RESUMO
In order for cytotoxic T cells to initiate immune responses, peptides derived from internalized antigens must be presented to the cytotoxic T cells on major histocompatibility complex (MHC) class I molecules. Here we show that dendritic cells, the only antigen-presenting cells that initiate immune responses efficiently, have developed a unique membrane transport pathway linking the lumen of endocytic compartments and the cytosol. Endosome-to-cytosol transport is restricted to dendritic cells, specific to internalized antigens and selective for the size of the transported molecules. Thus, in dendritic cells, internalized antigens gain access to the cytosolic antigen-processing machinery and to the conventional MHC class I antigen-presentation pathway.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos/metabolismo , Células Dendríticas/imunologia , Endocitose/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Animais , Antígenos/imunologia , Transporte Biológico , Células Cultivadas , Citosol/imunologia , Células Dendríticas/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/imunologiaRESUMO
The human cell line T2 has been reported to be class I assembly deficient, and accordingly expresses reduced amounts of HLA-A2 and no HLA-B5 at the cell surface. By immunoblotting we observe the steady-state class I heavy chain levels of T2 to be near normal when compared with the identical class I alleles of the wild-type cell line T1. In pulse chase experiments, formation of heavy chain beta 2-microglobulin complexes is observed for both HLA-A2 and HLA-B5. Culture at reduced temperatures (26 or 20 degrees C) does not increase the amount of class I molecules transported, unlike what has been reported for the class I assembly-deficient mouse mutant cell line RMA-S. The HLA-B5 and the HLA-A2 complexes formed by T2 are thermolabile in cell lysates, albeit to different degrees. The thermolability of HLA-B5 can be overcome by addition of HLA-B5-presentable peptides, obtained by trifluoroacetic acid extraction from an HLA-B5-positive cell line, underlining the necessity of peptide for class I stability and indicating that T2-derived class I complexes are devoid of peptide. Cytoplast fusion of T2 cells with RMA-S cells shows the defect in class I assembly of RMA-S to be similar to that of T2. Localization of class I molecules observed by immuno-electron microscopy reveals the accumulation in the T2 cell line of both HLA-B5 and HLA-A2 in the endoplasmic reticulum (ER). Class I molecules are present in all the cisternae of the Golgi complex of T2, but the ratio of HLA-A and -B locus products in the Golgi area differs significantly from that at the cell surface. We conclude that the requirement for peptide in transport of class I molecules manifests itself at a stage beyond the ER, most likely the Golgi area.
Assuntos
Antígenos de Histocompatibilidade Classe I/análise , Linhagem Celular , Antígeno HLA-A2/análise , Antígenos HLA-B/análise , Humanos , Microscopia Imunoeletrônica , Temperatura , Microglobulina beta-2/análiseRESUMO
The well defined, immature murine dendritic cell (DC) line D1 was used to study the role of DC maturation in CTL induction in vitro and in vivo. Maturation of D1 cells, characterized by markedly increased expression of MHC and costimulatory molecules, was induced by incubation with lipopolysaccharide, agonistic CD40 antibody, or specific CD4(+) T helper (Th) cells. Activated, but not immature, D1 cells efficiently primed alloreactive T cell responses in vitro. Similarly, priming of CTL immunity in vivo in CD4-depleted mice was only observed if these mice were immunized with activated D1 cells. This study provides formal evidence that activation of DCs, induced by Th-independent as well as Th-dependent stimuli, is essential for efficient induction of CTL responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos/farmacologia , Antígenos CD40/imunologia , Linhagem Celular , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Feminino , Lipopolissacarídeos/farmacologia , Ativação Linfocitária , Complexo Principal de Histocompatibilidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Células Th1/imunologiaRESUMO
Dendritic cells (DC) represent potent antigen-presenting cells for the induction of T cell-dependent immune responses. Previous work on antigen uptake and presentation by human DC is based largely on studies of blood DC that have been cultured for various periods of time before analysis. These cultured cells may therefore have undergone a maturation process from precursors that have different capacities for antigen capture and presentation. We have now used immunoelectron microscopy and antigen presentation assays to compare freshly isolated DC (f-DC) and cultured DC (c-DC). f-DC display a round appearance, whereas c-DC display characteristic long processes. c-DC express much more cell surface major histocompatibility complex (MHC) class II than f-DC. The uptake of colloidal gold-labeled bovine serum albumin (BSA), however, is greater in f-DC, as is the presentation of 65-kD heat shock protein to T cell clones. The most striking discovery is that the majority of MHC class II molecules in both f-DC and c-DC occur in intracellular vacuoles with a complex shape (multivesicular and multilaminar). These MHC class II enriched compartments (MIIC) represent the site to which BSA is transported within 30 min. Although MIIC appear as more dense structures with less MHC class II molecules in f-DC than c-DC, the marker characteristics are very similar. The MIIC in both types of DC are acidic, contain invariant chain, and express the recently described HLA-DM molecule that can contribute to antigen presentation. CD19+ peripheral blood B cells have fewer MIIC and surface MHC class II expression than DCs, while monocytes had low levels of MIIC and surface MHC class II. These results demonstrate in dendritic cells the elaborate development of MIIC expressing several of the components that are required for efficient antigen presentation.
