Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients
Identifieur interne : 000E96 ( Pmc/Corpus ); précédent : 000E95; suivant : 000E97Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients
Auteurs : Li-Tao Yang ; Hui Peng ; Zhao-Ling Zhu ; Gang Li ; Zi-Tong Huang ; Zhi-Xin Zhao ; Richard A. Koup ; Robert T. Bailer ; Chang-You WuSource :
- Clinical Immunology (Orlando, Fla.) [ 1521-6616 ] ; 2006.
Abstract
The role of cell-mediated immunity in human SARS-CoV infection is still not well understood. In this study, we found that memory T-cell responses against the spike (S) protein were persistent for more than 1 year after SARS-CoV infection by detecting the production of IFN-γ using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4+ and CD8+ T cells were involved in cellular responses against SARS-CoV infection. Interestingly, most of SARS-CoV S-specific memory CD4+ T cells were central memory cells expressing CD45RO+ CCR7+ CD62L−. However, the majority of memory CD8+ T cells revealed effector memory phenotype expressing CD45RO− CCR7− CD62L−. Thus, our study provides the evidence that SARS-CoV infection in humans can induce cellular immune response that is persistent for a long period of time. These data may have an important implication in the possibility of designing effective vaccine against SARS-CoV infection, specifically in defining T-cell populations that are implicated in protective immunity.
Url:
DOI: 10.1016/j.clim.2006.05.002
PubMed: 16781892
PubMed Central: 7106132
Links to Exploration step
PMC:7106132Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients</title><author><name sortKey="Yang, Li Tao" sort="Yang, Li Tao" uniqKey="Yang L" first="Li-Tao" last="Yang">Li-Tao Yang</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author><author><name sortKey="Peng, Hui" sort="Peng, Hui" uniqKey="Peng H" first="Hui" last="Peng">Hui Peng</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author><author><name sortKey="Zhu, Zhao Ling" sort="Zhu, Zhao Ling" uniqKey="Zhu Z" first="Zhao-Ling" last="Zhu">Zhao-Ling Zhu</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author><author><name sortKey="Li, Gang" sort="Li, Gang" uniqKey="Li G" first="Gang" last="Li">Gang Li</name><affiliation><nlm:aff id="aff2">Third affiliated hospital of the Sun Yat-sen University, Guangzhou 510630, China</nlm:aff></affiliation></author><author><name sortKey="Huang, Zi Tong" sort="Huang, Zi Tong" uniqKey="Huang Z" first="Zi-Tong" last="Huang">Zi-Tong Huang</name><affiliation><nlm:aff id="aff3">Second affiliated hospital of the Sun Yat-sen University, Guangzhou 510120, China</nlm:aff></affiliation></author><author><name sortKey="Zhao, Zhi Xin" sort="Zhao, Zhi Xin" uniqKey="Zhao Z" first="Zhi-Xin" last="Zhao">Zhi-Xin Zhao</name><affiliation><nlm:aff id="aff2">Third affiliated hospital of the Sun Yat-sen University, Guangzhou 510630, China</nlm:aff></affiliation></author><author><name sortKey="Koup, Richard A" sort="Koup, Richard A" uniqKey="Koup R" first="Richard A." last="Koup">Richard A. Koup</name><affiliation><nlm:aff id="aff4">Vaccine Research Center, NIAID/NIH, Bethesda, MD 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Bailer, Robert T" sort="Bailer, Robert T" uniqKey="Bailer R" first="Robert T." last="Bailer">Robert T. Bailer</name><affiliation><nlm:aff id="aff4">Vaccine Research Center, NIAID/NIH, Bethesda, MD 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Wu, Chang You" sort="Wu, Chang You" uniqKey="Wu C" first="Chang-You" last="Wu">Chang-You Wu</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">16781892</idno><idno type="pmc">7106132</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7106132</idno><idno type="RBID">PMC:7106132</idno><idno type="doi">10.1016/j.clim.2006.05.002</idno><date when="2006">2006</date><idno type="wicri:Area/Pmc/Corpus">000E96</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000E96</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients</title><author><name sortKey="Yang, Li Tao" sort="Yang, Li Tao" uniqKey="Yang L" first="Li-Tao" last="Yang">Li-Tao Yang</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author><author><name sortKey="Peng, Hui" sort="Peng, Hui" uniqKey="Peng H" first="Hui" last="Peng">Hui Peng</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author><author><name sortKey="Zhu, Zhao Ling" sort="Zhu, Zhao Ling" uniqKey="Zhu Z" first="Zhao-Ling" last="Zhu">Zhao-Ling Zhu</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author><author><name sortKey="Li, Gang" sort="Li, Gang" uniqKey="Li G" first="Gang" last="Li">Gang Li</name><affiliation><nlm:aff id="aff2">Third affiliated hospital of the Sun Yat-sen University, Guangzhou 510630, China</nlm:aff></affiliation></author><author><name sortKey="Huang, Zi Tong" sort="Huang, Zi Tong" uniqKey="Huang Z" first="Zi-Tong" last="Huang">Zi-Tong Huang</name><affiliation><nlm:aff id="aff3">Second affiliated hospital of the Sun Yat-sen University, Guangzhou 510120, China</nlm:aff></affiliation></author><author><name