Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing
Identifieur interne : 002581 ( Istex/Corpus ); précédent : 002580; suivant : 002582Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing
Auteurs : Neil Blake ; Steven Lee ; Irina Redchenko ; Wendy Thomas ; Neil Steven ; Alison Leese ; Patty Steigerwald-Mullen ; Michael G. Kurilla ; Lori Frappier ; Alan RickinsonSource :
- Immunity [ 1074-7613 ] ; 1997.
English descriptors
- Teeft :
- Acad, Adenovirus, Antigen processing, Assay, Autologous, Baculovirus, Cell clones, Cell line, Cell response, Cell responses, Chromium, Chromium release assay, Chromium release assays, Clone, Control targets, Control vaccinia, Ctls, Cytotoxic, Dmso, E1cd, Ebna1, Ebna1 coding sequence, Ebna1 epitope, Ebna1 epitope peptide, Ebna1 epitopes, Ebna1 fragments, Ebna1 protein, Ebna1 sequence, Endogenously, Endoplasmic reticulum, Epitope, Epitope ebna1, Exogenous, Exogenous antigen, Exogenously, Fusion protein, Growth medium, Healthy donors, Histocompatibility, Human cytomegalovirus, Immunol, Khanna, Kieff, Kurilla, Latent cycle proteins, Levitskaya, Lymphocyte, Lysis, Major histocompatibility, Minimal epitope, Molecular weight, Natl, Npc4, Nuclear antigen, Pathway, Peptide, Peripheral blood lymphocytes, Plasmid, Preexposed, Preincubated, Proc, Protein extracts, Recent work, Recombinant, Recombinant adenoviruses, Recombinant vaccinia vectors, Recombinant vaccinia viruses, Representative results, Rickinson, Sensitization, Smaller peptides, Solid bars, Standard chromium release assay, Steven, Striped bars, Synthetic epitope peptide, Synthetic peptides, Target cells, Unpublished data, Vaccinia, Viral, Virol.
Abstract
Abstract: Epstein-Barr virus (EBV)–induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.
Url:
DOI: 10.1016/S1074-7613(00)80397-0
Links to Exploration step
ISTEX:474D05875BF03CF5B9136B67E5EB8676C803526FLe document en format XML
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Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>ebna1</json:string><json:string>epitope</json:string><json:string>clone</json:string><json:string>recombinant</json:string><json:string>peptide</json:string><json:string>vaccinia</json:string><json:string>lysis</json:string><json:string>target 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data</json:string><json:string>endoplasmic reticulum</json:string><json:string>smaller peptides</json:string><json:string>peripheral blood lymphocytes</json:string><json:string>striped bars</json:string><json:string>chromium</json:string></teeft></keywords><author><json:item><name>Neil Blake</name><affiliations><json:string>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</json:string></affiliations></json:item><json:item><name>Steven Lee</name><affiliations><json:string>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</json:string></affiliations></json:item><json:item><name>Irina Redchenko</name><affiliations><json:string>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</json:string></affiliations></json:item><json:item><name>Wendy Thomas</name><affiliations><json:string>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</json:string></affiliations></json:item><json:item><name>Neil Steven</name><affiliations><json:string>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</json:string></affiliations></json:item><json:item><name>Alison Leese</name><affiliations><json:string>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</json:string></affiliations></json:item><json:item><name>Patty Steigerwald-Mullen</name><affiliations><json:string>Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia, 22901, USA</json:string></affiliations></json:item><json:item><name>Michael G Kurilla</name><affiliations><json:string>Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia, 22901, USA</json:string></affiliations></json:item><json:item><name>Lori Frappier</name><affiliations><json:string>Department of Medical Genetics, Microbiology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada</json:string></affiliations></json:item><json:item><name>Alan Rickinson</name><affiliations><json:string>E-mail: williamsd@cancer.bham.ac.uk</json:string><json:string>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</json:string></affiliations></json:item></author><arkIstex>ark:/67375/6H6-NQ31TRTZ-D</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Epstein-Barr virus (EBV)–induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.