Assuntos
Apresentação de Antígeno , Antígenos/metabolismo , Células Dendríticas/imunologia , Antígenos HLA-D/imunologia , Antígenos de Histocompatibilidade Classe II , Soroalbumina Bovina/metabolismo , Animais , Antígenos/imunologia , Células Sanguíneas/imunologia , Bovinos , Compartimento Celular , Separação Celular , Células Cultivadas , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Endocitose , Antígenos HLA-D/análise , Humanos , Concentração de Íons de Hidrogênio , Membranas Intracelulares/ultraestrutura , Microscopia Imunoeletrônica , Soroalbumina Bovina/imunologia , Vacúolos/imunologia , Vacúolos/ultraestruturaRESUMO
In most human and mouse antigen-presenting cells, the majority of intracellular major histocompatibility complex (MHC) class II molecules resides in late endocytic MHC class II compartments (MIICs), thought to function in antigen processing and peptide loading. However, in mouse A20 B cells, early endocytic class II-containing vesicles (CIIVs) have been reported to contain most of the intracellular MHC class II molecules and have also been implicated in formation of MHC class II-peptide complexes. To address this discrepancy, we have studied in great detail the endocytic pathways of both a human (6H5.DM) and a mouse (A20.Ab) B cell line. Using quantitative immunoelectron microscopy on cryosections of cells that had been pulse-chased with transferrin-HRP or BSA-gold as endocytic tracers, we have identified up to six endocytic subcompartments including an early MIIC type enriched in invariant chain, suggesting that it serves as an important entrance to the endocytic pathway for newly synthesized MHC class II/invariant chain complexes. In addition, early MIICs represented the earliest endocytic compartment containing MHC class II- peptide complexes, as shown by using an antibody against an abundant endogenous class II-peptide complex. The early MIIC exhibited several though not all of the characteristics reported for the CIIV and was situated just downstream of early endosomes. We have not encountered any special class II-containing endocytic structures besides those normally present in nonantigen-presenting cells. Our results therefore suggest that B cells use conventional endocytic compartments rather than having developed a unique compartment to accomplish MHC class II presentation.
Assuntos
Linfócitos B/fisiologia , Compartimento Celular , Endocitose , Antígenos de Histocompatibilidade Classe II/fisiologia , Animais , Anticorpos Monoclonais/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Compartimento Celular/imunologia , Linhagem Celular , Linhagem Celular Transformada , Endocitose/imunologia , Coloide de Ouro/metabolismo , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Peroxidase do Rábano Silvestre/metabolismo , Humanos , Cinética , Ativação Linfocitária , Camundongos , Soroalbumina Bovina/metabolismo , Transferrina/metabolismoRESUMO
Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.
Assuntos
Apresentação de Antígeno , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Vesículas Transportadoras/metabolismo , Animais , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Células Dendríticas/ultraestrutura , Endocitose/fisiologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Lipopolissacarídeos/farmacologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Transporte Proteico , Regulação para CimaRESUMO
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.