sortKey="Zhao, Zhi Xin" sort="Zhao, Zhi Xin" uniqKey="Zhao Z" first="Zhi-Xin" last="Zhao">Zhi-Xin Zhao</name><affiliation><nlm:aff id="aff2">Third affiliated hospital of the Sun Yat-sen University, Guangzhou 510630, China</nlm:aff></affiliation></author><author><name sortKey="Koup, Richard A" sort="Koup, Richard A" uniqKey="Koup R" first="Richard A." last="Koup">Richard A. Koup</name><affiliation><nlm:aff id="aff4">Vaccine Research Center, NIAID/NIH, Bethesda, MD 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Bailer, Robert T" sort="Bailer, Robert T" uniqKey="Bailer R" first="Robert T." last="Bailer">Robert T. Bailer</name><affiliation><nlm:aff id="aff4">Vaccine Research Center, NIAID/NIH, Bethesda, MD 20892, USA</nlm:aff></affiliation></author><author><name sortKey="Wu, Chang You" sort="Wu, Chang You" uniqKey="Wu C" first="Chang-You" last="Wu">Chang-You Wu</name><affiliation><nlm:aff id="aff1">Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</nlm:aff></affiliation></author></analytic><series><title level="j">Clinical Immunology (Orlando, Fla.)</title><idno type="ISSN">1521-6616</idno><idno type="eISSN">1521-7035</idno><imprint><date when="2006">2006</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>The role of cell-mediated immunity in human SARS-CoV infection is still not well understood. In this study, we found that memory T-cell responses against the spike (S) protein were persistent for more than 1 year after SARS-CoV infection by detecting the production of IFN-γ using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4<sup>+</sup> and CD8<sup>+</sup> T cells were involved in cellular responses against SARS-CoV infection. Interestingly, most of SARS-CoV S-specific memory CD4<sup>+</sup> T cells were central memory cells expressing CD45RO<sup>+</sup> CCR7<sup>+</sup> CD62L<sup>−</sup>. However, the majority of memory CD8<sup>+</sup> T cells revealed effector memory phenotype expressing CD45RO<sup>−</sup> CCR7<sup>−</sup> CD62L<sup>−</sup>. Thus, our study provides the evidence that SARS-CoV infection in humans can induce cellular immune response that is persistent for a long period of time. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Clin Immunol</journal-id><journal-id journal-id-type="iso-abbrev">Clin. Immunol</journal-id><journal-title-group><journal-title>Clinical Immunology (Orlando, Fla.)</journal-title></journal-title-group><issn pub-type="ppub">1521-6616</issn><issn pub-type="epub">1521-7035</issn><publisher><publisher-name>Elsevier Inc.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">16781892</article-id><article-id pub-id-type="pmc">7106132</article-id><article-id pub-id-type="publisher-id">S1521-6616(06)00735-2</article-id><article-id pub-id-type="doi">10.1016/j.clim.2006.05.002</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Yang</surname><given-names>Li-Tao</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Peng</surname><given-names>Hui</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Zhu</surname><given-names>Zhao-Ling</given-names></name><xref rid="aff1" ref-type="aff">a</xref></contrib><contrib contrib-type="author"><name><surname>Li</surname><given-names>Gang</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Huang</surname><given-names>Zi-Tong</given-names></name><xref rid="aff3" ref-type="aff">c</xref></contrib><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Zhi-Xin</given-names></name><xref rid="aff2" ref-type="aff">b</xref></contrib><contrib contrib-type="author"><name><surname>Koup</surname><given-names>Richard A.</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Bailer</surname><given-names>Robert T.</given-names></name><xref rid="aff4" ref-type="aff">d</xref></contrib><contrib contrib-type="author"><name><surname>Wu</surname><given-names>Chang-You</given-names></name><email>Changyou_wu@yahoo.com</email><xref rid="aff1" ref-type="aff">a</xref><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="aff1"><label>a</label>Department of Immunology, Zhongshan Medical School, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou 510080, China</aff><aff id="aff2"><label>b</label>Third affiliated hospital of the Sun Yat-sen University, Guangzhou 510630, China</aff><aff id="aff3"><label>c</label>Second affiliated hospital of the Sun Yat-sen University, Guangzhou 510120, China</aff><aff id="aff4"><label>d</label>Vaccine Research Center, NIAID/NIH, Bethesda, MD 20892, USA</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author. Fax: +86 20 87331552. <email>Changyou_wu@yahoo.com</email></corresp></author-notes><pub-date pub-type="pmc-release"><day>16</day><month>6</month><year>2006</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>8</month><year>2006</year></pub-date><pub-date pub-type="epub"><day>16</day><month>6</month><year>2006</year></pub-date><volume>120</volume><issue>2</issue><fpage>171</fpage><lpage>178</lpage><history><date date-type="received"><day>22</day><month>11</month><year>2005</year></date><date date-type="rev-recd"><day>21</day><month>4</month><year>2006</year></date><date date-type="accepted"><day>1</day><month>5</month><year>2006</year></date></history><permissions><copyright-statement>Copyright © 2006 Elsevier Inc. All rights reserved.