</abstract><qualityIndicators><score>8.368</score><pdfWordCount>8085</pdfWordCount><pdfCharCount>51073</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>12</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>114</abstractWordCount><abstractCharCount>768</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing</title><pmid><json:string>9430224</json:string></pmid><pii><json:string>S1074-7613(00)80397-0</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Immunity</title><language><json:string>unknown</json:string></language><publicationDate>1997</publicationDate><issn><json:string>1074-7613</json:string></issn><pii><json:string>S1074-7613(00)X0033-7</json:string></pii><volume>7</volume><issue>6</issue><pages><first>791</first><last>802</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1997</json:string></date><geogName></geogName><orgName><json:string>University of Homburg-Saar, Germany</json:string><json:string>Department of Medical Genetics and Microbiology</json:string><json:string>University of Birmingham, United Kingdom</json:string><json:string>Life Technologies</json:string><json:string>Canada Summary Epstein-Barr</json:string><json:string>University of Wisconsin</json:string><json:string>University of Toronto Toronto</json:string><json:string>Medical Research Council Career Development Award</json:string><json:string>University of Tokyo, Japan</json:string><json:string>Department of Pathology and Microbiology</json:string><json:string>Department of Clinical Oncology, Prince of Wales Hospital, Shatin, Hong Kong</json:string><json:string>University of Virginia Charlottesville</json:string><json:string>Medical Research Council</json:string><json:string>Cancer Research Campaign</json:string><json:string>United Kingdom</json:string></orgName><orgName_funder><json:string>Medical Research Council</json:string><json:string>Cancer Research Campaign</json:string><json:string>United Kingdom</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Lori Frappier</json:string><json:string>Irina Redchenko</json:string><json:string>Wendy Thomas</json:string><json:string>Patty Steigerwald-Mullen</json:string><json:string>Alison Leese</json:string><json:string>Carl Schildkraut</json:string><json:string>Alta Bioscience</json:string><json:string>Bill Sugden</json:string><json:string>Alan Rickinson</json:string><json:string>Neil Steven</json:string><json:string>GAr Domain</json:string><json:string>M. Takiguchi</json:string><json:string>To</json:string><json:string>Michael G. Kurilla</json:string><json:string>F. Grasser</json:string><json:string>Steven Lee</json:string><json:string>P. Johnson</json:string></persName><placeName><json:string>Bradford</json:string><json:string>San Diego</json:string><json:string>NY</json:string><json:string>CA</json:string><json:string>York</json:string></placeName><ref_url></ref_url><ref_bibl><json:string>Hennessy and Kieff, 1983</json:string><json:string>Falk et al., 1995</json:string><json:string>Levitskaya et al., 1995</json:string><json:string>Rock, 1996</json:string><json:string>Gavioli et al., 1992</json:string><json:string>Lee et al., 1995, 1997</json:string><json:string>Norbury et al., 1995</json:string><json:string>Khanna et al., 1992</json:string><json:string>Gilbert et al., 1996</json:string><json:string>Takiguchi et al., 1994</json:string><json:string>Henle et al., 1987</json:string><json:string>Khanna et al., 1997</json:string><json:string>Schirmbeck et al., 1995</json:string><json:string>Baer et al., 1984</json:string><json:string>Wilkinson and Akrigg, 1992</json:string><json:string>Rammensee et al., 1995</json:string><json:string>Murray et al., 1992</json:string><json:string>Kovacsovics-Bankowski et al., 1993</json:string><json:string>York and Rock, 1996</json:string><json:string>Rickinson and Kieff, 1996</json:string><json:string>Salter et al., 1985</json:string><json:string>Floettmann et al., 1996</json:string><json:string>Hill et al., 1995</json:string><json:string>Lee et al., 1997</json:string><json:string>Burrows et al., 1992b</json:string><json:string>Goldsmith et al., 1993</json:string><json:string>Ball, 1987</json:string><json:string>Levitskaya et al.