Assuntos
Apresentação de Antígeno , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Animais , Compartimento Celular , Linhagem Celular , Membrana Celular/imunologia , Retículo Endoplasmático/imunologia , Genes MHC da Classe II , Antígenos HLA-D/análise , Antígenos HLA-D/genética , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Células L , Camundongos , Microscopia Imunoeletrônica , Frações Subcelulares/imunologia , Células Tumorais CultivadasRESUMO
A previous analysis of yeast ribosomal protein L25 implicated an evolutionarily conserved motif of seven amino acids near the C terminus (positions 120 to 126) in specific binding of the protein to domain III of 26 S rRNA. We analyzed the effect of various point mutations in this amino acid sequence on the capacity of the protein to interact in vitro with its binding site on the rRNA. Most of the mutations tested, including some conservative replacements, strongly reduced or abolished rRNA binding, further supporting a pivotal role for the motif in the specific interaction between L25 and 26 S rRNA. We have also determined the ability of the various mutant L25 species to complement in vivo for the absence of wild-type protein in cells that conditionally express the chromosomal L25 gene. Surprisingly, up to a fivefold reduction in the in vitro binding capacity of L25 is tolerated without affecting the ability of the mutant protein to support (virtually) wild-type rates of 60 S subunit formation and cell growth. Mutations that completely abolish recognition of 26 S rRNA, however, block the formation of 60 S particles, demonstrating that binding of L25 to this rRNA is an essential step in the assembly of the large ribosomal subunit. Using the same combination of approaches we identified an element, located between positions 133 and 139, that is indispensable for the ability of L25 to support a normal rate of 60 S subunit formation, but plays a relatively minor role in determining the rRNA-binding capacity of the protein. In particular, the presence of a hydrophobic amino acid at position 135 was found to be highly important. These results indicate that the element in question is crucial for a step in the assembly of the 60 S subunit subsequent to association of L25 with 26 S rRNA.
Assuntos
Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Análise Mutacional de DNA , Dados de Sequência Molecular , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas Ribossômicas/química , Homologia de Sequência de AminoácidosRESUMO
Langerhans cells are the antigen-presenting cells of the skin, belonging to the family of dendritic cells, which present exogenous antigens in the context of major histocompatibility complex class II (MHC-II) molecules to CD4+ T lymphocytes. Langerhans cells are potent stimulators of different T-cell responses including primary immune responses. Culturing of Langerhans cells leads to modulation of their phenotype and function, as they seem more capable of activating T cells, whereas freshly isolated Langerhans cells are specialized in the endocytosing and processing of antigen. We studied the intracellular distribution of MHC-II molecules and invariant chain (I-chain) in resident Langerhans cells using immunogold labeling of ultrathin cryosections of human epidermis and found the majority of intracellular MHC-II molecules present on membranes of rough endoplasmic reticulum and in so-called MHC-II-enriched compartments (MIIC). The MIIC appeared to be negative for the cation-independent mannose 6-phosphate receptor and positive for the lysosomal enzyme beta-hexosaminidase and acquired the endocytotic tracer, cationized horseradish peroxidase, only after 60 min of internalization. Taken together, these data show that MIIC in Langerhans cells share characteristics with lysosomes. I-chain, which is associated with MHC-II molecules in early biosynthetic compartments, was found in the rough endoplasmic reticulum and Golgi complex, but was detected only occasionally in MIIC and at the plasma membrane. MIIC with internal membrane vesicles showed some I-chain labeling, suggesting that these are newly formed MIIC in which degradation of the I-chain is not yet complete.
Assuntos
Células de Langerhans/ultraestrutura , Lisossomos/genética , Feminino , Genes MHC da Classe II/fisiologia , Antígenos de Histocompatibilidade Classe II/análise , Peroxidase do Rábano Silvestre/análise , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Pele/enzimologia , Pele/imunologiaRESUMO
Application of the fluorescent contact sensitizer Rhodamin B on mouse epidermis was used to study the migration kinetics of Langerhans cells into the draining lymph nodes. The expression of the dendritic cell markers NLDC-145 and MIDC-8 was followed over time to determine the correlation between these markers and Langerhans cell migration. In contrast with its high expression on intraepidermal Langerhans cells, the expression of NLDC-145 on dendritic cells in the draining lymph node was low at 24 h but increased at later times; in contrast, MIDC-8 expression on dendritic cells decreased. Ten days after Rhodamin B application, antigen-bearing Langerhans cells were still present in the epidermis; application of another unrelated contact sensitizer to the epidermis at this time did not lead to migration of these residual Langerhans cells. These results indicate that not all antigen-bearing Langerhans cells migrate from the skin after application of a contact sensitizer, indicating that signals in addition to simple antigen binding are necessary for migration. During this migration from epidermis to lymph nodes Langerhans cells undergo phenotypic changes. The decreased expression of the endosomal antigens MIDC-8 and MOMA-2 correlates with differentiation from predominantly antigen-processing cells to predominantly antigen-presenting cells. The reduced expression of NLDC-145 is discussed in light of a Langerhans cell-independent pathway of antigen transportation from skin to lymph node.
