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The Journal of Immunology, 2007, 178: 7932-7942.
Copyright © 2007 by The American Association of Immunologists, Inc.
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A Transporter Associated with Antigen-Processing Independent Vacuolar Pathway for the MHC Class I-Mediated Presentation of Endogenous Transmembrane Proteins

Neeraj Tiwari1,*, Natalio Garbi*, Thomas Reinheckel{dagger}, Gerhard Moldenhauer*, Günter J. Hämmerling* and Frank Momburg2,*

* Department of Molecular Immunology, German Cancer Research Center, Heidelberg, Germany; and {dagger} Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University, Freiburg, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
MHC class I molecules present peptides derived from the ectodomains of endogenous transmembrane proteins; however, the processing of these Ags is incompletely understood. As model transmembrane Ags we investigated the processing of MHC-I-derived fusion proteins containing the N-terminally extended Kb-restricted OVA epitope SIINFEKL in the extracytoplasmic domain. In TAP-deficient, nonprofessional APCs, the epitope was cleaved out of various sequence contexts and presented to T cells. Ag presentation was inhibited by acidophilic amines and inhibitors of the vacuolar proton pump, indicating processing in endosomes. Endosomal aspartic-type cathepsins, and to some extent also the trans-Golgi network protease furin, were involved in processing. Clathrin-dependent and independent internalization from the cell surface targeted MHC-I fusion proteins to early and late endosomes, where SIINFEKL/Kb complexes were detected by immunofluorescence microscopy. Targeting of MHC-I fusion proteins to processing compartments was independent of sequence motifs in the cytoplasmic tail. Not only TAP-deficient cells, but also TAP-competent APCs used the vacuolar pathway for processing of MHC-I fusion proteins. Thus, endosomal processing of internalized endogenous transmembrane proteins represents a novel alternate pathway for the generation of MHC-I-binding peptides.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In the conventional pathway leading to MHC class I (MHC-I)3 peptide loading, cytosolic protein Ags are digested by proteasomes into oligopeptides (1, 2). Proteasomal products are translocated into the lumen of the endoplasmic reticulum (ER) by the ATP-dependent TAP peptide transporter for binding to MHC-I molecules (3, 4). In the cross-presentation pathway, peptides are derived from exogenous Ags after internalization into the endocytic route (5, 6). Internalized protein Ags can be shuttled from phagosomes to the cytosol, where MHC-I ligands are produced in a proteasome- and TAP-dependent fashion (6, 7). Alternatively, MHC-I-binding peptides can be processed out of exogenous soluble and particulate Ags in endocytic vesicles with no contribution of proteasomes and TAP (5, 8). Similar to MHC-II molecules, recycling MHC-I molecules can assemble with peptides in the acidic environment of late endosomes (9, 10). Cysteine proteases such as cathepsin S have been reported to contribute to the generation of MHC-I peptides in endosomes (11, 12).

The generation of MHC-I ligands from endogenously synthesized transmembrane proteins is incompletely understood. Misfolded transmembrane proteins may be retrotranslocated from the ER membrane to the cytosol and engage a proteasome/TAP-dependent pathway (13, 14, 15). However, MHC-I epitopes are processed out of protein ectodomains also in TAP-deficient cells (16, 17, 18, 19). To date, only the trans-Golgi network (TGN) proprotein convertase furin has been demonstrated to cleave a particular secreted viral Ag (20). Because of its restricted cleavage motif, furin cannot account for the final processing of all known TAP-independent MHC-I peptides. Intracellular compartments and proteases involved in this alternative processing pathway thus remain poorly characterized to date.

In this study, we provide evidence for a major role of an endosomal pathway for the generation of MHC-I-binding peptides that are processed out of endogenous transmembrane Ags after internalization from the cell surface. This pathway is operative in both TAP-deficient and TAP-sufficient APCs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Chemicals

Lactacystin, folimycin, and chlorophenol red-beta-D-galactoside (CPRG) were from Merck Biosciences. Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Dec-RVKR-CMK) and Boc-Arg-Val-Arg-Arg-7-amido-4-methylcoumarine (Boc-RVRR-AMC) were purchased from Bachem. All other chemicals were from Sigma-Aldrich. Peptides were synthesized by Fmoc chemistry by the peptide synthesis core facility of the German Cancer Research Center and were >95% pure by HPLC and mass spectrometry. Peptides were prepared as 10 mM stock solutions in DMSO.

Cell lines

MCB6TAP–/– is a H-2b+ methylcholanthrene-induced fibrosarcoma line generated in the TAP1–/– mouse (21) and was provided by H.-G. Ljunggren (Karolinska Institute, Stockholm, Sweden). MC6 and MC4 are methylcholanthrene-induced fibroblast lines from tapasin+/+ and tapasin–/– C57BL/6 mice (22), respectively. Kb-transfected P815 mastocytoma cells were from B. Arnold (German Cancer Research Center, Heidelberg, Germany). Ltk– is a fibroblast line (H-2k). TAP-deficient RMA-S T lymphoma cells (H-2b) have been described (23). RMA cells (H-2b) expressing OVA in the cytoplasm were a gift from L. Eisenbach (Weizman Institute, Rehovot, Israel). Mouse embryonal fibroblast lines (H-2b+) derived from cathepsin B–/– and cathepsin D–/– mice have been described (24).

Abs and Western blots

The Kb- and Kd-specific mAbs Y3 and 15.5.5S have been described (25, 26). mAb 25.D1-16, provided by R. Germain (National Institutes of Health, Bethesda, MD), recognizes Kb molecules complexed with SIINFEKL peptide (27). The anti-His6 mAb 13/45/31a and the anti-flag mAb 4E11-18 were gifts from H. Zentgraf (German Cancer Research Center, Heidelberg, Germany). Rabbit polyclonal Ab specific for mouse TAP2 (mTAP2.688) (22) has been described. Rabbit anti-mouse tapasin antiserum 2668 was donated by T. Hansen (Washington University, St. Louis, MO). Rabbit anti-mouse ERAP1 polyclonal Ab was raised against the N-terminal keyhole limpet hemocyanin-coupled ERAP1 peptide QNSDIESLKASNGC by Eurogentec. Western blot analyses of ERAP1-transfected cells as well as TAP1–/– and tapasin–/– fibroblasts were performed exactly as described (22, 28). Rabbit anti-EEA-1 and rat anti-LAMP-1 polyclonal Abs were from BD Biosciences. Anti-CD63 mAb C2418-09 was from USBiological. Rabbit anti-vesicle-associated membrane protein 8 (VAMP-8) has been described (29). A rabbit antiserum specific for the 270-kDa mannose-6-phosphate receptor (M6PR) was a gift from K. von Figura (University of Göttingen, Göttingen, Germany). Cy5-coupled goat anti-mouse IgG, Cy3-coupled goat anti-rabbit, goat anti-rat or goat anti-mouse IgG, and tetramethylrhodamine isothiocyanate-coupled goat anti-rabbit IgG secondary Abs were from Dianova.

DNA plasmids

Plasmids encoding Kb, Kd, and OVA cDNAs were provided by B. Arnold (German Cancer Research Center, Heidelberg, Germany) and Y. Reiss (Tel Aviv University, Tel Aviv, Israel). cDNAs coding for peptide-MHC-I fusion proteins were assembled stepwise in pBluescript KS II+ (Stratagene) using conventional PCR techniques and insertion of double-stranded oligonucleotides digested with appropriate restriction enzymes. Completed constructs were subcloned into the expression vector pcDNA3.1(+) (Invitrogen Life Technologies). A modified influenza virus HA1 leader sequence (MAKANLLVLLCALAAADA) was used to target the fusion proteins into the ER. This leader was followed by sequences coding for SIINFEKL, LX-SIINFEKL (with X being E, R, Y, F, Q, H, I, or P), OVA(V249-E256)-SIINFEKL, OVA(V120-E256)-SIINFEKL, RKKR-SIINFEKL, LCRKKR-SIINFEKL, and DYKDDDDKA-SIINFEKL, respectively. The linker FLG4SG4APGSG3S (sp19) was positioned between SIINFEKL and cDNA sequences encoding mature Kb or Kd. To study the influence of the C-terminal flank on processing, the residues FL in sp19 were replaced by DL, GL, RL, VL, PL, GG, and PG, respectively. Cytoplasmic tail point mutants of Kb (Y321P; T329A/S330A/S333A) and Kd (Y320P; T328A/S329A/S332A) and truncated tails of Kb/d (residual tail sequence RRRNTGG) were generated by PCR using reverse primers containing the mentioned mutations or a premature stop codon, respectively. To generate Kb and Kd fusion proteins tagged at the truncated cytoplasmic tail with enhanced GFP (EGFP), cDNAs containing an EcoRI site inserted 3' of RRRNTGG were ligated into the multicloning site of pEGFP-N1 (BD Biosciences). To generate His-tagged Kb fusion proteins, the blunted BspHI site (at residue H361) was ligated onto the Eco32I site in pcDNA6-V5/HisA (Invitrogen Life Technologies). A mouse ERAP1 cDNA clone was obtained from the German Resource Center for Genome Research. All constructs were confirmed by sequencing.