</json:string><json:string>Riddell et al., 1991</json:string><json:string>Wiertz et al., 1996a, 1996b</json:string><json:string>Steven et al., 1997</json:string><json:string>Tamaki et al., 1995</json:string><json:string>Burrows et al., 1992a</json:string><json:string>Tierney et al., 1994</json:string><json:string>Rickinson and Moss, 1997</json:string><json:string>Tigges et al., 1992</json:string><json:string>Ahn et al., 1997</json:string><json:string>McGrory et al., 1988</json:string><json:string>Pfeifer et al., 1993</json:string><json:string>Chen et al., 1995</json:string><json:string>Levitskaya et al. (1995)</json:string><json:string>Steven et al., 1996</json:string><json:string>Liu et al., 1995</json:string><json:string>Jones et al., 1996</json:string><json:string>Khanna et al., 1995, 1997</json:string><json:string>Gavioli et al., 1993</json:string><json:string>Grunhaus and Horwitz, 1992</json:string><json:string>Kay and McPherson, 1987</json:string><json:string>Ahn et al., 1996</json:string><json:string>Hengel et al., 1997</json:string><json:string>Ulmer et al., 1994</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-NQ31TRTZ-D</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - immunology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - clinical medicine</json:string><json:string>3 - immunology</json:string></scienceMetrix><scopus><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Infectious Diseases</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - Immunology</json:string><json:string>1 - Health Sciences</json:string><json:string>2 - Medicine</json:string><json:string>3 - Immunology and Allergy</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences medicales</json:string></inist></categories><publicationDate>1997</publicationDate><copyrightDate>1997</copyrightDate><doi><json:string>10.1016/S1074-7613(00)80397-0</json:string></doi><id>474D05875BF03CF5B9136B67E5EB8676C803526F</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-NQ31TRTZ-D/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-NQ31TRTZ-D/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-NQ31TRTZ-D/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>©1997 Cell Press</p></licence><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</p></availability><date>1997</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note><note type="content">Section title: Article</note><note type="content">Figure 1: Screening of EBV-Specific CTL Clones from Donors RT and IM52 (A) Schematic representation of the full-length EBNA1 protein and of the glycine–alanine–deleted form E1ΔGA. The GAr domain (striped box) and nuclear localization signal (nls, shaded boxes) are highlighted. Amino acid coordinates refer to the EBNA1 sequence in the reference EBV strain, B95.8. (B) Autologous target cells, infected overnight with a recombinant vaccinia virus expressing the indicated EBV latent protein, were labeled with (51Cr)O4 and used in standard chromium release assays with CTL clones derived from in vitro LCL stimulation of peripheral blood mononuclear cells from donors RT and IM52. Specific clones are illustrated to indicate the range of CTL responses generated. Results are expressed as percentage specific lysis seen in assays conducted at effector:target ratios of between 2:1 and 5:1.</note><note type="content">Figure 2: An E1ΔGA-Specific CTL Clone from Donor RT Is Restricted through HLA-B35.01 and Recognizes the Minimal Epitope Peptide HPVGEADYFEY (EBNA1 407–417) (A) CTL clone RT c28.90 (recognized as E1ΔGA-specific in screening assays of the kind illustrated in Figure 1) was tested in a chromium release assay against vacc-E1ΔGA–infected LCL targets matched at particular HLA class I loci, as indicated. As a control, the LCL targets were infected with a vaccinia vector expressing EBNA3B (vE3B). Note that the range of targets also included the TAP-negative T2:B35.01 cell line either infected with vacc-E1ΔGA or vE3B as described above, or preexposed to the EBNA1 407–417 epitope peptide at 2 × 10−8 M or to DMSO alone as a control. (B) RT c28.90 was tested against autologous LCL cells preincubated with the indicated molar concentration of synthetic peptides from within the EBNA1 region 406–417. The minimal epitope was defined as HPVGEADYFEY (EBNA1 407–417). All results are expressed as percentage specific lysis seen in a standard chromium release assay at an effector:target ratio of 5:1.</note><note type="content">Figure 3: E1ΔGA-Specific CTL Clones from Donor NPC4 Are Restricted through HLA-A2.03 and Recognize the Minimal Epitope Peptide VLKDAIKDL (EBNA1 574–582) (A) CTL clone NPC4 c11 (recognized as E1ΔGA-specific in screening assays of the kind illustrated in Figure 1) was tested in a chromium release assay against vacc-E1ΔGA–infected LCL targets matched at particular HLA class I loci as indicated. As a control, the same LCL targets were infected with a vaccinia vector expressing EBNA3C (vE3C). (B) A second E1ΔGA-specific clone, NPC c16, was tested against autologous LCL target cells preincubated with the indicated molar concentration of synthetic peptides within the EBNA1 region 573–582 or with an equivalent dilution of DMSO as the 'no peptide' control. The minimal epitope was defined as VLKDAIKDL (EBNA1 574–582). All results are expressed as percentage specific lysis seen in a standard chromium release assay at an effector:target ratio of 5:1.