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• W1569113943 abstract "The fate of viral glycopeptides as cytotoxic T lymphocyte (CTL) epitopes is unclear. We have dissected the mechanisms of antigen presentation and CTL recognition of the peptide GP392–400 (WLVTNGSYL) from the lymphocytic choriomeningitis virus (LCMV) and compared them with those of the previously reported GP92–101 antigen (CSANNSHHYI). Both GP392–400 and GP92–101 bear a glycosylation motif, are naturallyN-glycosylated in the mature viral glycoproteins, bind to major histocompatibility complex H-2Db molecules, and are immunogenic. However, post-translational modifications differentially affected GP92–101 and GP392–400. Upon N-glycosylation or de-N-glycosylation, a marked decrease in major histocompatibility complex binding was observed for GP392–400 but not for GP92–101. Further, under its N-glycosylated or de-N-glycosylated form, GP392–400 then lost its initial ability to generate a CTL response in mice, whereas GP92–101 was still immunogenic under the same conditions. The genetically encoded form of GP392–400, which on the basis of its immunogenicity could still be presented with H-2Db during the course of LCMV infection, does not in fact appear at the surface of LCMV-infected cells. Our results show that post-translational modifications of viral glycopeptides can have pleiotropic effects on their presentation to and recognition by CTL that contribute to either creation of neo-epitopes or destruction of potential epitopes. The fate of viral glycopeptides as cytotoxic T lymphocyte (CTL) epitopes is unclear. We have dissected the mechanisms of antigen presentation and CTL recognition of the peptide GP392–400 (WLVTNGSYL) from the lymphocytic choriomeningitis virus (LCMV) and compared them with those of the previously reported GP92–101 antigen (CSANNSHHYI). Both GP392–400 and GP92–101 bear a glycosylation motif, are naturallyN-glycosylated in the mature viral glycoproteins, bind to major histocompatibility complex H-2Db molecules, and are immunogenic. However, post-translational modifications differentially affected GP92–101 and GP392–400. Upon N-glycosylation or de-N-glycosylation, a marked decrease in major histocompatibility complex binding was observed for GP392–400 but not for GP92–101. Further, under its N-glycosylated or de-N-glycosylated form, GP392–400 then lost its initial ability to generate a CTL response in mice, whereas GP92–101 was still immunogenic under the same conditions. The genetically encoded form of GP392–400, which on the basis of its immunogenicity could still be presented with H-2Db during the course of LCMV infection, does not in fact appear at the surface of LCMV-infected cells. Our results show that post-translational modifications of viral glycopeptides can have pleiotropic effects on their presentation to and recognition by CTL that contribute to either creation of neo-epitopes or destruction of potential epitopes. major histocompatibility complex lymphocytic choriomeningitis virus cytotoxic T lymphocyte(s) N-(9-fluorenyl)methoxycarbonyl high pressure liquid chromatography bovine serum albumin phosphate-buffered saline effector:target Viral peptides presented by major histocompatibility complex (MHC)1 class I molecules at the surface of infected cells to CTL are key molecular signals allowing recognition of the infected cells and their subsequent destruction by the activated CTL. Those peptides commonly consist of octameric to undecameric antigenic peptides generated from viral proteins by a multistep, intracellular processing pathway involving components present in the cytoplasm and the endoplasmic reticulum. Peptides generated in the cytosol from the degradation of ubiquitinated proteins by the proteasome and possibly by nonproteasomal cytosolic proteases (1Nandi D. Marusina K. Monaco J. Curr. Top. Microbiol. Immunol. 1998; 232: 15-48PubMed Google Scholar, 2Groettrup M. Soza A. Kuckelkorn U. Kloetzel P.-M. Immunol. Today. 