Transfections

Stable transfections of P185 and RMA-S cells were done by electroporation of 107 cells with 10 µg of plasmid DNA using the Bio-Rad GenePulser (220 V, 960 µF) and selection with 0.8–1.0 mg/ml geneticin (Sigma-Aldrich). For transient transfections of MCB6TAP–/– and Ltk– cells, 104 cells/well were seeded in poly-L-lysine-coated U-bottom 96-well plates. Complexes of Lipofectamine 2000 (Invitrogen Life Technologies) and plasmid DNAs were made in Opti-MEM (Invitrogen Life Technologies) at a 1:3 ratio, according to the manufacturer’s instructions. Cells were usually transfected with 0.4 µg of DNA per well for 48 h. Transfection efficiencies were checked by MHC-I surface staining or by using EGFP-tagged MHC-I molecules. Up to 40% of the transfected cells showed expression. Mouse ERAP1-specific and control small interfering RNAs (siRNAs) were obtained from Qiagen and transfected into MC4 cells using oligofectamine, according to the manufacturer’s instructions.

T cell assays

The lacZ-inducible T cell hybridoma B3Z recognizing Kb molecules loaded with SIINFEKL peptide was a gift from N. Shastri (University of California, Berkeley, CA) (30). A total of 5 x 104 B3Z cells was cocultured with 104 stably or transiently transfected cells for 18 h at 37°C. The Ag-specific stimulation of B3Z was determined by addition of lacZ substrate buffer (9 mM MgCl2, 0.15 mM CPRG, 100 mM 2-ME, 0.125% Nonidet P-40 in PBS (pH 7.5)) for 4 h at 37°C. The cleaved CPRG was measured in an ELISA reader as absorbance at 595 nm with 630 nm as the reference wavelength. The data are shown as means of triplicate values and SEM from representative examples of two to four similar experiments. To remove pre-existing MHC-I-peptide complexes from the cell surface, APCs were preincubated in the presence of various inhibitors for 1 h, washed once in PBS, and incubated in acid wash buffer at pH 3.0 (39.8 ml of 0.1 M citric acid/10.2 ml of 0.2 M Na2HPO4/50 ml of water) for 3 min, followed by three washes in DMEM supplemented with 10% FCS. After acid stripping, the cells were allowed to recover in the presence of the same concentrations of inhibitors for 6–14 h. Subsequently, cells were washed in PBS and fixed in 0.05% glutaraldehyde in ice-cold Dulbecco’s PBS (D-PBS) for 4 min. The fixative was quenched with 200 mM glycine in PBS for 30 s, followed by two washes in PBS, two washes in complete DMEM, and addition of B3Z cells.

Enzyme inhibition assays

To assess the inhibition of cell-expressed furin by the furin inhibitor decanoyl-RVKR-CMK, 106 MCB6TAP–/– cells per sample were pretreated with 100 µM furin inhibitor or DMSO for 14 h, followed by homogenization in 50 mM Na acetate (pH 7.0)/5 mM CaCl2 using a Potter homogenizer and centrifugation at 15,000 x g for 10 min at 4°C. Triplicate samples of 100 µl of supernatant were reconstituted with 100 µM furin inhibitor or DMSO for control. Then 100 µM furin substrate Boc-RVRR-AMC was added for 4 h at room temperature. The reaction was stopped by addition of 50 µl of acetic acid. Samples were analyzed in a SpectraMax Gemini XS spectrometer (Molecular Devices) using 380 nm excitation and 525 nm emission wavelengths.

To assess the inhibition of autophagy by the phosphoinositide 3-kinase inhibitors wortmannin and 3-methyladenine, a dansylcadaverine uptake assay was performed essentially as described (31). MCB6TAP–/– cells (3.5 x 105/sample) were pretreated with 1.5 µM wortmannin or 7.5 mM 3-methyladenine for 3 h, followed by three washes in D-PBS. To induce starvation, samples were then incubated in D-PBS for 4 h at 37°C, or DMEM/10% FCS medium for control. Dansylcadaverine (50 µM) was added for 15 min at 37°C. After four washes in D-PBS, the cell pellets were lysed in 250 µl of TBS (pH 8.0)/0.1% Nonidet P-40. Lysates were analyzed in a SpectraMax Gemini XS spectrometer using 380-nm excitation and 525-nm emission wavelengths.

Immunofluorescence stainings

Flow cytometry of transfected cells was performed as described previously (28). For confocal immunofluorescence microscopy, transiently transfected MCB6TAP–/– cells were seeded in 48-well plates on coverslips coated with 0.1% (w/v) poly-L-lysine. Cells were fixed for 10 min in fixation buffer (4% paraformaldehyde in 150 mM NaCl, 200 mM HEPES (pH 7.4)) on ice, followed by two washes in 200 mM glycine buffer for 2 min. Fixed cells were permeabilized and blocked using PBS (pH 7.4) supplemented with 0.1% saponin (Sigma-Aldrich) and 7% horse serum for 1 h at room temperature. Primary Abs were diluted in 250 µl of PBS/saponin and incubated with the cells overnight at 4°C. After three washes in PBS/saponin, second Abs were added in 250 µl for 30 min at room temperature, followed by three washes and mounting. Immunofluorescence stainings were analyzed by confocal laser-scanning microscopy (LSM 510; Zeiss). Costaining coefficients were obtained from the analysis of 8–10 cells using LSM Image Examiner software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TAP-independent presentation of MHC-I-derived fusion proteins

The ectodomain of MHC-I molecules itself is known to harbor peptide epitopes presented by a variety of MHC-I allomorphs. We therefore used modified MHC-I H chains to study the TAP-independent processing and presentation of endogenous transmembrane proteins. The Kb-restricted OVA epitope SIINFEKL (SL8), or N-terminally extended versions of SL8, were fused through a linker sequence to the N-terminal end of the ectodomains of Kb and Kd H chains (Fig. 1A). This strategy targeted the epitope-carrying sequence to the ER lumen and excluded a contribution of cytosolic proteases to epitope processing when the fusion proteins were expressed in TAP-deficient cells.