Assuntos
Células Apresentadoras de Antígenos/citologia , Células de Langerhans/citologia , Linfonodos/fisiologia , Animais , Apresentação de Antígeno/genética , Movimento Celular/fisiologia , Drenagem , Feminino , Corantes Fluorescentes/farmacologia , Cinética , Camundongos , Fenótipo , Rodaminas/farmacologia , Pele/efeitos dos fármacosRESUMO
UNLABELLED: The cell biology of intracellular compartments and their interrelationships require detailed knowledge of the proteins that characterize the compartment and that are involved in the communication between them. To date, this can be best achieved by high resolution immunoelectron microscopy (IEM). Other methods, which make use of different embedding materials, such as EPON, Spurr's resin, LR white, or Lowicryls, also allow the detection of immunodeterminants. However, IEM is in many cases the optimum technique owing to better accessibility of the immunodeterminants to antibodies and the absence of denaturing solvents. In our laboratory for IEM we use immunogold labeling on cryosections. This technique combines optimal ultrastructure and good preservation of protein and/or lipid antigens. The ultrathin cryosections (50-100 nm) are prepared from small tissue blocks or cell pellets with a cryo-ultramicrotome. The sections are thawed, and labeled with antibodies, which are visualized with protein A-gold particles (PAG). We recommend the books by Larson (1) and Griffith (2), and chapters in Handbook of Experimental Immunology (3) and METHODS: a Companion to METHODS in Enzymology (4). The present chapter will describe the different aspects of IEM in detail, such as fixation procedures, the processing of samples, ultrathin cryosectioning, and immunogold labeling.
Assuntos
Apresentação de Antígeno/fisiologia , Células Dendríticas/imunologia , Animais , Linfócitos B/imunologia , Transporte Biológico Ativo , Células Dendríticas/ultraestrutura , Endocitose , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Macrófagos/imunologia , Camundongos , Frações Subcelulares/imunologiaAssuntos
Antígenos/metabolismo , Células Dendríticas/imunologia , Células-Tronco Hematopoéticas/imunologia , Animais , Apresentação de Antígeno , Compartimento Celular , Diferenciação Celular , Linhagem Celular , Células Dendríticas/citologia , Dextranos , Endocitose , Fluoresceína-5-Isotiocianato/análogos & derivados , Células-Tronco Hematopoéticas/citologia , Camundongos , Microscopia Imunoeletrônica , Ovalbumina , PinocitoseRESUMO
Exosomes are small membrane vesicles that are secreted by a multitude of cell types as a consequence of fusion of multivesicular late endosomes/lysosomes with the plasma membrane. Depending on their origin, exosomes can play roles in different physiological processes. Maturing reticulocytes externalize obsolete membrane proteins such as the transferrin receptor by means of exosomes, whereas activated platelets release exosomes whose function is not yet known. Exosomes are also secreted by cytotoxic T cells, and these might ensure specific and efficient targeting of cytolytic substances to target cells. Antigen presenting cells, such as B lymphocytes and dendritic cells, secrete MHC class-I- and class-II-carrying exosomes that stimulate T cell proliferation in vitro. In addition, dendritic-cell-derived exosomes, when used as a cell-free vaccine, can eradicate established murine tumors. Although the precise physiological target(s) and functions of exosomes remain largely to be resolved, follicular dendritic cells (accessory cells in the germinal centers of secondary lymphoid organs) have recently been shown to bind B-lymphocyte-derived exosomes at their cell surface, which supports the notion that exosomes play an immunoregulatory role. Finally, since exosomes are derived from multivesicular bodies, their molecular composition might provide clues to the mechanism of protein and lipid sorting in endosomes.