</copyright-statement><copyright-year>2006</copyright-year><copyright-holder>Elsevier Inc.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract><p>The role of cell-mediated immunity in human SARS-CoV infection is still not well understood. In this study, we found that memory T-cell responses against the spike (S) protein were persistent for more than 1 year after SARS-CoV infection by detecting the production of IFN-γ using ELISA and ELISpot assays. Flow cytometric analysis showed that both CD4<sup>+</sup> and CD8<sup>+</sup> T cells were involved in cellular responses against SARS-CoV infection. Interestingly, most of SARS-CoV S-specific memory CD4<sup>+</sup> T cells were central memory cells expressing CD45RO<sup>+</sup> CCR7<sup>+</sup> CD62L<sup>−</sup>. However, the majority of memory CD8<sup>+</sup> T cells revealed effector memory phenotype expressing CD45RO<sup>−</sup> CCR7<sup>−</sup> CD62L<sup>−</sup>. Thus, our study provides the evidence that SARS-CoV infection in humans can induce cellular immune response that is persistent for a long period of time. These data may have an important implication in the possibility of designing effective vaccine against SARS-CoV infection, specifically in defining T-cell populations that are implicated in protective immunity.</p></abstract><kwd-group><title>Keywords</title><kwd>SARS</kwd><kwd>SARS-CoV</kwd><kwd>S protein</kwd><kwd>IFN-γ</kwd><kwd>Cellular immune response</kwd><kwd>Memory T cells</kwd></kwd-group></article-meta></front><body><sec><title>Introduction</title><p>Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease that led to thousands of human infections and hundreds of deaths. The etiologic agent of the syndrome, a novel coronavirus termed SARS-associated coronavirus (SARS-CoV) has been identified <xref rid="bib1" ref-type="bibr">[1]</xref>, <xref rid="bib2" ref-type="bibr">[2]</xref>, <xref rid="bib3" ref-type="bibr">[3]</xref>. Although the epidemic was eventually controlled, the high morbidity and mortality make it extremely urgent to develop effective means to prevent this disease. Therefore, a successful vaccine against SARS-CoV remains a high priority. The unique opportunity of studying survivors of the infection allows for delineation of the T-cell populations implicated in the protective immunity, and those T cells would be the target population for expansion via a vaccine.</p><p>The spike (S) glycoprotein is a major structural protein of SARS-CoV and a potential target for SARS-specific humoral immunity and/or cell-mediated immune responses. Recent studies in animal models have demonstrated that vaccines based on the S protein of SARS-CoV seem to induce a considerable neutralizing antibody response <xref rid="bib4" ref-type="bibr">[4]</xref>, <xref rid="bib5" ref-type="bibr">[5]</xref>, <xref rid="bib6" ref-type="bibr">[6]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>, <xref rid="bib8" ref-type="bibr">[8]</xref> and provide protection via an inhibition of viral replication after challenge with live SARS-CoV <xref rid="bib6" ref-type="bibr">[6]</xref>, <xref rid="bib7" ref-type="bibr">[7]</xref>. In addition, SARS-CoV-specific IgG antibody can be detected at week 3 after the onset of syndrome in SARS patients and persistent for a long period of time <xref rid="bib9" ref-type="bibr">[9]</xref>, <xref rid="bib10" ref-type="bibr">[10]</xref>. Polyclonal immune sera from convalescent SARS patients were passively transferred to treat SARS patients during the outbreak in 2003 <xref rid="bib11" ref-type="bibr">[11]</xref>. These findings revealed that humoral immunity may provide immediate protection against SARS-CoV infection in mice and humans.</p><p>In addition to humoral immune responses, the cell-mediated response likely plays a major role in eliminating virus. Although it has been demonstrated that DNA vaccine encoding the S protein of SARS-CoV can induce antigen-specific T-cell responses in animal models <xref rid="bib4" ref-type="bibr">[4]</xref>, <xref rid="bib12" ref-type="bibr">[12]</xref>, the protective effect of T cells specific for the S protein in humans remains undefined <xref rid="bib13" ref-type="bibr">[13]</xref>. Thus, it is essential to determine whether recovered SARS patients maintained cell-mediated immunity against SARS-CoV and how long this immunity will persist. In addition, the phenotypic characteristics of memory CD4<sup>+</sup> or CD8<sup>+</sup> T cells specific for SARS-CoV have not been extensively studied. In this study, we analyzed CD4<sup>+</sup> and CD8<sup>+</sup> T-cell responses in peripheral blood of recovered SARS patients to a pool of 169 overlapping SARS-CoV S peptides and characterized the phenotype of memory T-cell subpopulations. Our study demonstrated that SARS-CoV S-specific memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells in peripheral blood of recovered SARS patients were persistent for more than 1 year after infection. These memory T cells revealed both effector and central memory phenotypes.