Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

Herpes simplex virus (HSV)-encoded glycoprotein B (gB) is the most abundant protein in the viral envelope and promotes fusion of the virus with the cellular membrane. In the present study, we found that gB impacts on the major histocompatibility complex (MHC)-II pathway of antigen presentation by fostering homotypic fusion of early endosomes and trapping MHC-II molecules in these altered endosomes. By using an overexpression approach, we demonstrated that transient expression of gB induces giant vesicles of early endosomal origin, which contained Rab5, early endosomal antigen 1 (EEA1), and large amounts of MHC-II molecules [human leukocyte antigen (HLA)-DR, and HLA-DM], but no CD63. In HSV-1-infected and stably transfected cell lines that expressed lower amounts of gB, giant endosomes were not observed, but strongly increased amounts of HLA-DR and HLA-DM were found in EEA1+ early endosomes. We used these giant vesicles as a model system and revealed that gB interacts with Rab5 and EEA1, and that gB-induced homotypic fusion of early endosomes to giant endosomes requires phosphatidylinositol 3-phosphate, the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptors, and the cytosolic gB sequence 889YTQVPN894 We conclude that gB expression alters trafficking of molecules of the HLA-II processing pathway, which leads to increased retention of MHC-II molecules in early endosomal compartments, thereby intercepting antigen presentation.-Niazy, N., Temme, S., Bocuk, D., Giesen, C., König, A., Temme, N., Ziegfeld, A., Gregers, T. F., Bakke, O., Lang, T., Eis-Hübinger, A. M., Koch, N. Misdirection of endosomal trafficking mediated by herpes simplex virus-encoded glycoprotein B.

The genome sequence of Atlantic cod reveals a unique immune system.

Atlantic cod (Gadus morhua) is a large, cold-adapted teleost that sustains long-standing commercial fisheries and incipient aquaculture. Here we present the genome sequence of Atlantic cod, showing evidence for complex thermal adaptations in its haemoglobin gene cluster and an unusual immune architecture compared to other sequenced vertebrates. The genome assembly was obtained exclusively by 454 sequencing of shotgun and paired-end libraries, and automated annotation identified 22,154 genes. The major histocompatibility complex (MHC) II is a conserved feature of the adaptive immune system of jawed vertebrates, but we show that Atlantic cod has lost the genes for MHC II, CD4 and invariant chain (Ii) that are essential for the function of this pathway. Nevertheless, Atlantic cod is not exceptionally susceptible to disease under natural conditions. We find a highly expanded number of MHC I genes and a unique composition of its Toll-like receptor (TLR) families. This indicates how the Atlantic cod immune system has evolved compensatory mechanisms in both adaptive and innate immunity in the absence of MHC II. These observations affect fundamental assumptions about the evolution of the adaptive immune system and its components in vertebrates.

Invariant chain as a vehicle to load antigenic peptides on human MHC class I for cytotoxic T-cell activation.

Protective T-cell responses depend on efficient presentation of antigen (Ag) in the context of major histocompatibility complex class I (MHCI) and class II (MHCII) molecules. Invariant chain (Ii) serves as a chaperone for MHCII molecules and mediates trafficking to the endosomal pathway. The genetic exchange of the class II-associated Ii peptide (CLIP) with antigenic peptides has proven efficient for loading of MHCII and activation of specific CD4(+) T cells. Here, we investigated if Ii could similarly activate human CD8(+) T cells when used as a vehicle for cytotoxic T-cell (CTL) epitopes. The results show that wild type Ii, and Ii in which CLIP was replaced by known CTL epitopes from the cancer targets MART-1 or CD20, coprecipitated with HLA-A*02:01 and mediated colocalization in the endosomal pathway. Furthermore, HLA-A*02:01-positive cells expressing CLIP-replaced Ii efficiently activated Ag-specific CD8(+) T cells in a TAP- and proteasome-independent manner. Finally, dendritic cells transfected with mRNA encoding IiMART-1 or IiCD20 primed naïve CD8(+) T cells. The results show that Ii carrying antigenic peptides in the CLIP region can promote efficient presentation of the epitopes to CTLs independently of the classical MHCI peptide loading machinery, facilitating novel vaccination strategies against cancer.

