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Heat Shock Protein-chaperoned Peptides but Not Free Peptides Introduced into the Cytosol Are Presented Efficiently by Major Histocompatibility Complex I Molecules

2001; Elsevier BV; Volume: 276; Issue: 20 Linguagem: Inglês

10.1074/jbc.m011547200

1083-351X

Robert Binder, Nathalie E. Blachère, Pramod K. Srivastava,

Escherichia coli research studies

The studies reported here bear on the events in the cytosol that lead to trafficking of peptides during antigen processing and presentation by major histocompatibility complex (MHC) I molecules. We have introduced free antigenic peptides or antigenic peptides bound to serum albumin or to cytosolic heat shock proteins hsp90 (and its endoplasmic reticular homologue gp96) or hsp70 into the cytosol of living cells and have monitored the presentation of the peptides by appropriate MHC I molecules. The experiments show that (i) free peptides or serum albumin-bound peptides, introduced into the cytosol, become ligands of MHC I molecules at a far lower efficiency than peptides chaperoned by any of the heat shock proteins tested and (ii) treatment of cells with deoxyspergualin, a drug that binds hsp70 and hsp90 with apparent specificity, abrogates the ability of cells to present antigenic peptides through MHC I molecules, and introduction of additional hsp70 into the cytosol overcomes this abrogation. These results suggest for the first time a functional role for cytosolic chaperones in antigen processing. The studies reported here bear on the events in the cytosol that lead to trafficking of peptides during antigen processing and presentation by major histocompatibility complex (MHC) I molecules. We have introduced free antigenic peptides or antigenic peptides bound to serum albumin or to cytosolic heat shock proteins hsp90 (and its endoplasmic reticular homologue gp96) or hsp70 into the cytosol of living cells and have monitored the presentation of the peptides by appropriate MHC I molecules. The experiments show that (i) free peptides or serum albumin-bound peptides, introduced into the cytosol, become ligands of MHC I molecules at a far lower efficiency than peptides chaperoned by any of the heat shock proteins tested and (ii) treatment of cells with deoxyspergualin, a drug that binds hsp70 and hsp90 with apparent specificity, abrogates the ability of cells to present antigenic peptides through MHC I molecules, and introduction of additional hsp70 into the cytosol overcomes this abrogation. These results suggest for the first time a functional role for cytosolic chaperones in antigen processing. endoplasmic reticulum brefeldin A deoxyspergualin HSP, heat shock protein Mixed Lymphocyte Tumor Culture nucleoprotein serum albumin transporter associated with antigen processing vesicular stomatitis virus Roswell Park Memorial Institute N-[-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (C43H83NO8S) major histocompatibility complex phosphate-buffered saline N-acetyl-l-leucinyl-l-leucinal-l-norleucinal cytotoxic T lymphocyte large T-antigen protein from SV40 leukocyte function antigen-1. Cellular proteins undergo degradation in the cytosol, and the resulting peptides are transported into the endoplasmic reticulum (ER),1 generally through transporter associated with antigen processing (TAP). Within the ER, the peptides are charged onto MHC I molecules. One of the key unresolved questions in this scheme pertains to the mechanism through which peptides are channeled to the TAP or other transporters. Although peptides are generated in the cytosol, there is little evidence that the cytosol harbors free peptides. It has been proposed that the peptides exist in association with peptide-binding proteins in the cytosol and the ER (1Srivastava P.K. Maki R.G. Curr. Top. Microbiol. Immunol. 