</p></sec><sec><title>Materials and methods</title><sec><title>Donors</title><p>Eight fully recovered SARS patients, 5 male and 3 female, age 25 to 34 years, worked at the Third Affiliated Hospital of the Sun Yat-sen University, Guangzhou, China. All were diagnosed with the infection of SARS-CoV between March and April, 2003. Controls for the study were consisted of five healthy donors, 3 male and 2 female, age 27 to 33 years, without any contact history with SARS-CoV patients, and had no detectable antibodies specific to SARS-CoV in their sera.</p></sec><sec><title>Reagents</title><p>A total of 169 peptides spanning the entire SARS-CoV S protein (each peptide was 15–17 amino acids in length and overlapped by 10 amino acids) were kindly provided by the Vaccine Research Center, NIAID, National Institutes of Health, USA. Anti-CD4 PerCP and anti-CD8 PerCP were purchased from BD Bioscience Immunocytometry Systems (San Jose, CA, USA). Mouse anti-human CD28, anti-human CD49d, anti-CD4 FITC, anti-CD8 PE-Cy7, anti-CD45RO FITC, anti-CCR7 PE, anti-CD62L PE, anti-IFN-γ PE, anti-IFN-γ APC, anti-IL-2 APC and isotypes matched control antibodies were obtained from BD Bioscience Pharmingen (San Jose, CA, USA). RPMI 1640 medium, penicillin–streptomycin and 2-mercaptoethanol were purchased from GIBCO (Grand Island, NY, USA).</p></sec><sec><title>Cell preparation</title><p>Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized venous blood that was mixed with an equal volume of Hank's balanced salt solution using Ficoll-Hypaque density gradient centrifugation. The cells were washed twice in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) (HyClone), 100 U penicillin/ml, 100 μg streptomycin/ml and 50 μM 2-mercaptoethanol. The cells were finally adjusted to a concentration of 2 × 10<sup>6</sup>/ml in complete RPMI 1640 medium.</p></sec><sec><title>Cell culture conditions and IFN-γ detection by ELISA</title><p>Fresh PBMCs were isolated from recovered SARS patients and healthy donors, suspended in complete RPMI 1640 medium and stimulated with a pool of 169 overlapping SARS-CoV S peptides in the presence of 1 μg/ml anti-CD28 and 1 μg/ml anti-CD49d mAbs. The final concentration of each peptide was 1 μg/ml. PBMCs were plated into a flat-bottomed 96-well plate at a total of 2 × 10<sup>5</sup> cells/well in triplicate and incubated for 72 h at 37°C with 5% CO<sub>2</sub>. The supernatants were harvested and assayed for IFN-γ production by ELISA according to the manufacturer's protocol (BD Pharmingen, San Diego, CA). In each experiment, PBMCs were cultured with 1 μg/ml anti-CD28 plus 1 μg/ml anti-CD49d in the absence of SARS-CoV S peptides as a negative control. In addition, the cells were stimulated with 20 ng/ml phorbol-12-mristate-13-acetate (PMA) (Sigma, St. Louis, MO, USA) and 1 μg/ml ionomycin (Sigma) as a positive control.</p></sec><sec><title>IFN-γ ELISpot assay</title><p>Human IFN-γ ELISpot assays were performed using a commercially available set (BD Pharmingen) according to the manufacturer's instructions. Briefly, 96-well nitrocellulose plates (Millipore) were coated overnight at 4°C with 5 μg/ml anti-human IFN-γ capture antibody. The wells were then washed and blocked for 2 h at room temperature with 10% FCS–RPMI 1640 medium. A total of 2 × 10<sup>5</sup> PBMCs were added to microwell in triplicate and incubated for 20 h with a pool of SARS-CoV S peptides (1 μg/ml), 1 μg/ml anti-CD28 and 1 μg/ml anti-CD49d mAbs. Similarly, the cells supplemented with anti-CD28 (1 μg/ml) and anti-CD49d (1 μg/ml) mAbs in the absence of SARS-CoV S peptides were considered as negative controls, and the cells were cultured with polyclonal stimuli (20 ng/ml PMA plus 1 μg/ml ionomycin) as positive controls. The plates were washed and subsequently incubated with 2 μg/ml biotinylated anti-human IFN-γ detection antibody for 2 h at room temperature. After washing, wells were developed for another 1 h with streptavidin–HRP followed by addition with AEC substrate reagent. Spot development was monitored, and substrate reaction was stopped by washing wells with water. The spots were counted automatically using Immunospot Image Analyzer (Cellular Technology Ltd, USA). A spot represents an IFN-γ-producing cell. The average of spots in triplicate wells was calculated and considered as the number of specific spot-forming cells (SFC)/2 × 10<sup>5</sup> cultured PBMCs.