1996; 17: 429-435Abstract Full Text PDF PubMed Scopus (200) Google Scholar) are then translocated via peptide-specific transporters (transporter associated with antigen processing) into the endoplasmic reticulum where they assemble with the MHC class I molecules and are eventually subjected to further trimming (3Elliott T. Willis A. Cerundolo W. Townsend A. J. Exp. Med. 1995; 181: 1481-1491Crossref PubMed Scopus (156) Google Scholar, 4Snyder H.L. Yewdell J.W. Bennink J.R. J. Exp. Med. 1994; 180: 2389-2394Crossref PubMed Scopus (161) Google Scholar). The capacity or contrastingly the inability of a given peptide to proceed through these steps has been shown to determine its presentation by MHC molecules (5Luckey C.J. King G.M. Marto J.A. Venketeswaran S. Maier B.F. Crotzer V.L. Colella T.A. Shabanowitz J. Hunt D.F. Engelhard V.H. J. Immunol. 1998; 161: 112-121PubMed Google Scholar, 6Morel S. Levy F. Burlet-Schiltz O. Brasseur F. Probst-Kepper M. Peitrequin A.L. Monsarrat B. Van Velthoven R. Cerottini J.C. Boon T. Gairin J.E. Van den Eynde B.J. Immunity. 2000; 12: 107-117Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 7Valmori D. Gileadi U. Servis C. Dunbar P.R. Cerottini J.C. Romero P. Cerundolo V. Levy F. J. Exp. Med. 1999; 189: 895-906Crossref PubMed Scopus (107) Google Scholar). In contrast to the well studied processing pathway of peptides derived from cytosolic proteins, very little is known about the ability of glycopeptides originating from viral glycoproteins to successfully pass through the above mentioned steps. For example based on the use of model peptides chemically glycosylated (8Speir J.A. Abdel-Motal U.M. Jondal M. Wilson I.A. Immunity. 1999; 10: 51-61Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 9Glithero A. Tormo J. Haurum J.S. Arsequell G. Valencia G. Edwards J. Springer S. Townsend A. Pao Y.L. Wormald M. Dwek R.A. Jones E.Y. Elliott T. Immunity. 1999; 10: 63-74Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 10Haurum J.S. Arsequell G. Lellouch A.C. Wong S.Y. Dwek R.A. McMichael A.J. Elliott T. J. Exp. Med. 1994; 180: 739-744Crossref PubMed Scopus (182) Google Scholar, 11Haurum J.S. Tan L. Arsequell G. Frodsham P. Lellouch A.C. Moss P.A. Dwek R.A. McMichael A.J. Elliott T. Eur. J. Immunol. 1995; 25: 3270-3276Crossref PubMed Scopus (66) Google Scholar, 12Abdel-Motal U. Berg L. Rosen A. Bengtsson M. Thorpe C. Kihlberg J. Dahmen J. Magnusson G. Karlsson K. Jondal M. Eur. J. immunol. 1996; 26: 544-551Crossref PubMed Scopus (84) Google Scholar) or affected by other post-translational modifications such as reduction of sulfhydryl groups (13Chen W. Yewdell J.W. Levine R.L. Bennink J.R. J. Exp. Med. 1999; 189: 1757-1764Crossref PubMed Scopus (98) Google Scholar), bond rearrangement (14Chen W. Ede N.J. Jackson D.C. McCluskey J. Purcell A.W. J. Immunol. 1996; 157: 1000-1005PubMed Google Scholar), or phosphorylation (15Andersen M.H. Bonfill J.E. Neisig A. Arsequell G. Sondergaard I. Valencia G. Neefjes J. Zeuthen J. Elliott T. Haurum J.S. J. Immunol. 1999; 163: 3812-3818PubMed Google Scholar), it has been proposed that these modifications could generate new epitopes and thus increase antigenic diversity (10Haurum J.S. Arsequell G. Lellouch A.C. Wong S.Y. Dwek R.A. McMichael A.J. Elliott T. J. Exp. Med. 1994; 180: 739-744Crossref PubMed Scopus (182) Google Scholar, 11Haurum J.S. Tan L. Arsequell G. Frodsham P. Lellouch A.C. Moss P.A. Dwek R.A. McMichael A.J. Elliott T. Eur. J. Immunol. 1995; 25: 3270-3276Crossref PubMed Scopus (66) Google Scholar, 16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 17Ferris R.L. Buck C. Hammond S.A. Woods A.S. Cotter R.J. Takiguchi M. Igarashi Y. Ichikawa Y. Siliciano R.F. J. Immunol. 1996; 156: 834-840PubMed Google Scholar, 18Skipper J.C.A. Hendrickson R.C. Gulden P.H. Brichard V. VanPel A. Chen Y. Shabanowitz J. Wolfel T. Slinguff C.L.J. Boon T. Hunt D.F. Engelhard V.H. J. Exp. Med. 1996; 183: 527-534Crossref PubMed Scopus (378) Google Scholar, 19Zarling A. Ficarro S. White F. Shabanowitz J. Hunt D. Engelhard V. J. Exp. Med. 2000; 192: 1755-1762Crossref PubMed Scopus (167) Google Scholar). However, given the failure to identify naturally processedN-glycopeptides as epitopes to date, the fate of viral glycopeptides as MHC class I-restricted antigens remains very unclear. Lymphocytic choriomeningitis virus (LCMV) infection of H-2bmice is cleared by CTL that recognize immunodominant (NP396–404, GP33–41/43, and GP376–386) (20Murali-Krishna K. Altman J.D. Suresh M. Sourdive D.J. Zajac A.J. Miller J.D. Slansky J. Ahmed R. Immunity. 