Figure 1
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  		FIGURE 1. Processing and presentation of fusion proteins containing the Kb-restricted OVA epitope SIINFEKL (SL8). A, Schematic representation of ER-targeted peptide MHC-I fusion proteins used in this study as model to analyze the processing and presentation of endogenous membrane-associated Ags. The SL8 epitope is embedded between N-terminal extensions and a spacer linking the peptides to the N terminus of Kb or Kd H chains. B, Stimulation of the B3Z T hybridoma by TAP-deficient cells expressing peptide-MHC-I fusion proteins. The MCB6TAP–/– fibroblast line, generated in the TAP1–/– mouse, was transiently transfected with plasmids encoding the indicated peptide-Kb fusion proteins before coincubation with B3Z. The chimeras contained the unextended SL8 epitope, SL8 N-terminally extended by the indicated naturally occurring flanks of the OVA sequence (L255-E256, V249-E256, V120-E256), or the artificial N-terminal extensions RKKR, DYKDDDDKA (flag), and GP-flag, respectively. Using the chromogenic substrate CPRG, induction of beta-galactosidase activity in B3Z cells resulting from TCR occupation was measured as absorption at 595 nm. Means of triplicate values and SEM are shown. C, Response of the T cell hybridoma B3Z to synthetic SIINFEKL peptide and extended analogues. P815.Kb cells were incubated for 30 min at 4°C with titrated amounts of synthetic SL8 peptide and the indicated N-terminally, C-terminally, or biterminally extended analogues before fixation and coculture with B3Z for 18 h. As compared with SL8, the response to extended SL8 analogues was reduced by >3 orders of magnitude. D, Titration of synthetic SL8 peptide added to MCB6TAP–/– cells for 30 min at 37°C before fixation (left), and titration of plasmids coding for the SL8-Kb and LE-SL8-Kb fusion proteins in 48-h transient transfections (right). E, B3Z responses to MCB6TAP–/– cells transfected with Kd-derived fusion proteins harboring the SL8 epitope. Untransfected cells were left untreated or incubated with 12.5 mg/ml OVA protein for 24 h. For control, cells were incubated with 150 nM synthetic SL8 peptide for 30 min. F, B3Z responses to TAP-deficient RMA-S T lymphoma cells transfected with the SL8-Kd fusion protein. Untransfected RMA-S cells were left untreated, incubated with 12.5 mg/ml OVA for 24 h, or with exogenous SL8 peptide (1 µM) for 30 min. G and H, B3Z responses to MCB6TAP–/– cells that were transiently transfected with the indicated peptide-Kb fusion proteins containing variations in N-terminal (G) or C-terminal (H) amino acids, respectively, flanking the SL8 epitope. Expression of the fusion proteins was monitored by Western blot using an His6-tag-specific mAb. –, no N-terminal extension.

 
TAP-deficient MCB6TAP–/– fibroblasts were transfected to express SL8-Kb fusion proteins containing N-terminal extensions derived from the natural OVA protein or appended by the sequences RKKR (a potential furin cleavage site), DYKDDDDKA (flag-tag), and GPDYKDDDDKA (GP-flag), respectively (Fig. 1B). The SL8/Kb-specific T cell hybridoma B3Z recognized all transfectants, albeit with varying efficiencies. Consistent with earlier reports (30, 32), we found that B3Z T cells recognized N- or C-terminally peptide analogues of the SL8 epitope ~3 orders of magnitude less efficiently than SL8 itself, and were unresponsive to biterminally extended SL8 (Fig. 1C). The stimulation of B3Z by Kb fusions containing SL8 embedded within N- and C-terminal flanking sequences thus indicated proteolytic processing of the fusion proteins and final trimming of the SL8 epitope. The presentation of exogenous SL8 peptide and transfected fusion proteins by MCB6TAP–/– cells was titrated in Fig. 1D. The 0.4 µg of DNA normally used for transient transfections was a subsaturating amount. Because Kd is unable to restrict the SL8 epitope, Kd-derived SL8 fusion proteins required processing and peptide loading of Kb molecules constitutively expressed in MCB6TAP–/– cells. (RKKR)-SL8-Kd molecules were efficiently presented to B3Z, eliciting responses that were equal to 150 nM SL8 peptide (Fig. 1E). In contrast to these membrane-bound endogenous Ags, exogenous OVA protein offered at a high concentration (12.5 mg/ml for 24 h) was poorly cross-presented. Likewise, TAP-deficient RMA-S T lymphoma cells presented the SL8-Kd conjugate, but did not significantly cross-present OVA protein (Fig. 1F). To study the effect of flanking residues on epitope processing, fusion proteins with the consensus sequences LX-SL8-sp19-Kb and RKKR-SL8-XL/G-sp17-Kb were expressed in MCB6TAP–/– fibroblasts. Various charged, polar and hydrophobic amino acids were tolerated as N-terminal (Fig. 1G) and C-terminal flanks (Fig. 1H). Major quantitative differences were noted, however, with B3Z responses being significantly reduced when a proline residue was located N- or C-terminally adjacent to the SL8 sequence. Using cells from the same transient transfections, similar expression levels of the different fusion proteins were monitored by Western blots detecting a hexa-histidine tag attached to the cytoplasmic tail of Kb fusion proteins.

Evidence for involvement of furin, but not ERAP1 in fusion protein processing

TAP-deficient MCB6TAP–/– cells still express tapasin (Fig. 2A) and can therefore build up a partial peptide-loading complex that might modulate the expression of TAP-independent MHC-I peptide complexes (28, 33). To study a potential role of tapasin, we used the tapasin–/– fibroblast line MC4 that also completely lacks TAP2 expression (Fig. 2A), which is most likely due to the lack of TAP stabilization in the absence of tapasin (22, 28). MC4 cells presented, however, N-terminally extended SL8 fusion proteins with no apparent defect as compared with MCB6TAP–/–, suggesting that tapasin played no specific role in fusion protein trafficking and processing. Next, we analyzed a potential role of the ER aminopeptidase ERAP1, which disfavors trimming of substrates with proline at position 2 (34). Down-modulation of ERAP1 by siRNA transfection of MC4 cells left the B3Z responses to SL8 fusion proteins either unaltered or slightly enhanced their presentation (Fig. 2B). Overexpression of ERAP1 in MCB6TAP–/– cells did not influence fusion protein presentation (Fig. 2C). For control of the mouse ERAP1 cDNA, TAP-proficient RMA cells expressing cytoplasmic OVA were supertransfected with mouse ERAP1. SL8 presentation was enhanced, suggesting improved peptide trimming in the presence of overexpressed ERAP1, whereas the aminopeptidase inhibitor leucine thiol (34) blocked this enhancement (Fig. 2D). The endoprotease furin, which cleaves proproteins at the C-terminal end of RXXR motifs, was described to be involved in Ag processing in the secretory pathway (20). After an acid wash to remove pre-existing SL8:Kb complexes, transfected MCB6TAP–/– cells were treated with the furin inhibitor Dec-RVKR-CMK. This treatment significantly reduced the presentation of fusion proteins carrying the N-terminal extensions RKKR and LCRKKR, but did not affect the presentation of the SL8-, LE-SL8-, and RKNKTR-SL8-Kb chimeras (Fig. 2E). In parallel, we assessed the capacity of Dec-RVKR-CMK to block cell-borne furin. Inhibitor-treated or control MCB6TAP–/– cells were homogenized, and residual furin activity was determined using a fluorogenic substrate. Furin activity was found to be largely inhibited (Fig. 2E). Thus, depending on the sequence context, the TGN protease furin appeared to contribute to processing of the SL8 epitope.


Figure 2
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  		FIGURE 2. Role for furin, but not for ERAP1 and tapasin in fusion protein processing. A, Western blot analysis of tapasin expression in the MC4 fibroblast line from the tapasin–/– mouse and in TAP-deficient MCB6TAP–/– cells (left panel). MC6 Tpn+/+ fibroblasts were used for control. Right panel, Western blot analysis of the same cells using anti-mouse TAP2 Abs. The asterisk denotes an unspecifically stained band. Bottom panel, B3Z responses to MC4 cells transiently transfected with the indicated N-terminally extended and nonextended SL8-Kb fusion proteins. B, B3Z responses to MC4 cells transfected for 24 h with ERAP1-specific siRNA or irrelevant siRNA for control and subsequently with the indicated fusion proteins for another 48 h (bottom panel). The Western blot shows ERAP1 expression in ERAP1 and control siRNA-treated MC4 cells (top panel). C, B3Z responses to MCB6TAP–/– cells transfected with the indicated SL8-Kb fusion proteins together with 0.4 µg of mouse ERAP1 cDNA or empty vector for control (bottom panel). The Western blot shows ERAP1 expression in these APCs using rabbit anti-ERAP1 Ab (top panel). D, TAP-proficient RMA T lymphoma cells expressing OVA in the cytoplasm (RMA.cytOVA) were supertransfected with mouse ERAP1 cDNA or pcDNA6 vector for control. B3Z responses were analyzed for cells treated with 50 µM leucine thiol or DMSO for control in the presence of 50 µM DTT (bottom panel). The Western blot shows ERAP1 expression in these APCs using rabbit anti-ERAP1 Ab (top panel). E, Forty-eight hours after transfection with the indicated fusion proteins, MCB6TAP–/– cells were acid stripped to remove pre-existing MHC-I peptide complexes and incubated in the presence of 100 µM furin inhibitor Dec-RVKR-CMK during a 14-h recovery period before fixation and coincubation with B3Z for another 18 h (left panel). Dec-RVKR-CMK significantly inhibited presentation of RKKR-S8L-Kb (p = 0.006, Student’s t test) and LCRKKR-S8L-Kb (p = 0.0004). To control for inhibition of cell-bound furin, cellular extracts of Dec-RVKR-CMK or DMSO-treated MCB6TAP–/– cells were assayed in triplicate using the fluorogenic substrate Boc-RVRR-AMC. Arbitrary fluorescence units were normalized to DMSO-treated cells (right panel).