</p></sec><sec><title>Cell surface, intracellular cytokine staining and flow cytometric analysis</title><p>PBMCs from recovered SARS patients and healthy individuals were stimulated with or without a mixture of 169 overlapping SARS-CoV S peptides in the presence of 1 μg/ml anti-CD28 and 1 μg/ml anti-CD49d mAbs in 15 ml tubes. After the first 1 h incubation, brefeldin A (Sigma) at a final concentration 10 μg/ml was added to cultures to enable intracellular protein to accumulate in all stimulations. After incubation for a total 5 h, the cells were washed, fixed, permeabilized using saponin (Sigma) and blocked with 25 μg/ml human IgG for 30 min at 4°C. The cells were then stained with anti-CD4, anti-CD8, anti-IFN-γ, anti-IL-2, anti-CD45RO, anti-CCR7 and anti-CD62L. After staining, all samples were washed twice in PBS buffer (containing 0.1% saponin, 0.1% BSA and 0.05% NaN<sub>3</sub>) and resuspended in 300 μl PBS for flow cytometry. More than 600,000 cells were acquired on a FACS Aria flow cytometer (Becton Dickinson, San Jose, CA), and FACS data were analyzed using CellQuest software (Becton Dickinson, San Jose, CA).</p></sec><sec><title>Statistical analyses</title><p>The levels of IFN-γ and numbers of IFN-γ-producing cells were compared respectively using Student's <italic>t</italic> test between recovered SARS patients and healthy donors in the same condition. Value of <italic>P</italic> < 0.05 was considered significant.</p></sec></sec><sec><title>Results</title><sec><title>Quantification of SARS-CoV S-peptide-specific memory T lymphocytes</title><p>We first focused on understanding whether recovered SARS individuals could generate and maintain memory T-cell responses against the S protein of SARS-CoV more than 1 year after SARS-CoV infection. Fresh PBMCs were isolated from fully recovered SARS patients and healthy donors and cultured in vitro with or without a pool of 169 peptides spanning the entire S protein of SARS-CoV in the presence of 1 μg/ml anti-CD28 and 1 μg/ml anti-CD49d mAbs. The final concentration of each peptide was 1 μg/ml. The PBMCs were cultured with polyclonal stimuli (20 ng/ml PMA plus 1 μg/ml ionomycin) as a positive control. After stimulation for 72 h, cell-free culture supernatants were harvested and assayed for the production of IFN-γ by ELISA. The results showed that PBMCs from five healthy donors secreted very low levels of IFN-γ (0.10–0.13 ng/ml) when cultured either with or without the SARS-CoV S peptides. Statistic analysis indicated that the production of IFN-γ between these two culture conditions was not significantly different (<italic>P</italic> > 0.05) (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A). In contrast, PBMCs from eight recovered SARS individuals produced significantly higher levels of IFN-γ (1.77 ± 0.98 ng/ml) when stimulated with SARS-CoV S peptides (<italic>P</italic> < 0.01) compared with the levels of IFN-γ from the cells cultured without SARS-CoV S peptides (0.19 ± 0.17 ng/ml) (<xref rid="fig1" ref-type="fig">Fig. 1</xref>A). In addition, no markedly difference in the production of IFN-γ between recovered patients and healthy individuals was observed when the PBMCs were stimulated with PMA plus ionomycin (<italic>P</italic> > 0.05) (data not shown).<fig id="fig1"><label>Figure 1</label><caption><p>SARS-CoV S-peptide-specific IFN-γ production by PBMCs. Fresh PBMCs were isolated from fully recovered SARS patients and normal individuals and cultured in vitro with or without a pool of 169 SARS-CoV S peptides in the presence of anti-CD28 (1 μg/ml) and anti-CD49d mAbs (1 μg/ml). After incubation for 72 h, the level of IFN-γ in culture supernatants was assayed by ELISA (A). The frequency of IFN-γ-secreting cells was detected by ELISpot assay at the single cell level after stimulation for 20 h (B). Student's <italic>t</italic> test was used for statistical analysis. The significance was set at <italic>P</italic> < 0.01 (***). ns, not significant.</p></caption><graphic xlink:href="gr1_lrg"></graphic></fig></p><p>To determine the frequency of SARS-CoV S-specific T cells at the single cell level, fresh PBMCs isolated from recovered SARS patients and uninfected healthy individuals were cultured in the presence or absence of SARS-CoV S peptides. The frequency of IFN-γ-producing cells was detected by ELISpot assay after stimulation for 20 h. As shown in <xref rid="fig1" ref-type="fig">Fig. 1</xref>B, 2–5 IFN-γ-positive spots in 2 × 10<sup>5</sup> PBMCs were detected in cells without stimulation, either in healthy donors or recovered SARS individuals. The SARS-CoV S peptides, however, elicited a strong antigen-specific T-cell response in recovered SARS patients with an average number of spot-forming cells (SFC) 49/2 × 10<sup>5</sup> PBMCs (range 18 to 109) (<xref rid="fig1" ref-type="fig">Fig. 1</xref>B), which were significantly higher (<italic>P</italic> < 0.01) than spot-producing cells in healthy donors (1–6 spots in 2 × 10<sup>5</sup> PBMCs) when PBMCs were stimulated with SARS-CoV S peptides. Moreover, the PBMCs from recovered SARS patients revealed similar frequency of IFN-γ-forming cells (328/2 × 10<sup>5</sup> PBMCs) compared with the PBMCs from healthy individuals (299/2 × 10<sup>5</sup> PBMCs) when stimulated with PMA plus ionomycin (<italic>P</italic> > 0.05) (data not shown). Taken together, SARS-CoV S-specific memory T cells were persistent in peripheral blood of recovered SARS individuals and displayed the capacity to secrete IFN-γ upon subsequent stimulation with SARS-CoV S peptides in vitro.