The fusion of early endosomes induces molecular-motor-driven tubule formation and fission.

Organelles in the endocytic pathway interact and communicate through the crucial mechanisms of fusion and fission. However, any specific link between fusion and fission has not yet been determined. To study the endosomal interactions with high spatial and temporal resolution, we enlarged the endosomes by two mechanistically different methods: by expression of the MHC-class-II-associated chaperone invariant chain (Ii; or CD74) or Rab5, both of which increased the fusion rate of early endosomes and resulted in enlarged endosomes. Fast homotypic fusions were studied, and immediately after the fusion a highly active and specific tubule formation and fission was observed. These explosive tubule formations following fusion seemed to be a direct effect of fusion. The tubule formations were dependent on microtubule interactions, and specifically controlled by Kif16b and dynein. Our results show that fusion of endosomes is a rapid process that destabilizes the membrane and instantly induces molecular-motor-driven tubule formation and fission.

The human-specific invariant chain isoform Iip35 modulates Iip33 trafficking and function.

The invariant chain (Ii) is a multifunctional protein, which has an essential role in the assembly and transport of major histocompatibility complex class II (MHC II) molecules. From a single gene, Ii is synthesized as four different isoforms: Iip33, Iip35, Iip41 and Iip43. Iip35 and Iip43 are specific to humans, and are formed due to an upstream alternative translation site, resulting in an N-terminal extension of 16 amino acids. This extension harbors a strong endoplasmic reticulum (ER) retention motif. Consequently, Iip35 or Iip43 expressed alone are retained in the ER, whereas Iip33 and Iip41 rapidly traffic to the endosomal pathway. Endogenously expressed, the four isoforms form mixed heterotrimers in the ER; however, mainly due to the absence of the Iip35/p43 isoforms in mice, little is known about how they influence general Ii function. In this study, we have co-expressed Iip33 and Iip35 in human cells with and without MHC II to gain a better understanding of how Iip35 isoform influences the cellular properties of Iip33. We find that Iip35 significantly affects the properties of Iip33. In the presence of Iip35, the transport of Iip33 out of the ER is delayed, its half-life is dramatically prolonged and its ability to induce enlarged endosomes and delayed endosomal maturation is abrogated.

Invariant chain increases the half-life of MHC II by delaying endosomal maturation.

Mounting adaptive immune responses requires the cell surface expression of major histocompatibility class II molecules (MHC II) loaded with antigenic peptide. However, in the absence of antigenic stimuli, the surface population of MHC II is highly dynamic and exhibits a high turnover. Several studies have focused on the regulation of MHC II, and it is now recognized that ubiquitination is one key mechanism operating in the turnover of MHC II in B cells and dendritic cells. Here, we describe how the invariant chain (Ii) can prolong the half-life of MHC II through its action on the endocytic pathway. We find that in cells expressing intermediate-to-high levels of Ii, the half-life of MHC II is increased, with MHC II accumulating in slowly-maturing endosomes. The accumulation in endosomes is not due to retention of new MHC II directed from the endoplasmatic reticulum, as also mature, not Ii associated, MHC II is preserved. We suggest that this alternative endocytic pathway induced by Ii would serve to enhance the rate, quantity and diversity of MHC II antigen presentation by concentrating MHC II into specialized compartments and reducing the need for new MHC II synthesis upon antigen encounter.

EDEM is involved in retrotranslocation of ricin from the endoplasmic reticulum to the cytosol.

The plant toxin ricin is transported retrogradely from the cell surface to the endoplasmic reticulum (ER) from where the enzymatically active part is retrotranslocated to the cytosol, presumably by the same mechanism as used by misfolded proteins. The ER degradation enhancing alpha-mannosidase I-like protein, EDEM, is responsible for directing aberrant proteins for ER-associated protein degradation. In this study, we have investigated whether EDEM is involved in ricin retrotranslocation. Overexpression of EDEM strongly protects against ricin. However, when the interaction between EDEM and misfolded proteins is inhibited by kifunensin, EDEM promotes retrotranslocation of ricin from the ER to the cytosol. Furthermore, puromycin, which inhibits synthesis and thereby transport of proteins into the ER, counteracted the protection seen in EDEM-transfected cells. Coimmunoprecipitation studies revealed that ricin can interact with EDEM and with Sec61alpha, and both kifunensin and puromycin increase these interactions. Importantly, vector-based RNA interference against EDEM, which leads to reduction of the cellular level of EDEM, decreased retrotranslocation of ricin A-chain to the cytosol. In conclusion, our results indicate that EDEM is involved in retrotranslocation of ricin from the ER to the cytosol.