1998; 8: 177-187Abstract Full Text Full Text PDF PubMed Scopus (1712) Google Scholar, 21Schulz M. Aichele P. Wollenweider M. Bobe F.W. Cardinaux F. Hengartner H. Zinkernagel R.M. Eur. J. Immunol. 1989; 19: 1657-1667Crossref PubMed Scopus (111) Google Scholar, 22Gairin J.E. Mazarguil H. Hudrisier D. Oldstone M.B.A. J. Virol. 1995; 69: 2297-2305Crossref PubMed Google Scholar, 23Klavinskis L.S. Whitton J.L. Joly E. Oldstone M.B. Virology. 1990; 178: 393-400Crossref PubMed Scopus (101) Google Scholar, 24Oldstone M.B.A. Whitton J.L. Lewicki H. Tishon A. J. Exp. Med. 1988; 168: 559-570Crossref PubMed Scopus (126) Google Scholar) or subdominant (GP92–101) epitopes presented in the context of a MHC class I H-2Dband/or H-2Kb molecule (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 20Murali-Krishna K. Altman J.D. Suresh M. Sourdive D.J. Zajac A.J. Miller J.D. Slansky J. Ahmed R. Immunity. 1998; 8: 177-187Abstract Full Text Full Text PDF PubMed Scopus (1712) Google Scholar, 25Hudrisier D. Mazarguil H. Laval F. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1996; 271: 17829-17836Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Vandermost R. Muralikrishna K. Whitton J. Oseroff C. Alexander J. Southwood S. Sidney J. Chesnut R. Sette A. Ahmed R. Virology. 1998; 240: 158-167Crossref PubMed Scopus (138) Google Scholar). The presence of glycopeptides derived from the LCMV glycoproteins bearing the H-2Db binding motif makes LCMV infection a model of choice to study the fate of glycopeptides as CTL epitopes. We recently showed that the LCMV GP92–101 subdominant epitope (CSANNSHHYI), which bears the glycosylation motif NNS within its sequence, was post-translationally modified in infected cells and presented at the cell surface by H-2Db molecules in two different forms, a nonglycosylated form (CSANNSHHYI) and a de-N-glycosylated form (CSADNSHHYI) resulting from the N-glycanase-mediated de-N-glycosylation of a previously N-glycosylated peptide sequence (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). An additional H-2Db-restricted glycopeptide GP392–400 (WLVTNGSYL) was previously identified in the LCMV GP2 protein (25Hudrisier D. Mazarguil H. Laval F. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1996; 271: 17829-17836Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). It shares similar biochemical properties with GP92–101. Like GP92–101, GP392–400: (i) bears a NXS glycosylation motif, (ii) is N-glycosylated in the mature LCMV GP2 glycoprotein (27Wright K.E. Spiro R.C. Burns J.W. Buchmeier M.J. Virology. 1990; 177: 175-183Crossref PubMed Scopus (71) Google Scholar), and (iii) binds efficiently to H-2Db (25Hudrisier D. Mazarguil H. Laval F. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1996; 271: 17829-17836Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 28Oldstone M.B.A. Lewicki H. Borrow P. Hudrisier D. Gairin J.E. J. Virol. 1995; 69: 7423-7429Crossref PubMed Google Scholar). In contrast to GP92–101, no CTL response against GP392–400 has been detected (28Oldstone M.B.A. Lewicki H. Borrow P. Hudrisier D. Gairin J.E. J. Virol. 