1991; 167: 109-123Crossref PubMed Scopus (169) Google Scholar, 2Srivastava P.K. Udono H. Blachere N.E. Li Z. Immunogenetics. 1994; 39: 93-98Crossref PubMed Scopus (501) Google Scholar). Because heat shock proteins (HSPs) are known to chaperone a wide array of molecules (3Ellis R.J. Curr. Biol. 1999; 9: R137-R139Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and because immunological and structural evidence exists that HSPs chaperone antigenic peptides (see Ref. 4Srivastava P.K. Menoret A. Basu S. Binder R.J. McQuade K.L. Immunity. 1998; 8: 1-20Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar for review), it was suggested that HSPs are the peptide-binding proteins that transport peptides (1Srivastava P.K. Maki R.G. Curr. Top. Microbiol. Immunol. 1991; 167: 109-123Crossref PubMed Scopus (169) Google Scholar, 2Srivastava P.K. Udono H. Blachere N.E. Li Z. Immunogenetics. 1994; 39: 93-98Crossref PubMed Scopus (501) Google Scholar). This view has received little formal attention in the form of support or rejection, although no alternative mechanisms of peptide traffic have been suggested. Nonetheless, evidence has continued to accumulate that (a) HSPs are associated with peptides from a wide spectrum of antigens, including tumor antigens (5Udono H. Srivastava P.K. J. Exp. Med. 1993; 178: 1391-1396Crossref PubMed Scopus (565) Google Scholar, 6Ishii T. Udono H. Yamano T. Ohta H. Uenaka A. Ono T. Hizuta A. Tanaka N. Srivastava P.K. Nakayama E. J. Immunol. 1999; 162: 1303-1309PubMed Google Scholar), viral antigens (7Nieland T.J.F. Tan M.C.A. Muijen M.M. Koning F. Kruisbeek A.M. van Bleek G.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6135-6139Crossref PubMed Scopus (192) Google Scholar), model antigens (8Arnold D. Faath S. Rammensee H.-G. Schild H. J. Exp. Med. 1995; 182: 885-889Crossref PubMed Scopus (293) Google Scholar, 9Arnold D. Wahl C. Faath S. Rammensee H.-G. Schild H. J. Exp. Med. 1997; 186: 461-466Crossref PubMed Scopus (74) Google Scholar, 10Breloer M. Marti T. Fleischer B. von Bonin A. Eur. J. Immunol. 1998; 28: 1016-1021Crossref PubMed Scopus (88) Google Scholar), and minor H antigens (8Arnold D. Faath S. Rammensee H.-G. Schild H. J. Exp. Med. 1995; 182: 885-889Crossref PubMed Scopus (293) Google Scholar), and that (b) the repertoire of peptides associated with the HSP of the ER is dependent upon the functional status of TAP (9Arnold D. Wahl C. Faath S. Rammensee H.-G. Schild H. J. Exp. Med. 1997; 186: 461-466Crossref PubMed Scopus (74) Google Scholar). In this report, we address the issue functionally and ask if the chaperoning of peptides in the cytosol by HSPs confers on the HSP-chaperoned peptides any advantage not available to unchaperoned peptides in terms of their presentability by MHC I molecules. EL4 cells were treated with brefeldin A (BFA) at two different concentrations in succession to respectively block the MHC I pathway of antigen presentation (6.0 μg/ml for 3 h) and to maintain the block (0.6 μg/ml for up to 12 h). Maintenance of the BFA block did not affect CTL function during the CTL assay. EL4 cells, untreated or treated with BFA at these concentrations, were analyzed by FACScan to show maximal decreases (∼40%) in surface expression of MHC I after 20 h (data not shown). BFA-treated cells were loaded with protein and used as targets in the CTL assay, as described, in the presence of BFA. The T-Ag-transformed cell lines SVB6 and PS-C3H were obtained from Prof. S. S. Tevethia and have been previously described (11Tevethia S.S. Lewis M. Tanaka Y. Milici B.K. Maloy W.L. Anderson R. J. Virol. 1990; 64: 1192-1200Crossref PubMed Google Scholar) The VSVNP-transfected EL4 cell line, N1, was obtained from Dr. Lynn Puddington and has been previously described (12Puddington L. Bevan M.J. Rose J.K. Lefrancois L. J. Virol. 