Distinct subsets of CD1d-restricted T cells recognize self-antigens loaded in different cellular compartments.

Although recent studies have indicated that the major histocompatibility complex-like, beta2-microglobulin-associated CD1 molecules might function to present a novel chemical class of antigens, lipids and glycolipids, to alpha/beta T cells, little is known about the T cell subsets that interact with CD1. A subset of CD1d-autoreactive, natural killer (NK)1.1 receptor-expressing alpha/beta T cells has recently been identified. These cells, which include both CD4(-)CD8(-) and CD4(+) T cells, preferentially use an invariant Valpha14-Jalpha281 T cell receptor (TCR) alpha chain paired with a Vbeta8 TCR beta chain in mice, or the homologous Valpha24-JalphaQ/Vbeta11 in humans. This cell subset can explosively release key cytokines such as interleukin (IL)-4 and interferon (IFN)-gamma upon TCR engagement and may regulate a variety of infectious and autoimmune conditions. Here, we report the existence of a second subset of CD1d-restricted CD4(+) T cells that do not express the NK1.1 receptor or the Valpha14 TCR. Like the Valpha14(+) NK1.1(+) T cells, these T cells exhibit a high frequency of autoreactivity to CD1d, use a restricted albeit distinct set of TCR gene families, and contribute to the early burst of IL-4 and IFN-gamma induced by intravenous injection of anti-CD3. However, the Valpha14(+) NK1.1(+) and Valpha14(-) NK1.1(-) T cells differ markedly in their requirements for self-antigen presentation. Antigen presentation to the Valpha14(+) NK1.1(+) cells requires endosomal targeting of CD1d through a tail-encoded tyrosine-based motif, whereas antigen presentation to the Valpha14(-) NK1.1(-) cells does not. These experiments suggest the existence of two phenotypically different subsets of CD1d-restricted T cells that survey self-antigens loaded in distinct cellular compartments.

An alpha-helical signal in the cytosolic domain of the interleukin 2 receptor beta chain mediates sorting towards degradation after endocytosis.

High-affinity IL2 receptors consist of three components, the alpha, beta, and gamma chains that are associated in a noncovalent manner. Both the beta and gamma chains belong to the cytokine receptor superfamily. Interleukin 2 (IL2) binds to high-affinity receptors on the cell surface and IL2-receptor complexes are internalized. After endocytosis, the components of this multimolecular receptor have different intracellular fates: one of the chains, alpha, recycles to the plasma membrane, while the others, beta and gamma, are routed towards late endocytic compartments and are degraded. We show here that the cytosolic domain of the beta chain contains a 10-amino acid sequence which codes for a sorting signal. When transferred to a normally recycling receptor, this sequence diverts it from recycling. The structure of a 17-amino acid segment of the beta chain including this sequence has been studied by nuclear magnetic resonance and circular dichroism spectroscopy, which revealed that the 10 amino acids corresponding to the sorting signal form an amphipathic alpha helix. This work thus describes a novel, highly structured signal, which is sufficient for sorting towards degradation compartments after endocytosis.

The tyrosine kinase substrate eps15 is constitutively associated with the plasma membrane adaptor AP-2.

The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase. Its functions are not yet delineated and this work provides evidence for its possible role in endocytosis. A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the alpha- and beta-adaptins of the AP-2 complex on the basis of their NH2-terminal sequence and their immunoreactivity with anti-alpha- and anti-beta-adaptin antibodies but not with anti-gamma-adaptin antibody. In addition, the anti-eps15 antibody coimmunoprecipitated metabolically labeled polypeptides with molecular mass of 50 and 17 kD, comparable to those of the two other components of the AP-2 complex, mu2 and sigma 2. Constitutive association of eps15 with AP-2 was confirmed by two sets of experiments. First, eps15 was detected in immunoprecipitates of anti-alpha- and anti-beta-adaptin antibodies. Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferase eps15 fusion protein. The association of eps15 with AP-2 was ubiquitous and conserved between species, since it was observed in human lymphocytes and epithelial cells and in murine NIH3T3 fibroblasts. Our results are in keeping with a recent study showing homology between the NH2-terminal domains of eps15 and the product of the gene END3, involved in clathrin-mediated endocytosis of the pheromone alpha factor in Saccharomyces cerevisiae, and suggest a possible role for eps15 in clathrin-mediated endocytosis in mammals.

Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains.

Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and IL15 have common beta and gamma chains, while those for IL2, 4, 7, and 9 have a common gamma chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the alpha, beta, and gamma chains had different intracellular localizations after being endocytosed together. The alpha chain was always in transferrin-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the beta and gamma chains were excluded from transferrin-positive organelles and did not colocalize with alpha. Furthermore, beta could be found in rab7-positive vesicles. These differences suggest that the alpha chain recycles to the plasma membrane, while the beta and gamma chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The beta and gamma chains are very short-lived polypeptides since their half-life on the surface is only approximately 1 h, whereas alpha is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.

Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin.

It has been recently shown (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson, 1983, Cell, 33:273-285) that after a hypotonic shock followed by incubation in a K+-free medium, human fibroblasts arrest their coated pit formation and therefore arrest receptor-mediated endocytosis of low density lipoprotein. We have used this technique to study the endocytosis of transferrin, diphtheria toxin, and ricin toxin by three cell lines (Vero, Wi38/SV40, and Hep2 cells). Only Hep2 cells totally arrested internalization of [125I]transferrin, a ligand transported by coated pits and coated vesicles, after intracellular K+ depletion. Immunofluorescence studies using anti-clathrin antibodies showed that clathrin associated with the plasma membrane disappeared in Hep2 cells when the level of intracellular K+ was low. In the absence of functional coated pits, diphtheria toxin was unable to intoxicate Hep2 cells but the activity of ricin toxin was unaffected by this treatment. By measuring the rate of internalization of [125I]ricin toxin by Hep2 cells, with and without functional coated pits, we have shown that this labeled ligand was transported in both cases inside the cells. Hep2 cells with active coated pits internalized twice as much [125I]ricin toxin as Hep2 cells without coated pits. Entry of ricin toxin inside the cells was a slow process (8% of the bound toxin per 10 min at 37 degrees C) when compared to transferrin internalization (50% of the bound transferrin per 10 min at 37 degrees C). Using the indirect immunofluorescence technique on permeabilized cells, we have shown that Hep2 cells depleted in intracellular K+ accumulated ricin toxin in compartments that were predominantly localized around the cell nucleus. Our study indicates that in addition to the pathway of coated pits and coated vesicles used by diphtheria toxin and transferrin, another system of endocytosis for receptor-bound molecules takes place at the level of the cell membrane and is used by ricin toxin to enter the cytosol.

AP-2/Eps15 interaction is required for receptor-mediated endocytosis.

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2-binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2-binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2-binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane-coated pits.

Involvement of the ubiquitin/proteasome system in sorting of the interleukin 2 receptor beta chain to late endocytic compartments.

Down-regulation of cell surface growth factor receptors plays a key role in the tight control of cellular responses. Recent reports suggest that the ubiquitin system, in addition to participating in degradation by the proteasome of cytosolic and nuclear proteins, might also be involved in the down-regulation of various membrane receptors. We have previously characterized a signal in the cytosolic part of the interleukin 2 receptor beta chain (IL2Rbeta) responsible for its targeting to late endosomes/lysosomes. In this report, the role of the ubiquitin/proteasome system on the intracellular fate of IL2Rbeta was investigated. Inactivation of the cellular ubiquitination machinery in ts20 cells, which express a thermolabile ubiquitin-activating enzyme E1, leads to a significant decrease in the degradation rate of IL2Rbeta, with little effect on its internalization. In addition, we show that a fraction of IL2Rbeta can be monoubiquitinated. Furthermore, mutation of the lysine residues of the cytosolic region of a chimeric receptor carrying the IL2Rbeta targeting signal resulted in a decreased degradation rate. When cells expressing IL2Rbeta were treated either by proteasome or lysosome inhibitors, a significant decrease in receptor degradation was observed. Our data show that ubiquitination is required for the sorting of IL2Rbeta toward degradation. They also indicate that impairment of proteasome function might more generally affect intracellular routing.

Presentation of antigens derived from microorganisms residing in host-cell vacuoles.

Antigens presented by major histocompatibility complex molecules have been classified into those presented by 'endogenous' and 'exogenous' pathways. Some microorganisms reside within host-cell vacuoles that appear to avoid both pathways. Novel presentation mechanisms are being unraveled for these microorganisms, and their antigens, rather than being just peptides, can also consist of lipids or DNA fragments.

Clathrin-lndependent endocytosis and signalling of interleukin 2 receptors IL-2R endocytosis and signalling.

Interleukin 2 receptors (IL-2R) belong to the cytokine receptor family and share subunits with other members of the family. They are essential in T cell activation and in maintaining homeostatic immune responses. These receptors do not have an intrinsic kinase activity and use multiple signalling pathways. Their endocytic pathway is different from that of classic growth factor receptors in that it does not follow the classic clathrin-coated pit and vesicle route. After uptake, one of the IL-2R chains, alpha, recycles to the plasma membrane, whereas the two other chains, beta and gamma, are targeted to late endosomes/lysosomes and degraded. This involves ubiquitination of the receptor as a sorting signal. Links between the signalling events, internalisation and intracellular sorting of these receptors are reviewed.

Epitope tagging of DAB389IL-2: new insights into C-domain delivery to the cytosol of target cells.

The fusion toxin DAB389IL-2 is composed of the catalytic (C) and transmembrane (T) domains of native diphtheria toxin to which human interleukin-2 (IL-2) has been genetically fused (1,2). Following binding to the IL-2 receptor, the fusion toxin is internalized by receptor mediated endocytosis, and upon acidification of the endocytic vesicle, the T domain spontaneously inserts into the membrane, and facilitates the delivery of the C domain to the cytosol (3,4). In order to further study the process by which the C domain is delivered to the target cell cytosol, we genetically fused an eleven amino acid epitope derived from the vesicular stomatitis virus (VSV) G protein to the N-terminal end of DAB389IL-2. The epitope labelled fusion toxin, VSV-G-DAB389IL-2, was found to retain IL-2 receptor specific binding and cytotoxic activity. Target cells were incubated for various times in the presence of VSV-G-DAB389, fixed and then treated with anti-VSV G and FITC conjugated secondary antibody. Laser scanning confocal microscopy was used to determine the location of the fluorescent signal. The VSV-G epitope tagged fusion toxin was found only to be associated with small vesicles that were situated adjacent to the plasma membrane. These results suggest that the C domain of the fusion toxin is associated with an early intracellular compartment and is rapidly delivered to the cytosol. Since channel formation by the T domain is necessary for the delivery of the C domain, it follows that T domain insertion into the membrane also occurs early in the intoxication pathway.

Characterization of three monoclonal antibodies specifically directed against the ligand binding site area of the p55 subunit of the mouse interleukin-2 receptor.

Three new rat monoclonal antibodies (MAbs) (5A2, 125A8 and 135D5) directed against the mouse interleukin-2 receptor (IL-2R) were isolated. They were obtained after immunization of LOU rats with 14.1.6 T helper cell clones. These three MAbs recognize the p55 subunit of the IL-2R and compete with the binding of previously characterized MAbs AMT13 and 3C7 specific for this p55 subunit [Moreau et al. (1987) Eur. J. Immun. 15, 723-727]. They recognize the same (or closely related epitopes) since they reciprocally compete with each other's binding. Scatchard plot analysis of the data from inhibition experiments clearly indicate that they recognize with very high affinity the ligand binding site area of the p55 subunit of the IL-2R. The properties of the Fab fragment prepared from 5A2 and 135D5 indicate that at saturation one intact IgG molecule binds two IL-2R molecules.

Both the alpha and beta chains of high-affinity interleukin 2 receptors are located in intracellular vesicles when their ligand is endocytosed.

The growth factor interleukin 2 (IL2) binds to high and low-affinity receptors (Kd approximately 10-100 pM and 10 nM, respectively) present on activated T lymphocytes. High-affinity receptors are composed of two non-convalently linked polypeptides, alpha and beta of 55 and 70 kDa. These two polypeptides do not share any sequence homology, but each of them, in the absence of the other, binds IL2: alpha with a Kd approximately 10 nM and beta with a Kd approximately 1 nM. When these two chains are associated in lymphocytes, they form high-affinity receptors that mediate IL2 endocytosis and degradation, and transduce IL2 signaling. On cells that physiologically express IL2 receptors, such as activated T lymphocytes, both high and low affinity-receptors are present simultaneously on the cell surface, and low-affinity receptors (alpha without beta) are, in most instances, more abundant by a factor 5 to 10 than high-affinity receptors (alpha associated to beta). Low-affinity receptors bind IL2 but do not induce its internalization and signaling. The physiological role of the complexity of this receptor system is not fully understood. In the present study, we have investigated directly the fate of the high-affinity receptors when the ligand is endocytosed. By confocal microscopy, using two monoclonal antibodies specific for alpha and for beta, respectively, we show that each of these two polypeptides is located in intracellular endocytic compartments. Therefore, when the alpha chain is part of high-affinity receptors, it is endocytosed, as opposed to when it is part of low-affinity receptors and is not endocytosed.(ABSTRACT TRUNCATED AT 250 WORDS)

Type III secretion system in Chlamydia species: identified members and candidates.

Chlamydia trachomatis and Chlamydia pneumoniae genomes contain genes coding for type III secretion apparatuses. Like other pathogens, Chlamydia probably uses this system to secrete proteins in the host cell. With the aim of identifying such proteins, we analyzed the organization of Chlamydia type III secretion genes.

Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.

Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor.

A variety of ligands and macromolecules enter cells by receptor-mediated endocytosis. Ligands bind to their receptors on the cell surface and ligand-receptor complexes are localized in specialized regions of the plasma membrane called coated pits. Coated pits invaginate and give rise to intracellular coated vesicles containing ligand-receptor complexes which are thus internalized. Transferrin, a major serum glycoprotein which transports iron into cells, enters cells by this pathway. It binds to its receptor on the cell surface, transferrin-receptor complexes cluster in coated pits and are internalized in coated vesicles. Coated vesicles then lose their clathrin coat and fuse with endosomes, an organelle with an internal pH of about 5-5.5. Most ligands dissociate from their receptors in endosomes and they finally end up in lysosomes where they are degraded, while their receptors remain bound to membrane structures and recycle to the cell surface. Transferrin has a different fate: in endosomes iron dissociates from transferrin but apotransferrin remains bound to its receptor because of its high affinity for the receptor at acid pH. Apotransferrin thus recycles back to the plasma membrane still bound to its receptor. When the ligand-receptor complex reaches the plasma membrane or a compartment at neutral pH, apotransferrin dissociates from its receptor with a half-life of 18 s because of its low affinity for its receptor at neutral pH. The receptor is then ready for a new cycle of internalization, while apotransferrin enters the circulation, reloads iron in the appropriate organs and is ready for a new cycle of iron transport.

Lipopeptide presentation pathway in dendritic cells.

Lipopeptides are currently being evaluated as candidate vaccines in human volunteers. They elicit cytotoxic responses from CD8(+) T lymphocytes, whereas peptides without a lipidic moiety usually do not. The exact processing and presentation pathways leading to association with MHC class I molecules has not yet been defined. This is of particular interest in dendritic cells, which are required for primary T cell stimulation. We have tracked lipopeptides derived from an HLA-A2.1-restricted HIV-1 Reverse Transcriptase epitope, by N-terminal addition of an N-epsilon-palmitoyl-lysine. Entry of the lipopeptides into human monocyte-derived dendritic cells (MDC) was mediated by endocytosis, as assessed by colocalization using analogs labelled with rhodamine, and by confocal microscopy. This internalization in DC induced functional stimulation of CD8(+) T lymphocytes specific for the epitopes, quantified by Interferon-gamma ELISPOT assays. The peptide alone was not visualized inside the DC and was only presented through direct surface association to HLA-A*0201. Therefore, lipopeptides provide a model system to define precisely the cross-presentation pathways that lead exogenous proteins to associate with class I MHC molecules within dendritic cells. Using this approach, cross-presentation pathways can be better defined and vaccine lipopeptides can be further optimized for MHC class I association in human dendritic cells.

Fluorescent labelling of intracellular bacteria in living host cells.

The fluorescent reagent, CellTracker, labels metabolically-active cells and was used here to label Chlamydia in vivo during their exponential phase of growth in infected cells. HeLa cells infected with C. psittaci were labelled with the CellTracker reagents between 15 and 48 h post-infection. The fluorescent label accumulated in the host-cell membrane compartment (inclusion) within which Chlamydia reside and replicate, and was also incorporated by the bacteria. Labelling with the CellTracker affected neither the growth nor the differentiation of the chlamydiae, and labelled chlamydiae isolated from infected cells were infectious. Our results demonstrate that the CellTracker could become a valuable tool for in vivo labelling of obligate intracellular parasites for which no genetic tools exist.

Refolding of a bifunctional enzyme and its monofunctional fragment.

The renaturation of the bifunctional enzyme aspartokinase II-homoserine dehydrogenase II has been studied by using the reappearance of its two activities. The same kinetics of renaturation are obtained for the dehydrogenase (EC 1.1.1.3) and the kinase activity (EC 2.7.2.4). The mechanism of refolding of the enzyme apparently involves two steps, a folding step occurring within a monomer and a subsequent dimerization step. The reappearance of the two activities depends on this dimerization step, suggesting that monomeric species are inactive. A proteolytic fragment possessing full dehydrogenase activity is shown to be able to renature, as judged by the recovery of its activity. In this case also, the refolding depends on the formation of dimeric species. However, the refolding of this fragment is much faster than that of the dehydrogenase region in the intact enzyme. These results suggest that, although the dehydrogenase region can refold by itself when isolated as a fragment, refolding of this same region in the whole protein involves interactions with the remainder of the protein.