1995; 69: 7423-7429Crossref PubMed Google Scholar), and its cellular status remains unknown. The contrasting immunogenic properties of GP92–101 and GP392–400 suggest that the common biochemical events or intracellular mechanisms encountered by these two viral glycopeptides along the processing and presentation pathway lead to different fates as MHC class I-restricted CTL epitopes. This would not have been predicted on the basis of their biochemical properties. The aim of our study was therefore to explore and dissect the intracellular molecular mechanisms that may differentially affect the processing pathways of these two viral glycopeptides and explain their different antigenic properties. CTL were obtained from C57BL/6 mice immunized subcutaneously at the base of the tail with 50–100 μg of the indicated peptides mixed with 5 μg of P30 T-helper epitope from tetanus toxoid in incomplete Freund's adjuvant. After 1 week, draining lymph nodes were removed, and CTL were restimulated at weekly intervals with irradiated (2,500 rads) C57BL/6 splenocytes and irradiated (10,000 rads) peptide-pulsed (1 μm) RMA cells in the presence of 30 IU/ml interleukin-2 (EL4 supernatant). Murine H-2b mutant RMA-S cells were used in binding experiments. The murine H-2b cell lines RMA and MC57 were used inin vitro cytotoxicity assays. The cells were grown in Dulbecco's modified Eagle's medium (RMA, RMA-S, and MC57) containing 5% bovine serum, l-glutamine (2 mm), and antibiotics (10 units/ml penicillin and 10 μg/ml streptomycin). The LCMV Armstrong strain (LCMV Arm) was used to infect mice or cells. The peptides were synthesized by the solid phase method using Fmoc chemistry. For Asn-glycosylated peptides, Fmoc-Asn(Ac3AcNH-βGlc)-OH, a commercially available derivative of Asn bearing GlcNAc (Novabiochem) was used. After standard protocols for solid phase synthesis, cleavage, and deprotection, the glycopeptide was de-O-acetylated with 0.1m sodium hydroxide as described previously (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The peptides were purified by HPLC on a RP300-C8 reversed phase column (Brownlee Lab), and their identities were confirmed by electrospray ionization mass spectrometry. RMA or MC57 cells were incubated for 1 h at 37 °C in medium containing 10-fold dilutions of peptides. MC57 cells were infected at a multiplicity of infection of 2 with LCMV Arm 48 h prior to the assay. RMA (peptide-pulsed or not) or MC57 (LCMV-infected or not) cells were51Cr-labeled and used as targets (5 × 103/well) in chromium release assays. CTL (1.5 × 104/well or at the indicated E/T ratio) were added, and after 4 h of incubation at 37 °C, the 51Cr content of supernatants was determined. The specific lysis was calculated as 100 × [(experimental − spontaneous release)/(total − spontaneous release)]. The binding studies were performed as described previously (25Hudrisier D. Mazarguil H. Laval F. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1996; 271: 17829-17836Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 29Hudrisier D. Mazarguil H. Oldstone M.B.A. Gairin J.E. Mol. Immunol. 1995; 32: 895-907Crossref PubMed Scopus (19) Google Scholar). Briefly, RMA-S cells (5 × 105 cells/well) previously incubated at 26 °C for 40 h were placed in U-bottomed 96-well plates for 4 h at 37 °C with increasing concentrations (0–10−4m) of unlabeled peptides. The cells were then washed twice with BSA/PBS and incubated at 4 °C for 45 min. with the 28–14-8S anti-H-2Db monoclonal antibody (hybridoma supernatant) (30Ozato K. Hansen T.H. Sachs D.H. J. Immunol. 1980; 125: 2473-2477PubMed Google Scholar). After two washes in BSA/PBS, an anti-mouse IgG antibody conjugated to fluorescein isothiocyanate (Sigma) was added for 45 min. Then the cells were washed twice in BSA/PBS and analyzed by flow cytometry. The peptides were extracted from the surface of LCMV-infected MC57 (H-2b) cells as described previously (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Briefly, cells (1–2 × 109) were washed in PBS and then resuspended in 0.1 m citrate/phosphate buffer at pH 3.0 for 2 min. The eluted material was desalted on a SepPak column, transferred onto a Centricon 10, and centrifuged at 3500 rpm for 30 min at 4 °C. Material less than 10 kDa was vacuum concentrated then resuspended in 20 μl of 0.08% trifluoroacetic acid. The peptides were separated by HPLC (Waters 600S controller system) on a reversed phase C18 column (7 μm, 2.1 × 100 mm, Aquapore, BrownleeTM) according to the following procedure: solution A, 0.08% trifluoroacetic acid in H2O; solution B, 0.08% trifluoroacetic acid in CH3CN; a 