1986; 60: 708-717Crossref PubMed Google Scholar). EL4 cells and the TAP-dysfunctional cell line, RMA-S, were obtained from Prof. S. Nathenson. The RMA cell line has been previously described (13Lunggren H.-G. Karre K. J. Exp. Med. 1985; 162: 1745-1759Crossref PubMed Scopus (646) Google Scholar). All chemicals were purchased from Sigma Chemical Co. unless otherwise specified. HL-1 and RPMI media, together with pyruvate, glutamine, penicillin-streptomycin, and non-essential amino acids were purchased from Life Technologies, Inc. RPMI containing 5% fetal calf serum (Intergen) and 1% each of pyruvate, glutamine, penicillin-streptomycin, and non-essential amino acids is subsequently referred to as complete RPMI. HSPs were detected by immunoblotting with specific antibodies: gp96 (rat monoclonal antibody SPA-850, clone 9G10); cytosolic hsp70 (mouse monoclonal antibody SPA-820, clone N27F3-4 recognizes constitutive hsp73 and inducible hsp72); hsp90 (rat monoclonal antibody SPA-845, clone 1R2D12p90). All these antibodies were purchased from StressGen Biotechnologies Corp., Victoria, Canada. Anti-Kb, anti-Db, anti-Dd, or anti-LFA-1 (clones AF6-88-5, KH95, 34-2-12, and 2D7, respectively)-fluorescein-conjugated monoclonal antibodies were obtained from PharMingen (San Diego, CA). To prepare proteins (gp96, hsp70, hsp90, or SA; complexed or not) for loading, the indicated amount of protein was incubated with DOTAP (N-[-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate (C43H83NO8S)) (Roche Molecular Biochemicals) at a 3:2 ratio (microgram amounts) for 15 min at room temperature. In all loading experiments, 1.5 × 106 cells (EL4, RMA, or RMA-S) were washed three times with serum-free HL-1 media and then incubated in 1 ml of HL-1 media with a protein:DOTAP combination for 4–4.5 h at 37 °C. Control cells were either mock-loaded by incubating 1.5 × 106 cells in the same amount of DOTAP alone or were incubated with protein alone in the absence of DOTAP (pulsed cells). After loading (or mock loading for controls), cells were washed three times with HL-1 media and once with complete RPMI. Where indicated, loaded, mock-loaded, or pulsed cells were used as targets in CTL assays. Loading efficiencies of gp96, hsp70, hsp90, or serum albumin alone were the same, and inter-experimental values did not vary significantly. Free peptides were loaded into cells using the same protocol. CTL assays were carried out as follows. Briefly 2 × 103 51chromium (supplied as Na2CrO4; ICN) labeled target cells in 100 μl of complete RPMI were added to various dilutions of T-Ag or VSV8-specific CTL in 100 μl of complete RPMI, as indicated by the effector-to-target ratios. Effectors and targets were cultured in v-bottomed 96-well plates for 4 h. Supernatants (50 μl) were harvested, mixed with scintillation fluid, and counted in a 1450 MicroBeta Trilux liquid scintillation counter (Wallac Inc.). Percent specific 51Cr release was measured as follows, Exerimental51Cr release−spontaneous51Cr releaseMaximum51Cr release−spontaneous51Cr release×100%Equation 1 Maximum and spontaneous releases were measured by culturing 2 × 103 labeled target cells in lysis buffer (0.5% Nonidet P-40, 10 mm Tris, 1 mm EDTA, 150 mm NaCl) and complete RPMI, respectively, for 4 h. VSV8-specific CTL were obtained by dual immunizations of C57BL/6 mice, 1 week apart, with N1 cells. Spleen cells were harvested 1 week after the second immunization, restimulated in culture with irradiated N1 cells, and cloned by limiting dilution (14Heike M. Blachere N.E. Wolfel T. Meyer zum Buschenfelde K.H. Storkel S. Srivastava P.K. J. Immunother. Emphas. Tumor Immunol. 