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J J Neefjes

Publications and source records attributed to J J Neefjes.

At least 19 recordsLinked to original sources
PubMed1997

Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells.

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.

Amino Acid Sequence↗
PubMed1997

Mannose receptor mediated uptake of antigens strongly enhances HLA-class II restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.

Antigen Presentation↗
PubMed1997

Interleukin-10 down-regulates MHC class II alphabeta peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling.

Interleukin-10 (IL-10) inhibits antigen-specific T cell responses when human monocytes are used as antigen-presenting cells. This is correlated with a down-regulation of MHC class II molecules on the surface of the monocyte. Here we show that IL-10 does not affect MHC class II transcription, polypeptide synthesis, subunit assembly, or antigenic peptide loading. Instead, newly synthesized mature MHC class II molecules are localized to the MHC class II loading compartment but are prevented from reaching the plasma membrane. In addition, treatment of monocytes with IL-10 leads to an accumulation of internalized MHC class II complexes in intracellular vesicles. These results indicate that IL-10 affects antigen presentation by regulating MHC exocytosis and recycling.

Biological Transport↗
PubMed1996

Assembled pre-B cell receptor complexes are retained in the endoplasmic reticulum by a mechanism that is not selective for the pseudo-light chain.

The pre-B cell receptor (BCR) complex, consisting of micro heavy chain, a pseudo-light chain, and the Mb-1/B29 heterodimer, directs the transition to the mature B cell stage. Plasma membrane expression of the pre-BCR is extremely low, despite its presumed signaling function. We have compared assembly and intracellular transport of the pre-BCR complex with that of the BCR complex in mature B cells. Synthesis and assembly rate of pre-BCR and BCR components are comparable. However, the pre-BCR is subject to a highly efficient retention mechanism, which only allows exit of a few percent of the complexes from the endoplasmic reticulum (ER). This small transported pool of pre-BCR complexes is significantly enriched for protein-tyrosine kinase activity, as compared with the ER-localized receptor pool. Accordingly, the Src-related tyrosine kinase Lyn was found in the transported glycoprotein fraction but not in association with ER-localized glycoproteins. Upon introduction of a conventional light chain into pre-B cells, plasma membrane receptor levels increased, but the efficiency of intracellular transport of the receptor complex was not restored to that in mature B cells. This indicates that the ER retention mechanism is not selective for the pseudo-light chain and may be inherent to pre-B cells. We propose that this retention mechanism contributes to the regulation of pre-BCR-mediated signal transduction.

Biological Transport↗
PubMed1996

Translocation of long peptides by transporters associated with antigen processing (TAP).

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8-11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8-12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT.. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10(5) peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Overexpression of the ABC transporter TAP in multidrug-resistant human cancer cell lines.

Multidrug resistance (MDR) to anti-cancer drugs has been associated with the overexpression of P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP), both being members of the ATP-binding cassette (ABC) superfamily of transporters. We investigated whether in addition to P-gp and MRP, another ABC transporter, the transporter associated with antigen processing (TAP), is associated with MDR. TAP plays a major role in MHC class I-restricted antigen presentation by mediating peptide translocation over the endoplasmic reticulum membrane. TAP1 and P-gp share a significant degree of homology among their transmembrane domains, which are thought to be the primary determinants of substrate specificity, and both can apparently mediate the translocation of peptides. Using immunocytochemistry and Western blot, TAP was overexpressed in parallel with MHC class I in several MDR human cancer cell lines. TAP was overexpressed more frequently in MRP-positive MDR cell lines (three out of three) than in P-gp positive MDR cells (two out of five). Reversal of resistance resulted in a decrease in TAP levels. Transfection of the TAP genes into TAP-deficient lymphoblastoid T2 cells conferred mild resistance to etoposide, vincristine and doxorubicin (2- to 2.5-fold). Furthermore, etoposide and vincristine inhibited TAP-dependent peptide translocation to the endoplasmic reticulum. Collectively, our results suggest that TAP may modestly contribute to the MDR phenotype, in particular in MRP- overexpressing MDR cells. Further insight into the role of TAP in MDR will require the study of other transfectants, as well as the investigation of TAP expression in P-gp and MRP-negative MDR cancer cell lines.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Major histocompatibility complex class II compartments in human B lymphoblastoid cells are distinct from early endosomes.