60-min gradient, 5–60% B; flow rate: 400 μl/min. The fractions (200 μl) were collected in U-bottomed 96-well plates, lyophilized, reconstituted in PBS, and stored at −80 °C before analysis. The three-dimensional models for LCMV GP92–101 (CSANNSHHYI) and LCMV GP392–400 (WLVTNGSYL) interacting with H-2Db have been produced using the online modeling facility SwissModel (GlaxoWellcome) at the following address: www.expasy.ch/spdbv/. Peptides bearing aN-glycosylation motif NX(S/T) can theoretically exist in three different forms: a nonglycosylated one bearing an unmodified NX(S/T) sequence, a N-glycosylated one in which a glycan moiety is attached to Asn, and a de-N-glycosylated one resulting in the conversion of Asn to Asp. To analyze their fate as CTL epitopes, we synthesized peptides corresponding to the nonglycosylated (genetically encoded),N-glycosylated, and de-N-glycosylated forms of LCMV GP392–400 (named GP392–400, [GlcNAc-N396]-GP392–400, and [D396]-GP392–400 respectively), and of LCMV GP92–101 (named GP92–101, [GlcNAc-N95]-GP92–101, and [D95]-GP92–101 respectively). The N-acetyl-d-glucosamine moiety was chosen to mimic the glycosylated form because it is the first carbohydrate attached to Asn and common to all eukaryotic glycosylation processes. Previous studies have failed to reveal a response to LCMV GP392–400 following LCMV infection of mice (28Oldstone M.B.A. Lewicki H. Borrow P. Hudrisier D. Gairin J.E. J. Virol. 1995; 69: 7423-7429Crossref PubMed Google Scholar), but in these studies the fact that GP392–400 bears a N-glycan in the mature viral protein was not taken into account. Our first aim was therefore to analyze the ability of the different unmodified (GP392–400) and post-translationally modified ([GlcNAc-N396]-GP392–400 and [D396]-GP392–400) forms of LCMV GP392–400 to generate a CTL response upon immunization. After several rounds of in vitrorestimulation, CTL from peptide-immunized B6 mice were obtained against the GP392–400 form but not against the [GlcNAc-N396]-GP392–400 and [D396]-GP392–400 forms. This indicated that only GP392–400 was immunogenic, a result that contrasted with those obtained with LCMV GP92–101, where all three forms (GP92–101, [GlcNAc-N95]-GP92–101, and [D95]-GP92–101) were found to be immunogenic under the same experimental conditions (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). As shown in Fig.1, anti-GP392–400 CTL specifically recognized the H-2b RMA (panel A) or MC57 (panel B) cells pulsed with GP392–400, in an effector:target (E:T) ratio-dependent manner. Unpulsed targets were not lysed (RMA) or were lysed weakly at the highest E:T ratios (MC57). A CTL clone was then derived from the LCMV GP392–400-specific CTL line by limiting dilution. We tested the ability of these CTL to kill target cells coated with the immunizing peptide GP392–400 or its post-translationally modified forms [GlcNAc-N396]-GP392–400 or [D396]-GP392–400. Both the CTL line (Fig.2A) and the CTL clone (Fig.2B) recognized and lysed GP392–400-pulsed target cells with comparable efficacy (half-maximal lysis in the pm range). In contrast, 3–4-log higher concentrations of [GlcNAc-N396]-GP392–400 or of [D396]-GP392–400 (half-maximal lysis in the 10−9–10−8m range) were required to obtain the same lysis by both the CTL line and the clone. In a comparative analysis shown in Fig. 2C, LCMV GP92–101-specific CTL generated against the GP92–101 peptide recognized the [D95]-GP92–101 form more efficiently than the GP92–101 form used for the immunization (half-maximal lysis at 10−12 and 10−10m, respectively). Higher concentrations of [GlcNAc-N95]-GP92–101 (half-maximal lysis in the 10−8m range, comparable with that of [GlcNAc-N396]-GP392–400 by anti-GP392–400 specific CTL) were necessary to sensitize target cells to CTL killing, confirming previous results (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Because one of the factors determining the immunogenicity of peptides is their ability to be presented by MHC class-I molecules (31vandenBurg S.H. Visseren M.J.W. Brandt R.M.P. Kast W.M. Melief C.J.M. J. Immunol. 