Modulation of the Major Histocompatibility Complex Class II–Associated Peptide Repertoire by Human Histocompatibility Leukocyte Antigen (Hla)-Do

Antigen presentation by major histocompatibility complex class II molecules is essential for antibody production and T cell activation. For most class II alleles, peptide binding depends on the catalytic action of human histocompatibility leukocyte antigens (HLA)-DM. HLA-DO is selectively expressed in B cells and impedes the activity of DM, yet its physiological role remains unclear. Cell surface iodination assays and mass spectrometry of major histocompatibility complex class II–eluted peptides show that DO affects the antigenic peptide repertoire of class II. DO generates both quantitative and qualitative differences, and inhibits presentation of large-sized peptides. DO function was investigated under various pH conditions in in vitro peptide exchange assays and in antigen presentation assays using DO− and DO+ transfectant cell lines as antigen-presenting cells, in which effective acidification of the endocytic pathway was prevented with bafilomycin A1, an inhibitor of vacuolar ATPases. DO effectively inhibits antigen presentation of peptides that are loaded onto class II in endosomal compartments that are not very acidic. Thus, DO appears to be a unique, cell type–specific modulator mastering the class II–mediated immune response induced by B cells. DO may serve to increase the threshold for nonspecific B cell activation, restricting class II–peptide binding to late endosomal compartments, thereby affecting the peptide repertoire

The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity

SummaryFOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity

A Critical Phenylalanine Residue in the Respiratory Syncytial Virus Fusion Protein Cytoplasmic Tail Mediates Assembly of Internal Viral Proteins into Viral Filaments and Particles

Respiratory syncytial virus (RSV) is a single-stranded RNA virus in the Paramyxoviridae family that assembles into filamentous structures at the apical surface of polarized epithelial cells. These filaments contain viral genomic RNA and structural proteins, including the fusion (F) protein, matrix (M) protein, nucleoprotein (N), and phosphoprotein (P), while excluding F-actin. It is known that the F protein cytoplasmic tail (FCT) is necessary for filament formation, but the mechanism by which the FCT mediates assembly into filaments is not clear. We hypothesized that the FCT is necessary for interactions with other viral proteins in order to form filaments. In order to test this idea, we expressed the F protein with cytoplasmic tail (CT) truncations or specific point mutations and determined the abilities of these variant F proteins to form filaments independent of viral infection when coexpressed with M, N, and P. Deletion of the terminal three FCT residues (amino acids Phe-Ser-Asn) or mutation of the Phe residue resulted in a loss of filament formation but did not affect F-protein expression or trafficking to the cell surface. Filament formation could be restored by addition of residues Phe-Ser-Asn to an FCT deletion mutant and was unaffected by mutations to Ser or Asn residues. Second, deletion of residues Phe-Ser-Asn or mutation of the Phe residue resulted in a loss of M, N, and P incorporation into virus-like particles. These data suggest that a C-terminal Phe residue in the FCT mediates assembly through incorporation of internal virion proteins into virus filaments at the cell surface

CTCF binds to sites in the major histocompatibility complex that are rapidly reconfigured in response to interferon-gamma

Activation of the major histocompatibility complex (MHC) by interferon-gamma (IFN−γ) is a fundamental step in the adaptive immune response to pathogens. Here, we show that reorganization of chromatin loop domains in the MHC is evident within the first 30 min of IFN−γ treatment of fibroblasts, and that further dynamic alterations occur up to 6 h. These very rapid changes occur at genomic sites which are occupied by CTCF and are close to IFN−γ-inducible MHC genes. Early responses to IFN−γ are thus initiated independently of CIITA, the master regulator of MHC class II genes and prepare the MHC for subsequent induction of transcription

Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages

Signal integration between activating Fc receptors and inhibitory signal regulatory protein α (SIRPα) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fcγ receptor I (FcγRI), FcγRII, and SIRPα are not homogeneously distributed at macrophage surfaces but are organized in discrete nanoclusters, with a mean radius of 71 ± 11 nm, 60 ± 6 nm, and 48 ± 3 nm, respectively. Nanoclusters of FcγRI, but not FcγRII, are constitutively associated with nanoclusters of SIRPα, within 62 ± 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of FcγRI and SIRPα nanoclusters to be 197 ± 3 nm apart. Co-ligation of SIRPα with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, FcγRI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometer- and micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration

Dynamics of filamentous viral RNPs prior to egress

The final step in the maturation of paramyxoviruses, orthomyxoviruses and viruses of several other families, entails the budding of the viral nucleocapsid through the plasma membrane of the host cell. Many medically important viruses, such as influenza, parainfluenza, respiratory syncytial virus (RSV) and Ebola, can form filamentous particles when budding. Although filamentous virions have been previously studied, details of how viral filaments bud from the plasma membrane remain largely unknown. Using molecular beacon (MB)-fluorescent probes to image the viral genomic RNA (vRNA) of human RSV (hRSV) in live Vero cells, the dynamics of assembled viral filaments was observed to consist of three primary types of motion prior to egress from the plasma membrane: (i) filament projection and rotation, (ii) migration and (iii) non-directed motion. In addition, from information gained by imaging the 3D distribution of cellular vRNA, observing and characterizing vRNA dynamics, imaging vRNA/Myosin Va colocalization, and studying the effects of cytochalasin D (actin depolymerizing agent) exposure, a model for filamentous virion egress is presented

Nanoclusters of the resting T cell antigen receptor (TCR) localize to non-raft domains

© 2014 Elsevier B.V. In the last decade an increasing number of plasma membrane (PM) proteins have been shown to be non-randomly distributed but instead forming submicron-sized oligomers called nanoclusters. Nanoclusters exist independently of the ligand-bound state of the receptors and their existence implies a high degree of lateral organisation of the PM and its proteins. The mechanisms that drive receptor nanoclustering are largely unknown. One well-defined example of a transmembrane receptor that forms nanoclusters is the T cell antigen receptor (TCR), a multisubunit protein complex whose nanoclustering influences its activity. Membrane lipids, namely cholesterol and sphingomyelin, have been shown to contribute to TCR nanoclustering. However, the identity of the membrane microdomain in which the TCR resides remains controversial. Using a GFP-labeled TCR we show here that the resting TCR localized in the disordered domain of giant PM vesicles (GPMVs) and PM spheres (PMSs) and that single and nanoclustered TCRs are found in the high-density fractions in sucrose gradients. Both findings are indicative of non-raft localization. We discuss possible mechanisms of TCR nanoclustering in T cells. This article is part of a Special Issue entitled: Nanoscale membrane organisation and signalling.German Research Foundation (GSC-4, the Spemann Graduate School and EXC294, the BIOSS Center for Biological Signalling Studies, by the German Research Foundation grant SCH 976/2-1, and by the European Union through grant FP7/2007-2013 SYBILLAPeer Reviewe

Induction of alternatively activated macrophages enhances pathogenesis during severe acute respiratory syndrome coronavirus infection

Infection with severe acute respiratory syndrome coronavirus (SARS-CoV) causes acute lung injury (ALI) that often leads to severe lung disease. A mouse model of acute SARS-CoV infection has been helpful in understanding the host response to infection; however, there are still unanswered questions concerning SARS-CoV pathogenesis. We have shown that STAT1 plays an important role in the severity of SARS-CoV pathogenesis and that it is independent of the role of STAT1 in interferon signaling. Mice lacking STAT1 have greater weight loss, severe lung pathology with pre-pulmonary-fibrosis-like lesions, and an altered immune response following infection with SARS-CoV. We hypothesized that STAT1 plays a role in the polarization of the immune response, specifically in macrophages, resulting in a worsened outcome. To test this, we created bone marrow chimeras and cell-type-specific knockouts of STAT1 to identify which cell type(s) is critical to protection from severe lung disease after SARS-CoV infection. Bone marrow chimera experiments demonstrated that hematopoietic cells are responsible for the pathogenesis in STAT1(−/−) mice, and because of an induction of alternatively activated (AA) macrophages after infection, we hypothesized that the AA macrophages were critical for disease severity. Mice with STAT1 in either monocytes and macrophages (LysM/STAT1) or ciliated lung epithelial cells (FoxJ1/STAT1) deleted were created. Following infection, LysM/STAT1 mice display severe lung pathology, while FoxJ1/STAT1 mice display normal lung pathology. We hypothesized that AA macrophages were responsible for this STAT1-dependent pathology and therefore created STAT1/STAT6(−/−) double-knockout mice. STAT6 is essential for the development of AA macrophages. Infection of the double-knockout mice displayed a lack of lung disease and prefibrotic lesions, suggesting that AA macrophage production may be the cause of STAT1-dependent lung disease. We propose that the control of AA macrophages by STAT1 is critical to regulating immune pathologies and for protection from long-term progression to fibrotic lung disease in a mouse model of SARS-CoV infection

Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells

Signaling from immunotyrosine-based inhibitory motifs (ITIMs) neutralizes activating signals by inducing a retraction of NK cells from the surface of stimulatory cells

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells