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

Abstract

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 cytosolic hsp70 monoclonal antibody clone and hsp90 (rat monoclonal antibody clone All these were purchased from or and monoclonal were obtained from proteins or or for the of protein was with methylsulfate at a for at In cells or were with HL-1 and in of HL-1 with a for h at cells were by cells in the of or were with protein in the of for cells were with HL-1 and with complete RPMI. or cells were used as targets in CTL of or serum albumin were the and did not peptides were loaded into cells the CTL were as as cells in of complete RPMI were to of or CTL in of complete as by the and targets were in for were with and in a specific was as and were by cells in and complete for CTL were obtained by of with cells were after the in with cells, and by M. Blachere N.E. T. S. Srivastava P.K. J. Tumor Immunol. 1994; PubMed Scopus Google Scholar). The of the CTL clone was tested by and antibody This CTL clone was to specific for the peptide bound to A with SVB6 cells, was used to the CTL This clone was to specific for the peptide previously (11Tevethia S.S. Lewis M. Tanaka Y. Milici B.K. Maloy W.L. Anderson R. J. Virol. 1990; 64: 1192-1200Crossref PubMed Google Scholar). cells were and to in with or for h at or of the cells at were at for additional One of the cells not treated with at for h was at in the presence of for were at for with or and analyzed on a FACScan purchased from were with with and with antibody were a virus was obtained from A or EL4 cells were with of cell for h at in RPMI and to in RPMI with fetal calf serum for were in gp96 was from these cells as described The peptides were used the MHC I MHC were by to as by The MHC I peptide is to I of the (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 MHC I molecules on cells and targets for by the All peptides to MHC as by their to MHC molecules on the surface of cells and their to cells for by gp96 or hsp90 was at the with peptide at a of in of for at and for a at The peptide used for was 1% of the gp96 or hsp90 molecules was loaded with the peptides by this N.E. Li Z. R. Basu S. Udono H. Srivastava P.K. J. Exp. Med. 1997; 186: PubMed Scopus Google Scholar). The of hsp70 or was with peptide at a of in of at for used for was peptides, were with in the efficiency of peptides were In with peptides, peptides were to proteins and by and (data not shown). The efficiency of or to complex peptides was EL4 cells in complete RPMI were treated for h with of the in or with In other EL4 cells were treated with in for In the treated cells were in the presence of the during with FACScan of cells than in cell surface MHC I expression after 20 h of MHC I hsp70 and gp96 were from cells to previously described Menoret A. Srivastava P.K. J. Immunol. 1997; PubMed Scopus Google Scholar, P.K. Scholar). hsp90 was to the of M. PubMed Scopus Google Scholar) with minor were obtained from cell and to a purchased from and to the was a from Dr. S. at Co. was in and in at a of at of with or peptide of was to the of CTL a at each of the was and tested for its ability to and EL4 cells in a assay. The was used to or free peptides into the of the and were used to that gp96 the cytosolic of the cells Although the cytosolic HSPs are of in this gp96 was used as a because its the of the into which the were as become from the The cells were with each of two of cells with to of with results in of not C. S. R. R. Microbiol. 1998; 167: PubMed Scopus Google Scholar). The were to the which were analyzed for the presence of gp96 by The gp96 is detected in the of cells not in the cells and because gp96 is a of the ER which to gp96 is not detected in cells treated with gp96 or gp96 The gp96 is in the cells is if it has been introduced with into cells from additional tested for the cytosolic chaperone hsp70 of gp96 (and by other into cells into the cytosolic results were obtained with introduction of peptides by of introduced proteins through that of the protein is introduced into the cytosol and that of this 5% is detected in a cytosolic of the cells (data not shown). in the the cytosolic chaperones hsp90 and hsp70 are of for the studies described through this the ER chaperone gp96 was used in because (i) gp96 was used for the that proteins into the cytosol, (ii) gp96 is of (see P.K. Maki R.G. Curr. Top. Microbiol. Immunol. 1991; 167: 109-123Crossref PubMed Scopus (169) Google Scholar) to the cytosolic chaperone and immunological and structural on is 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). from the T-Ag-transformed cell line and chaperoning peptides was loaded into EL4 cells by of T peptides by MHC I molecules of EL4 cells was monitored by specific of cells a CTL clone specific for of the (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 The peptides were present in gp96 and were to in this of of the loaded cells with of gp96 loaded into the was with than of In EL4 cells were in the of to with to if there is of peptides gp96 and surface MHC I molecules on EL4 surface was hsp70 or hsp90 were loaded into EL4 cells in of the loaded cells by was hsp70 or hsp90 was loaded that to hsp70 or hsp90 their chaperoned peptides to become ligands for MHC I molecules. of cells was dependent on the of HSP loaded by Although peptides chaperoned by HSPs become ligands for by MHC I molecules, the efficiency of was different for each (∼40%) of cells, and of and were of HSP, which no was detected were and for and A antigenic the was used to the of the in the nucleoprotein peptides chaperoned by gp96 or hsp70 from the VSVNP-transfected cell line (12Puddington L. Bevan M.J. Rose J.K. Lefrancois L. J. Virol. 1986; 60: 708-717Crossref PubMed Google Scholar) are and by CTL after the HSPs are introduced into the cytosol of EL4 cells by that by of cells loaded with is of obtained by treatment of hsp70 N.E. Li Z. R. Basu S. Udono H. Srivastava P.K. J. Exp. Med. 1997; 186: PubMed Scopus Google Scholar), were into EL4 of EL4 cells loaded with hsp70 was HSP not peptides did not loaded cells to with any of HSP The results that presentation and cell are and and processing of the It is to and presentation of specific antigenic peptides in the efficiency of of specific HSP-chaperoned peptides, HSPs with known of antigenic peptides or their were The I of the SV40 protein or peptides on the or were to HSPs or or a peptide-binding protein serum albumin and with to peptide or free peptides or were loaded into EL4 cells with In experiments and each of the peptides were used to of each with in the cytosol of the This that of the of each introduced in the cells by from the cytosol (data not shown). The of this for the results with each antigenic The cells into which the or peptides were introduced were monitored for by 3 It was that (i) a of free peptide was for to of the EL4 cells to that for of peptide chaperoned by (ii) peptides chaperoned by which binds peptides were not by MHC I molecules, that HSPs a role different from the peptides, and MHC I are generated from peptides chaperoned by HSPs of are on the or The cytosolic have been as the of peptide ligands for MHC I molecules Immunol. Full Text PDF PubMed Scopus Google Scholar, K.L. PubMed Scopus Google Scholar). Because of cells with the results in presentation the peptides by MHC we tested the for for of HSP-chaperoned peptides. Because HSPs are from cells after the peptides have been generated through and have been to chaperone MHC I peptide 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, T.J.F. Tan M.C.A. Muijen M.M. 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Biol. 1997; Full Text Full Text PDF Scopus Google Scholar), we with the the of HSP-chaperoned peptide we used in of the The peptides used 3 were to or the were loaded into EL4 cells, not treated or treated with to It was that (i) treatment with of peptides, (ii) treatment of cells with of MHC peptides chaperoned by hsp70 or in of the MHC I peptide to gp96 was not by These suggest during other than degradation of peptides. suggest that peptides chaperoned by the ER HSP, are by a different mechanism from that of peptides chaperoned by the cytosolic hsp70 and The structural for this is not of of functional by on of and peptides chaperoned by are of EL4 cells were treated with to with HSP peptide in as described was monitored in a by a CTL were obtained in a at of was as the specific obtained in the of This was in a are of EL4 cells were treated with to with HSP peptide in as described was monitored in a by a CTL were obtained in a at of was as the specific obtained in the of This was generated in the cytosol are transported by TAP into the endoplasmic reticulum for association with MHC I molecules B. M.J. 1994; PubMed Scopus Google Scholar, K. K. 1994; PubMed Scopus Google Scholar, M.J. T. 1995; PubMed Scopus Google Scholar, B. B. T. Immunity. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Eur. J. Immunol. 1997; PubMed Scopus Google Scholar). The for TAP in of HSP-chaperoned peptides was tested by peptide in cells RMA and in TAP-dysfunctional cells cells were not by CTL after loaded with gp96 or hsp70 at any of HSP used In RMA cells, functional TAP molecules, did the HSP-chaperoned peptides as by the of RMA cells RMA cells with HSPs in the of were not to that was not to of peptides. A for TAP for presentation of with the of Bevan and Bevan M.J. J. Exp. Med. PubMed Scopus Google Scholar) that by MHC I in the of functional molecules in a of the and results that the are not cells as cells that to transport peptides into the It is of antigenic peptides, as by introduction of the of the the become A the peptides generated in cells are transported by introduction of the peptides with HSPs into the MHC I molecules are loaded with peptides in the ER, are transported to the cell surface the by vesicular A (BFA) is a known of vesicular traffic J. J. PubMed Scopus Google Scholar). EL4 cells were not treated or treated with BFA for h to and during of or The loaded cells were tested for by It was that BFA of peptides chaperoned by and hsp90 This was by BFA-treated EL4 cells in the of BFA for 3 h to with is a drug to with hsp70 and hsp90 B. PubMed Scopus Google Scholar, K. M. 1994; PubMed Scopus Google Scholar). The drug cells and with We to the of to of lead to of hsp70 and which to chaperone the generated antigenic peptides into the presentation This was tested in a of a first was to lymphocyte tumor of cells cells with the (12Puddington L. Bevan M.J. Rose J.K. Lefrancois L. J. Virol. 1986; 60: 708-717Crossref PubMed Google and CTL The generated in the presence and of were tested in a for and of with was to the of this if peptide were to the of peptide to from SV40 T antigen with the as the did not the These that treatment with in a in the of the on if was at the of the or the the were and were in with or and were tested for their ability to treatment for as as h no on the The of on cells was monitored cells a of specific MHC which have a and because a of MHC are of Y. M. Immunity. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), a was no specific MHC EL4 cells were treated not with for h as to of hsp70 and hsp90 molecules. were with The and viral cells, which or not been to were used to as described in a (see The cells were to to the at the cells as additional in these EL4 cells, with or treatment with were with and these were tested for the ability to with was to have no on the ability of cells treatment of cells with no on viral and expression of viral proteins as by of cells with protein antibody to a These results show that treatment with with a in the which is for of the specific MHC although the block is not in of MHC I molecules Although has been to with hsp70 and hsp90 B. PubMed Scopus Google Scholar), the that the are not to the HSPs to of with the role of hsp70 was tested in of the it is to molecules into the cytosol of living cell with the of This was used to or as a has been previously to peptides N.E. Li Z. R. Basu S. Udono H. Srivastava P.K. J. Exp. Med. 1997; 186: PubMed Scopus Google into the cells h after with The cells were used to the as The that introduction of hsp70 not the in ability in The ability of to block the trafficking of peptides for the cell surface MHC I molecules was tested by assay. MHC of peptides are on the cell surface at are at C. J. A. Karre K. 1990; PubMed Scopus Google Scholar). The MHC detected by and These were used to the presence of MHC I molecules on the cell surface of and untreated It was that treatment of EL4 cells with at lead to a in the of as by the specific antibody no was if the EL4 cells at were to the cells a of than the cells, that a large of molecules of cells at were of peptides and in the EL4 cells the expression in and untreated cells was monitored and to by treatment with that was not the pathway as by the at These support for the that HSPs are for transport of antigenic peptides in the cytosol and that with this The studies reported here on trafficking of peptides in the cytosol, to presentation of peptides by the MHC I molecules. free peptides introduced into the cytosol are as with HSP-chaperoned peptides. This the that the trafficking of peptides in the cytosol not by by including chaperoning (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, 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). This is because of The of peptides available in a cell the of for from a is to presentation by MHC I molecules, if the peptide were to The of peptide has a of if it were chaperoned by HSP as chaperoning by a is for peptide because peptides are not any than free peptides. The structural that the for and efficiency of chaperoning in presentation for each HSP, as of peptide association with other proteins are not studies through which These structural at for the in for hsp90 and hsp70 gp96 In because hsp70 and hsp90 are cytosolic their with in the appropriate to their chaperoning In the gp96 its peptides into into the cytosol, for by with different and to the first two of studies show that of hsp70 (and with transport of antigenic peptides to MHC I molecules. binds to cytosolic hsp70 and hsp90 the H. 1998; PubMed Scopus Google Scholar). Because in the cytosol 1993; PubMed Scopus Google Scholar) and because the ER gp96 not have the the of to the cytosolic of to HSPs not affect the ability of peptides to to or from these HSPs and its on HSPs H. 1998; PubMed Scopus Google Scholar and not shown). the of this is the first of role for hsp70 molecules in antigen presentation by MHC I molecules. results in this the of H. M. Immunol. 1998; PubMed Scopus Google Scholar) that of antigen cells with hsp70 the cells presentation results at a role for of proteins or peptides into MHC I HSP-chaperoned peptides, generated from the processing including in the cells from which are for the peptides chaperoned by hsp70 or hsp90 are the MHC their the presence of functional This is with the in to peptides from are in of the peptides into the TAP pathway through a the 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). of cells with the ability of the to or with of the as TAP and block peptide The in or in the of peptides from the a that This to in a results in of on the cytosolic events in antigen processing. B. A. N. A. A. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar) have that of or is for of protein in studies in a form of the virus nucleoprotein as the U. M. J. C. J. Biol. 1999; PubMed Scopus (192) Google Scholar) have specific in the cytosol antigenic peptides are that is chaperoned to these by and that the undergo degradation by in at these to of antigenic peptides. In view of results that free antigenic peptides in the cytosol are and that HSP-chaperoned peptides are we to the model by U. M. J. C. J. Biol. 1999; PubMed Scopus (192) Google Scholar) to suggest that (and is not in the of this by chaperoning or to this in chaperoning the resulting antigenic peptides to the TAP We have suggested previously that of these including the transport of peptides to in a which we the 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). We Dr. Basu of for the