1996; 156: 3308-3314PubMed Google Scholar, 32Hudrisier D. Riond J. Gairin J. J. Virol. 2001; 75: 2468-2471Crossref PubMed Scopus (6) Google Scholar), we hypothesized that post-translational modifications could differentially affect the MHC class I presentation and could result in different immunogenic properties of the LCMV viral glycopeptides. Peptide binding to H-2Db was measured in a previously described MHC stabilization assay on RMA-S cells (25Hudrisier D. Mazarguil H. Laval F. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1996; 271: 17829-17836Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 29Hudrisier D. Mazarguil H. Oldstone M.B.A. Gairin J.E. Mol. Immunol. 1995; 32: 895-907Crossref PubMed Scopus (19) Google Scholar). As shown in Fig.3A, GP392–400 bound efficiently to H-2Db (half-maximal binding in the 0.1–0.2 μm range), in agreement with previous observations (25Hudrisier D. Mazarguil H. Laval F. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1996; 271: 17829-17836Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In contrast binding of the two modified analogs to H-2Dbwas strongly reduced; [D396]-GP392–400 showed a 2-log decrease in H-2Db binding properties (half-maximal binding at 10 μm). Half-maximal binding was even not measurable for [GlcNAc-N396]-GP392–400 (only ∼25% maximal binding was observed at the highest concentration tested (10−4m)). Deficiency in MHC binding of these two peptides is therefore likely one of the reasons that may explain their lack of immunogenicity. A different situation was observed with the LCMV GP92–101 subdominant antigen for which the three forms bound efficiently to H-2Db (Fig. 3B). GP92–101 and [D95]-GP92–101 showed superimposed curves (half-maximal binding at <0.1 μm), whereas the [GlcNAc-N395]-GP92–101 form exhibited a 1-log higher binding (half-maximal binding at <0.01 μm). The differences in the efficiency with which they are recognized by CTL may thus likely reflect differences in TCR recognition rather than MHC binding (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). It can be deduced from the experiments presented above that only the Asn396 form of GP392–400 is likely to be presented by H-2Db at the surface of infected cells, whereas all three forms of GP92–101 could be presented. The cytolytic activity of the anti-GP392–400 CTL was thus tested against LCMV-infected or uninfected MC57 target cells. Fig.4 shows that neither the CTL line (panel A) nor the CTL clone (panel B) generated against GP392–400 were able to kill LCMV-infected cells, even at the highest E:T ratio tested. In comparison (panel C), efficient killing was observed when the CTL line generated against the unmodified form of the subdominant LCMV epitope GP92–101 was used, in accord with a previous study (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Further, when HPLC-fractionated material eluted from the surface of LCMV-infected MC57 cells was used to pulse uninfected targets, no lysis by anti-GP392–400 CTL was observed (Fig.5B). This observation contrasted that made with cells coated with mock-extracted synthetic GP392–400 peptide (Fig. 5A) and results obtained with anti-GP92–101 CTL directed against cells pulsed with the same pool of peptides extracted from LCMV-infected MC57 cells (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Because a very low number of copies (100 or less) of an antigenic peptide present at the cell surface is sufficient to trigger CTL lysis, we can conclude from these results that GP392–400 is not naturally presented at the surface of LCMV-infected cells. This observation contrasts with that made with LCMV GP92–101 for which we found that both the genetically encoded GP92–101 and post-translationally modified [D95]-GP92–101 forms were co-presented to CTL at the surface of LCMV-infected cells, whereas the N-glycosylated form [GlcNAc-N95]-GP92–101 was absent (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar).Figure 5Recognition of acid-eluted peptides from LCMV-infected MC57 cells by anti-GP392–400-specific