Disentangling the immune response and host-pathogen interactions in Francisella noatunensis infected Atlantic cod.

The genetic repertoire underlying teleost immunity has been shown to be highly variable. A rare example is Atlantic cod and its relatives Gadiformes that lacks a hallmark of vertebrate immunity: Major Histocompatibility Complex class II. No immunological studies so far have fully unraveled the functionality of this particular immune system. Through global transcriptomic profiling, we investigate the immune response and host-pathogen interaction of Atlantic cod infected with the facultative intracellular bacterium Francisella noatunensis. We find that Atlantic cod displays an overall classic innate immune response with inflammation, acute-phase proteins and cell recruitment through up-regulation of e.g. IL1B, fibrinogen, cathelicidin, hepcidin and several chemotactic cytokines such as the neutrophil attractants CXCL1 and CXCL8. In terms of adaptive immunity, we observe up-regulation of interferon gamma followed by up-regulation of several MHCI transcripts and genes related to antigen transport and loading. Finally, we find up-regulation of immunoglobulins and down-regulation of T-cell and NK-like cell markers. Our analyses also uncover some contradictory transcriptional findings such as up-regulation of anti-inflammatory IL10 as well as down-regulation of the NADPH oxidase complex and myeloperoxidase. This we interpret as the result of host-pathogen interactions where F. noatunensis modulates the immune response. In summary, our results suggest that Atlantic cod mounts a classic innate immune response as well as a neutrophil-driven response. In terms of adaptive immunity, both endogenous and exogenous antigens are being presented on MHCI and antibody production is likely enabled through direct B-cell stimulation with possible neutrophil help. Collectively, we have obtained novel insight in the orchestration of the Atlantic cod immune system and determined likely targets of F. noatunensis host-pathogen interactions.

The cytoplasmic tail of invariant chain regulates endosome fusion and morphology.

The major histocompatibility complex class II associated invariant chain (Ii) has been shown to inhibit endocytic transport and to increase the size of endosomes. We have recently found that this property has a significant impact on antigen processing and presentation. Here, we show in a cell-free endosome fusion assay that expression of Ii can increase fusion after phosphatidylinositol 3-kinase activity is blocked by wortmannin. In live cells wortmannin was also not able to block formation of the Ii-induced enlarged endosomes. The effects of Ii on endosomal transport and morphology depend on elements within the cytoplasmic tail. Data from mutagenesis analysis and nuclear magnetic resonance-based structure calculations of the Ii cytoplasmic tail demonstrate that free negative charges that are not involved in internal salt bridges are essential for both interactions between the tails and for the formation of enlarged endosomes. This correlation indicates that it is interactions between the Ii cytoplasmic tails that are involved in endosome fusion. The combined data from live cells, cell-free assays, and molecular dynamic simulations suggest that Ii molecules on different vesicles can promote endosome docking and fusion and thereby control endosomal traffic of membrane proteins and endosomal content.

Evolutionary redesign of the Atlantic cod (Gadus morhua L.) Toll-like receptor repertoire by gene losses and expansions.

Genome sequencing of the teleost Atlantic cod demonstrated loss of the Major Histocompatibility Complex (MHC) class II, an extreme gene expansion of MHC class I and gene expansions and losses in the innate pattern recognition receptor (PRR) family of Toll-like receptors (TLR). In a comparative genomic setting, using an improved version of the genome, we characterize PRRs in Atlantic cod with emphasis on TLRs demonstrating the loss of TLR1/6, TLR2 and TLR5 and expansion of TLR7, TLR8, TLR9, TLR22 and TLR25. We find that Atlantic cod TLR expansions are strongly influenced by diversifying selection likely to increase the detectable ligand repertoire through neo- and subfunctionalization. Using RNAseq we find that Atlantic cod TLRs display likely tissue or developmental stage-specific expression patterns. In a broader perspective, a comprehensive vertebrate TLR phylogeny reveals that the Atlantic cod TLR repertoire is extreme with regards to losses and expansions compared to other teleosts. In addition we identify a substantial shift in TLR repertoires following the evolutionary transition from an aquatic vertebrate (fish) to a terrestrial (tetrapod) life style. Collectively, our findings provide new insight into the function and evolution of TLRs in Atlantic cod as well as the evolutionary history of vertebrate innate immunity.