 
Fusion proteins are internalized from the cell surface and processed in a pH-dependent manner

To analyze whether autophagy of ER-derived vesicles was involved in fusion protein, we used the PI3K inhibitors, 3-methyladenine and wortmannin, which block the autophagy response. In acid wash/reappearance assays, these inhibitors had no reducing effect (Fig. 3A). To control for the autophagy blocking effect of wortmannin and 3-methyladenine, we performed a dansylcadaverine accumulation assay (31). After starvation in saline, MCB6TAP–/– cells showed an increased uptake of dansylcadaverine, which was inhibited by the PI3K inhibitors (Fig. 3A). These experiments argue against a significant contribution of autophagy to fusion protein processing.


Figure 3
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  		FIGURE 3. Fusion protein processing requires surface internalization and is pH dependent. A, B3Z responses to MCB6TAP–/– cells transiently transfected with the indicated Kd- and Kb-derived peptide fusion proteins. After a wash with isotonic acid buffer to remove pre-existing SL8:Kb complexes, the autophagy inhibitors 3-methyladenine (7.5 mM), wortmannin (1.5 µM), or DMSO as solvent control were added during a 11-h recovery period, followed by fixation and coculture with B3Z (top panel). To control for autophagy inhibition, MCB6TAP–/– were either deprived for amino acids by culture in D-PBS, or left in serum-containing medium for control. The presence of autophagy inhibitors during starvation, or serum, blocks cellular accumulation of the autofluorescent drug dansylcadaverine (bottom panel). Following cell lysis, intracellular dansylcadaverine is quantitated by fluorescence photometry. Arbitrary fluorescence units were normalized to cells in culture medium treated with DMSO. B, Cytofluorometric analysis of MCB6TAP–/–/SL8-Kd (top) and RMA-S/SL8-Kd (bottom) using the anti-Kd mAb 15.5.5S, and MCB6TAP–/–/flag-SL8-Kb (middle) using the anti-flag mAb 4E11-18 (black lines). Untransfected MCB6TAP–/– and RMA-S cells stained with the same Abs for control are shown with gray lines. C, Acid wash recovery experiments as in A. Filipin complex (10 µg/ml) 10 µg/ml chlorpromazine, or DMSO for control were added during a 11-h recovery period, followed by fixation and coculture with B3Z. D, Acid wash recovery experiments as in A. CHX (50 µM) (CHX chase) or DMSO for control was added during a 11-h recovery period, followed by fixation and coculture with B3Z. For control of the function of CHK 50 µM CHX was added already during the transfection period (Transf.+CHX). E, top panel, MCB6TAP–/– cells were transiently transfected for 48 h with SL8-Kb, RKKR-SL8-Kb, and SL8-Kd preincubated with acidification inhibitors for 60 min, followed by acid denaturation of surface MHC-I molecules and recovery during 14 h in the presence of inhibitors (100 µM chloroquine, 250 µM primaquine, 2.5 nM folimycin, 0.5 µM bafilomycin A1) before fixation and coculture with B3Z. Bottom panel, Transiently transfected MCB6TAP–/– cells were treated with acidification inhibitors as above and, in addition, pulsed with 1 µM or 0.1 µM SIINFEKL peptide for 30 min at room temperature before fixation and coculture with B3Z. F, RMA-S cells expressing SL8-Kd were preincubated with inhibitors, acid stripped, and allowed to recover for 6 h in the presence of 150 µM primaquine, 5 nM folimycin, or 250 nM bafilomycin A1, respectively, before fixation and coculture with B3Z T cells.

 
Because transfected peptide-Kd/b fusion proteins were detectable on the cell surface of MCB6TAP–/– and RMA-S cells by cytofluorometry (Fig. 3B), it was conceivable that they underwent internalization from the plasma membrane into endolysosomal vesicles for processing. To explore this possibility, we used the sterol-binding agent filipin as well as the cationic amphiphilic compound chlorpromazine to block the clathrin-independent and -dependent internalization pathways, respectively (35, 36). In acid wash/reappearance assays, both chlorpromazine and filipin significantly inhibited the presentation of (RKKR)SL8-Kd and Kb fusion proteins, indicating that clathrin-dependent as well as clathrin-independent internalization preceded processing (Fig. 3C). The protein synthesis inhibitor cycloheximide (CHX) did not affect presentation in reappearance assays, whereas CHX suppressed the de novo expression and presentation of peptide-MHC-I fusion proteins when present during the transfection period (Fig. 3D). This finding suggested that epitope processing and formation of SL8:Kb complexes were independent of newly synthesized fusion protein emerging out of the ER during the reappearance period, but rather relied on pre-existing molecules.

The presentation of Kb and Kd fusion proteins was substantially inhibited when transfected MCB6TAP–/– or RMA-S cells were allowed to recover in the presence of the acidophilic amines, chloroquine and primaquine, as well as the inhibitors of the V-type H+-ATPase, bafilomycin A1 and folimycin. Because these pH-neutralizing compounds interfere with the function of endolysosomal hydrolases, the results suggest that the fusion proteins underwent processing in acidic endosomal compartments (Fig. 3, E, top panel, and F). In addition to increasing endosomal pH, primaquine could have interfered with the recycling of peptide-loaded MHC-I molecules from endosomes to the cell surface (37). For control of adverse effects on protein trafficking, transiently transfected MCB6TAP–/– cells were acid denatured, treated with pH-neutralizing compounds, and incubated with synthetic SL8 peptide before fixation. The capacity of MCB6TAP–/– cells to present exogenous SL8 peptide remained unimpaired (Fig. 3E, bottom panel), suggesting that the reappearance of peptide-receptive Kb molecules was not affected by the inhibitors, including primaquine.

The endosomal processing pathway is also used by TAP-proficient cells

To investigate the question whether the endosomal processing pathway was also operative in murine TAP-proficient cells, Ltk– cells (H-2k) were transiently transfected with (RRKR)-SL8-Kb and (RKKR)-SL8-Kd fusion proteins. Because of the Kb-restricted presentation of the SL8 epitope, presentation of Kd-derived fusion proteins strictly required cotransfection of unconjugated Kb, whereas the Kb-derived fusion proteins were self-sufficient (Fig. 4A). B3Z responses against the (RKKR)-SL8-Kd and (RKKR)-SL8-Kb fusion proteins were substantially blocked by the vacuolar ATPase inhibitor folimycin (Fig. 4A). In the same line, the presentation of the flag-SL8-Kb fusion protein by TAP-proficient P815 cells was completely blocked by bafilomycin (Fig. 4B). By contrast, the proteasome inhibitor lactacystin did not affect the reappearance of SL8:Kb complexes after surface stripping. Lactacystin blocked, however, the presentation of SL8:Kb complexes by TAP-proficient RMA cells expressing OVA in the cytoplasm (Fig. 4C), indicating successful proteasome inhibition under the assay conditions used.


Figure 4
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  		FIGURE 4. A, Ltk– cells transiently transfected with (RKKR)-SL8-Kb and (RKKR)-SL8-Kd fusion proteins were pretreated with folimycin for 1 h, acid stripped, and allowed to recover for 6 h in the presence of 5 nM folimycin or DMSO for control, followed by fixation and coculture with B3Z. Where indicated, unconjugated Kb was cotransfected. B, P815 cells expressing the flag-SL8-Kb fusion protein were acid stripped and allowed to recover for 6 h in the presence of 10 µM lactacystin or 250 nM bafilomycin A1, before fixation and coculture with B3Z. DMSO was used as solvent control during the reappearance period. Another control sample was fixed immediately after the acid stripping (No recovery). C, The proteasome inhibitor lactacystin blocks the reappearance of SL8:Kb complexes on the surface of acid-washed RMA cells expressing cytoplasmic OVA, but not of P815.flag-SL8-Kb cells. P815.flag-SL8-Kb and RMA.cytOVA cells were fixed immediately after acid stripping (No recovery) or allowed to recover for 6 h in the presence of 10 µM lactacystin or DMSO for control.

