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J Neefjes

Publications and source records attributed to J Neefjes.

At least 55 records · Page 3Linked to original sources
PubMed1997

The proteasome-specific inhibitor lactacystin blocks presentation of cytotoxic T lymphocyte epitopes in human and murine cells.

We describe the effect of the proteasome specific inhibitor lactacystin on the metabolic stability of influenza nucleoprotein (NP) and on the generation of antigens presented by human and murine class I molecules of the major histocompatibility complex to cytotoxic T lymphocytes (CTL). We show that cells treated with lactacystin fail to present influenza antigens to influenza-specific CTL, but retain the capacity to present defined epitopes expressed as peptides intracellularly by recombinant vaccinia viruses. This block in antigen presentation can be overcome by expressing the viral protein within the lumen of the endoplasmic reticulum, confirming the specificity of lactacystin for cytosolic proteases. We also show that the effect of lactacystin on antigen presentation correlates with the block of breakdown of a rapidly degraded form of the influenza NP linked to ubiquitin. These results demonstrate that proteasome-dependent degradation plays an important role in the cytosolic generation of CTL epitopes.

Acetylcysteine↗
PubMed1997

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics.

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of approximately 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

TAP-translocated peptides specifically bind proteins in the endoplasmic reticulum, including gp96, protein disulfide isomerase and calreticulin.

The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (TAP) associates with peptides in the cytosol and translocates these into the ER lumen. Here, MHC class I molecules bind a subset of these peptides and the remainder is either removed or degraded, or may be retained in the ER in association with other proteins. We have visualized peptide-binding proteins in the ER using radioactive peptides with a photoreactive group. Besides TAP, two proteins were identified as gp96 and protein disulfide isomerase (PDI). Calreticulin, previously found in complex with TAP, only binds glycosylated peptides. In addition, two as yet unidentified, ER luminal glycoproteins (gp120 and gp170) were visualized. The effects of peptide size and sequence on binding to the ER-resident proteins were studied by using partially degenerated peptides with photoreactive side chains. All identified proteins were able to bind peptides within the size range of peptides translocated by TAP, from 8 to more than 20 amino acids. Whereas PDI associated with all peptides tested, gp96 and gp120 showed a clear sequence preference for non-charged amino acids at positions 2 and 9 in 9mer peptides. Thus various ER proteins, other than the MHC class I heterodimer and TAP, are able to interact with peptides albeit with a different substrate selectivity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

High-resolution density gradient electrophoresis of proteins and subcellular organelles.

Following a concept developed by Bier et al. (Electrophoresis 1993, 14, 1011-1018), binary mixtures of amphoteric buffers with low conductivity and a good buffering capacity permit rapid rate zonal separation of proteins on a density gradient electrophoresis apparatus (7 cm, x 2.2 cm). At pH 8.66 and 250 V, beta-lactoglobulin (Mr 36600) was separated into the A and B isoforms within 44 min; human transferrin (Mr 76000-81000) was separated into its sialylated glycoforms and carbonic anhydrase (Mr 30000) separated into its isoenzymes. From these results we arrive at the term high-performance density gradient electrophoresis. Compartments belonging to the endosomal system were separated by density gradient electrophoresis. Early endosomes, recycling vesicles, intermediate endosomes, late endosomes and lysomes became well-separated after 80 min at 10 mA using [125I]transferrin and horseradish peroxidase as reporter molecules in pulse-chase regimes. Mixtures of Bier buffers and standard electrophoresis media permitted very short separation times (19 min at 10 mA) for the endosomal compartments. Concommittantly, endoplasmic reticulum and proteasomes were well resolved.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Hierarchy of Epstein-Barr virus-specific cytotoxic T-cell responses in individuals carrying different subtypes of an HLA allele: implications for epitope-based antiviral vaccines.