</note><note type="content">Figure 4: Partial Recognition of vacc-E1/vacc-T7 Coinfected Target Cells by E1ΔGA-Specific CTL Clones from Donors RT and NPC4 (A) CTL clone RT c28 was tested against a standard B35.01-matched target LCL that had been infected with vacc-E1ΔGA (vE1ΔGA), with vacc-TK (vTK−) as a control or coinfected with vacc-E1 (vE1) and vacc-T7 (vT7) viruses. Control targets included the same LCL preexposed to the EBNA1 407–417 epitope peptide at 2 × 10−8 M or to DMSO alone. Results are expressed as percentage specific lysis served at effector:target ratios of 5:1 (solid bars) and 2:1 (striped bars). (B) CTL clones NPC4 c16 (solid bars) and c11 (striped bars) were tested against an A2.03-matched target LCL infected with the same vaccinia recombinants as in (A). Control targets included the same LCL preexposed to the EBNA1 574–582 epitope peptide at 2 × 10−8 M or to DMSO alone. Results are expressed as percentage specific lysis observed at an effector:target ratio of 5:1 for both clones. (C) Immunoblot of protein extracts from human fibroblasts either mock-infected or infected at 10 moi with the following recombinant vaccinia viruses: vacc-TK (vTK−), vacc-E1 plus vacc-T7 (vE1/vT7), vacc-E1ΔGA (vE1ΔGA), or vacc-E1 alone (vE1). Cells were harvested 20 hr after infection and extracts (105 cell equivalents/track) resolved on a 7.5% SDS-PAGE gel along with reference extracts (106 cell equivalents/track) from the EBV-positive B95.8 LCL and from the BJAB cell line as controls. The blot was probed with the EBNA1-specific MAb 1H4–1 and developed using enhanced chemiluminescence (Amersham).</note><note type="content">Figure 5: Nonrecognition of RAdE1-Infected Target Cells by E1ΔGA-Specific CTL Clones from Donors RT and IM52 (A) CTL clone RT c28.28 and (B) CTL clone IM52 c66 were tested against a standard B35.01-matched target LCL that had been infected with recombinant adenoviruses expressing E1ΔGA (RAdE1ΔGA), EBNA1 (RAdE1), or the control RAd35. Control targets included the same LCL preexposed to the EBNA1 407–417 epitope peptide at 2 × 10−8 M or to DMSO alone. Results are expressed as percentage specific lysis observed at effector:target ratios of 5:1 (solid bars) and 2:1 (striped bars). (C) Immunoblot of protein extracts from A549 cells infected at 200 moi with RAdE1, RAdE1ΔGA, or the control RAd35. Cells were harvested 20 hr after infection and extracts (2 × 105 cell equivalents/track) resolved by 7.5% SDS-PAGE along with B95.8 LCL and BJAB reference extracts (106 cell equivalents/track); the blot was then probed with MAb 1H4–1 as described for Figure 4C.</note><note type="content">Figure 6: EBNA1 Epitope–Specific CTL Clones Recognize B35.01-Matched LCL Target Cells Exogenously Loaded with an EBNA1/MBP Fusion Protein (A) Target cells were preincubated in 1 ml AIM-V serum free medium for 12 hr at 37°C with bacterially expressed MBP or with the MBP fusion proteins E1NX or E1CD at 2 × 10−7 M and then washed extensively and used in a chromium release assay with CTL clone RT c28.58 specific for the EBNA1 407–417 epitope. As controls, target cells were similarly preexposed for 12 hr to the EBNA1 peptide 407–417 at 2 × 10−7 M or to DMSO alone. (B) Target cells were preloaded for 12 hr at 37°C with different concentrations of E1CD and then washed extensively and used in a chromium release assay with CTL clone RT c83 specific for the EBNA1 407–417 epitope. Peptide- and DMSO-treated control targets are as in (A). (C) Target cells were preloaded with E1CD at 2 × 10−7 M in 1 ml of AIM-V media for the indicated time period and then washed extensively before being used in a chromium release assay with CTL clone RT c83. As controls, target cells were preexposed for 45 min to the EBNA1 epitope peptide 407–417 at 2 × 10−8 M or to DMSO alone. (D) Target cells were preincubated for 12 hr with MBP or E1CD at 2 × 10−7 M in 1 ml AIM-V media either at 37°C or at 4°C, before extensive washing and use in a chromium release assay with CTL clone RT c83. As controls, target cells were preexposed for 12 hr at 4°C to the EBNA1 epitope peptide 407–417 at 2 × 10−8 M or to DMSO alone. Results throughout are expressed as percentage specific lysis seen at an effector:target ratio of 5:1.