No abstract

MHC class I molecules usually present peptides derived from endogenous antigens that are bound in the endoplasmic reticulum. Loading of exogenous antigens on class I molecules, e.g., in cross-priming, sometimes occurs, but the intracellular location where interaction between the antigenic fragment and class I takes place is unclear. Here we show that measles virus F protein can be presented by class I in transporters associated with antigen processing-independent, NH 4 Clsensitive manner, suggesting that class I molecules are able to interact and bind antigen in acidic compartments, like class II molecules. Studies on intracellular transport of green fluorescent protein-tagged class I molecules in living cells confirmed that a small fraction of class I molecules indeed enters classical MHC class II compartments (MIICs) and is transported in MIICs back to the plasma membrane. Fractionation studies show that class I complexes in MIICs contain peptides. The pH in MIIC (around 5.0) is such that efficient peptide exchange can occur. We thus present evidence for a pathway for class I loading that is shared with class II molecules.MHC molecules display antigenic peptides on the cell surface for surveillance by T lymphocytes. MHC class I molecules present peptides to CD8 ϩ cytotoxic T cells, whereas MHC class II molecules present peptides to CD4 ϩ Th cells. The current dogma is that antigens from the extracellular fluid enter the exogenous processing pathway by endocytosis and are partially degraded in acidic endosomal or lysosomal structures to yield peptides that bind MHC class II molecules. This type of processing is inhibited by reagents that prevent endosomal acidification (chloroquine, NH 4 Cl) (1). In the endogenous processing pathway intracellular proteins are degraded in the cytosol by the proteasome complex, generating peptides that are transported from the cytoplasm into the lumen of the endoplasmic reticulum (ER) by the transporters associated with antigen processing (TAP), where they bind to nascent MHC class I heavy chain-␤ 2 -microglobulin (␤ 2 m) heterodimers. Fully assembled class I͞peptide complexes exit the ER and are transported through the Golgi to the cell surface by the constitutive secretory route. This processing pathway can be blocked by proteasome inhibitors or Brefeldin A (BFA), an inhibitor of anterograde ER-Golgi transport, but not by lysosomotropic agents. Thus, in general endogenous antigens are presented by MHC class I molecules, and exogenous antigens are displayed at the cell surface by MHC class II molecules. However accumulating evidence has shown that this dichotomy in presentation of antigen from endogenous and exogenous origin is not absolute. It was demonstrated that cytotoxic T lymphocyte (CTL) responses can be primed in vitro and in vivo with exogenous antigen (reviewed in refs. 2 and 3). At least two fundamentally different pathways for presentation of exogenous antigens by MHC class I molecules in vitro have been described: one involving access of exogenous antigen to...

Human immunodeficiency virus (HIV), the causative agent of AIDS, infects human lymphocytes and monocytes. An interaction between the viral envelope gp 120 and CD4 protein is required to initiate an infectious cycle. HIV infection in vitro induces syncytium formation by cell-to-cell fusion; this aspect of viral cytopathogenicity is even more dependent on gp120-CD4 interactions. That gp120 is extremely heavily glycosylated (31-36 N-linked glycans per molecule), suggests involvement of N-linked glycans in the gp120-CD4 interaction. We therefore investigated the effects of castanospermine, 1-deoxynojirimycin (dNM) and 1-deoxymannojirimycin (dMM), three trimming glycosidase inhibitors which perturb N-linked glycan structure, on induction of the formation of syncytium between HIV-infected and CD4-expressing cells. The glucosidase inhibitors castanospermine and dNM, but not the mannosidase inhibitor dMM, inhibited syncytium formation and interfered with infectivity. The potential of glucosidase inhibitors as anti-HIV therapeutic agents deserves further investigation, especially because dNM and related compounds show little toxicity in vitro and in vivo.

Abstract. Newly synthesized MHC class II molecules are sorted to lysosomal structures where peptide loading can occur. Beyond this point in biosynthesis, no MHC class II molecules have been detected at locations other than the cell surface. We studied this step in intracellular transport by visualizing MHC class II molecules in living cells. For this purpose we stably expressed a modified HLA-DR1 13 chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (13-GFP) in class II-expressing Mel JuSo cells. This modification of the class II 13 chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ 13-GFP chimera was studied in living cells at 37°C. We visualize rapid movement of acidic class II/13-GFP containing vesicles from lysosomal compartments to the plasma membrane and show that fusion of these vesicles with the plasma membrane occurs. Furthermore, we show that this transport route does not intersect the earlier endosomal pathway. MHC class II molecules present peptides to CD4 ÷ T cells. Most bound peptides are derived from antigens degraded in the endosomal pathway. To allow association with these peptides, class II molecules are targeted to endosomal compartments by the invariant chain (or Ii) 1 (7,8). Here, Ii is degraded and Ii-degradation products are exchanged with antigenic peptides, a process catalyzed by 41,47,48). The endosomal compartments where class II molecules are loaded with peptide may be considered "special" lysosomes with a multilamellar and/or multivesicular appearance and were originally termed MIIC for MHC class II--containing compartments (31). This "unique" morphology appears to be induced by the expression of class II molecules (4). Although earlier endosomal compartments have been noted as "specialized class II loading compartments" as well (1, 50), HLA-DM and class II molecules are generally located in compartments with lysosomal proteins like CD63, lamp-l, and cathepsin D (13,24,32,42).

The HLA-DM genes encode an unconventional HLA (human leukocyte antigen) class II molecule that is required for appropriate binding of peptide to classical HLA class II products. In the absence of DM, other class II molecules are unstable upon electrophoresis in sodium dodecyl sulfate and are largely associated with a nested set of peptides derived from the invariant chain called CLIP, for class II-associated invariant chain peptides. DMA and DMB associated and accumulated in multilaminar, intracellular compartments with classical class II molecules, but were found infrequently, if at all, at the cell surface. Thus, DM may facilitate peptide binding to class II molecules within these intracellular compartments.

HLA-DO is a negative modulator of HLA-DM. By stably associating with HLA-DM, the catalytic action of HLA-DM on class II peptide loading is inhibited. HLA-DO thus affects the peptide repertoire that is eventually presented to the immune system by MHC class II molecules.

No abstract

Dendritic cells (DC) efficiently take up antigens by macropinocytosis and mannose receptor-mediated endocytosis. Here we show that endocytosis of mannose receptor-antigen complexes takes place via small coated vesicles, while non-mannosylated antigens were mainly present in larger vesicles. Shortly after internalization the mannose receptor and its ligand appeared in the larger vesicles. Within 10 min, the mannosylated and non-mannosylated antigens co-localized with typical markers for major histocompatibility complex class II-enriched compartments and lysosomes. In contrast, the mannose receptor appeared not to reach these compartments, suggesting that it releases its ligand in an earlier endosomal structure. Moreover, we demonstrate that mannosylation of protein antigen and peptides resulted in a 200-10,000-fold enhanced potency to stimulate HLA class II-restricted peptide-specific T cell clones compared to non-mannosylated peptides. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DC which may be applicable in vaccine design.