 
Aspartic-type cathepsins are involved in fusion protein processing

To further characterize endosomal proteases involved in fusion protein processing, cells were treated with inhibitors of either cysteine-type or aspartic-type cathepsins in acid wash/reappearance assays. Pepstatin A caused a ~50% inhibition of the B3Z response to RMA-S cells expressing the SL8-Kd conjugate, suggesting that aspartic-type cathepsins contributed to processing, whereas no inhibition was observed using the potent membrane-permeable inhibitor of cathepsin S, E-64d (Fig. 5A). Inhibition of cysteine proteases by leupeptin or cystatin also had only a minor effect on the presentation of SL8:Kb complexes by RMA-S cells (Fig. 5B). Consistently, pepstatin A inhibited presentation of fusion proteins by fibroblasts deficient for the cysteine protease cathepsin B (Fig. 5C). Furthermore, the presentation of peptide-Kb/d fusion proteins was reduced in cathepsin D-deficient fibroblasts as compared with wild-type B6 and cathepsin B-deficient fibroblasts, whereas synthetic SL8 peptide was presented with equal efficiency (Fig. 5D).


Figure 5
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  		FIGURE 5. Involvement of aspartic-type cathepsins in fusion protein processing. A, RMA-S.SL8-Kd cells were acid stripped and allowed to recover for 6 h in the presence of the aspartic-type cathepsin inhibitor pepstatin A (200 µM) or the cysteine-type cathepsin inhibitor E-64d (200 µM) before fixation and coculture with B3Z. B, Reappearance assay with acid-stripped RMA-S.SL8-Kd cells that had recovered during 6 h in the presence of the cysteine-type cathepsin inhibitors leupeptin (200 µM) or cystatin (75 µg/ml). DMSO was used for sham treatment. C, Cathepsin B–/– mouse embryonal fibroblasts were transiently transfected with the indicated Kd- and Kb-derived peptide conjugates. Pepstatin (200 µM), or DMSO for control, was added during the 9-h reappearance time after acid stripping. D, Wild-type B6, cathepsin B–/–, and cathepsin D–/– mouse embryonal fibroblasts (MEF) were transfected for 48 h with the indicated fusion proteins before fixation and coculture with B3Z. To control for the efficacy of Kb-restricted presentation, untransfected cells were pulsed with 125 nM SIINFEKL peptide before addition of B3Z.

 
Targeting to processing compartments is independent of the cytoplasmic tail of MHC-I

Within the cytoplasmic tail of MHC-I molecules, a putative Tyr-based internalization motif (38), Ser phosphorylation sites (39, 40), and Lys residues as acceptors for ubiquitin conjugation (41) have been implicated in the internalization of MHC-I from the cell surface, their targeting to endolysomal subcompartments, and recycling. To analyze the endosomal targeting of MHC-I-peptide fusion proteins in TAP-deficient, nonprofessional APCs, we either substituted the conserved tyrosine residue and potential phosphorylation sites of the cytoplasmic tail in Kb and Kd fusion proteins, or truncated the cytoplasmic tail to the residual amino acids RRRNTGG. Neither point mutation of serine/threonine and tyrosine residues (Fig. 6, A and C), nor extensive truncation of the cytoplasmic tail (Fig. 6B), nor fusion of the truncated tail with enhanced GFP (EGFP) impaired the presentation of peptide-fusion proteins by MCB6TAP–/– cells. As shown for RMA-S cells expressing SL8-Kd(Y320P), SL8 presentation was blocked by folimycin, indicating that in the absence of the putative internalization motif the processing remained endosomal (Fig. 6C). Likewise, Ltk– cells transfected with tail-truncated (RKKR)SL8-Kd together with tailless Kb were able to present SL8:Kb complexes, and also this presentation was inhibited by folimycin (Fig. 6D). This result suggests that not only the internalization of peptide-tagged MHC-I to endosomal processing compartments, but also the recycling of peptide-loaded Kb are independent of sequence motifs in the cytoplasmic tail. Consistently, cathepsin B-deficient fibroblasts presented (RKKR)SL8-conjugated Kd and Kb fusion proteins with truncated tails, and also in this case pepstatin A markedly inhibited B3Z responses (Fig. 6E). The presentation of tailless (RKKR)SL8-conjugated Kd and Kb fusion proteins could be blocked by filipin and chlorpromazine (data not shown), indicating that they were internalized from the cell surface into processing compartments.


Figure 6
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  		FIGURE 6. A, B3Z responses to MCB6TAP–/– cells transiently transfected with SL8-Kd and RKKR-SL8-Kb conjugates containing either the wild-type cytoplasmic tail or cytoplasmic tails with mutated Tyr and Ser/Thr residues, respectively. B, B3Z responses to MCB6TAP–/– cells transiently transfected with (RKKR)-SL8-Kd/b fusion proteins with wild-type cytoplasmic tail or a cytoplasmic tail truncated to the seven residues RRRNTGG (tr.tail), and with (RKKR)-SL8-Kb/d molecules in which the truncated cytoplasmic tail was fused with EGFP. C, RMA-S cells transfected with SL8-Kd(Y320P) were preincubated with folimycin for 60 min, acid stripped, and allowed to recover in the presence of 2.5 nM folimycin during 11 h, followed by fixation and coculture with B3Z. D, Ltk– cells were transiently transfected with SL8-Kd or RKKR-SL8-Kd together with unconjugated Kb also having a truncated tail. The transfectants were pretreated with folimycin and recovered during 6 h before fixation and coculture with B3Z. E, Fibroblasts derived from the cathepsin B–/– mouse were transiently transfected with the indicated Kd- and Kb-derived peptide conjugates with truncated tails. Pepstatin (200 µm), or DMSO for control, was added during the 9-h reappearance time after acid stripping.

 
SIINFEKL-loaded Kb molecules appear in early and late endosomes

To investigate the intracellular trafficking of fusion proteins and the sites of SL8:Kb complex formation, confocal laser-scanning microscopy was performed with MCB6TAP1–/– cells transiently transfected with EGFP-tagged SL8-Kd. EGFP appeared in a reticular ER-like staining as well as in punctate vesicular structures. We performed stainings of SL8-Kd-EGFP-transfected cells with the early endosomal marker EEA-1, and the late endosomal markers CD63, LAMP-1, and VAMP-8, respectively. EGFP staining was colocalized, averaged over 8–10 cells for which the costaining was quantitated, with ~10% of the vesicles staining for EEA-1, ~59% CD63+ vesicles, ~22% LAMP-1+ vesicles (Fig. 7A), and ~35% VAMP-8+ vesicles (data not shown), respectively. The Ab 25.D1-16 specifically recognizing SL8:Kb complexes (27) predominantly stained vesicular structures (Fig. 7B), in agreement with the formation of SL8:Kb complexes in endosomes. We noted an overlap of the 25.D1-16 staining with ~5% of M6PR+ late endosomes (Fig. 7B) and ~2% of EEA-1+ early endosomes (data not shown).