</note><note type="content">Figure 7: Exogenously Loaded EBNA1 Is Processed in a TAP-Independent Manner (A) B35.01-matched LCL target cells were preincubated with either bacterially expressed proteins (MBP, E1NX, or E1CD) or baculovirus-expressed proteins (b451-641, b1-641, or b330-641) at 2 × 10−7 in 1 ml of AIM-V serum for 12 hr at 37°C and then washed extensively and used in chromium release assays with CTL clones RT c83 (top) and RT c84 (bottom) specific for the EBNA1 407–417 epitope. (B) TAP-negative T2:B35.01 target cells were preincubated with either bacterially expressed proteins (MBP, E1NX, or E1CD) or baculovirus expressed proteins (b451-641, b1-641, or b330-641), washed, and used in chromium release assays exactly as described for Figure 7A, with CTL clones RT c83 (top) and RT c28.90 (bottom) specific for the EBNA1 407–417 epitope. Results are expressed as percentage specific lysis seen at an effector:target ratio of 5:1. Note that each assay included as control targets the same target line preexposed as described above to the EBNA1 407–417 epitope at 2 × 10−8 M or to DMSO alone. nt, not tested.</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing</title><author xml:id="author-0000"><persName><forename type="first">Neil</forename><surname>Blake</surname></persName><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Steven</forename><surname>Lee</surname></persName><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation></author><author xml:id="author-0002"><persName><forename type="first">Irina</forename><surname>Redchenko</surname></persName><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation></author><author xml:id="author-0003"><persName><forename type="first">Wendy</forename><surname>Thomas</surname></persName><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation></author><author xml:id="author-0004"><persName><forename type="first">Neil</forename><surname>Steven</surname></persName><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation></author><author xml:id="author-0005"><persName><forename type="first">Alison</forename><surname>Leese</surname></persName><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation></author><author xml:id="author-0006"><persName><forename type="first">Patty</forename><surname>Steigerwald-Mullen</surname></persName><affiliation>Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia, 22901, USA</affiliation></author><author xml:id="author-0007"><persName><forename type="first">Michael G</forename><surname>Kurilla</surname></persName><affiliation>Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia, 22901, USA</affiliation></author><author xml:id="author-0008"><persName><forename type="first">Lori</forename><surname>Frappier</surname></persName><affiliation>Department of Medical Genetics, Microbiology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada</affiliation></author><author xml:id="author-0009"><persName><forename type="first">Alan</forename><surname>Rickinson</surname></persName><email>williamsd@cancer.bham.ac.uk</email><note type="correspondence"><p>Corresponding author: Alan Rickinson, 44 121 414 4492 (phone), 44 121 414 4486 (fax)</p></note><affiliation>To whom correspondence should be addressed (e-mail: williamsd@cancer.bham.ac.uk).</affiliation><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation></author><idno type="istex">474D05875BF03CF5B9136B67E5EB8676C803526F</idno><idno type="ark">ark:/67375/6H6-NQ31TRTZ-D</idno><idno type="DOI">10.1016/S1074-7613(00)80397-0</idno><idno type="PII">S1074-7613(00)80397-0</idno></analytic><monogr><title level="j">Immunity</title><title level="j" type="abbrev">IMMUNI</title><idno type="pISSN">1074-7613</idno><idno type="PII">S1074-7613(00)X0033-7</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1997"></date><biblScope unit="volume">7</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="791">791</biblScope><biblScope unit="page" to="802">802</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1997</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Epstein-Barr virus (EBV)–induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.</p></abstract></profileDesc><revisionDesc><change when="1997-10-20">Modified</change><change when="1997">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-NQ31TRTZ-D/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="gr1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="gr2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="gr3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="gr4"></istex:entity><istex:entity SYSTEM="gr5" NDATA="IMAGE" name="gr5"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="gr6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="gr7"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>IMMUNI</jid><aid>831</aid><ce:pii>S1074-7613(00)80397-0</ce:pii><ce:doi>10.1016/S1074-7613(00)80397-0</ce:doi><ce:copyright type="other" year="1997">Cell