1994; 15: 165-174Crossref PubMed Scopus (13) Google Scholar). The specificity of the CTL clone was tested by cold target inhibition and antibody blocking experiments. This CTL clone was shown to be specific for the VSV8 peptide (NH2-RGYVYQGL-COOH) bound to Kbmolecules. A similar strategy, with SVB6 cells, was used to obtain the T-Ag-specific CTL clone. This clone was shown to be specific for the 9-mer peptide (NH2-AINNYAQKL-COOH), previously named epitope 1 (11Tevethia S.S. Lewis M. Tanaka Y. Milici B.K. Maloy W.L. Anderson R. J. Virol. 1990; 64: 1192-1200Crossref PubMed Google Scholar). N1 cells were irradiated (5000 rads) and allowed to recover in AIM V medium with or without DSG for 48 h at 37 °C or 25 °C. Half of the cells incubating at 25 °C were then placed at 37 °C for an additional 8 h. One group of the cells not treated with DSG but incubated at 25 °C for 48 h was placed at 37 °C in the presence of DSG for 8 h. Cells (1 × 106) were then stained at 4 °C for 40 min with anti-Kb, anti-Db, anti-Dd, or anti-LFA fluorescein-conjugated antibodies and analyzed on a FACScan flow cytometer purchased from Becton Dickinson (San Jose, CA). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% saponin and probed with anti-VSV glycoprotein Cy3-coupled antibody (Sigma). Cells were visualized using a Zeiss LSM confocal microscope. Vesicular stomatis virus (VSV) was obtained from Advanced Biotechnology (Columbia, MD). Meth A or EL4 cells were incubated with 10 plaque forming units of VSV per cell for 1 h at 37 °C in plain RPMI and allowed to recover in RPMI with 10% fetal calf serum for 4 h. Cells were washed three times in PBS (10 mmphosphate buffer, 150 mm NaCl, 2.7 mm KCL, pH 7.4). gp96 was then purified from these cells as described below. The following peptides were used (underlinedsequences represent the precise MHC I binding epitope): unextended MHC binding 9-mer, NH2-AINNYAQKL-COOH; T-Ag 20-mer (N terminus extended), NH2-FFLTPHRHRVS- AINNYAQKL-COOH; T-Ag 20-mer (N+C termini extended), NH2-RHRVSAINNYAQKLCTFSFL-COOH; T-Ag 20-mer (C terminus extended), NH2-AINNYAQKLCTFSFLICKGV-COOH. Peptides were synthesized by Genemed to >95% purity as determined by high pressure liquid chromatography. The unextended MHC I binding 9-mer peptide is identical to epitope I of the T-Ag (11Tevethia S.S. Lewis M. Tanaka Y. Milici B.K. Maloy W.L. Anderson R. J. Virol. 1990; 64: 1192-1200Crossref PubMed Google Scholar). The T-Ag 9-mer stabilized MHC I molecules on RMA-S cells and sensitized targets for lysis by the T-Ag-specific CTL. All three T-Ag 20-mer peptides failed to bind MHC H-2Db as determined by their inability to stabilize empty MHC molecules on the surface of RMA-S cells and their inability to sensitize target cells for lysis by T-Ag-specific CTL. Purified gp96 or hsp90 was incubated, at the indicated amount, with peptide at a protein-to-peptide molar ratio of 1:50 in 700 μl of PBS for 10 min at 50 °C and incubated for a further 30 min at room temperature. The peptide concentration used for complexing was 10−6 m. Approximately 1% of the gp96 or hsp90 molecules was loaded with the exogenous peptides by this method (15Blachere N.E. Li Z. Chandawarkar R.Y. Suto R. Jaikaria N.S. Basu S. Udono H. Srivastava P.K. J. Exp. Med. 1997; 186: 1315-1322Crossref PubMed Scopus (491) Google Scholar). The indicated amount of purified hsp70 or SA was incubated with peptide at a protein-to-peptide molar ratio of 1:50 in 300 μl of PBS at 37 °C for 1 h. Peptide concentration used for complexing was 10−6 m. To remove free, uncomplexed peptides, complexes were washed extensively with PBS in an Ultrafree-4 centrifugal device, Biomax 10K NMWL membrane (Millipore Corp.). To determine the efficiency of complexing, peptides were labeled with125I (ICN) using IODO-BEADs (Pierce). In parallel with unlabeled peptides, 