Figure 7
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  		FIGURE 7. Detection of SL8-loaded Kb molecules in late endosomal vesicles by confocal laser-scanning immunofluorescence microscopy. A, MCB6TAP–/– cells transiently transfected with SL8-Kd-EGFP were stained with Abs against the early endosomal marker EEA-1 (top), the late endosomal marker anti-CD63 (middle), and the late endosomal/lysosomal marker LAMP-1 (bottom), and Cy3-conjugated secondary anti-IgG Abs. Vesicles colabeled for EGFP and the indicated markers are depicted in white. B, MCB6TAP–/– cells transiently transfected with SL8-Kd-EGFP were analyzed for EGFP expression (green). The cells were stained with 25.D1-16 recognizing SL8-loaded Kb molecules (Cy5-labeled goat anti-mouse IgG secondary Ab) together with rabbit Abs against the late endosomal marker mannose-6-phosphate receptor (tetramethylrhodamine isothiocyanate-labeled goat anti-rabbit IgG secondary Ab). In the merged 25.D1-16/{alpha}M6PR staining, magenta to white color indicates colocalization. Shown are representative examples from three experiments with similar results. The scale bars represent 10 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TAP-independent processing and presentation of proteins in the secretory pathway have been described (16, 17, 18, 19); however, little is known about processing compartments and proteases involved. A database of MHC ligands (42) lists 22 allelic MHC-I molecules that bind 25 different peptides derived from the ectodomains of other MHC-I allomorphs. We therefore reasoned that MHC-I molecules themselves might be well-suited carriers of peptide epitopes. As model Ags for endogenous membrane-associated proteins, we studied the processing of chimeric Kb and Kd MHC-I molecule proteins that contained the OVA-derived epitope SIINFEKL covalently linked to the ectodomain.

We found that high levels of SL8:Kb complexes were produced from most of the studied N-terminally extended SL8-MHC-I fusion proteins. Processing and presentation of fusion proteins were observed for TAP-deficient fibroblasts as well as TAP-deficient RMA-S T lymphoma cells, and are hence seen in different types of nonprofessional APCs. In accord with earlier reports (43, 44, 45, 46, 47) regarding the TAP-independent processing of soluble or bacterial-derived OVA, we found that TAP-deficient RMA-S and MCB6TAP–/– cells did not significantly process and present exogenous OVA protein. We hypothesize that the comparatively much more efficient TAP-independent processing of membrane-bound Ags results from a spatial vicinity of recycling MHC-I molecules and epitope-carrying fusion proteins. Ag processing close to endosomal membranes may result in higher local peptide concentrations supporting peptide exchange by recycling MHC-I molecules.

Without exception, the generation of SL8:Kb complexes from N-terminally extended Kd and Kb peptide fusions was blocked by acidophilic amines and inhibitors of the vacuolar proton pump. These pH-neutralizing drugs interfere with vesicular trafficking in the endosomal pathway as well as with the function of endolysosomal proteases (48, 49, 50). After acid stripping of the surface, neither newly synthesized fusion proteins exiting the ER nor autophagy of ER vesicles played a detectable role during subsequent SL8 epitope processing. By contrast, reagents inhibiting clathrin-dependent and -independent internalization clearly blocked the presentation of peptide-MHC-I fusion proteins. Endocytosis of MHC-I molecules by either mechanism has been described (35, 51, 52, 53). Together, these findings suggest that SL8 was predominantly cleaved out of fusion proteins that were internalized from the plasma membrane into endosomal vesicles.

Interestingly, this internalization did not require sequence motifs in the cytoplasmic tail of MHC-I because no loss in the presentation of Kd- and Kb-derived fusion proteins was observed after deletion of potential motifs either by sequence mutation or truncation of the cytoplasmic tail. Although a truncated tail was sufficient for MHC-I internalization in the human Jurkat T cell line (54), our results are in apparent conflict with a recent report showing that in dendritic cells, Kb molecules with mutated tyrosine residue failed to cross-present exogenous soluble OVA and to produce SL8:Kb complexes in late endosomes (38). Dendritic cells may, however, use cell type-specific mechanisms to target Kb to late endosomes that are dispensable for endosomal trafficking of MHC-I in transformed fibroblasts.

In the fusion protein SL8-Kb, the SL8 peptide may bind to Kb without endoproteolytic cleavage of the linker sequence, as suggested by former work using SL8-beta2-microglobulin-Kb and peptide-beta2-microglobulin fusion proteins (55, 56, 57). Our experiments do not exclude that a minor fraction of SL8-Kb single-chain molecules in fact bound and presented SL8 in its unprocessed form; however, the similar sensitivity of SL8-Kb and SL8-Kd to internalization inhibitors and bafilomycin suggests that both conjugates underwent pH-dependent processing in endosomal vesicles. The MHC-I-associated ER chaperone tapasin was completely dispensable for the presentation of peptides processed out of MHC-I-derived chimeras. No evidence was also obtained for an involvement of the ER aminopeptidase ERAP1. As suggested by the partial blockade of MHC-I fusion protein presentation by a potent furin inhibitor, the proprotein convertase furin was involved in the processing if respective recognition sites were present. Furin is active in TGN vesicles that have an acidic pH ~5.0 (58, 59). The efficient blockade of Ag presentation by neutralizing agents is therefore not in conflict with furin contributing to MHC-I ectodomain processing.

In TAP-competent cells, antigenic peptides were previously shown to be processed out of membrane-associated proteins in a proteasome-dependent pathway (13, 14, 15). In this study, SL8-MHC-I fusion proteins were efficiently processed and presented by various TAP-competent cells, including P815 and L cells (see Fig. 4), as well as HeLa and COS-7 cells (N. Tiwari and F. Momburg, unpublished results). Although the proteasome-specific inhibitor lactacystin exerted no detectable inhibition, proton pump inhibitors strongly suppressed the generation of the SL8 epitope from Kb and Kd fusion proteins. Thus, also in TAP-proficient cells, the vacuolar route rather than ER dislocation and cytosolic degradation seems to be an important source for antigenic peptides derived from glycoprotein ectodomains.

Cathepsin S and other cysteine proteases have been implicated in the cross-presentation of exogenous OVA administered through biodegradable microspheres to TAP-deficient dendritic cells (12) and through OVA-recombinant Escherichia coli to TAP-deficient macrophages (11). In the present study, the inhibitor of aspartic proteases, pepstatin A, partially blocked B3Z responses to SL8-Kd in reappearance assays, whereas various inhibitors of cathepsin S, B, and other cysteine proteases did not significantly affect presentation. Wild-type levels of Ag presentation by cathepsin B–/– fibroblasts suggested that the presence of this most abundant cysteine-type cathepsin was not critically required for processing. The inhibition of fusion protein presentation by pepstatin A in cathepsin B–/– cells and the reduced presentation in cathepsin D–/– cells, however, confirmed the importance of aspartic-type cathepsins for processing in these TAP-competent, nonprofessional APCs.

EGFP-tagged SL8-Kd fusion proteins, which require processing and transfer of the SL8 epitope onto Kb molecules for presentation, clearly colocalized with vesicles staining for late and early endosomal markers, supporting the presence of internalized fusion proteins throughout endosomal system, where they can be subjected to proteolytic degradation. The unique specificity of mAb 25.D1-16 furthermore allowed us to investigate by confocal microscopy intracellular compartments containing SL8:Kb complexes. We observed partial costaining of the 25.D1-16+ epitope with vesicles labeled for the late endosomal marker M6PR or the early endosomal marker EEA-1. This is in agreement with previous studies demonstrating the presence of MHC-I molecules in early endosomes and in multivesicular late endosomes, respectively, using different cell types for the analysis (9, 10, 60). The low pH of late endosomes was reported to favor peptide exchange on recycling MHC-I molecules (9, 43, 58). A larger fraction of 25.D1-16+ vesicles, however, did not label for M6PR. Thus, it seems possible that recycling Kb molecules were loaded with the SL8 peptide also in less acidic early endosomes. Alternatively, the 25.D1-16 staining of vesicles located in the cellular periphery may have resulted from the recycling into early endosomes of SL8:Kb complexes that were assembled in late endosomes.

Taken together, evidence is provided for the targeting of epitope-carrying MHC-I-derived fusion proteins to endosomal vesicles for degradation, where a T cell epitope was cleaved out of various sequence contexts and loaded onto recycling Kb molecules. This endosomal processing pathway functioned not only in TAP-deficient, but also in wild-type nonprofessional APCs. The vacuolar processing of membrane-associated Ags showed an unexpected efficacy. Therefore, membrane-associated proteins may be a source of TAP-independent Ags in tumor or virus-infected cells with down-modulated components of the proteasome/TAP pathway.


