{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,3]],"date-time":"2025-10-03T22:32:22Z","timestamp":1759530742066},"reference-count":55,"publisher":"Portland Press Ltd.","issue":"15","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2017,8,1]]},"abstract":"<jats:p>T-cell responses have been demonstrated to be essential for preventing Mycobacterium tuberculosis infection. The Th1-cytokines produced by T cells, such as INF-\u03b3, IL-2, and TNF-\u03b1, not only limit the invasion of M. tuberculosis but also eliminate the pathogen at the site of infection. Bacillus Calmette\u2013Gu\u00e9rin (BCG) is known to induce Th1-type responses but the protection is inadequate. Identification of immunogenic components, in addition to those expressed in BCG, and induction of a broad spectrum of Th1-type responses provide options for generating sufficient adaptive immunity. Here, we studied human pulmonary T-cell responses induced by the M. tuberculosis-specific antigen Rv3615c, a protein with a similar size and sequence homology to ESAT-6 and CFP-10, which induced dominant CD4+ T-cell responses in human tuberculosis (TB) models. We characterized T-cell responses including cytokine profiling, kinetics of activation, expansion, differentiation, TCR usage, and signaling of activation induced by Rv3615c compared with other M. tuberculosis-specific antigens. The expanded CD4+ T cells induced by Rv3615c predominately produced Th1, but less Th2 and Th17, cytokines and displayed effector\/memory phenotypes (CD45RO+CD27\u2212CD127\u2212CCR7\u2212). The magnitude of expansion and cytokine production was comparable to those induced by well-characterized the 6 kDa early secreted antigenic target (ESAT-6), the 10 kDa culture filtrate protein (CFP-10) and BCG. Rv3615c contained multiple epitopes Rv3615c1\u201315, Rv3615c6\u201320, Rv3615c66\u201380, Rv3615c71\u201385 and Rv3615c76\u201390 that activated CD4+ T cells. The Rv3615c-specific CD4+ T cells shared biased of T-cell receptor variable region of \u03b2 chain (TCR V\u03b2) 1, 2, 4, 5.1, 7.1, 7.2 and\/or 22 chains to promote their differentiation and proliferation respectively, by triggering a signaling cascade. Our data suggest that Rv3615c is a major target of Th1-type responses and can be a highly immunodominant antigen specific for M. tuberculosis infection.<\/jats:p>","DOI":"10.1042\/cs20170205","type":"journal-article","created":{"date-parts":[[2017,6,7]],"date-time":"2017-06-07T00:47:32Z","timestamp":1496796452000},"page":"1859-1876","update-policy":"http:\/\/dx.doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":11,"title":["<i>Mycobacterium tuberculosis<\/i>Rv3615c is a highly immunodominant antigen and specifically induces potent Th1-type immune responses in tuberculosis pleurisy"],"prefix":"10.1042","volume":"131","author":[{"given":"Jiangping","family":"Li","sequence":"first","affiliation":[{"name":"Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan","family":"Shen","sequence":"additional","affiliation":[{"name":"Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Suihua","family":"Lao","sequence":"additional","affiliation":[{"name":"The Section of ICU, Chest Hospital, Guangzhou 510095, PR China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaomin","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jie","family":"Liu","sequence":"additional","affiliation":[{"name":"Laboratory of Infectious Diseases and Vaccine, West China School of Medicine, West China Hospital, Sichuan University, Chengdu 610041, PR China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Changyou","family":"Wu","sequence":"additional","affiliation":[{"name":"Institute of Immunology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080, PR China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","published-online":{"date-parts":[[2017,7,5]]},"reference":[{"key":"2021113011051270300_B1","doi-asserted-by":"crossref","first-page":"1211","DOI":"10.1016\/S0140-6736(15)00151-8","article-title":"Tuberculosis","volume":"387","author":"Dheda","year":"2016","journal-title":"Lancet"},{"key":"2021113011051270300_B2","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1038\/503469e","article-title":"TB