Major histocompatibility complex class I-restricted Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) in healthy virus carriers constitute a primary effector arm of the immune system in controlling the proliferation of virus-infected B cells in vivo. These CTLs generally recognize target epitopes included within the latent antigens of the virus. For example, CTLs from HLA B44+ healthy virus carriers often recognize peptide EENLLDFVRF [corrected] from EBV nuclear antigen 6. However, the strength of this response directly correlates with the HLA B44 subtype expressed by the individual donor. Indeed, HLA B*4405+ virus carriers consistently show a very high frequency of CTL precursors for the EENLLDFVRF [corrected] epitope, while a much weaker response is seen in HLA B*4403+ and HLA B*4402+ individuals. This disparity is not due to an intrinsic difference in the CTLs generated by individuals carrying different subtypes of HLA B44. In fact, virus-specific CTLs recognize EENLLDFVRF [corrected] peptide-sensitized HLA B*4405+ target cells more efficiently than B*4402+ or B*4403+ target cells irrespective of the HLA B44 subtype expressed by the donors from whom these effectors were isolated. This effect is evident whether the CTL epitope is endogenously processed or exogenously presented. In addition, a comparison of the intracellular transport kinetics of different B44 subtypes revealed that the B*4405 allele is rapidly assembled and arrives in the trans-Golgi compartment at a faster rate than B*4402 or B*4403. Based on these results, we propose that HLA class I alleles that are capable of binding peptides more efficiently from the intracellular pool, and are rapidly assembled and transported, may confer a protective advantage against viral infection.

Alleles↗
PubMed1996

Point mutations in the alpha 2 domain of HLA-A2.1 define a functionally relevant interaction with TAP.

BACKGROUND: Glycoproteins encoded by the major histocompatibility complex class I region (MHC class I) present peptide antigens to cytotoxic T cells (CTLs). Peptides are delivered to the site of MHC class I assembly by the transporter associated with antigen processing (TAP), and cell lines that lack this transporter are unable to present endogenous antigens to CTLs. Although it has been shown that a fraction of newly synthesized class I molecules are in physical association with TAP, it is not known whether this interaction is functionally relevant, or where on the class I molecule the TAP binding site might be. RESULTS: C1R cells transfected with a mutant HLA-A2.1 heavy chain (HC), where threonine at position 134 in the alpha 2 domain is changed to lysine (T134K), are unable to present endogenous antigens to CTLs. We have studied the biochemistry of this mutant in C1R cells, and found that a large pool of unstable empty class I HC-beta 2m (beta-2 microglobulin) heterodimers exist that are rapidly transported to the cell surface. The T134K mutant seemed to bind peptide antigens and assemble with beta 2m as efficiently as wild-type HLA-A2.1. However, we show here that the inefficiency with which T134K presents intracellular antigen is associated with its inability to interact with the TAP heterodimer. CONCLUSIONS: These experiments establish that the class I-TAP interaction is obligatory for the presentation of peptide epitopes delivered to the endoplasmic reticulum (ER) by TAP. Wild-type HLA-A2.1 molecules in TAP-deficient cells are retained in the ER, whereas T134K is rapidly released to the cell surface, but is unstable, suggesting a role for the TAP complex as an intracellular checkpoint that only affects the release of class I molecules with stably bound peptide ligands.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Allele-specific differences in the interaction of MHC class I molecules with transporters associated with antigen processing.

MHC class I molecules bind peptides that are translocated from the cytosol into the endoplasmic reticulum by the peptide transporter associated with antigen processing (TAP). Class I heterodimers have been shown to associate with TAP and are released when loaded with peptide. Here, we show the existence of two pools of class I heterodimers, one associated with TAP and one that is free. Whereas the free pool is recognized by the class I-specific Ab W6/32, the TAP-associated pool is not. Analysis of several class I alleles shows binding to TAP with different efficiencies, even at the earliest time points of MHC class I assembly. Most HLA-A and -C alleles tested interacted efficiently with TAP, whereas a considerable number of HLA-B alleles associated very inefficiently or not at all with TAP. This was also observed in cells with nonfunctional TAP. Sequence comparison of the different class I alleles allowed the definition of amino acids in the peptide binding groove that might be involved in TAP association. Binding of peptides to two different pools of class I heterodimers may ensure efficient peptide association in an environment where peptides have a short life span.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

Two Listeria monocytogenes CTL epitopes are processed from the same antigen with different efficiencies.

Listeria monocytogenes is an intracellular bacterium that elicits MHC class I-restricted CTL in infected mice. A major CTL specificity is the nonamer peptide p60 217-225, which is derived from the bacterial murein hydrolase p60 and presented by the H-2Kd MHC class I molecule. In this report, we identify a second H-2Kd presented peptide, encompassing residues 449-457 of p60, that is detected by L. monocytogenes-specific CTL. Both p60-derived CTL epitopes are good competitors for H-2Kd binding and TAP (transporter associated with Ag processing) transport. CTL clone WP11.12 lyses L. monocytogenes infected cells and recognizes naturally processed p60 449-457 acid eluted from L. monocytogenes-infected macrophages. Although both epitopes derive from the same Ag and bind the same allelic form of MHC class I, quantitative analysis reveals that the amount of p60 449-457 in infected cells is approximately 10-fold greater than the amount of p60 217-225. Shuffling p60 217-225 into position 449-457 decreases its processing efficiency, indicating that the large number of p60 449-457 epitopes cannot be entirely attributed to epitope-flanking sequences. Our findings indicate that CTL epitopes can be processed from Ags with markedly different kinetics and efficiencies. Intrinsic qualities of an epitope and its location within a protein influence the efficiency of Ag processing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1996

A conditionally immortalized dendritic cell line which differentiates in contact with T cells or T cell-derived cytokines.