Unraveling the evolution of the Atlantic cod's (Gadus morhua L.) alternative immune strategy.

Genes encoding the major histocompatibility complex (MHC) have been thought to play a vital role in the adaptive immune system in all vertebrates. The discovery that Atlantic cod (Gadus morhua) has lost important components of the MHC II pathway, accompanied by an unusually high number of MHC I genes, shed new light on the evolution and plasticity of the immune system of teleosts as well as in higher vertebrates. The overall aim of this study was to further investigate the highly expanded repertoire of MHC I genes using a cDNA approach to obtain sequence information of both the binding domains and the sorting signaling potential in the cytoplasmic tail. Here we report a novel combination of two endosomal sorting motifs, one tyrosine-based associated with exogenous peptide presentation by cross-presenting MHCI molecules, and one dileucine-based associated with normal MHC II functionality. The two signal motifs were identified in the cytoplasmic tail in a subset of the genes. This indicates that these genes have evolved MHC II-like functionality, allowing a more versatile use of MHC I through cross-presentation. Such an alternative immune strategy may have arisen through adaptive radiation and acquisition of new gene function as a response to changes in the habitat of its ancestral lineage.

BiP negatively affects ricin transport.

The AB plant toxin ricin binds both glycoproteins and glycolipids at the cell surface via its B subunit. After binding, ricin is endocytosed and then transported retrogradely through the Golgi to the endoplasmic reticulum (ER). In the ER, the A subunit is retrotranslocated to the cytosol in a chaperone-dependent process, which is not fully explored. Recently two separate siRNA screens have demonstrated that ER chaperones have implications for ricin toxicity. ER associated degradation (ERAD) involves translocation of misfolded proteins from ER to cytosol and it is conceivable that protein toxins exploit this pathway. The ER chaperone BiP is an important ER regulator and has been implicated in toxicity mediated by cholera and Shiga toxin. In this study, we have investigated the role of BiP in ricin translocation to the cytosol. We first show that overexpression of BiP inhibited ricin translocation and protected cells against the toxin. Furthermore, shRNA-mediated depletion of BiP enhanced toxin translocation resulting in increased cytotoxicity. BiP-dependent inhibition of ricin toxicity was independent of ER stress. Our findings suggest that in contrast to what was shown with the Shiga toxin, the presence of BiP does not facilitate, but rather inhibits the entry of ricin into the cytosol.

Differential regulation of Rab GTPase expression in monocyte-derived dendritic cells upon lipopolysaccharide activation: a correlation to maturation-dependent functional properties.

The regulation of Rab expression to modulate cellular function has recently been proposed. Dendritic cells are a prototypic example of cells that drastically alter their function in response to environmental cues by reducing endocytosis, secreting cytokines, changing surface protein repertoires and altering morphology and migration. This is not a binary event, but is subject to fluctuations through the activation process, termed maturation. Consequently, DCs transiently increase endocytosis and production of major histocompatibility complex class II molecules, and secrete inflammatory cytokines in infected tissues before migrating to secondary lymph nodes and releasing T cell polarizing factors. All these cellular processes rely on intracellular membrane transport, which is regulated by Rab family GTPases and their diverse effectors. Here we examine how the Rabs likely to be involved in these functions are regulated throughout DC maturation. We find that Rab expression is altered upon lipopolysaccharide-induced activation, and discuss how this correlates to the reported functions of these cells during maturation.

The Mitogen-activated protein kinase p38 links Shiga Toxin-dependent signaling and trafficking.

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+) dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus.

A mutant human IgG molecule with only one C1q binding site can activate complement and induce lysis of target cells.