In human B lymphoblastoid cell lines, the majority of major histocompatibility complex (MHC) class II heterodimers are located on the cell surface and in endocytic compartments, while invariant chain (Ii)-associated class II molecules represent biosynthetic intermediates which are present mostly in the endoplasmic reticulum and Golgi complex. To investigate the origin of the MHC class II-positive compartments and their relation to early endosomes, the intracellular distribution of MHC class II molecules and Ii in relation to endocytic tracers was studied in human lymphoblastoid B cells by immunoelectronmicroscopy on ultrathin cryosections. Cross-linking of surface immunoglobulins, followed by a brief period of internalization of the immune complexes, did not alter the intracellular distribution of MHC class II molecules. While early endosomes were abundantly labeled for the cross-linked immunoglobulins, < 1% of total MHC class II molecules were detectable in early endosomes. MHC class II- and Ii-positive structures associated with the trans-Golgi network can be reached by endocytosed bovine serum albumin (BSA)-gold conjugates after 30 min of internalization. Prolonged exposure to BSA-gold allowed visualization of later endocytic compartments, in which a progressive loss of Ii was observed: first the lumenal portion, and then the cytoplasmic portion of Ii escaped detection, culminating in the formation of MHC class II-positive compartments (MIIC) devoid of Ii. The loss of Ii also correlated with a transition from a multivesicular to a multilaminar, electron-dense MIIC. The intracellular compartments in which class II molecules reside (MIIC) are therefore a heterogeneous set of structures, part of the later aspects of the endocytic pathway.

Antigen-Presenting Cells↗
PubMed1995

MHC class II compartments and the kinetics of antigen presentation in activated mouse spleen dendritic cells.

MHC class II (MHC-II) molecules bind fragments of exogenous Ags in an intracellular endocytotic compartment. In view of divergent data on the MHC-II distribution in different cell lines, it was of interest to localize MHC-II molecules in a natural and the most potent APC type, the dendritic cell (DC). By using immunogold labeling of ultrathin cryosections of cultured mouse spleen DC, we found that MHC-II molecules were present abundantly at the plasma membrane and in intracellular compartments containing internal membrane vesicles and/or membrane sheets. The majority of these compartments was situated late in the endocytotic route, as demonstrated by the late appearance (after a lag of 30 min) of internalized exogenous tracer. These compartments contained the lysosomal enzymes cathepsin D and beta-hexosaminidase, but lacked the late endosomal marker cation-dependent mannose-6-phosphate receptor. We conclude that most of the intracellular MHC-II molecules in cultured spleen DC reside in a compartment with (pre)lysosomal characteristics, resembling the so-called MHC-II-enriched compartments (MIIC), originally described in B cells. We also investigated whether the presence of MHC-II molecules in endocytotic compartments was related to the kinetics of Ag processing and presentation by these cells. Pulse-chase endocytosis experiments with hen egg lysozyme (HEL) as a model Ag showed that activated spleen DC were able to efficiently process and present this Ag to an HEL-specific T hybridoma cell line. However, presentation started only after a lag of 2 h and was maximal after 6 h. The difference in time between the arrival of Ag in proteolytic endocytotic compartments, in particular MIIC, and effective Ag presentation is discussed in the context of DC maturation.

Animals↗
PubMed1995

Major differences in transporter associated with antigen presentation (TAP)-dependent translocation of MHC class I-presentable peptides and the effect of flanking sequences.

The MHC-encoded transporter associated with Ag presentation (TAP) translocates peptides from the cytosol to the ER lumen, where association with MHC class I molecules occurs. The MHC class I/peptide complex is subsequently transported to the cell surface for presentation to CD8+T cells. We studied TAP-dependent translocation of defined MHC class I presentable murine peptides by competition for translocation of a radiolabeled model peptide, to address whether efficient peptide presentation by MHC class I molecules is preceded by equal efficient peptide translocation by TAP. Surprisingly, we observed that four immunodominant viral peptides of 16 peptides tested were very inefficiently transported by TAP. Inefficient translocation could be overcome by substitution of a proline residue present at position 3 in the peptides. Furthermore, addition of natural flanking amino acids directly surrounding a poorly transported peptide could considerably improve translocation by TAP. Our data suggest that some peptides are efficiently transported by TAP in their optimal size for MHC class I binding, whereas other peptides are transported as larger peptide fragments that need further trimming in the ER for MHC class I binding.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Accumulation of HLA-DM, a regulator of antigen presentation, in MHC class II compartments.

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.

Animals↗
PubMed1994

Peptide size selection by the major histocompatibility complex-encoded peptide transporter.

The major histocompatibility complex (MHC)-encoded heterodimeric TAP1/TAP2 transporter (TAP) translocates cytosolic peptides into the lumen of the endoplasmic reticulum (ER), where peptides of 8 to 11 amino acids long associate with MHC class I molecules. We have studied the selectivity of peptide translocation by TAP in streptolysin O-permeabilized cells using glycosylatable, radioiodinated model peptides to detect import into the ER lumen. TAP-dependent translocation of a radiolabeled nonamer peptide was most efficiently inhibited by unlabeled 9- to 11-mer peptides. Peptides between 7 and 40 amino acids long all could inhibit transport, the longer peptides being least effective. Also, peptides shorter than eight amino acids were inefficiently translocated. The use of directly labeled length variants in translocation assays and TLC analysis of the transported material revealed two pathways for translocation: short peptides (7 to 13 amino acids long) were translocated without prior modification. In contrast, transport of longer peptides was not effective. Instead such peptides were clipped by cytosolic peptidases before efficient transport. Our data suggest that TAP preferentially translocates peptides of appropriate length for class I binding. Furthermore, TAP-translocated peptides were rapidly released from the ER unless they were trapped there by being glycosylated or by binding to MHC class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Selectivity of MHC-encoded peptide transporters from human, mouse and rat.