</p></sec><sec><title>Subpopulation of SARS-CoV S-peptide-specific memory T cells</title><p>We next analyzed the subpopulation of IFN-γ-producing T cells by flow cytometry. PBMCs were isolated and stimulated with a pool of SARS-CoV S peptides (1 μg/ml), 1 μg/ml anti-CD28 and 1 μg/ml anti-CD49d mAbs in the presence of 10 μg/ml brefeldin A. After stimulation for 5 h, the cell surface markers and intracellular cytokines were stained with anti-CD4 FITC, anti-CD8 PerCP, anti-IFN-γ PE, anti-IL-2 APC and their corresponding isotype controls. The expression of IFN-γ and IL-2 was assessed when cells were gated on CD4<sup>+</sup> or CD8<sup>+</sup> T cells. The results showed that both CD4<sup>+</sup> and CD8<sup>+</sup> T cells from 5 recovered SARS patients failed to secret IFN-γ and IL-2 in the absence of SARS-CoV S peptides. However, addition of SARS-CoV S peptides to cultures resulted in the expression of IFN-γ and IL-2 in both CD4<sup>+</sup> and CD8<sup>+</sup> T-cell populations. Two representative examples, patient 2 and patient 5, are shown in <xref rid="fig2" ref-type="fig">Figs. 2</xref>A and B. The percentage of IFN-γ and IL-2-producing CD4<sup>+</sup> and CD8<sup>+</sup> T cells from 5 recovered SARS patients in SARS-CoV S-peptide-stimulated cultures was significantly higher than that from unstimulated cells (<italic>P</italic> < 0.05) (<xref rid="fig2" ref-type="fig">Fig. 2</xref>C), whereas no difference was observed in the proportion of IL-2-producing CD8 T cells between those two groups (<italic>P</italic> > 0.05). Taken together, the results demonstrated that both memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells participated in cellular immune responses to SARS-CoV S antigen in humans.<fig id="fig2"><label>Figure 2</label><caption><p>Expression of intracellular IFN-γ and IL-2 in PBMCs of recovered SARS patients after stimulation with SARS-CoV S peptides in vitro. Fresh PBMCs from recovered SARS patients were cultured for 5 h in vitro with a mixture of 169 SARS-CoV S peptides and stained with anti-CD4, CD8, IFN-γ and IL-2 mAbs. The expression of IFN-γ and IL-2 on CD4<sup>+</sup> (A) and CD8<sup>+</sup> (B) T cells was analyzed. The numbers in each quadrant were indicated as percentage. Data were representative of two of five recovered SARS individuals with similar results. The percentage (mean ± SE) of IFN-γ or IL-2-secreting cells from 5 independent experiments was shown within CD4 or CD8 T cells (C). Student's <italic>t</italic> test was used for statistical analysis. Statistical significance was set at <italic>P</italic> < 0.05 (*), ns, not significant.</p></caption><graphic xlink:href="gr2_lrg"></graphic></fig></p></sec><sec><title>Correlation of the expression of IFN-γ and IL-2 by CD4 T lymphocytes</title><p>We next analyzed the expression of IFN-γ and IL-2 in CD4<sup>+</sup> T cells following stimulation of PBMCs from SARS recovered patients with a pool of SARS-CoV S peptides. Based on the expression of IFN-γ and IL-2, antigen-specific CD4<sup>+</sup> T cells could be divided into three distinct subpopulations: IFN-γ<sup>+</sup> IL-2<sup>−</sup>, IFN-γ<sup>+</sup> IL-2<sup>+</sup> and IFN-γ<sup>−</sup> IL-2<sup>+</sup> (two examples, patient 2 and patient 5, shown in <xref rid="fig3" ref-type="fig">Fig. 3</xref>A). We further analyzed the simultaneous expression of high or low levels of IFN-γ and IL-2 at the single cell (two examples, patient 2 and patient 5, shown in <xref rid="fig3" ref-type="fig">Figs. 3</xref>B, C). A total of four patients have been assessed for cytokine production. A representative of patient 2 where approximately 61–72% CD4<sup>+</sup> IFN-γ<sup>high</sup> cells showed high level expression of IL-2 and 67–81% CD4<sup>+</sup> IFN-γ<sup>low</sup> cells was IL-2<sup>low</sup>. 64–76% of CD4<sup>+</sup> IL-2<sup>high</sup> displayed phenotype of IFN-γ<sup>high</sup> and 55–65% CD4<sup>+</sup> IL-2<sup>low</sup> were IFN-γ<sup>low</sup>. Similar tendency was also found in the mean fluorescence intensity (MFI). However, in patient 5, IL-2<sup>low</sup> cells accounted for only 41% of CD4<sup>+</sup> IFN-γ<sup>low</sup> but still showed similar results as seen in other 4 patients. These results suggested that the majority of IFN-γ<sup>+</sup> IL-2<sup>+</sup> T cells were confined to IFN-γ<sup>high</sup> IL-2<sup>high</sup> subset.<fig id="fig3"><label>Figure 3</label><caption><p>The correlation of intensity of IFN-γ and IL-2 within SARS-CoV S-specific CD4 T cells. The subsets of SARS-CoV S-specific CD4<sup>+</sup> T cells based on the expression of IFN-γ and IL-2 (A), the expression of IL-2 in both IFN-γ<sup>high</sup> or IFN-γ<sup>low</sup> subpopulations (B) and frequency as well as intensity of IFN-γ-producing cells in IL-2<sup>high</sup> and IL-2<sup>low</sup> subsets (C) (A, top panels, unstimulated cells; bottom panels, S-peptide-stimulated cells; B–C, left panels top quadrant, unstimulated cells; left panels bottom quadrant, S-peptide-stimulated cells). Percentage of indicated cells and the mean fluorescent intensity (MFI) were shown. Data were representative of two of five recovered SARS individuals.