   Acknowledgments
 
We thank Nitin Agarwal for his help with the confocal microscopy, Petra Ludwig for help with Western blots, and Tobias Dick for critical comments.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Current address: Institut National de la Santé et de la Recherche Médicale U699, Faculté de Médicine Xavier Bichat, 16 rue Henri Huchard, 75018 Paris, France. Back

2 Address correspondence and reprint requests to Dr. Frank Momburg, Department of Molecular Immunology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail address: f.momburg{at}dkfz.de Back

3 Abbreviations used in this paper: MHC-I, MHC class I; ER, endoplasmic reticulum; CHX, cycloheximide; CPRG, chlorophenol red-beta-D-galactoside; D-PBS, Dulbecco’s PBS; EEA-1, early endosomal Ag 1; EGFP, enhanced GFP; ERAP1, ER-associated aminopeptidase 1; LAMP-1, lysosome-associated membrane protein 1; M6PR, mannose 6-phosphate receptor; siRNA, small interfering RNA; TGN, trans-Golgi network; VAMP-8, vesicle-associated membrane protein 8. Back

Received for publication November 23, 2005. Accepted for publication March 27, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Kloetzel, P. M.. 2004. Generation of major histocompatibility complex class I antigens: functional interplay between proteasomes and TPPII. Nat. Immunol. 5: 661-669. [Medline]
  2. Rock, K. L., I. A. York, T. Saric, A. L. Goldberg. 2002. Protein degradation and the generation of MHC class I-presented peptides. Adv. Immunol. 80: 1-70. [Medline]
  3. Momburg, F., G. J. Hämmerling. 1998. Generation and TAP-mediated transport of peptides for major histocompatibility complex class I molecules. Adv. Immunol. 68: 191-256. [Medline]
  4. Grommé, M., J. Neefjes. 2002. Antigen degradation or presentation by MHC class I molecules via classical and non-classical pathways. Mol. Immunol. 39: 181-202. [Medline]
  5. Yewdell, J. W., C. C. Norbury, J. R. Bennink. 1999. Mechanisms of exogenous antigen presentation by MHC class I molecules in vitro and in vivo: implications for generating CD8+ T cell responses to infectious agents, tumors, transplants, and vaccines. Adv. Immunol. 73: 1-77. [Medline]
  6. Ackerman, A. L., P. Cresswell. 2004. Cellular mechanisms governing cross-presentation of exogenous antigens. Nat. Immunol. 5: 678-684. [Medline]
  7. Ramirez, M. C., L. J. Sigal. 2004. The multiple routes of MHC-I cross-presentation. Trends Microbiol. 12: 204-207. [Medline]
  8. Jondal, M., R. Schirmbeck, J. Reimann. 1996. MHC class I-restricted CTL responses to exogenous antigens. Immunity 5: 295-302. [Medline]
  9. Grommé, M., F. G. Uytdehaag, H. Janssen, J. Calafat, R. S. van Binnendijk, M. J. Kenter, A. Tulp, D. Verwoerd, J. Neefjes. 1999. Recycling MHC class I molecules and endosomal peptide loading. Proc. Natl. Acad. Sci. USA 96: 10326-10331. [Abstract/Free Full Text]
  10. Kleijmeer, M. J., J. M. Escola, F. G. UytdeHaag, E. Jakobson, J. M. Griffith, A. D. Osterhaus, W. Stoorvogel, C. J. Melief, C. Rabouille, H. J. Geuze. 2001. Antigen loading of MHC class I molecules in the endocytic tract. Traffic 2: 124-137. [Medline]
  11. Campbell, D. J., T. Serwold, N. Shastri. 2000. Bacterial proteins can be processed by macrophages in a transporter associated with antigen processing-independent, cysteine protease-dependent manner for presentation by MHC class I molecules. J. Immunol. 16: 168-175.
  12. Shen, L., L. J. Sigal, M. Boes, K. L. Rock. 2004. Important role of cathepsin S in generating peptides for TAP-independent MHC class I crosspresentation in vivo. Immunity 21: 155-165. [Medline]
  13. Bacik, I., H. L. Snyder, L. C. Anton, G. Russ, W. Chen, J. R. Bennink, L. Urge, L. Otvos, B. Dudkowska, L. Eisenlohr, J. W. Yewdell. 1997. Introduction of a glycosylation site into a secreted protein provides evidence for an alternative antigen processing pathway: transport of precursors of major histocompatibility complex class I-restricted peptides from the endoplasmic reticulum to the cytosol. J. Exp. Med. 186: 479-487. [Abstract/Free Full Text]
  14. Mosse, C. A., L. Meadows, C. J. Luckey, D. J. Kittlesen, E. L. Huczko, C. L. Slingluff, J. Shabanowitz, D. F. Hunt, V. H. Engelhard. 1998. The class I antigen-processing pathway for the membrane protein tyrosinase involves translation in the endoplasmic reticulum and processing in the cytosol. J. Exp. Med. 187: 37-48. [Abstract/Free Full Text]
  15. Selby, M., A. Erickson, C. Dong, S. Cooper, P. Parham, M. Houghton, C. M. Walker. 1999. Hepatitis C virus envelope glycoprotein E1 originates in the endoplasmic reticulum and requires cytoplasmic processing for presentation by class I MHC molecules. J. Immunol. 162: 669-676. [Abstract/Free Full Text]
  16. Hammond, S. A., R. C. Bollinger, T. W. Tobery, R. F. Siliciano. 1993. Transporter-independent processing of HIV-1 envelope protein for recognition by CD8+ T cells. Nature 364: 158-161. [Medline]
  17. Elliott, T., A. Willis, V. Cerundolo, A. Townsend. 1995. Processing of major histocompatibility class I-restricted antigens in the endoplasmic reticulum. J. Exp. Med. 181: 1481-1491. [Abstract/Free Full Text]
  18. Snyder, H. L., I. Bacik, J. R. Bennink, G. Kearns, T. W. Behrens, T. Bächi, M. Orlowski, J. W. Yewdell. 1997. Two novel routes of transporter associated with antigen processing (TAP)-independent major histocompatibility complex class I antigen processing. J. Exp. Med. 186: 1087-1098. [Abstract/Free Full Text]
  19. Snyder, H. L., I. Bacik, J. W. Yewdell, T. W. Behrens, J. R. Bennink. 1998. Promiscuous liberation of MHC-class I-binding peptides from the C termini of membrane and soluble proteins in the secretory pathway. Eur. J. Immunol. 28: 1339-1346. [Medline]
  20. Gil-Torregrosa, B. C., A. R. Castaño, M. Del Val. 1998. Major histocompatibility complex class I viral antigen processing in the secretory pathway defined by the trans-Golgi network protease furin. J. Exp. Med. 188: 1105-1116. [Abstract/Free Full Text]
  21. Van Kaer, L., P. G. Ashton-Rickardt, H. L. Ploegh, S. Tonegawa. 1992. TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4–8+ T cells. Cell 71: 1205-1214. [Medline]
  22. Garbi, N., N. Tiwari, F. Momburg, G. J. Hämmerling. 2003. A major role for tapasin as a stabilizer of the TAP peptide transporter and consequences for MHC class I expression. Eur. J. Immunol. 33: 264-273. [Medline]
  23. Ljunggren, H. G., K. Kärre. 1985. Host resistance directed selectively against H-2-deficient lymphoma variants: analysis of the mechanism. J. Exp. Med. 162: 1745-1759. [Abstract/Free Full Text]
  24. Deussing, J., W. Roth, P. Saftig, C. Peters, H. L. Ploegh, J. A. Villadangos. 1998. Cathepsins B and D are dispensable for major histocompatibility complex class II-mediated antigen presentation. Proc. Natl. Acad. Sci. USA 95: 4516-4521. [Abstract/Free Full Text]
  25. Jones, B., C. A. Janeway, Jr. 1981. Cooperative interaction of B lymphocytes with antigen-specific helper T lymphocytes is MHC restricted. Nature 292: 547-549. [Medline]
  26. Ozato, K., P. Henkart, C. Jensen, D. H. Sachs. 1981. Spatially distinct allodeterminants of the H-2Kk molecule as detected by monoclonal anti-H-2 antibodies. J. Immunol. 126: 1780-1785. [Abstract]
  27. Porgador, A., J. W. Yewdell, Y. Deng, J. R. Bennink, R. N. Germain. 1997. Localization, quantitation, and in situ detection of specific peptide-MHC class I complexes using a monoclonal antibody. Immunity 6: 715-726. [Medline]