vaccine failure was predictable","volume":"503","author":"Beverley","year":"2013","journal-title":"Nature"},{"key":"2021113011051270300_B3","doi-asserted-by":"crossref","first-page":"542","DOI":"10.1016\/j.immuni.2010.09.011","article-title":"Induction of immunity to human immunodeficiency virus type-1 by vaccination","volume":"33","author":"McElrath","year":"2010","journal-title":"Immunity"},{"key":"2021113011051270300_B4","doi-asserted-by":"crossref","first-page":"S1","DOI":"10.1038\/502S1a","article-title":"Tuberculosis","volume":"502","author":"Scully","year":"2013","journal-title":"Nature"},{"key":"2021113011051270300_B5","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1038\/mi.2014.136","article-title":"Immune requirements for protective Th17 recall responses to Mycobacterium tuberculosis challenge","volume":"8","author":"Monin","year":"2015","journal-title":"Mucosal Immunol."},{"key":"2021113011051270300_B6","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1038\/nature13489","article-title":"Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk","volume":"511","author":"Mayer-Barber","year":"2014","journal-title":"Nature"},{"key":"2021113011051270300_B7","doi-asserted-by":"crossref","first-page":"8533","DOI":"10.1038\/ncomms9533","article-title":"Mucosal vaccination with attenuated Mycobacterium tuberculosis induces strong central memory responses and protects against tuberculosis","volume":"6","author":"Kaushal","year":"2015","journal-title":"Nat. Commun."},{"key":"2021113011051270300_B8","doi-asserted-by":"crossref","first-page":"1996","DOI":"10.1093\/infdis\/jiw467","article-title":"Induction of an Immune-Protective T-Cell Repertoire With Diverse Genetic Coverage by a Novel Viral-Vectored Tuberculosis Vaccine in Humans","volume":"214","author":"Jeyanathan","year":"2016","journal-title":"J. Infect. Dis."},{"key":"2021113011051270300_B9","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.1128\/jb.178.5.1274-1282.1996","article-title":"Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis","volume":"178","author":"Mahairas","year":"1996","journal-title":"J. Bacteriol."},{"key":"2021113011051270300_B10","doi-asserted-by":"crossref","first-page":"537","DOI":"10.1038\/31159","article-title":"Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence","volume":"393","author":"Cole","year":"1998","journal-title":"Nature"},{"key":"2021113011051270300_B11","doi-asserted-by":"crossref","first-page":"1520","DOI":"10.1126\/science.284.5419.1520","article-title":"Comparative genomics of BCG vaccines by whole-genome DNA microarray","volume":"284","author":"Behr","year":"1999","journal-title":"Science"},{"key":"2021113011051270300_B12","doi-asserted-by":"crossref","first-page":"12420","DOI":"10.1073\/pnas.1635213100","article-title":"The primary mechanism of attenuation of bacillus Calmette-Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue","volume":"100","author":"Hsu","year":"2003","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"2021113011051270300_B13","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1086\/345862","article-title":"Deletion of RD1 from Mycobacterium tuberculosis Mimics Bacille Calmette-Gu\u00e9rin Attenuation","volume":"187","author":"Lewis","year":"2003","journal-title":"J. Infect. Dis."},{"key":"2021113011051270300_B14","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/j.coi.2009.05.017","article-title":"Tuberculosis vaccine research: the impact of immunology","volume":"21","author":"Barker","year":"2009","journal-title":"Curr. Opin. Immunol."