</p></caption><graphic xlink:href="gr3_lrg"></graphic></fig></p></sec><sec><title>Characterization of memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells specific for SARS-CoV S peptides</title><p>With regard to memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells in humans, multiple phenotypes and a broad spectrum of functions have been observed in different viral infections. In this study, we performed phenotypic characterization of antiviral T-cell responses specific for SARS-CoV S peptides on the basis of their ability to secrete IFN-γ and the expression of CD45RO, CCR7 and CD62L. Consistent with previous reports, our results indicated that the majority of total CD4<sup>+</sup> T cells (<xref rid="fig4" ref-type="fig">Fig. 4</xref>A) expressed high levels of CCR7 (85%) (<xref rid="fig4" ref-type="fig">Fig. 4</xref>C) and CD62L (77%) (<xref rid="fig4" ref-type="fig">Fig. 4</xref>D), whereas approximately 40% of CD4<sup>+</sup> T cells expressed CD45RO (<xref rid="fig4" ref-type="fig">Figs. 4</xref>C, D). SARS-CoV S-specific IFN-γ-producing CD4<sup>+</sup> T cells (<xref rid="fig4" ref-type="fig">Fig. 4</xref>B) had an increased expression of CD45RO (<xref rid="fig4" ref-type="fig">Fig. 4</xref>C) but had decreased expression of CD62L (<xref rid="fig4" ref-type="fig">Fig. 4</xref>D) compared with total CD4<sup>+</sup> T cells. Furthermore, the majority of antigen-specific CD4<sup>+</sup> IFN-γ<sup>+</sup> cells were CD45RO<sup>+</sup> CCR7<sup>+</sup> (<xref rid="fig4" ref-type="fig">Fig. 4</xref>C) or CD45RO<sup>+</sup> CD62L<sup>−</sup> (<xref rid="fig4" ref-type="fig">Fig. 4</xref>D). In contrast, a total of CD8<sup>+</sup> T cells (<xref rid="fig5" ref-type="fig">Fig. 5</xref>A) expressed low level of CCR7 (<xref rid="fig5" ref-type="fig">Fig. 5</xref>C) and CD62L (<xref rid="fig5" ref-type="fig">Fig. 5</xref>D) compared with total CD4<sup>+</sup> T cells. Moreover, IFN-γ-positive CD8<sup>+</sup> T cells (<xref rid="fig5" ref-type="fig">Fig. 5</xref>B) were predominantly CD45RO<sup>−</sup> (78%) and mainly defined as CD45RO<sup>−</sup> CCR7<sup>−</sup> CD62L<sup>−</sup> (<xref rid="fig5" ref-type="fig">Figs. 5</xref>C, D). These results emphasized the heterogeneity of memory T cells and also demonstrated difference between memory CD4<sup>+</sup> and CD8<sup>+</sup> T-cell populations.<fig id="fig4"><label>Figure 4</label><caption><p>Phenotypic analyses of memory CD4 T cells specific for SARS-CoV S peptides. Total CD4<sup>+</sup> (A) or CD4<sup>+</sup> IFN-γ<sup>+</sup> cells (B) were analyzed for the expression of CD45RO and CCR7 (C) or CD45RO and CD62L (D). The numbers in each quadrant were indicated as percentage. Data were representative for 3 separate experiments with similar results.</p></caption><graphic xlink:href="gr4_lrg"></graphic></fig><fig id="fig5"><label>Figure 5</label><caption><p>Characteristics of SARS-CoV S peptides-specific memory CD8 T cells. Total CD8<sup>+</sup> (A) or CD8<sup>+</sup> IFN-γ<sup>+</sup> (B) were analyzed for the expression of CD45RO and CCR7 expression (C) or CD45RO and CD62L (D). Data were representative of 1 of 3 independent experiments with similar results.</p></caption><graphic xlink:href="gr5_lrg"></graphic></fig></p></sec></sec><sec><title>Discussion</title><p>In the current study, we found that antigen-specific memory T cells were capable of secreting high levels of IFN-γ by ELISA assay upon stimulation in vitro with a pool of SARS-CoV S peptides. These memory T cells persisted for more than 1 year after recovery in peripheral blood of SARS individuals. These data were further confirmed by IFN-γ ELISpot assay showing a high frequency of SARS-CoV S-specific IFN-γ-producing T cells in recovered SARS patients. Our results are consistent with the report that two newly identified HLA-A*0201-restricted CD8<sup>+</sup> T-cell epitopes from SARS-CoV S protein, S1203 and S978, elicit memory T-cell responses in PBMCs from HLA-A*0201<sup>+</sup> recovered SARS patients up to 3 months post-infection <xref rid="bib13" ref-type="bibr">[13]</xref>, <xref rid="bib14" ref-type="bibr">[14]</xref>.