Press</ce:copyright></item-info><head><ce:dochead><ce:textfn>Article</ce:textfn></ce:dochead><ce:title>Human CD8<ce:sup>+</ce:sup> T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing</ce:title><ce:author-group><ce:author><ce:given-name>Neil</ce:given-name><ce:surname>Blake</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Steven</ce:given-name><ce:surname>Lee</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Irina</ce:given-name><ce:surname>Redchenko</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Wendy</ce:given-name><ce:surname>Thomas</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Neil</ce:given-name><ce:surname>Steven</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Alison</ce:given-name><ce:surname>Leese</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Patty</ce:given-name><ce:surname>Steigerwald-Mullen</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Michael G</ce:given-name><ce:surname>Kurilla</ce:surname><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Lori</ce:given-name><ce:surname>Frappier</ce:surname><ce:cross-ref refid="AFF3"><ce:sup>3</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Alan</ce:given-name><ce:surname>Rickinson</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="FN1">§</ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>williamsd@cancer.bham.ac.uk</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia, 22901, USA</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>3</ce:label><ce:textfn>Department of Medical Genetics, Microbiology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author: Alan Rickinson, 44 121 414 4492 (phone), 44 121 414 4486 (fax)</ce:text></ce:correspondence></ce:author-group><ce:date-received day="12" month="8" year="1997"></ce:date-received><ce:date-revised day="20" month="10" year="1997"></ce:date-revised><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Epstein-Barr virus (EBV)–induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8<ce:sup>+</ce:sup> CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.</ce:simple-para></ce:abstract-sec></ce:abstract></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Human CD8</title></titleInfo><name type="personal"><namePart type="given">Neil</namePart><namePart type="family">Blake</namePart><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Steven</namePart><namePart type="family">Lee</namePart><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Irina</namePart><namePart type="family">Redchenko</namePart><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Wendy</namePart><namePart type="family">Thomas</namePart><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Neil</namePart><namePart type="family">Steven</namePart><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Alison</namePart><namePart type="family">Leese</namePart><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Patty</namePart><namePart type="family">Steigerwald-Mullen</namePart><affiliation>Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia, 22901, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Michael G</namePart><namePart type="family">Kurilla</namePart><affiliation>Department of Pathology and Microbiology, University of Virginia, Charlottesville, Virginia, 22901, USA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Lori</namePart><namePart type="family">Frappier</namePart><affiliation>Department of Medical Genetics, Microbiology, University of Toronto, Toronto, Ontario, M5S 1A8, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Alan</namePart><namePart type="family">Rickinson</namePart><affiliation>E-mail: williamsd@cancer.bham.ac.uk</affiliation><affiliation>CRC Institute for Cancer Studies, University of Birmingham, Birmingham, B15 2TA, United Kingdom</affiliation><description>Corresponding author: Alan Rickinson, 44 121 414 4492 (phone), 44 121 414 4486 (fax)</description><description>To whom correspondence should be addressed (e-mail: williamsd@cancer.bham.ac.uk).</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1997</dateIssued><dateModified encoding="w3cdtf">1997-10-20</dateModified><copyrightDate encoding="w3cdtf">1997</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Epstein-Barr virus (EBV)–induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.