A conditionally immortalized dendritic cell line was established from bone marrow of mice transgenic for a thermolabile mutant of the SV40 large T antigen under the control of the class I Kb promoter. At the permissive temperature of 33 degrees-37 degrees C, the line divides in the absence of granulocyte/macrophage colony stimulating factor. It shares a number of cell surface markers with bone marrow macrophages, but unlike macrophages, is constitutively major histocompatibility complex (MHC) class II+, negative for nonspecific esterase and unable to phagocytose sheep red blood cells. The cells show characteristic dendrites, an abundance of acidic vesicles and are highly active in endocytosis. If maintained at 33 degrees C, the dendritic cell line processes and presents exogenous protein to MHC class II-restricted T cell hybrids and acts as potent mixed lymphocyte reaction stimulator, but fails to activate naive, resting T cells. Transfer to 39 degrees C arrests growth and results in up-regulation of surface markers such as B7.1, CD40 and intercellular adhesion molecule-1. Further up-regulation of cell surface markers and acquisition of functional maturity occur following contact with T cells and their cognate antigen or in culture with a cytokine mixture derived from activated T cells.

Animals↗
PubMed1996

High resolution density gradient electrophoresis of cellular organelles.

A density gradient electrophoresis apparatus made of Perspex was constructed, with a separation column (7 x 2.2 cm) containing a 0-5% linear Ficoll gradient. The useful separation path is 6 cm. A specially designed gradient mixer is described which fits over the application cone. This cone permits precise gradient and sample introduction as well as undisturbed fractionation after electrophoresis. A bottom circular palladium cathode is separated hydrodynamically but not electrically from the density gradient by a cellophane membrane, merely secured by an O-ring. The top circular platinum anode allows for upward electrophoresis (80-100 min at 10 mA). The markedly higher resolution of subcellular organelles was compared with separations obtained earlier with a small, but much more difficult to fabricate, prototype. Moreover, ease of manipulation was greatly improved. A wide separation distance was obtained between plasma membrane, endoplasmatic reticulum as well as between two populations of lysosomes. Even early, middle, and late endosomes could be separated with high resolution. Soluble isoenzymes could be separated as well and were far away from the vesicle-enclosed enzymes.

Cellulose↗
PubMed1996

Association between HLA-DM and HLA-DR in vivo.

The exchange of HLA class II-associated invariant chain peptides (CLIP) for cognate peptide is catalyzed by HLA-DM under acidic conditions in vitro by an unknown mechanism. Here, we show an association between HLA-DM and HLA-DR in vivo by coprecipitation of the two heterodimers. The association is favored at low pH and in the nonionic detergent digitonin. Most DM-DR complexes are isolated from dense subcellular fractions. Recovery of HLA-DM by the conformation-dependent DR3 monoclonal antibody 16.23 suggests an association with HLA-DR heterodimers beyond the stage at which CLIP is released. The additional N-linked glycan on mutant DR3 molecules isolated from the 10.24.6 cell line, which interferes with DM-enhanced CLIP release from DR3 in vitro, also affects the DM-DR interaction.

HLA-D Antigens↗
PubMed1996

A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation.

CTL epitope (KSPWFTTL) encoded by AKV/MCF type of murine leukemia virus (MuLV) differs from the sequence in Friend/Moloney/Rauscher (FMR) type in one residue (RSPWFTTL). CTL experiments indicated defective processing of the FMR peptide in tumor cells. Proteasome-mediated digestion of AKV/MCF-type 26-mer peptides resulted in the early generation and higher levels of epitope-containing fragments than digestion of FMR-type peptides, explained by prominent cleavage next to R in the FMR sequence. The fragments were identified as 10- and 11-mer peptides and were efficiently translocated by TAP. The naturally presented AKV/MCF peptide is the 8-mer, indicating ER peptide trimming. In conclusion, a single residue exchange can cause CTL epitope destruction by specific proteasomal cleavage.