CTL. Each HPLC fraction corresponding to mock-extracted synthetic LCMV GP396–400 control peptide (solid circles, A) or to material eluted from the surface of LCMV-infected MC57 cells (2 × 108 eq) (open circles, B) was tested for its capacity to sensitize RMA cells to lysis by anti-GP392–400 CTL in a classical CTL assay as described under “Experimental Procedures.” The arrows numbered1, 2, and 3 indicate the retention times of synthetic peptides [GlcNAc-N396]-GP392–400, GP392–400, and [D396]-GP392–400, respectively. The results are expressed as percentages of specific lysis and are representative of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, by analyzing two H-2Db-restricted LCMV glycopeptides, GP392–400 and GP92–101 (25Hudrisier D. Mazarguil H. Laval F. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1996; 271: 17829-17836Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Vandermost R. Muralikrishna K. Whitton J. Oseroff C. Alexander J. Southwood S. Sidney J. Chesnut R. Sette A. Ahmed R. Virology. 1998; 240: 158-167Crossref PubMed Scopus (138) Google Scholar), we showed that post-translational modifications of MHC class I-restricted viral peptides that bear a glycosylation motif NXS can have pleiotropic effects on their presentation by MHC and CTL recognition, depending on the peptide sequence (TableI). We previously reported that synthetic peptides corresponding to the unmodified (genetically encoded),N-glycosylated or de-N-glycosylated analogs of GP92–101 share the ability to bind strongly to H-2Db, are both immunogenic, and are naturally processed and presented at the surface of infected cells (16Hudrisier D. Riond J. Mazarguil H. Oldstone M.B.A. Gairin J.E. J. Biol. Chem. 1999; 274: 36274-36280Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). We now show that post-translational modifications of so called “high affinity peptides” do not always have a positive effect on the diversity of antigen presentation. The results presented in this paper show that N-glycosylation or de-N-glycosylation of GP392–400 led to peptides with reduced MHC binding properties at physiological concentrations, whereas unmodified GP392–400 efficiently bound to this molecule. This may be one of the reasons why only the unmodified form of GP392–400 is immunogenic in C56BL/6 mice and could generate epitope-specific CTL that efficiently lysed peptide-pulsed target cells. However GP392–400-specific CTL did not recognize LCMV-infected cells, showing that GP392–400 is not presented by H-2Db in a natural situation. The lack of presentation of the [GlcNAc-N396]-GP392–400 and [D396]-GP392–400 forms can be easily explained by our observation that those peptides are poor H-2Db binders and show no immunogenicity. Indeed above a certain threshold of binding to the MHC, the immunogenicity of peptides rapidly drops, preventing these peptides from acting as T cell epitopes (33Barber L.D. Parham P. J. Exp. Med. 1994; 180: 1191-1194Crossref PubMed Scopus (29) Google Scholar). That LCMV-infected cells were not recognized by anti-GP392–400 CTL tells us that not only [GlcNAc-N396]-GP392–400 and [D396]-GP392–400 but also GP392–400, despite its high H-2Db binding properties, were absent from the infected cell surface.Table ISummary of the cellular, biochemical, and immunological properties of the different forms of the viral glycopeptides LCMV GP392–400 and GP92–101SequencePeptideCellular statusCTL epitopeH-2D1-bPeptides were considered as immunogenic or not when a CTL response was generated or not following injection of synthetic peptide as described under “Experimental Procedures.”binding1-aBinding was considered as efficient, moderate, or weak when measured half-maximal binding values were ≤1 μm, ≤100 μm, or >100 μm, respectively.Immunogenicity1-bPeptides were considered as immunogenic or not when a CTL response was generated or not following injection of synthetic peptide as described under “Experimental Procedures.”Presented by LCMV-infected cellsG" @default.
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