Major histocompatibility complex (MHC) class I molecules present peptides from degraded intracellular antigens to CD8+ T cells. These peptides are translocated in an ATP-dependent fashion into the lumen of the endoplasmic reticulum (ER) for binding to class I molecules by means of the MHC-encoded transporters associated with antigen processing, TAP1 and TAP2. These are members of a family of proteins containing an ATP-binding cassette and form heterodimers in the ER membrane. Defects in the genes encoding TAP1 or TAP2 account for impaired class I assembly and antigen presentation in several human and rodent cell lines. Whereas MHC class I molecules select peptides according to binding motifs, it is not clear to what extent the TAP1-TAP2 transporters have peptide sequence and length specificity. Previous studies of the rat MHC class I molecule RT1Aa, suggested a specific conveyance of peptides by rat TAP1-TAP2. Here we substitute the amino- and carboxy-terminal and the penultimate amino-acid residues of model peptides to show that these residues influence the efficiency of transport. Human TAP and rat TAPa translocated peptides with hydrophobic and basic C termini, whereas mouse TAP and rat TAPu preferred peptides with hydrophobic C termini. This pattern correlates with the predominant peptide binding profiles of mouse and human class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Targeting major histocompatibility complex class II molecules to the cell surface by invariant chain allows antigen presentation upon recycling.

We studied the functional consequences of targeting class II molecules to either the cell surface or to endocytic structures by expressing HLA-DR1 in human kidney cells in the presence or absence of different forms of the invariant chain (Ii). Transfectants expressing class II molecules in the absence of Ii present influenza virus efficiently and co-expression of full length Ii does not further increase antigen presentation. Chimeric Ii containing the cytoplasmic domain of the transferrin receptor (Tfr-Ii) delivers class II molecules associated with Tfr-Ii to endosomal compartments, but this does not result in efficient antigen presentation. When class II molecules are targeted to the cell surface by Ii lacking either 15 (delta 15Ii) or 23 (delta 23Ii) amino acids from the cytoplasmic domain, a fraction of free class II molecules is also observed. Whereas delta 15Ii did not affect antigen presentation by class II molecules, delta 23Ii inhibited, but did not abrogate, the response. We show that class II molecules expressed in the presence of delta 23Ii can be internalized, followed by degradation of delta 23Ii and return of free class II alpha beta heterodimers to the cell surface. A fraction of the resulting free class II molecules is sodium dodecyl sulfate stable, indicating that internalization and reappearance of class II molecules at the cell surface can be an alternative route for antigen presentation. In all transfectants, class II molecules were found in endocytic compartments that labeled for CD63 and resembled the multilaminar MIIC compartments found in B cell lines. Ii is not required for endosomal targeting of class II molecules. The number of class II molecules observed in the multilaminar compartments correlates with the efficiency of antigen presentation.

Antigen Presentation↗
PubMed1994

Peptide selection by MHC-encoded TAP transporters.

With the discovery of MHC-encoded peptide transporters (TAP) came the identification of a new class of molecules within the immune system. TAP belongs to a large family of ATP-binding, multimembrane-spanning transporters that are expressed in a diversity of cells, from prokaryotic to mammalian, and show specificity for a variety of different substrates. TAP represents the solution to a major topological problem in immunology, namely the translocation of peptides, generated by cytosolic degradation of antigens, into the lumen of the endoplasmic reticulum where they associate with newly synthesized MHC class I molecules. A novel assay allows us to determine the requirements for the TAP-mediated peptide transport. First results indicate that TAP preselects peptides according to sequence and length in a way that is compatible with the characteristics of peptides isolated from class I molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

Folding and assembly of major histocompatibility complex class I heterodimers in the endoplasmic reticulum of intact cells precedes the binding of peptide.

Major histocompatibility complex (MHC) class I molecules are heterotrimers consisting of a polymorphic H chain, beta 2-microglobulin (beta 2m) and peptide. Peptides are thought to associate early during biosynthesis but the order of assembly of class I molecules from their component subunits in intact cells is not settled. We have studied the assembly of MHC class I molecules in intact cells with or without peptide transporters. MHC class I H chain/beta 2m heterodimers can be efficiently recovered only 4 min after translation and are preceded by a folding intermediate. Approximately 2 min after their formation, the class I heterodimers are loaded with peptides resulting in stable class I heterotrimers. In these in vivo studies, no evidence was obtained that peptide binding to the H chain preceded the association with beta 2m. In contrast, nonassembled class I H chains could be recovered immediately after translation, but this pool did not participate in the formation of class I molecules.

Biological Transport↗