</abstract><note type="content">Section title: Article</note><note type="content">Figure 1: Screening of EBV-Specific CTL Clones from Donors RT and IM52 (A) Schematic representation of the full-length EBNA1 protein and of the glycine–alanine–deleted form E1ΔGA. The GAr domain (striped box) and nuclear localization signal (nls, shaded boxes) are highlighted. Amino acid coordinates refer to the EBNA1 sequence in the reference EBV strain, B95.8. (B) Autologous target cells, infected overnight with a recombinant vaccinia virus expressing the indicated EBV latent protein, were labeled with (51Cr)O4 and used in standard chromium release assays with CTL clones derived from in vitro LCL stimulation of peripheral blood mononuclear cells from donors RT and IM52. Specific clones are illustrated to indicate the range of CTL responses generated. Results are expressed as percentage specific lysis seen in assays conducted at effector:target ratios of between 2:1 and 5:1.</note><note type="content">Figure 2: An E1ΔGA-Specific CTL Clone from Donor RT Is Restricted through HLA-B35.01 and Recognizes the Minimal Epitope Peptide HPVGEADYFEY (EBNA1 407–417) (A) CTL clone RT c28.90 (recognized as E1ΔGA-specific in screening assays of the kind illustrated in Figure 1) was tested in a chromium release assay against vacc-E1ΔGA–infected LCL targets matched at particular HLA class I loci, as indicated. As a control, the LCL targets were infected with a vaccinia vector expressing EBNA3B (vE3B). Note that the range of targets also included the TAP-negative T2:B35.01 cell line either infected with vacc-E1ΔGA or vE3B as described above, or preexposed to the EBNA1 407–417 epitope peptide at 2 × 10−8 M or to DMSO alone as a control. (B) RT c28.90 was tested against autologous LCL cells preincubated with the indicated molar concentration of synthetic peptides from within the EBNA1 region 406–417. The minimal epitope was defined as HPVGEADYFEY (EBNA1 407–417). All results are expressed as percentage specific lysis seen in a standard chromium release assay at an effector:target ratio of 5:1.</note><note type="content">Figure 3: E1ΔGA-Specific CTL Clones from Donor NPC4 Are Restricted through HLA-A2.03 and Recognize the Minimal Epitope Peptide VLKDAIKDL (EBNA1 574–582) (A) CTL clone NPC4 c11 (recognized as E1ΔGA-specific in screening assays of the kind illustrated in Figure 1) was tested in a chromium release assay against vacc-E1ΔGA–infected LCL targets matched at particular HLA class I loci as indicated. As a control, the same LCL targets were infected with a vaccinia vector expressing EBNA3C (vE3C). (B) A second E1ΔGA-specific clone, NPC c16, was tested against autologous LCL target cells preincubated with the indicated molar concentration of synthetic peptides within the EBNA1 region 573–582 or with an equivalent dilution of DMSO as the 'no peptide' control. The minimal epitope was defined as VLKDAIKDL (EBNA1 574–582). All results are expressed as percentage specific lysis seen in a standard chromium release assay at an effector:target ratio of 5:1.</note><note type="content">Figure 4: Partial Recognition of vacc-E1/vacc-T7 Coinfected Target Cells by E1ΔGA-Specific CTL Clones from Donors RT and NPC4 (A) CTL clone RT c28 was tested against a standard B35.01-matched target LCL that had been infected with vacc-E1ΔGA (vE1ΔGA), with vacc-TK (vTK−) as a control or coinfected with vacc-E1 (vE1) and vacc-T7 (vT7) viruses. Control targets included the same LCL preexposed to the EBNA1 407–417 epitope peptide at 2 × 10−8 M or to DMSO alone. Results are expressed as percentage specific lysis served at effector:target ratios of 5:1 (solid bars) and 2:1 (striped bars). (B) CTL clones NPC4 c16 (solid bars) and c11 (striped bars) were tested against an A2.03-matched target LCL infected with the same vaccinia recombinants as in (A). Control targets included the same LCL preexposed to the EBNA1 574–582 epitope peptide at 2 × 10−8 M or to DMSO alone. Results are expressed as percentage specific lysis observed at an effector:target ratio of 5:1 for both clones. (C) Immunoblot of protein extracts from human fibroblasts either mock-infected or infected at 10 moi with the following recombinant vaccinia viruses: vacc-TK (vTK−), vacc-E1 plus vacc-T7 (vE1/vT7), vacc-E1ΔGA (vE1ΔGA), or vacc-E1 alone (vE1). Cells were harvested 20 hr after infection and extracts (105 cell equivalents/track) resolved on a 7.5% SDS-PAGE gel along with reference extracts (106 cell equivalents/track) from the EBV-positive B95.8 LCL and from the BJAB cell line as controls. The blot was probed with the EBNA1-specific MAb 1H4–1 and developed using enhanced chemiluminescence (Amersham).