125I-labeled peptides were complexed to proteins and checked by SDS-polyacrylamide gel electrophoresis and autoradiography (data not shown). The efficiency of gp96, hsp70, hsp90, or SA to complex peptides was comparable. EL4 cells (107) in complete RPMI were treated for 2 h with 100 μm of the proteasome inhibitorN-acetyl-l-leucinyl-l-leucinal-l-norleucinal (LLnL) in Me2SO or with 0.002% Me2SO alone. In other experiments, EL4 cells were treated with 100 μmlactacystin dissolved in Me2SO for 1 h. In both cases, the treated cells were constantly in the presence of the inhibitor during loading with protein. FACScan analysis of inhibitor-treated cells showed greater than 35% decrease in cell surface MHC I expression after 20 h confirming inhibition of MHC I trafficking. hsp70 and gp96 were purified from cells according to previously described methods (16Peng P. Menoret A. Srivastava P.K. J. Immunol. Methods. 1997; 204: 13-21Crossref PubMed Scopus (111) Google Scholar, 17Srivastava P.K. Immunology Methods Manual. Academic Press Ltd., New York1997: 739-747Google Scholar). hsp90 was purified according to the protocol of Denis (18Denis M. Anal. Biochem. 1988; 173: 405-411Crossref PubMed Scopus (27) Google Scholar) with minor modifications. Briefly, 100,000 × g supernatants were obtained from cell lysates and applied to a Mono Q column (Mono Q HR 16/10, purchased from Amersham Pharmacia Biotech and attached to the BIOCAD, Perseptive Biosytems). 15-Deoxyspergualin (DSG) was a gift from Dr. S. Nadler at Bristol-Myers Squibb Co. (Wallingford, CT). Lyophilized DSG was dissolved in PBS and stored in aliquots at a concentration of 10 mg/ml at −130 °C. Twenty micrograms per ml of DSG, with or without peptide (final concentration of 1 μm), was added to the MLTC of VSV CTL clones. After a 5-day incubation at 37 °C, each well of the MLTC was harvested and tested for its ability to lyse51Cr-labeled N1 and EL4 cells in a 4-h 51Cr release assay. The cationic liposome, DOTAP, was used to introduce HSP·peptide complexes or free peptides into the cytosol. Distinct properties of the detergents Nonidet P-40 and Saponin were used to demonstrate that DOTAP-loaded gp96 enters the soluble, non-vesicular, cytosolic compartment of the cells (Fig.1). Although the cytosolic HSPs are of primary interest in this study, gp96 was used as a test case, because its distinct non-cytosolic localization (in the ER) permitted determination of the compartment into which the HSP·peptide complexes were being introduced, as will become clear from the following. The gp96/DOTAP-loaded cells were lysed with each of two detergents. Lysis of live cells with 0.5% Nonidet P-40 leads to solubilization of all non-nuclear membranes, whereas lysis with 0.01% Saponin results in solubilization of plasma membranes but not internal membranes (19Forster C. Marienfeld S. Wilhelm R. Kramer R. FEMS Microbiol. Lett. 1998; 167: 209-214Crossref PubMed Scopus (14) Google Scholar). The lysates were centrifuged to obtain the solubilized components, which were analyzed for the presence of gp96 by immunoblotting: The internal resident gp96 is detected in the Nonidet P-40-lysates of non-loaded cells (Fig. 1, lane 1) but not in the Saponin-lysed non-loaded cells (Fig. 1, lanes 2 and 3), because gp96 is a luminal component of the ER compartment, which remains impervious to Saponin. gp96 is not detected in cells treated with DOTAP without gp96 (lane 2) or gp96 without DOTAP (lane 3). The only instance where gp96 is detectable in the Saponin-solubilized cells is if it has been introduced along with DOTAP into cells (lane 4), i.e. from an exogenous source. As additional controls, all samples tested predictably positive for the cytosolic chaperone hsp70 (Fig. 1, bottom panel,lanes 1–4). Thus, DOTAP-mediated