeleton, and the intracellular traffic of several vesicular organelles. T cell polarization is key for both T cell activation lead- ing to T cell proliferation and differentiation, and for T cell effector functions such as polarized secretion of cytokines by helper T cells, or polarized delivery of lytic granules by cytotoxic T cells. Efficient targeting of lytic granules by cytotoxic T cells is a crucial event for the con- trol and elimination of infected or tumor cells. Understanding how lytic granule delivery is regulated and quantifying its efficiency under physiological and pathological conditions may help to improve immune responses against infection and cancer

Adenomatous Polyposis Coli Modulates Actin and Microtubule Cytoskeleton at the Immunological Synapse to Tune CTL Functions

International audienceAdenomatous polyposis coli (Apc) is a cell polarity regulator and a tumor suppressor associated with familial adenomatous polyposis and colorectal cancer. Apc involvement in T lymphocyte functions and antitumor immunity remains poorly understood. Investigating Apc-depleted human CD8 T cells and CD8 T cells from ApcMin/+ mutant mice, we found that Apc regulates actin and microtubule cytoskeleton remodeling at the immunological synapse, controlling synapse morphology and stability and lytic granule dynamics, including targeting and fusion at the synapse. Ultimately, Apc tunes cytotoxic T cell activity, leading to tumor cell killing. Furthermore, Apc modulates early TCR signaling and nuclear translocation of the NFAT transcription factor with mild consequences on the expression of some differentiation markers. In contrast, no differences in the production of effector cytokines were observed. These results, together with our previous findings on Apc function in regulatory T cells, indicate that Apc mutations may cause a dual damage, first unbalancing epithelial cell differentiation and growth driving epithelial neoplasms and, second, impairing T cell-mediated antitumor immunity at several levels

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

Thymocytes subpopulation frequencies in control or SLP76-S376A knock-in mice.

<p>Data represent percentage of the indicated subpopulation (average ± SD; n = 8).</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

HIV-1 Nef Hijacks Lck and Rac1 Endosomal Traffic To Dually Modulate Signaling-Mediated and Actin Cytoskeleton–Mediated T Cell Functions

International audienceEndosomal traffic of TCR and signaling molecules regulates immunological synapse formation and T cell activation. We recently showed that Rab11 endosomes regulate the subcellular localization of the tyrosine kinase Lck and of the GTPase Rac1 and control their functions in TCR signaling and actin cytoskeleton remodeling. HIV-1 infection of T cells alters their endosomal traffic, activation capacity, and actin cytoskeleton organization. The viral protein Nef is pivotal for these modifications. We hypothesized that HIV-1 Nef could jointly alter Lck and Rac1 endosomal traffic and concomitantly modulate their functions. In this study, we show that HIV-1 infection of human T cells sequesters both Lck and Rac1 in a pericentrosomal compartment in an Nef-dependent manner. Strikingly, the Nef-induced Lck compartment contains signaling-competent forms (phosphorylated on key Tyr residues) of Lck and some of its downstream effectors, TCRζ, ZAP70, SLP76, and Vav1, avoiding the proximal LAT adaptor. Importantly, Nef-induced concentration of signaling molecules was concomitant with the upregulation of several early and late T cell activation genes. Moreover, preventing the concentration of the Nef-induced Lck compartment by depleting the Rab11 effector FIP3 counteracted Nef-induced gene expression upregulation. In addition, Nef extensively sequesters Rac1 and downregulates Rac1-dependent actin cytoskeleton remodeling, thus reducing T cell spreading. Therefore, by modifying their endosomal traffic, Nef hijacks signaling and actin cytoskeleton regulators to dually modulate their functional outputs. Our data shed new light into the molecular mechanisms that modify T cell physiology during HIV-1 infection

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