Assuntos
Transporte Biológico , Endossomos/metabolismo , Transdução de Sinais , Vesículas Transportadoras/fisiologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas Foliculares/metabolismo , Células Dendríticas Foliculares/ultraestrutura , Humanos , Lisossomos/metabolismo , Complexo Principal de Histocompatibilidade , Ativação Plaquetária , Transporte Proteico , Reticulócitos/metabolismoRESUMO
Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Proteínas de Transporte/análise , Retículo Endoplasmático/química , Complexo de Golgi/química , Complexo Principal de Histocompatibilidade , Proteínas de Membrana/análise , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Antígenos de Histocompatibilidade Classe I/análise , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Mutagênese , Peptídeos/imunologia , Peptídeos/metabolismo , TransfecçãoRESUMO
Follicular dendritic cells (FDCs) present in lymphoid follicles play a critical role in germinal center reactions. They trap native Ags in the form of immune complexes providing a source for continuous stimulation of specific B lymphocytes. FDCs have been reported to express MHC class II molecules, suggesting an additional role in the presentation of not only native, but also processed Ag in the form of peptide-loaded MHC class II. Adoptive bone marrow transfer experiments have shown that MHC class II molecules are only passively acquired. Up to now the origin of these MHC class II molecules was not clear. Here we show by cryoimmunogold electron microscopy that MHC class II molecules are not present at the plasma membrane of FDCs. In contrast, microvesicles attached to the FDC surface contain MHC class II and other surface proteins not expressed by FDCs themselves. The size and marker profiles of these microvesicles resemble exosomes. Exosomes, which are secreted internal vesicles from multivesicular endosomes, have been shown earlier to stimulate proliferation of specific T lymphocytes in vitro, but their target in vivo remained a matter of speculation. We demonstrate here that isolated exosomes in vitro bind specifically to FDCs and not to other cell types, suggesting that FDCs might be a physiological target for exosomes.
Assuntos
Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Endossomos/imunologia , Endossomos/metabolismo , Antígenos de Histocompatibilidade Classe II/biossíntese , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/ultraestrutura , Sítios de Ligação/imunologia , Linhagem Celular , Membrana Celular/imunologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Separação Celular , Células Dendríticas Foliculares/ultraestrutura , Endossomos/fisiologia , Endossomos/ultraestrutura , Humanos , Ativação Linfocitária , Microscopia Imunoeletrônica , Tonsila PalatinaRESUMO
Evidence suggests that peptide loading onto MHC class II molecules occurs in a specialized late endocytic compartment (MIIC) where HLA-DM predominantly resides and in which MHC class II transiently accumulates before transport to the cell surface. We examined the targeting signals and compartments involved in the intracellular trafficking of human HLA-DM by expressing hybrid molecules comprising the cytoplasmic domain of DMB and luminal and transmembrane domains of CD8 in HeLa cells. A tyrosine-based tetrapeptide motif present in the cytoplasmic domain of DMB targeted hybrid molecules to intracellular vesicles. Mutation of the tyrosine residue to alanine resulted in redistribution of hybrid molecules to the cell surface. Correct intracellular targeting of HLA-DM was crucial for normal function in B cells. Immunoelectron microscopy on ultrathin cryosections showed that CD8-DMB molecules accumulated in late endocytic compartments sharing characteristics with lysosomes, like MHC class II compartments in APCs. Thus far, the exit of DMB from the Golgi complex has not been elucidated. Interestingly, we found that although the mannose 6-phosphate receptor and CD8-DMB contain similar tyrosine signals, no co-localization was observed in the trans-Golgi network, suggesting that these proteins are differentially sorted at this site. Co-transfection of CD8-DMB, HLA-DR alpha, HLA-DR beta, and an invariant chain revealed that HLA-DR molecules accumulated together with CD8-DMB in these lysosomal compartments. The similarity of these lysosomal-like compartments in wild-type and transfected cells suggests that they are part of the normal endocytic pathway in non-APCs.
Assuntos
Endossomos/metabolismo , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidade Classe II , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Antígenos CD8/metabolismo , Compartimento Celular , Endossomos/ultraestrutura , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Antígenos HLA-D/química , Células HeLa , Humanos , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Plasmídeos/química , Receptor IGF Tipo 2/metabolismo , TransfecçãoRESUMO
Association of major histocompatibility complex (MHC) class II molecules with peptides occurs in a series of endocytic vacuoles, termed MHC class II-enriched compartments (MIICs). Morphological criteria have defined several types of MIICs, including multivesicular MIICs, which are composed of 50-60-nm vesicles surrounded by a limiting membrane. Multivesicular MIICs can fuse with the plasma membrane, thereby releasing their internal vesicles into the extracellular space. The externalized vesicles, termed exosomes, carry MHC class II and can stimulate T-cells in vitro. In this study, we show that exosomes are enriched in the co-stimulatory molecule CD86 and in several tetraspan proteins, including CD37, CD53, CD63, CD81, and CD82. Interestingly, subcellular localization of these molecules revealed that they were concentrated on the internal membranes of multivesicular MIICs. In contrast to the tetraspans, other membrane proteins of MIICs, such as HLA-DM, Lamp-1, and Lamp-2, were mainly localized to the limiting membrane and were hardly detectable on the internal membranes of MIICs nor on exosomes. Because internal vesicles of multivesicular MIICs are thought to originate from inward budding of the limiting membrane, the differential distribution of membrane proteins on the internal and limiting membranes of MIICs has to be driven by active protein sorting.
Assuntos
Linfócitos B/fisiologia , Endossomos/fisiologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígeno B7-2 , Exocitose/fisiologia , Antígenos HLA-D/metabolismo , Humanos , Imuno-Histoquímica , Proteínas de Membrana Lisossomal , Fusão de Membrana/fisiologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/análise , Microscopia Imunoeletrônica , Linfócitos T/fisiologiaRESUMO
Cryoimmobilization is regarded as the most reliable method to preserve cellular ultrastructure for electron microscopic analysis, because it is both fast (milliseconds) and avoids the use of harmful chemicals on living cells. For immunolabelling studies samples have to be dehydrated by freeze-substitution and embedded in a resin. Strangely, although most of the lipids are maintained, intracellular membranes such as endoplasmic reticulum, Golgi and mitochondrial membranes are often poorly contrasted and hardly visible. By contrast, Tokuyasu cryosectioning, based on chemical fixation with aldehydes is the best established and generally most efficient method for localization of proteins by immunogold labelling. Despite the invasive character of the aldehyde fixation, the Tokuyasu method yields a reasonably good ultrastructural preservation in combination with excellent membrane contrast. In some cases, however, dramatic differences in cellular ultrastructure, especially of membranous structures, could be revealed by comparison of the chemical with the cryofixation method. To make use of the advantages of the two different approaches a more general and quantitative knowledge of the influence of aldehyde fixation on ultrastructure is needed. Therefore, we have measured the size and shape of endosomes and lysosomes in high-pressure frozen and aldehyde-fixed cells and found that aldehyde fixation causes a significant deformation and reduction of endosomal volume without affecting the membrane length. There was no considerable influence on the lysosomes. Ultrastructural changes caused by aldehyde fixation are most dramatic for endosomes with tubular extensions, as could be visualized with electron tomography. The implications for the interpretation of immunogold localization studies on chemically fixed cells are discussed.
Assuntos
Aldeídos/química , Endossomos/ultraestrutura , Lisossomos/ultraestrutura , Fixação de Tecidos/métodos , Linfócitos B/ultraestrutura , Linhagem Celular Transformada , Criopreservação/métodos , Substituição ao Congelamento , Humanos , Pressão , Tomografia/métodos , Células Tumorais CultivadasRESUMO
Major histocompatibility complex (MHC) class I molecules bind antigenic peptides that are translocated from the cytosol into the endoplasmic reticulum by the transporter associated with antigen processing. MHC class I loading independent of this transporter also exists and involves peptides derived from exogenously acquired antigens. Thus far, a detailed characterization of the intracellular compartments involved in this pathway is lacking. In the present study, we have used the model system in which peptides derived from measles virus protein F are presented to cytotoxic T cells by B-lymphoblastoid cells that lack the peptide transporter. Inhibition of T cell activation by the lysosomotropic drug ammoniumchloride indicated that endocytic compartments were involved in the class I presentation of this antigen. Using immunoelectron microscopy, we demonstrate that class I molecules and virus protein F co-localized in multivesicular endosomes and lysosomes. Surprisingly, these compartments expressed high levels of class II molecules, and further characterization identified them as MHC class II compartments. In addition, we show that class I molecules co-localized with class II molecules on purified exosomes, the internal vesicles of multivesicular endosomes that are secreted upon fusion of these endosomes with the plasma membrane. Finally, dendritic cells, crucial for the induction of primary immune responses, also displayed class I in endosomes and on exosomes.