delivery of gp96 (and by deduction other proteins) into cells introduces them into the cytosolic compartment. Similar results were obtained with introduction of labeled peptides by DOTAP. Quantitative analysis of exogenously introduced radiolabeled proteins through DOTAP indicated that ∼5% of the DOTAP-loaded protein is introduced into the cytosol and that >96% of this 5% is detected in a soluble, non-vesicular, cytosolic compartment of the cells (data not shown). As discussed in the previous section, the cytosolic chaperones hsp90 and hsp70 are of primary interest for the studies described all through this report. However, the ER chaperone gp96 was also used in all studies, primarily because (i) gp96 was used for the demonstration that DOTAP introduces proteins into the cytosol, (ii) gp96 is highly homologous (protein sequence homology of 50%) (see Ref.1Srivastava P.K. Maki R.G. Curr. Top. Microbiol. Immunol. 1991; 167: 109-123Crossref PubMed Scopus (169) Google Scholar) to the cytosolic chaperone hsp90, and (iii) considerable immunological and structural information on gp96-peptide interaction is already available (see Ref. 4Srivastava P.K. Menoret A. Basu S. Binder R.J. McQuade K.L. Immunity. 1998; 8: 1-20Abstract Full Text Full Text PDF PubMed Scopus (490) Google Scholar). gp96, purified from the T-Ag-transformed cell line SVB6, and chaperoning T-Ag-derived peptides was loaded into EL4 cells by DOTAP. Presentation of T antigen-derived peptides by MHC I molecules of EL4 cells was monitored by specific lysis of DOTAP-loaded cells using a CTL clone specific for epitope 1 of the T-Ag (11Tevethia S.S. Lewis M. Tanaka Y. Milici B.K. Maloy W.L. Anderson R. J. Virol. 1990; 64: 1192-1200Crossref PubMed Google Scholar) as described under "Experimental Procedures." The T-Ag-derived peptides were present in gp96 preparations and were observed to be efficiently re-presented in this assay (Fig. 2 A). Percentage of lysis of the loaded cells increased with increasing amounts of gp96 loaded into the cells. No lysis was observed with less than 25 μg of gp96. In parallel control experiments, EL4 cells were pulsed, in the absence of DOTAP (as opposed to loaded) with gp96, to determine if there is extracellular exchange of peptides between gp96 and surface MHC I molecules on EL4 cells. No surface charging was detected. SVB6-derived hsp70 or hsp90 preparations were also loaded into EL4 cells in increasing doses. Antigen-specific recognition of the loaded cells by CTLs was observed when either hsp70 or hsp90 was loaded (Fig.2 A), indicating that similar to gp96, hsp70 or hsp90 donate their chaperoned peptides to become ligands for MHC I molecules. Again, lysis of cells was dependent on the amount of HSP loaded by DOTAP. Although peptides chaperoned by all three HSPs could become ligands for re-presentation by MHC I molecules, the efficiency of doing so was different for each HSP. For comparable lysis (∼40%) of HSP-loaded cells, 100, 250, and 500 μg of gp96, hsp70, and hsp90, respectively, were required. Approximate amounts of HSP, below which no lysis was detected were 25, 180, and 400 μg for gp96, hsp70, and hsp90, respectively. A second, well characterized antigenic system, the Vesicular Stomatitis Virus (VSV) system, was used to test the generality of the observation in the T-Ag system. VSV nucleoprotein (VSVNP) derived peptides chaperoned by gp96 or hsp70 (purified from the VSVNP-transfected cell line N1 (12Puddington L. Bevan M.J. Rose J.K. Lefrancois L. J. Virol. 