</p><p>To date, most studies have been focused on mapping of CD8<sup>+</sup> T-cell epitopes from patients who were fully recovered from SARS infection <xref rid="bib13" ref-type="bibr">[13]</xref>, <xref rid="bib14" ref-type="bibr">[14]</xref>, <xref rid="bib15" ref-type="bibr">[15]</xref>. However, little is known about memory CD4<sup>+</sup> T-cell responses to SARS-CoV. Consistent with observation in mouse model <xref rid="bib4" ref-type="bibr">[4]</xref>, we found that both memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells participated in cellular responses to SARS-CoV. It is known that naive CD4<sup>+</sup> T cells can be divided into two major subpopulations, Th1 and Th2, based on cytokine production and biological functions <xref rid="bib16" ref-type="bibr">[16]</xref>. The Th1 cells are composed of IFN-γ<sup>+</sup> and IFN-γ<sup>−</sup> cells. The IFN-γ<sup>−</sup> Th1 cells may differentiate into memory cells in vivo <xref rid="bib17" ref-type="bibr">[17]</xref>. Based on the production of IFN-γ and IL-2, SARS-CoV S-specific CD4<sup>+</sup> T cells could be divided into three distinct populations: single IFN-γ-secreting cells, double IFN-γ/IL-2-secreting cells and single IL-2-secreting cells. Interestingly, IFN-γ/IL-2 double positive CD4<sup>+</sup> T cells were mainly restricted to IFN-γ<sup>high</sup> IL-2<sup>high</sup> subset. These results further demonstrated the heterogeneity of memory Th1 cells and also indicated that distinct subpopulations of memory CD4<sup>+</sup> T cells have different capacities to develop into short (IFN-γ<sup>+</sup>) and long-lived (IFN-γ<sup>+</sup> IL-2<sup>+</sup> and IL-2<sup>+</sup>) memory cells.</p><p>It is generally accepted that CD45RO expression defines to activated or memory T cells. Here, we found that most of SARS-CoV S-specific memory CD4<sup>+</sup> T cells were CD45RO<sup>+</sup>, whereas the majority of memory CD8<sup>+</sup> T cells displayed CD45RO<sup>−</sup> phenotype. Recently, SSp-1, an HLA-A*0201-restricted CD8<sup>+</sup> T-cell epitope, was identified from SARS-CoV S protein. The SSp-1-specific CTL cells displayed the CD45RA<sup>+</sup> CCR7<sup>−</sup> CD62L<sup>−</sup> phenotype, after PBMCs from recovered SARS patients were stimulated in vitro with heat-inactivated SARS-CoV for 7 days <xref rid="bib18" ref-type="bibr">[18]</xref>. Consistent with these observations, we found that the majority of SARS-CoV S-specific memory CD8 T cells were confined to CD45RO<sup>−</sup> CCR7<sup>−</sup> CD62L<sup>−</sup> population.</p><p>Our results also demonstrated that the majority of memory CD8<sup>+</sup> T cells exhibited effector memory phenotype of CD62L<sup>−</sup>. This observation is in agreement with recent studies showing that only central memory CD8<sup>+</sup> T cells produce IL-2 following a short-peptide stimulation in lymphocytic choriomeningitis virus (LCMV)-infected mouse model <xref rid="bib19" ref-type="bibr">[19]</xref>. Generally, the expression of CD62L is associated with the expression of CCR7 <xref rid="bib20" ref-type="bibr">[20]</xref>. Surprisingly, SARS-CoV S-specific memory CD4<sup>+</sup> T cells expressed a mixed CCR7<sup>+</sup> CD62L<sup>−</sup> phenotype that differs from phenotypes of classical CCR7<sup>+</sup> CD62L<sup>+</sup> T<sub>CM</sub> and CCR7<sup>−</sup> CD62L<sup>−</sup> T<sub>EM</sub> cells. Some groups have also found memory CD4<sup>+</sup> or CD8<sup>+</sup> T cells expressing a mixed CCR7<sup>+</sup> CD62L<sup>−</sup> phenotype in mouse model <xref rid="bib21" ref-type="bibr">[21]</xref>, <xref rid="bib22" ref-type="bibr">[22]</xref>. These studies suggest that the expression of CCR7 and CD62L in memory CD4<sup>+</sup> or CD8<sup>+</sup> T cells may overlap only partially.</p><p>Based on the expression of chemokine receptor CCR7, memory T cells can be divided into two distinct subsets: central memory T cells (T<sub>CM</sub>) expressing CCR7 and effector memory T cells (T<sub>EM</sub>), which lack the expression of CCR7. In our study, the majority of IFN-γ-producing CD4<sup>+</sup> T cells were CCR7<sup>+</sup>. This is supported by evidence that memory Th1 cells are mainly CCR7<sup>+</sup> in mice <xref rid="bib23" ref-type="bibr">[23]</xref>. Recent report has identified two functional distinct populations of memory CD8<sup>+</sup> T cells: double IFN-γ/IL-2-secreting cells and single IFN-γ-secreting cells. Virus-specific IFN-γ/IL-2-secreting CD8<sup>+</sup> T cells were CD45RA<sup>−</sup> CCR7<sup>−</sup>, whereas single IFN-γ-secreting CD8<sup>+</sup> T cells were either CD45RA<sup>−</sup> CCR7<sup>−</sup> or CD45RA<sup>+</sup> CCR7<sup>−</sup><xref rid="bib24" ref-type="bibr">[24]</xref>. Consistent with observations in HIV infection, we showed that SARS-CoV S-specific CD8<sup>+</sup> T cells produced IFN-γ but not IL-2 and predominantly displayed CD45RO<sup>−</sup> CCR7<sup>−</sup> phenotype which might represent terminally differentiated effector memory T cells <xref rid="bib25" ref-type="bibr">[25]</xref>. This may reflect difference in the inherent stability of CD4<sup>+</sup> and CD8<sup>+</sup> memory T cells that are CCR7<sup>−</sup> in normal steady-state conditions <xref rid="bib26" ref-type="bibr">[26]</xref>.</p><p>In conclusion, SARS-CoV infection elicits a cellular response mediated by both CD4<sup>+</sup> and CD8<sup>+</sup> T cells to the S protein. SARS-CoV S-specific memory CD4<sup>+</sup> and CD8<sup>+</sup> T cells are persistent for more than 1 year in peripheral blood of recovered SARS patients. This study provided an opportunity to delineate the memory T-cell response in survivors of a disease with high morbidity and mortality. 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