  28. Tan, P., H. Kropshofer, O. Mandelboim, N. Bulbuc, G. J. Hämmerling, F. Momburg. 2002. Recruitment of MHC class I molecules by tapasin into the transporter associated with antigen processing-associated complex is essential for optimal peptide loading. J. Immunol. 168: 1950-1960. [Abstract/Free Full Text]
  29. Paumet, F., J. Le Mao, S. Martin, T. Galli, B. David, U. Blank, M. Roa. 2000. Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment. J. Immunol. 164: 5850-5857. [Abstract/Free Full Text]
  30. Shastri, N., T. Serwold, F. Gonzalez. 1995. Presentation of endogenous peptide/MHC class I complexes is profoundly influenced by specific C-terminal flanking residues. J. Immunol. 155: 4339-4346. [Abstract]
  31. Munafó, D. B., M. I. Colombo. 2001. A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J. Cell Sci. 114: 3619-3629. [Medline]
  32. Serwold, T., N. Shastri. 1999. Specific proteolytic cleavages limit the diversity of the pool of peptides available to MHC class I molecules in living cells. J. Immunol. 162: 4712-4719. [Abstract/Free Full Text]
  33. Fromm, S. V., S. Duady-Ben Yaakov, C. Schechter, R. Ehrlich. 2002. Assembly and cell surface expression of TAP-independent, chloroquine-sensitive and interferon-{gamma}-inducible class I MHC complexes in transformed fibroblast cell lines are regulated by tapasin. Cell. Immunol. 215: 207-218. [Medline]
  34. Serwold, T., S. Gaw, N. Shastri. 2001. ER aminopeptidases generate a unique pool of peptides for MHC class I molecules. Nat. Immunol. 2: 644-651. [Medline]
  35. Naslavsky, N., R. Weigert, J. G. Donaldson. 2004. Characterization of a nonclathrin endocytic pathway: membrane cargo and lipid requirements. Mol. Biol. Cell 15: 3542-3552. [Abstract/Free Full Text]
  36. Orlandi, P. A., P. H. Fishman. 1998. Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J. Cell Biol. 141: 905-915. [Abstract/Free Full Text]
  37. Van Weert, A. W., H. J. Geuze, B. Groothuis, W. Stoorvogel. 2000. Primaquine interferes with membrane recycling from endosomes to the plasma membrane through a direct interaction with endosomes which does not involve neutralization of endosomal pH nor osmotic swelling of endosomes. Eur. J. Cell Biol. 79: 394-399. [Medline]
  38. Lizée, G., G. Basha, J. Tiong, J. P. Julien, M. Tian, K. E. Biron, W. A. Jefferies. 2003. Control of dendritic cell cross-presentation by the major histocompatibility complex class I cytoplasmic domain. Nat. Immunol. 4: 1065-1073. [Medline]
  39. Capps, G. G., M. Van Kampen, C. L. Ward, M. C. Zuñiga. 1989. Endocytosis of the class I major histocompatibility antigen via a phorbol myristate acetate-inducible pathway is a cell-specific phenomenon and requires the cytoplasmic domain. J. Cell Biol. 108: 1317-1329. [Abstract/Free Full Text]
  40. Vega, M. A., J. L. Strominger. 1989. Constitutive endocytosis of HLA class I antigens requires a specific portion of the intracytoplasmic tail that shares structural features with other endocytosed molecules. Proc. Natl. Acad. Sci. USA 86: 2688-2692. [Abstract/Free Full Text]
  41. Bartee, E., M. Mansouri, B. T. Hovey Nerenberg, K. Gouveia, K. Früh. 2004. Down-regulation of major histocompatibility complex class I by human ubiquitin ligases related to viral immune evasion proteins. J. Virol. 78: 1109-1120. [Abstract/Free Full Text]
  42. Rammensee, H., J. Bachmann, N. P. Emmerich, O. A. Bachor, S. Stevanovic. 1999. SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics 50: 213-219. [Medline]
  43. Chefalo, P. J., C. V. Harding. 2001. Processing of exogenous antigens for presentation by class I MHC molecules involves post-Golgi peptide exchange influenced by peptide-MHC complex stability and acidic pH. J. Immunol. 167: 1274-1282. [Abstract/Free Full Text]
  44. Chefalo, P. J., A. G. Grandea, III, L. Van Kaer, C. V. Harding. 2003. Tapasin–/– and TAP1–/– macrophages are deficient in vacuolar alternate class I MHC (MHC-I) processing due to decreased MHC-I stability at phagolysosomal pH. J. Immunol. 170: 5825-5833. [Abstract/Free Full Text]
  45. Kovacsovics-Bankowski, M., K. L. Rock. 1995. A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules. Science 267: 243-246. [Abstract/Free Full Text]
  46. Reis e Sousa, C., R. N. Germain. 1995. Major histocompatibility complex class I presentation of peptides derived from soluble exogenous antigen by a subset of cells engaged in phagocytosis. J. Exp. Med. 182: 841-851. [Abstract/Free Full Text]
  47. Wick, M. J., J. D. Pfeifer. 1996. Major histocompatibility complex class I presentation of ovalbumin peptide 257–264 from exogenous sources: protein context influences the degree of TAP-independent presentation. Eur. J. Immunol. 26: 2790-2799. [Medline]
  48. Fineschi, B., J. Miller. 1997. Endosomal proteases and antigen processing. Trends Biol. Sci. 22: 377-382.
  49. Gruenberg, J.. 2001. The endocytic pathway: a mosaic of domains. Nat. Rev. Mol. Cell Biol. 2: 721-730. [Medline]
  50. Trombetta, E. S., I. Mellman. 2005. Cell biology of antigen processing in vitro and in vivo. Annu. Rev. Immunol. 23: 975-1028. [Medline]
  51. Machy, P., A. Truneh, D. Gennaro, S. Hoffstein. 1987. Major histocompatibility complex class I molecules internalized via coated pits in T lymphocytes. Nature 328: 724-726. [Medline]
  52. Dasgupta, J. D., S. Watkins, H. Slayter, E. J. Yunis. 1988. Receptor-like nature of class I HLA: endocytosis via coated pits. J. Immunol. 141: 2577-2580. [Abstract]
  53. Naslavsky, N., R. Weigert, J. G. Donaldson. 2003. Convergence of non-clathrin- and clathrin-derived endosomes involves Arf6 inactivation and changes in phosphoinositides. Mol. Biol. Cell 14: 417-431. [Abstract/Free Full Text]
  54. Gur, H., T. D. Geppert, P. E. Lipsky. 1997. Structural analysis of class I MHC molecules: the cytoplasmic domain is not required for cytoskeletal association, aggregation and internalization. Mol. Immunol. 34: 125-132. [Medline]
  55. Mottez, E., P. Langlade-Demoyen, H. Gournier, F. Martinon, J. Maryanski, P. Kourilsky, J. P. Abastado. 1995. Cells expressing a major histocompatibility complex class I molecule with a single covalently bound peptide are highly immunogenic. J. Exp. Med. 181: 493-502. [Abstract/Free Full Text]
  56. Uger, R. A., B. H. Barber. 1998. Creating CTL targets with epitope-linked beta2-microglobulin constructs. J. Immunol. 160: 1598-1605. [Abstract/Free Full Text]
  57. Yu, Y. Y., N. Netuschil, L. Lybarger, J. M. Connolly, T. H. Hansen. 2002. Cutting edge: single-chain trimers of MHC class I molecules form stable structures that potently stimulate antigen-specific T cells and B cells. J. Immunol. 168: 3145-3149. [Abstract/Free Full Text]
  58. Stryhn, A., L. Ø. Pedersen, T. Romme, A. C. Olsen, M. H. Nissen, C. J. Thorpe, S. Buus. 1996. pH dependence of MHC class I-restricted peptide presentation. J. Immunol. 156: 4191-4197. [Abstract]
  59. Anderson, R. G., R. K. Pathak. 1985. Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell 40: 635-643. [Medline]
  60. Chiu, I., D. M. Davis, J. L. Strominger. 1999. Trafficking of spontaneously endocytosed MHC proteins. Proc. Natl. Acad. Sci. USA 96: 13944-13949. [Abstract/Free Full Text]



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