1986; 60: 708-717Crossref PubMed Google Scholar) are effectively re-presented and recognized by VSVNP-specific CTL after the respective HSPs are introduced into the cytosol of EL4 cells by DOTAP (Fig. 2 B). To demonstrate that lysis by VSVNP-specific CTL, of cells loaded with HSPs, is peptide-dependent, equivalent amounts of peptide-free hsp70, obtained by ATP treatment of N1-derived hsp70 preparations (15Blachere N.E. Li Z. Chandawarkar R.Y. Suto R. Jaikaria N.S. Basu S. Udono H. Srivastava P.K. J. Exp. Med. 1997; 186: 1315-1322Crossref PubMed Scopus (491) Google Scholar), were delivered into EL4 cells. No lysis of EL4 cells loaded with peptide-depleted hsp70 preparations was observed (Fig. 2 B). Furthermore, HSP preparations not carrying VSVNP-derived peptides (EL4-derived HSPs) (Fig. 2 B), did not render loaded cells susceptible to VSVNP-specific CTLs, with any amount of HSP loaded. The results imply that presentation and consequent cell lysis are both peptide-dependent and -specific and require intracellular processing of the HSP·peptide complexes. It is difficult to monitor and quantify presentation of specific antigenic peptides in naturally derived HSP·peptide complexes. To quantitate the efficiency of re-presentation of specific HSP-chaperoned peptides, HSPs reconstitutedin vitro with known quantity of antigenic peptides or their extended versions were used. The Db-restricted 9-mer epitope I of the SV40 T-Ag protein (NH2-AINNYAQKL-COOH), or 20-mer peptides extended on the NH2 terminus, COOH terminus, or both termini (Fig.3 A) were complexed to HSPs gp96, hsp90, or hsp70, or a control peptide-binding protein serum albumin (SA) (15 and "Experimental Procedures"). Peptides thus complexed (∼10−8 m with respect to peptide concentration) or free peptides (10−6 or 10−4 m) were loaded into EL4 cells with DOTAP. In parallel, experiments using radiolabeled HSPs, SA, and each of the peptides were used to determine how much of each moiety administered with DOTAP could be recovered in the cytosol of the cells. This exercise demonstrated that ∼6–8% of the quantity of each moiety introduced in the cells by DOTAP could be recovered from the cytosol (data not shown). The constancy of this number allows for valid comparisons among the results with each antigenic moiety. The cells into which the HSPs, SA, or peptides were introduced were then monitored for lysis by T-Ag-specific CTLs (Fig. 3 B). It was observed that (i) a concentration of 10−4 m free peptide was required for loading to observe lysis of the EL4 cells comparable to that observed for 10−8 m concentration of peptide when chaperoned by HSPs, (ii) peptides chaperoned by SA, which binds peptides efficiently ("Experimental Procedures"), were not re-presented by MHC I molecules, suggesting that HSPs play a role different from simply carrying the peptides, and (iii) MHC I epitopes are generated from peptides chaperoned by HSPs regardless of whether they are extended on the NH2, COOH, or both termini. The cytosolic proteasomes have been implicated as the primary producers of peptide ligands for MHC I molecules (for review see Refs. 20Monaco J.J. Immunol. Today. 1992; 13: 173-179Abstract Full Text PDF PubMed Scopus (398) Google Scholar, 21Goldberg A.L. Rock K.L. Nature. 1992; 357: 375-379Crossref PubMed Scopus (504) Google Scholar). Because DOTAP-mediated loading of cells with the HSP·peptide complexes results in presentation the peptides by MHC I, we tested the requirement for proteasomal activity for re-presentation of HSP-chaperoned peptides. Because HSPs are purified from cells after the peptides have been generated through protease activity and also have been shown to chaperone precise MHC I peptide epitopes (6Ishii T. Udono H. Yamano T. Ohta H. Uenaka A. Ono T. Hizuta A. Tanaka N. Srivastava P.K. Nakayama E. J. Immunol. 1999; 162: 1303-1309PubMed Google Scholar, 7Nieland T.J.F. Tan M.C.A. Muijen M.M. Koning F. Kruisbeek A.M. van Bleek G.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6135-6139Crossref PubMed Scopus (192) Google Scholar, 10Breloer M. Marti T. Fleischer B. von Bonin A. Eur. J. Immunol. 