ATP-Binding Cassette Transporters↗
PubMed1996

Direct vesicular transport of MHC class II molecules from lysosomal structures to the cell surface.

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 beta chain with the Green Fluorescent Protein (GFP) coupled to its cytoplasmic tail (beta-GFP) in class II-expressing Mel JuSo cells. This modification of the class II beta chain does not affect assembly, intracellular distribution, and peptide loading of the MHC class II complex. Transport of the class II/ beta-GFP chimera was studied in living cells at 37 degrees C. We visualize rapid movement of acidic class II/beta-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.

Biological Transport↗
PubMed1996

HLA-DM and MHC class II molecules co-distribute with peptidase-containing lysosomal subcompartments.

MHC class II molecules associate with peptides in the endocytic pathway. Different endosomal locations for peptide loading of class II molecules, varying from early endosomes (EE) to lysosomes, have been assigned on the basis of subcellular fractionation experiments. We have determined the intracellular location of HLA-DM, a molecule that supports peptide loading of class II molecules, by separating vesicles from the melanoma cell line Mel JuSo on the basis of buoying density and surface charge. In both fractionations, HLA-DM co-fractionated with a lysosomal compartment containing beta-hexosaminidase (beta-hex) activity and not with endosomes. Further analysis showed that HLA-DM mainly co-fractionated with a sub-lysosomal structure characterized by a relative low density and containing both pro- and mature cathepsin D and MHC class II molecules. Fluid phase markers first enter this compartment before entering high-density lysosomes that contain exclusively mature cathepsin D, some HLA-DM and no detectable MC class II molecules. Finally we determined the intracellular location of neutral and acidic peptidases. Whereas neutral peptidase activity was detected in the endoplasmic reticulum and/or plasma membrane fractions, acidic peptidase activity exclusively migrated at the position of HLA-DM containing lysosomal vesicles. Our results show that class II molecules co-migrate with HLA-DM, pro- and mature cathepsin D, beta-hex and acidic peptidase activity. HLA-DM, cathepsin d and class II molecules were not observed at the position of EE. Our data suggest that HLA-DM-mediated peptide loading of class II molecules occurs in a lysosomal subcompartment.

Cathepsin D↗
PubMed1995

Analysis of the fine specificity of rat, mouse and human TAP peptide transporters.

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Restriction of self-antigen presentation to cytolytic T lymphocytes by mouse peptide pumps.

Transport of an immunogenic self-peptide from the second domain of the mouse major histocompatibility complex (MHC) H-2Kd class I molecule is blocked at the TAP1-TAP2 peptide pump level due to its amino acid sequence and is not presented to cytolytic T lymphocytes (CTL). We demonstrate that first, TAP1-TAP2 pumps can restrict antigen presentation by selecting against internal peptide motifs which are not involved in peptide binding to MHC class I molecules. Second, some molecules targeted to the endoplasmic reticulum are processed for MHC class I presentation in the cytosol. Third, some abundantly expressed immunogenic self-peptides are cytosolically sequestered. The advantage for the host, in terms of the peripheral T cell repertoire is that the spared CTL can be used to recognize foreign antigens. It is, however, anticipated that this advantage will be exploited by pathogens to evade immune surveillance by similar strategies.

ATP-Binding Cassette Transporters↗
PubMed1994

Trimming of TAP-translocated peptides in the endoplasmic reticulum and in the cytosol during recycling.

Cytosolic peptides are translocated to the endoplasmic reticulum (ER) lumen by the transporters associated with antigen processing (TAP), where major histocompatibility complex (MHC) class I molecules associate with peptides of about 8-10 amino acids. TAP translocates peptides of 9-13 amino acids with the highest relative affinity but also longer and shorter peptides. The fate of the peptides that fail to associate with class I molecules because of incorrect sequence or length, is unknown. Here we show that the bulk of the translocated peptides are rapidly released from the ER by a mechanism that requires adenosine triphosphate (ATP) and that could not be inhibited by GTP gamma S. TAP does not appear to be involved in this process. Whereas free peptides are slowly trimmed in the ER lumen, they are rapidly degraded in the cytosol. A fraction of the peptides released from the ER escapes complete degradation in the cytosol and recycles back to the ER in a TAP-dependent fashion. These results suggest that peptides that are too long for binding to class I molecules in the ER can be trimmed further in the ER lumen or, alternatively, can be transported back to the cytosol where a fraction of the peptides is trimmed to a size suitable for association to MHC class I molecules and recycles back to the ER.

ATP-Binding Cassette Transporters↗