There are potentially two binding sites for C1q on IgG, one on each C(H)2 domain of the gamma heavy chains, close to the lower hinge region. It is not clear whether the presence and involvement of both the C1q binding sites is necessary to induce the activation signal of human IgG. In order to clarify this issue, we made a hybrid mutant IgG1/IgG3 molecule where the IgG1 half of the molecule was made unable to activate complement through the introduction of a P329A mutation. The IgG3 half of the molecule was mutated to harbor a hinge region identical to that of IgG1, and for detection a peptide tag derived from p21ras was introduced into the FG loop of the C(H)1 domain. The hybrid IgG1P329A/IgG3h1-ras molecules were isolated by Protein A affinity chromatography and shown to activate complement and induce complement-mediated lysis at the same levels as wild-type IgG1 and IgG3h1-ras molecules. Thus, one C1q binding site per IgG is sufficient to induce activation. Wild-type human IgG molecules might also normally expose only one C1q binding site as already shown for interaction with FcgammaR, were IgG expose one binding site per molecule.

The cytoplasmic tail of invariant chain modulates antigen processing and presentation.

The MHC class II-associated invariant chain (Ii) has several important functions in antigen presentation. In this study, we have examined the effect of Iip33 expression on endocytic transport and antigen presentation. We find that degradation of both endocytosed antigen and Ii itself is delayed in cells expressing high levels of Ii, whereas a mutant Ii with an altered charge distribution in the cytoplasmic tail was unable to exert this effect. Furthermore, the Ii mutant did not enhance the presentation of an Ii-dependent MHC class II-restricted epitope to the same extent as the wild type. In a parallel study, we investigated the effect of charge in the cytoplasmic tail of Ii. We find that due to exposed negative charges, it promotes endosome fusion events, and we suggest that this causes endosomal retention (Nordeng et al., Mol. Biol. Cell 2002). Together, the data reveal an additional property of the Iip33 cytoplasmic tail that contributes to the modulation of antigen processing and presentation.

MHC class II loading of high or low affinity peptides directed by Ii/peptide fusion constructs: implications for T cell activation.

CD4(+) T cells recognize peptides presented on the cell surface of antigen presenting cells in the MHC class II context. The biosynthesis and transport of MHC class II molecules depend on the type II transmembrane invariant chain (Ii) and are tightly regulated processes. Ii is known to bind to the MHC class II peptide-binding groove via its class II-associated Ii peptide (CLIP) region early in the biosynthetic pathway to prevent premature peptide binding. In this study we have genetically exchanged CLIP with peptides of either high or low affinity for the class II peptide binding groove and utilized the properties of Ii to manipulate MHC class II loading. An inducible promoter controlled expression of the Ii/peptide fusion constructs, and presentation at different expression levels was studied. Both peptides were excised from Ii and presented on MHC class II molecules as shown by liquid chromatography-tandem mass spectrometry, but the high affinity peptide was presented more efficiently than the low affinity peptide. Both peptides were efficient in eliciting T cell responses at high Ii/peptide concentration independent of the duration of T cell stimulation. The peptides were also able to elicit an IL-2 response at low expression levels; however, the kinetic differed as the T cells required longer duration of T cell contact to reach a significant T cell response. This probably reflects the number of class II/peptide complexes at the cell surface and is discussed.

Uncoating ATPase Hsc70 is recruited by invariant chain and controls the size of endocytic compartments.

Targeting of class II major histocompatibility complex molecules to endocytic compartments is mediated by their association with the invariant chain (Ii). Although the identity of certain sorting signals located in Ii's cytoplasmic tail is known, proteins that interact with Ii's cytoplasmic tail in living cells remain to be identified. Synthesis of a biotinylated trimeric Ii cytoplasmic tail allowed the retrieval of two proteins that interact with this domain. We identify one of them as the 70-kDa heat-shock cognate protein (hsc70), the uncoating ATPase of clathrin-coated vesicles, and the other as its mitochondrial homologue, the glucose-regulated protein grp75. Expression of Ii in COS cells results in the formation of large endocytic compartments. We observe extensive colocalization of hsc70 with Ii in these macrosomes. Expression of a dominant-negative (K71M) green fluorescent protein-tagged version of hsc70 counteracted the ability of Ii to modify the endocytic pathway, demonstrating an interaction in vivo of Ii with hsc70 as part of the machinery responsible for macrosome formation.