1998; 28: 1016-1021Crossref PubMed Scopus (88) Google Scholar), we expected that re-presentation of HSP-chaperoned peptides would not require further proteasomal action. EL4 cells were treated with the proteasome inhibitor, N-acetyl-Leu-Leu-norleucinal (LLnL) for 1 h prior to and during loading with either the endoplasmic reticulum (ER) chaperone gp96 or the cytosolic chaperone hsp70 derived from N1 cells. Surprisingly, re-presentation of VSVNP peptides chaperoned by gp96 or hsp70 was inhibited by LLnL (Fig.4 A), suggesting that re-presentation of HSP-chaperoned peptides requires functional protease activity. Control, LLnL-untreated EL4 cells loaded with gp96 or hsp70 in an identical manner were able to re-present VSVNP-derived peptides. Because LLnL has been shown to have inhibitory effects on proteases other than the proteasome (22Craiu A. Gaczynska M. Akopian T. Gramm C.F. Fenteany G. Goldberg A.L. Rock K.L. J. Biol. Chem. 1997; 172: 13437-13445Abstract Full Text Full Text PDF Scopus (349) Google Scholar), we replaced LLnL with the proteasome-specific inhibitor, lactacystin. To examine the proteasome dependence of HSP-chaperoned peptide re-presentation more precisely, we used HSP·peptide complexes reconstituted in vitro instead of the naturally derived complexes. The four T-Ag-derived peptides used earlier (Fig. 3 A) were complexed separately to gp96, hsp70, hsp90, or the non-HSP, SA. HSP·peptide complexes, reconstitutedin vitro, were loaded independently but identically, into EL4 cells, not treated or treated with lactacystin prior to loading. It was observed (Table I) that (i) treatment with lactacystin inhibited re-presentation of all theextended peptides, (ii) surprisingly, treatment of cells with lactacystin inhibited re-presentation of even theprecise unextended MHC I-binding peptides when chaperoned by hsp70 or hsp90; (iii) in another surprise, re-presentation of the precise MHC I binding peptide complexed to gp96 was not inhibited by lactacystin. These observations suggest that, during re-presentation, proteasomes may contribute function(s) other than proteolytic degradation of extended peptides. They also suggest that peptides chaperoned by the ER HSP, gp96, are processed by a different mechanism from that of peptides chaperoned by the cytosolic hsp70 and hsp90. The structural basis for this difference is not yet clear.Table IInfluence of inhibition of functional proteasomes by lactacystin on re-presentation of precise and extended peptides chaperoned by HSPsChaperonePeptidesN-extended, 20-merN+C-extended, 20-merC-extended, 20-merDb-binding, 9-mer%gp9660.196.081.00hsp9061.298.098.271.1hsp7090.198.365.346.1Data are percent inhibition of re-presentation. EL4 cells were treated with lactacystin prior to loading with HSP · peptide complexes reconstituted in vitro as described under "Experimental Procedures." Re-presentation was monitored in a 51Cr release assay by a T-Ag-specific CTL clone. Data were obtained in a cytotoxicity assay done at an effector:target ratio of 5:1. Percent inhibition was calculated, assuming as 100% the specific cytotoxicity obtained in the absence of lactacystin. This was approximately 60%. Open table in a new tab Data are percent inhibition of re-presentation. EL4 cells were treated with lactacystin prior to loading with HSP · peptide complexes reconstituted in vitro as described under "Experimental Procedures." Re-presentation was monitored in a 51Cr release assay by a T-Ag-specific CTL clone. Data were obtained in a cytotoxicity assay done at an effector:target ratio of 5:1. Percent inhibition was calculated, assuming as 100% the specific cytotoxicity obtained in the absence of lactacystin. This was approxima