Etienne Gagnon

Phagosomes are competent organelles for antigen cross-presentation

The ability to process microbial antigens and present them at the surface of cells is an important aspect of our innate ability to clear infections. It is generally accepted that antigens in the cytoplasm are loaded in the endoplasmic reticulum and presented at the cell surface on major histocompatibility complex (MHC) class I molecules, whereas peptides present in endo/phagocytic compartments are presented on MHC class II molecules. Despite the apparent segregation of the class I and class II pathways, antigens from intracellular pathogens including mycobacteria, Escherichia coli, Salmonella typhimurium, Brucella abortus and Leishmania, have been shown to elicit an MHC class-I-dependent CD8+ T-cell response, a process referred to as cross-presentation. The cellular mechanisms allowing the cross-presentation pathway are poorly understood. Here we show that phagosomes display the elements and properties needed to be self-sufficient for the cross-presentation of exogenous antigens, a newly ascribed function linked to phagocytosis mediated by the endoplasmic reticulum.

Endoplasmic Reticulum-Mediated Phagocytosis Is a Mechanism of Entry into Macrophages

Phagocytosis is a key aspect of our innate ability to fight infectious diseases. In this study, we have found that fusion of the endoplasmic reticulum (ER) with the macrophage plasmalemma, underneath phagocytic cups, is a source of membrane for phagosome formation in macrophages. Successive waves of ER become associated with maturing phagosomes during phagolysosome biogenesis. Thus, the ER appears to possess unexpectedly pluripotent fusion properties. ER-mediated phagocytosis is regulated in part by phosphatidylinositol 3-kinase and used for the internalization of inert particles and intracellular pathogens, regardless of their final trafficking in the host. In neutrophils, where pathogens are rapidly killed, the ER is not used as a major source of membrane for phagocytosis. We propose that intracellular pathogens have evolved to adapt and exploit ER-mediated phagocytosis to avoid destruction in host cells.

Regulation of T Cell Receptor Activation by Dynamic Membrane Binding of the CD3ɛ Cytoplasmic Tyrosine-Based Motif

Many immune system receptors signal through cytoplasmic tyrosine-based motifs (ITAMs), but how receptor ligation results in ITAM phosphorylation remains unknown. Live-cell imaging studies showed a close interaction of the CD3epsilon cytoplasmic domain of the T cell receptor (TCR) with the plasma membrane through fluorescence resonance energy transfer between a C-terminal fluorescent protein and a membrane fluorophore. Electrostatic interactions between basic CD3epsilon residues and acidic phospholipids enriched in the inner leaflet of the plasma membrane were required for binding. The nuclear magnetic resonance structure of the lipid-bound state of this cytoplasmic domain revealed deep insertion of the two key tyrosines into the hydrophobic core of the lipid bilayer. Receptor ligation thus needs to result in unbinding of the CD3epsilon ITAM from the membrane to render these tyrosines accessible to Src kinases. Sequestration of key tyrosines into the lipid bilayer represents a previously unrecognized mechanism for control of receptor activation.

Structural Biology of the T-cell Receptor: Insights into Receptor Assembly, Ligand Recognition, and Initiation of Signaling

The T-cell receptor (TCR)-CD3 complex serves as a central paradigm for general principles of receptor assembly, ligand recognition, and signaling in the immune system. There is no other receptor system that matches the diversity of both receptor and ligand components. The recent expansion of the immunological structural database is beginning to identify key principles of MHC and peptide recognition. The multicomponent assembly of the TCR complex illustrates general principles used by many receptors in the immune system, which rely on basic and acidic transmembrane residues to guide assembly. The intrinsic binding of the cytoplasmic domains of the CD3epsilon and zeta chains to the inner leaflet of the plasma membrane represents a novel mechanism for control of receptor activation: Insertion of critical CD3epsilon tyrosines into the hydrophobic membrane core prevents their phosphorylation before receptor engagement.

Phagocytosis: the convoluted way from nutrition to adaptive immunity.

Summary:  Phagocytosis, the process by which cells internalize large particulate materials from their milieu and sequester them in phagosomes, plays a role in a variety of cell functions ranging from nutrition in ameba to innate and adaptive immunity in mammals. Recent findings revealed unexpected characteristics of phagosomes, highlighting how this complex organelle may have evolved, from Dictyostelium to human, to become a key player in our ability to mount an efficient immune response against a variety of intracellular pathogens.

