NS1 Specific CD8(+) T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes

NFATc1 controls the cytotoxicity of CD8+ T cells

NFAT nuclear translocation has been shown to be required for CD8+ T cell cytokine production in response to viral infection. Here the authors show NFATc1 controls the cytotoxicity and metabolic switching of activated CD8+ T cells required for optimal response to bacteria and tumor cells

Efficient preparation of Arabidopsis pollen tubes for ultrastructural analysis using chemical and cryo-fixation

The pollen tube (PT) serves as a model system for investigating plant cell growth and morphogenesis. Ultrastructural studies are indispensable to complement data from physiological and genetic analyses, yet an effective method is lacking for PTs of the model plant Arabidopsis thaliana. Methods: Here, we present reliable approaches for ultrastructural studies of Arabidopsis PTs, as well as an efficient technique for immunogold detection of cell wall epitopes. Using different fixation and embedding strategies, we show the amount of PT ultrastructural details that can be obtained by the different methods. Results: Dozens of cross-sections can be obtained simultaneously by the approach, which facilitates and shortens the time for evaluation. In addition to in vitro-grown PTs, our study follows the route of PTs from germination, growth along the pistil, to the penetration of the dense stylar tissue, which requires considerable mechanical forces. To this end, PTs have different strategies from growing between cells but also between the protoplast and the cell wall and even within each other, where they share a partly common cell wall. The separation of PT cell walls in an outer and an inner layer reported for many plant species is less clear in Arabidopsis PTs, where these cell wall substructures are connected by a distinct transition zone. Conclusions: The major advancement of this method is the effective production of a large number of longitudinal and cross-sections that permits obtaining a detailed and representative picture of pollen tube structures in an unprecedented way. This is particularly important when comparing PTs of wild type and mutants to identify even subtle alterations in cytoarchitecture. Arabidopsis is an excellent plant for genetic manipulation, yet the PTs, several-times smaller compared to tobacco or lily, represent a technical challenge. This study reveals a method to overcome this problem and make Arabidopsis PTs more amenable to a combination of genetic and ultrastructural analyses

Methodological advances in imaging intravital axonal transport.

Axonal transport is the active process whereby neurons transport cargoes such as organelles and proteins anterogradely from the cell body to the axon terminal and retrogradely in the opposite direction. Bi-directional transport in axons is absolutely essential for the functioning and survival of neurons and appears to be negatively impacted by both aging and diseases of the nervous system, such as Alzheimer's disease and amyotrophic lateral sclerosis. The movement of individual cargoes along axons has been studied in vitro in live neurons and tissue explants for a number of years; however, it is currently unclear as to whether these systems faithfully and consistently replicate the in vivo situation. A number of intravital techniques originally developed for studying diverse biological events have recently been adapted to monitor axonal transport in real-time in a range of live organisms and are providing novel insight into this dynamic process. Here, we highlight these methodological advances in intravital imaging of axonal transport, outlining key strengths and limitations while discussing findings, possible improvements, and outstanding questions

Cytomegalovirus-based vaccine expressing Ebola virus glycoprotein protects nonhuman primates from Ebola virus infection.

Ebolaviruses pose significant public health problems due to their high lethality, unpredictable emergence, and localization to the poorest areas of the world. In addition to implementation of standard public health control procedures, a number of experimental human vaccines are being explored as a further means for outbreak control. Recombinant cytomegalovirus (CMV)-based vectors are a novel vaccine platform that have been shown to induce substantial levels of durable, but primarily T-cell-biased responses against the encoded heterologous target antigen. Herein, we demonstrate the ability of rhesus CMV (RhCMV) expressing Ebola virus (EBOV) glycoprotein (GP) to provide protective immunity to rhesus macaques against lethal EBOV challenge. Surprisingly, vaccination was associated with high levels of GP-specific antibodies, but with no detectable GP-directed cellular immunity

Systemic hematogenous maintenance of memory inflation by MCMV infection.

Several low-grade persistent viral infections induce and sustain very large numbers of virus-specific effector T cells. This was first described as a response to cytomegalovirus (CMV), a herpesvirus that establishes a life-long persistent/latent infection, and sustains the largest known effector T cell populations in healthy people. These T cells remain functional and traffic systemically, which has led to the recent exploration of CMV as a persistent vaccine vector. However, the maintenance of this remarkable response is not understood. Current models propose that reservoirs of viral antigen and/or latently infected cells in lymph nodes stimulate T cell proliferation and effector differentiation, followed by migration of progeny to non-lymphoid tissues where they control CMV reactivation. We tested this model using murine CMV (MCMV), a natural mouse pathogen and homologue of human CMV (HCMV). While T cells within draining lymph nodes divided at a higher rate than cells elsewhere, antigen-dependent proliferation of MCMV-specific effector T cells was observed systemically. Strikingly, inhibition of T cell egress from lymph nodes failed to eliminate systemic T cell division, and did not prevent the maintenance of the inflationary populations. In fact, we found that the vast majority of inflationary cells, including most cells undergoing antigen-driven division, had not migrated into the parenchyma of non-lymphoid tissues but were instead exposed to the blood supply. Indeed, the immunodominance and effector phenotype of inflationary cells, both of which are primary hallmarks of memory inflation, were largely confined to blood-localized T cells. Together these results support a new model of MCMV-driven memory inflation in which most immune surveillance occurs in circulation, and in which most inflationary effector T cells are produced in response to viral antigen presented by cells that are accessible to the blood supply

Regulation of immunity during visceral Leishmania infection

Unicellular eukaryotes of the genus Leishmania are collectively responsible for a heterogeneous group of diseases known as leishmaniasis. The visceral form of leishmaniasis, caused by L. donovani or L. infantum, is a devastating condition, claiming 20,000 to 40,000 lives annually, with particular incidence in some of the poorest regions of the world. Immunity to Leishmania depends on the development of protective type I immune responses capable of activating infected phagocytes to kill intracellular amastigotes. However, despite the induction of protective responses, disease progresses due to a multitude of factors that impede an optimal response. These include the action of suppressive cytokines, exhaustion of specific T cells, loss of lymphoid tissue architecture and a defective humoral response. We will review how these responses are orchestrated during the course of infection, including both early and chronic stages, focusing on the spleen and the liver, which are the main target organs of visceral Leishmania in the host. A comprehensive understanding of the immune events that occur during visceral Leishmania infection is crucial for the implementation of immunotherapeutic approaches that complement the current anti-Leishmania chemotherapy and the development of effective vaccines to prevent disease.The research leading to these results has received funding from the European Community’s Seventh Framework Programme under grant agreement No.602773 (Project KINDRED). VR is supported by a post-doctoral fellowship granted by the KINDReD consortium. RS thanks the Foundation for Science and Technology (FCT) for an Investigator Grant (IF/00021/2014). This work was supported by grants to JE from ANR (LEISH-APO, France), Partenariat Hubert Curien (PHC) (program Volubilis, MA/11/262). JE acknowledges the support of the Canada Research Chair Program

Imaging Trans-Cellular Neurexin-Neuroligin Interactions by Enzymatic Probe Ligation

Neurexin and neuroligin are transmembrane adhesion proteins that play an important role in organizing the neuronal synaptic cleft. Our lab previously reported a method for imaging the trans-synaptic binding of neurexin and neuroligin called BLINC (Biotin Labeling of INtercellular Contacts). In BLINC, biotin ligase (BirA) is fused to one protein while its 15-amino acid acceptor peptide substrate (AP) is fused to the binding partner. When the two fusion proteins interact across cellular junctions, BirA catalyzes the site-specific biotinylation of AP, which can be read out by staining with streptavidin-fluorophore conjugates. Here, we report that BLINC in neurons cannot be reproduced using the reporter constructs and labeling protocol previously described. We uncover the technical reasons for the lack of reproducibilty and then re-design the BLINC reporters and labeling protocol to achieve neurexin-neuroligin BLINC imaging in neuron cultures. In addition, we introduce a new method, based on lipoic acid ligase instead of biotin ligase, to image trans-cellular neurexin-neuroligin interactions in human embryonic kidney cells and in neuron cultures. This method, called ID-PRIME for Interaction-Dependent PRobe Incorporation Mediated by Enzymes, is more robust than BLINC due to higher surface expression of lipoic acid ligase fusion constructs, gives stronger and more localized labeling, and is more versatile than BLINC in terms of signal readout. ID-PRIME expands the toolkit of methods available to study trans-cellular protein-protein interactions in living systems.National Institutes of Health (U.S.) (DP1 OD003961

Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis

Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non–cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte–mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity

Epigenetic and transcriptional signatures of stable versus plastic differentiation of proinflammatory gd T cell subsets

Two distinct subsets of γδ T cells that produce interleukin 17 (IL-17) (CD27(-) γδ T cells) or interferon-γ (IFN-γ) (CD27(+) γδ T cells) develop in the mouse thymus, but the molecular determinants of their functional potential in the periphery remain unknown. Here we conducted a genome-wide characterization of the methylation patterns of histone H3, along with analysis of mRNA encoding transcription factors, to identify the regulatory networks of peripheral IFN-γ-producing or IL-17-producing γδ T cell subsets in vivo. We found that CD27(+) γδ T cells were committed to the expression of Ifng but not Il17, whereas CD27(-) γδ T cells displayed permissive chromatin configurations at loci encoding both cytokines and their regulatory transcription factors and differentiated into cells that produced both IL-17 and IFN-γ in a tumor microenvironment