</note><note type="content">Figure 5: Nonrecognition of RAdE1-Infected Target Cells by E1ΔGA-Specific CTL Clones from Donors RT and IM52 (A) CTL clone RT c28.28 and (B) CTL clone IM52 c66 were tested against a standard B35.01-matched target LCL that had been infected with recombinant adenoviruses expressing E1ΔGA (RAdE1ΔGA), EBNA1 (RAdE1), or the control RAd35. Control targets included the same LCL preexposed to the EBNA1 407–417 epitope peptide at 2 × 10−8 M or to DMSO alone. Results are expressed as percentage specific lysis observed at effector:target ratios of 5:1 (solid bars) and 2:1 (striped bars). (C) Immunoblot of protein extracts from A549 cells infected at 200 moi with RAdE1, RAdE1ΔGA, or the control RAd35. Cells were harvested 20 hr after infection and extracts (2 × 105 cell equivalents/track) resolved by 7.5% SDS-PAGE along with B95.8 LCL and BJAB reference extracts (106 cell equivalents/track); the blot was then probed with MAb 1H4–1 as described for Figure 4C.</note><note type="content">Figure 6: EBNA1 Epitope–Specific CTL Clones Recognize B35.01-Matched LCL Target Cells Exogenously Loaded with an EBNA1/MBP Fusion Protein (A) Target cells were preincubated in 1 ml AIM-V serum free medium for 12 hr at 37°C with bacterially expressed MBP or with the MBP fusion proteins E1NX or E1CD at 2 × 10−7 M and then washed extensively and used in a chromium release assay with CTL clone RT c28.58 specific for the EBNA1 407–417 epitope. As controls, target cells were similarly preexposed for 12 hr to the EBNA1 peptide 407–417 at 2 × 10−7 M or to DMSO alone. (B) Target cells were preloaded for 12 hr at 37°C with different concentrations of E1CD and then washed extensively and used in a chromium release assay with CTL clone RT c83 specific for the EBNA1 407–417 epitope. Peptide- and DMSO-treated control targets are as in (A). (C) Target cells were preloaded with E1CD at 2 × 10−7 M in 1 ml of AIM-V media for the indicated time period and then washed extensively before being used in a chromium release assay with CTL clone RT c83. As controls, target cells were preexposed for 45 min to the EBNA1 epitope peptide 407–417 at 2 × 10−8 M or to DMSO alone. (D) Target cells were preincubated for 12 hr with MBP or E1CD at 2 × 10−7 M in 1 ml AIM-V media either at 37°C or at 4°C, before extensive washing and use in a chromium release assay with CTL clone RT c83. As controls, target cells were preexposed for 12 hr at 4°C to the EBNA1 epitope peptide 407–417 at 2 × 10−8 M or to DMSO alone. Results throughout are expressed as percentage specific lysis seen at an effector:target ratio of 5:1.</note><note type="content">Figure 7: Exogenously Loaded EBNA1 Is Processed in a TAP-Independent Manner (A) B35.01-matched LCL target cells were preincubated with either bacterially expressed proteins (MBP, E1NX, or E1CD) or baculovirus-expressed proteins (b451-641, b1-641, or b330-641) at 2 × 10−7 in 1 ml of AIM-V serum for 12 hr at 37°C and then washed extensively and used in chromium release assays with CTL clones RT c83 (top) and RT c84 (bottom) specific for the EBNA1 407–417 epitope. (B) TAP-negative T2:B35.01 target cells were preincubated with either bacterially expressed proteins (MBP, E1NX, or E1CD) or baculovirus expressed proteins (b451-641, b1-641, or b330-641), washed, and used in chromium release assays exactly as described for Figure 7A, with CTL clones RT c83 (top) and RT c28.90 (bottom) specific for the EBNA1 407–417 epitope. Results are expressed as percentage specific lysis seen at an effector:target ratio of 5:1. Note that each assay included as control targets the same target line preexposed as described above to the EBNA1 407–417 epitope at 2 × 10−8 M or to DMSO alone. nt, not tested.</note><relatedItem type="host"><titleInfo><title>Immunity</title></titleInfo><titleInfo type="abbreviated"><title>IMMUNI</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1997</dateIssued></originInfo><identifier type="ISSN">1074-7613</identifier><identifier type="PII">S1074-7613(00)X0033-7</identifier><part><date>1997</date><detail type="volume"><number>7</number><caption>vol.</caption></detail><detail type="issue"><number>6</number><caption>no.</caption></detail><extent unit="issue-pages"><start>727</start><end>896</end></extent><extent unit="pages"><start>791</start><end>802</end></extent></part></relatedItem><identifier type="istex">474D05875BF03CF5B9136B67E5EB8676C803526F</identifier><identifier type="ark">ark:/67375/6H6-NQ31TRTZ-D</identifier><identifier type="DOI">10.1016/S1074-7613(00)80397-0</identifier><identifier type="PII">S1074-7613(00)80397-0</identifier><accessCondition type="use and reproduction" contentType="copyright">©1997 Cell Press</accessCondition><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource><recordOrigin>Cell Press, ©1997</recordOrigin></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-NQ31TRTZ-D/record.json</uri></json:item></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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