As the human tetraspanin CD81 binds hepatitis C virus (HCV) envelope glycoprotein E2, we addressed the role CD81 may play in cellular trafficking of HCV envelope proteins. Studies on HCV life cycle are complicated by the lack of a robust cell culture system; we therefore transfected mammalian cells with HCV E1-E2 cDNA, with or without human CD81 (huCD81) cDNA. In the absence of huCD81, HCV envelope proteins are almost completely retained in the endoplasmic reticulum. Instead, when huCD81 is present, a fraction of HCV envelope proteins passes through the Golgi apparatus, matures acquiring complex sugars and is found extracellularly associated with exosomes. These are 60-100-nm membrane vesicles enriched in tetraspanins, released into the extracellular milieu by many cell types and having fusogenic activity. We also report that human plasma contains exosomes and that in HCV patients, viral RNA is associated with these circulating vesicles. We propose that the HCV-CD81 complex leaves cells in the form of exosomes, circulates in this form and exploits the fusogenic capabilities of these vesicles to infect cells even in the presence of neutralizing antibodies.
Invariant natural killer T (NKT) cells are a highly conserved subset of T lymphocytes expressing a semi-invariant T cell receptor (TCR), which is restricted to CD1d and specific for the glycosphingolipid antigen α-galactosylceramide. Their ability to secrete a variety of cytokines, which in turn modulate the activation of cells of both innate and acquired immune responses, suggests that invariant NKT cells exert a regulatory role mainly via indirect mechanisms. A relevant question is whether invariant NKT cells can directly help B cells. We document here that human invariant NKT cells are as efficient as conventional CD4+ Th0 lymphocytes in promoting proliferation of autologous memory and naive B lymphocytes in vitro, and in inducing immunoglobulin production. Help to B cells by invariant NKT cells is CD1d-dependent and delivered also in the absence of α-galactosylceramide, suggesting that NKT cells recognize an endogenous ligand presented by CD1d on B cells. The two major subsets of invariant NKT cells, CD4+ and double negative (CD4−CD8−), express comparable levels of CD40 ligand and cytokines, but differ in helper functions. Indeed, both subsets induce similar levels of B cell proliferation, whereas CD4+ NKT cells induce higher levels of immunoglobulin production. These results suggest a direct role for invariant NKT cells in regulating B lymphocyte proliferation and effector functions.
The mouse hemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (alpha-D-N-acetylgalactosamine, or alpha-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attached at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67-76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.
TLR7 and TLR8 are intracellular sensors activated by single-stranded RNA species generated during viral infections. Various synthetic small molecules can also activate TLR7 or TLR8 or both through an unknown mechanism. Notably, direct interaction between small molecules and TLR7 or TLR8 has never been shown. To shed light on how small molecule agonists target TLRs, we labeled 2 imidazoquinolines, resiquimod and imiquimod, and one adenine-based compound, SM360320, IntroductionSince the discovery of type I IFN (IFN-I) in 1957 and its potential for treatment of viral infections and cancer, 1 several small molecule inducers of this factor have been identified. Many of these compounds are now known to be TLR7 or TLR8 or both agonists. Among them are imidazoquinolines, 2,3 such as resiquimod (R848) that activates both TLR7 and TLR8 in humans, and imiquimod (R837) that activates TLR7 only. 4,5 Imidazoquinolines have been largely tested in humans, and R837, formulated as a cream (Aldara), is licensed for topical treatment of genital warts, basal cell carcinoma, and actinic keratosis. 6 Similar to R837, purinelike molecules, such as 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (SM360320 or 1V136), are TLR7 agonists. 7 TLR7 and TLR8 are phylogenetically related, located on the X chromosome in most mammals, and probably derive from a gene duplication event. TLR7 and TLR8 are both activated by various singlestranded RNAs (ssRNAs) from viruses, 8 synthetic guanosine-or uridine-rich ssRNAs, 9 and synthetic small molecules. TLR9, together with TLR7 and TLR8, form a subfamily of TLRs that is based on genomic structure and sequence homology. 10 The natural agonist of TLR9 is unmethylated, CpG-containing DNA of bacterial or viral origin that can be mimicked by synthetic ssCpG oligonucleotides that can be classified as A-type (CpG-A), B-type (CpG-B), or C-type (CpG-C) on the basis of their different sequence motifs and biologic activities. 11 Several reports have suggested that TLR7, TLR8, and TLR9 can interact directly with nucleic acids [12][13][14][15] ; however, structural evidence confirming this hypothesis is not available yet. No convincing evidence of direct binding of imidazoquinolines or purine-like molecules to TLR7 or TLR8 exists either.TLR7 and TLR9 are the only TLRs expressed by plasmacytoid dendritic cells (pDCs), which are a rare subset of circulating DCs that are considered as a frontline defense against viral infections. pDCs produce most of the systemic IFN-I (ie, IFN-␣ and -) after viral infection and rapidly activate T cells with the use of presynthesized MHC class I and II molecules stored in their early and late endosomal compartments, respectively. 16,17 Although nucleic acid sensing provides protection from intracellular infection, nucleic acids are not exclusively from pathogens; therefore, a balance between self versus foreign responsiveness is necessary. Indeed, recognition of self nucleic acids by TLR7 and TLR9 and the resultant IFN-I production have been linked to autoimmune disorders such as lupu...
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