Alternative RISC assembly: Binding and repression of microRNA–mRNA duplexes by human Ago proteins

MicroRNAs (miRNAs) are small noncoding RNAs that post-transcriptionally regulate protein output from the majority of human mRNAs. In contrast to the consensus view that all miRNAs are associated with Argonaute (Ago) proteins, we determine that miRNAs are expressed in a 13-fold excess relative to Agos in HeLa cells and that miRNAs are bound to mRNAs in a sevenfold excess relative to Agos, implying the existence of miRNA-mRNA duplexes not stoichiometrically bound by Agos. We show that all four human Agos can repress miRNA-mRNA duplexes, but only Ago2 can cleave small interfering RNA-mRNA duplexes in vitro. We visualize direct Ago binding to miRNA-mRNA duplexes in live cells using fluorescence lifetime imaging microscopy. In contrast to the consensus view that Agos bind miRNA duplexes, these data demonstrate that Agos can bind and repress miRNA-mRNA duplexes and support a model of catalytic Ago function in translational repression.

Local changes in lipid environment of TCR microclusters regulate membrane binding by the CD3ε cytoplasmic domain

The CD3ε and ζ cytoplasmic domains of the T cell receptor bind to the inner leaflet of the plasma membrane (PM), and a previous nuclear magnetic resonance structure showed that both tyrosines of the CD3ε immunoreceptor tyrosine-based activation motif partition into the bilayer. Electrostatic interactions between acidic phospholipids and clusters of basic CD3ε residues were previously shown to be essential for CD3ε and ζ membrane binding. Phosphatidylserine (PS) is the most abundant negatively charged lipid on the inner leaflet of the PM and makes a major contribution to membrane binding by the CD3ε cytoplasmic domain. Here, we show that TCR triggering by peptide–MHC complexes induces dissociation of the CD3ε cytoplasmic domain from the plasma membrane. Release of the CD3ε cytoplasmic domain from the membrane is accompanied by a substantial focal reduction in negative charge and available PS in TCR microclusters. These changes in the lipid composition of TCR microclusters even occur when TCR signaling is blocked with a Src kinase inhibitor. Local changes in the lipid composition of TCR microclusters thus render the CD3ε cytoplasmic domain accessible during early stages of T cell activation.

Self-reactive human CD4 T cell clones form unusual immunological synapses.

Compared with influenza-specific T cells, self-reactive T cells from patients with multiple sclerosis or type 1 diabetes fail to slow down and do not form normal immunological synapses upon encounter with cognate self-peptide presented by MHC.

Quantitative proteomics reveals that only a subset of the endoplasmic reticulum contributes to the phagosome

Phagosomes, by killing and degrading pathogens for antigen presentation, are organelles implicated in key aspects of innate and adaptive immunity. Although it has been well established that phagosomes consist of membranes from the plasma membrane, endosomes, and lysosomes, the notion that the endoplasmic reticulum (ER) membrane could play an important role in the formation of the phagosome is debated. However, a method to accurately estimate the contribution of potential source organelles and contaminants to the phagosome proteome has been lacking. Herein, we have developed a proteomic approach for objectively quantifying the contribution of various organelles to the early and late phagosomes by comparing these fractions to their total membrane and postnuclear supernatant of origin in the J774A.1 murine macrophage cell line. Using quantitative label-free mass spectrometry, the abundance of peptides corresponding to hundreds of proteins was estimated and attributed to one of five organelles (e.g. plasma membrane, endosomes/lysosomes, ER, Golgi, and mitochondria). These data in combination with a stable isotope labeling in cell culture method designed to detect potential contaminant sources revealed that the ER is part of the phagosomal membrane and contributes ∼20% of the early phagosome proteome. In addition, only a subset of ER proteins is recruited to the phagosome, suggesting that a specific subdomain(s) of the ER might be involved in phagocytosis. Western blotting and immunofluorescence substantially validated this conclusion; we were able to demonstrate that the fraction of the ER in which the ER marker GFP-KDEL accumulates is excluded from the phagosomes, whereas that containing the mVenus-Syntaxin 18 is recruited. These results highlight promising new avenues for the description of the pathogenic mechanisms used by Leishmania, Brucella, and Legionella spp., which thrive in ER-rich phagosomes.

β-Selection-Induced Proliferation Is Required for αβ T Cell Differentiation

Proliferation and differentiation are tightly coordinated to produce an appropriate number of differentiated cells and often exhibit an antagonistic relationship. Developing T cells, which arise in the thymus from a minute number of bone-marrow-derived progenitors, undergo a major expansion upon pre-T cell receptor (TCR) expression. The burst of proliferation coincides with differentiation toward the αβ T cell lineage-but the two processes were previously thought to be independent from one another, although both were driven by signaling from pre-TCR and Notch receptors. Here we report that proliferation at this step was not only absolutely required for differentiation but also that its ectopic activation was sufficient to substantially rescue differentiation in the absence of Notch signaling. Consistently, pharmacological inhibition of the cell cycle machinery also blocked differentiation in vivo. Thus the proliferation step is strictly required prior to differentiation of immature thymocytes.