},{"key":"2021113011051270300_B15","doi-asserted-by":"crossref","first-page":"3571","DOI":"10.1016\/j.vaccine.2010.02.094","article-title":"Ag85B\u2013ESAT-6 adjuvanted with IC31\u00ae promotes strong and long-lived Mycobacterium tuberculosis specific T cell responses in na\u00efve human volunteers","volume":"28","author":"van Dissel","year":"2010","journal-title":"Vaccine"},{"key":"2021113011051270300_B16","doi-asserted-by":"crossref","first-page":"824","DOI":"10.1164\/ajrccm.163.4.2009100","article-title":"Rapid detection of Mycobacterium tuberculosis infection by enumeration of antigen-specific T cells","volume":"163","author":"Lalvani","year":"2001","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"2021113011051270300_B17","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1164\/rccm.200402-179OC","article-title":"Specific detection of tuberculosis infection","volume":"170","author":"Mori","year":"2004","journal-title":"Am. J. Respir. Crit. 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Microbiol."},{"key":"2021113011051270300_B21","doi-asserted-by":"crossref","first-page":"4025","DOI":"10.1128\/JB.184.14.4025-4032.2002","article-title":"Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes","volume":"184","author":"Fisher","year":"2002","journal-title":"J. Bacteriol."},{"key":"2021113011051270300_B22","doi-asserted-by":"crossref","first-page":"3371","DOI":"10.1128\/IAI.70.7.3371-3381.2002","article-title":"ideR, An essential gene in mycobacterium tuberculosis: role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response","volume":"70","author":"Rodriguez","year":"2002","journal-title":"Infect. 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U.S.A."},{"key":"2021113011051270300_B25","doi-asserted-by":"crossref","first-page":"3238","DOI":"10.1096\/fj.13-247056","article-title":"Tuberculosis antigen-induced expression of IFN-alpha in tuberculosis patients inhibits production of IL-1beta","volume":"28","author":"Ma","year":"2014","journal-title":"FASEB J."},{"key":"2021113011051270300_B26","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1007\/s10875-014-0090-8","article-title":"Mycobacterium tuberculosis-specific memory NKT cells in patients with tuberculous pleurisy","volume":"34","author":"Li","year":"2014","journal-title":"J. Clin. Immunol."},{"key":"2021113011051270300_B27","doi-asserted-by":"crossref","first-page":"28633","DOI":"10.18632\/oncotarget.5764","article-title":"Antigen-specific human NKT cells from tuberculosis patients produce IL-21 to help B cells for the production of immunoglobulins","volume":"6","author":"Wu","year":"2015","journal-title":"Oncotarget"},{"key":"2021113011051270300_B28","doi-asserted-by":"crossref","first-page":"3358","DOI":"10.1128\/IAI.00014-11","article-title":"Mycobacterium tuberculosis culture filtrate protein 10-specific effector\/memory CD4(+) and CD8(+) T cells in tubercular pleural fluid, with biased usage of T cell receptor Vbeta chains","volume":"79","author":"Qiao","year":"2011","journal-title":"Infect. 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Dis."},{"key":"2021113011051270300_B32","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1128\/CMR.18.4.687-702.2005","article-title":"Vaccines for tuberculosis: novel concepts and recent progress","volume":"18","author":"Doherty","year":"2005","journal-title":"Clin. Microbiol. Rev."},{"key":"2021113011051270300_B33","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1038\/7701","article-title":"A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection","volume":"21","author":"Jouanguy","year":"1999","journal-title":"Nat. Genet."},{"key":"2021113011051270300_B34","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1016\/j.tube.2003.08.010","article-title":"Immunology of tuberculosis and implications in vaccine development","volume":"84","author":"Flynn","year":"2004","journal-title":"Tuberculosis"},{"key":"2021113011051270300_B35","doi-asserted-by":"crossref","first-page":"369","DOI":"10.1038\/ni1449","article-title":"IL-23 and IL-17 in the establishment of protective pulmonary CD4+ T cell responses after vaccination and during Mycobacterium tuberculosis challenge","volume":"8","author":"Khader","year":"2007","journal-title":"Nat. Immunol."},{"key":"2021113011051270300_B36","doi-asserted-by":"crossref","first-page":"e1004099","DOI":"10.1371\/journal.ppat.1004099","article-title":"Unexpected role for IL-17 in protective immunity against hypervirulent Mycobacterium tuberculosis HN878 infection","volume":"10","author":"Gopal","year":"2014","journal-title":"PLoS Pathog."},{"key":"2021113011051270300_B37","doi-asserted-by":"crossref","first-page":"8047","DOI":"10.4049\/jimmunol.0801592","article-title":"Tuberculosis subunit vaccination provides long-term protective immunity characterized by multifunctional CD4 memory T cells","volume":"182","author":"Lindenstrom","year":"2009","journal-title":"J. Immunol."},{"key":"2021113011051270300_B38","doi-asserted-by":"crossref","first-page":"2902","DOI":"10.1016\/j.vaccine.2011.02.010","article-title":"Vaccine-induced anti-tuberculosis protective immunity in mice correlates with the magnitude and quality of multifunctional CD4 T cells","volume":"29","author":"Derrick","year":"2011","journal-title":"Vaccine"},{"key":"2021113011051270300_B39","doi-asserted-by":"crossref","first-page":"e23700","DOI":"10.1371\/journal.pone.0023700","article-title":"Identification of M. tuberculosis-specific Th1 cells expressing CD69 generated in vivo in pleural fluid cells from patients with tuberculous pleurisy","volume":"6","author":"Li","year":"2011","journal-title":"PLoS ONE"},{"key":"2021113011051270300_B40","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1016\/j.tube.2013.12.007","article-title":"Mycobacterium tuberculosis-specific polyfunctional cytotoxic CD8+ T cells express CD69","volume":"94","author":"Li","year":"2014","journal-title":"Tuberculosis (Edinb)"},{"key":"2021113011051270300_B41","doi-asserted-by":"crossref","first-page":"293","DOI":"10.1038\/nm.1935","article-title":"Effector memory T cell responses are associated with protection of rhesus monkeys from mucosal simian immunodeficiency virus challenge","volume":"15","author":"Hansen","year":"2009","journal-title":"Nat. Med."},{"key":"2021113011051270300_B42","doi-asserted-by":"crossref","first-page":"630","DOI":"10.1126\/science.1235454","article-title":"Disparate individual fates compose robust CD8+ T cell immunity","volume":"340","author":"Buchholz","year":"2013","journal-title":"Science"},{"key":"2021113011051270300_B43","doi-asserted-by":"crossref","first-page":"469","DOI":"10.1038\/nrmicro1419","article-title":"Advances in tuberculosis vaccine strategies","volume":"4","author":"Skeiky","year":"2006","journal-title":"Nat. Rev. Microbiol."},{"key":"2021113011051270300_B44","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1002\/(SICI)1097-0320(19960315)26:1<8::AID-CYTO2>3.0.CO;2-E","article-title":"Age- and sex-related changes in lymphocyte subpopulations of healthy Asian subjects: from birth to adulthood","volume":"26","author":"Lee","year":"1996","journal-title":"Cytometry"},{"key":"2021113011051270300_B45","doi-asserted-by":"crossref","first-page":"1433","DOI":"10.1084\/jem.20051365","article-title":"Selective accumulation of differentiated CD8+ T cells specific for respiratory viruses in the human lung","volume":"202","author":"de Bree","year":"2005","journal-title":"J. Exp. Med."},{"key":"2021113011051270300_B46","doi-asserted-by":"crossref","first-page":"1813","DOI":"10.4049\/jimmunol.166.3.1813","article-title":"Activated antigen-specific CD8+ T cells persist in the lungs following recovery from respiratory virus infections","volume":"166","author":"Hogan","year":"2001","journal-title":"J. Immunol."},{"key":"2021113011051270300_B47","doi-asserted-by":"crossref","first-page":"2795","DOI":"10.4049\/jimmunol.149.8.2795","article-title":"Maturation- and differentiation-dependent responsiveness of human CD4+ T helper subsets","volume":"149","author":"de Jong","year":"1992","journal-title":"J. Immunol."},{"key":"2021113011051270300_B48","doi-asserted-by":"crossref","first-page":"3161","DOI":"10.4049\/jimmunol.150.8.3161","article-title":"CD28 engagement by B7\/BB-1 induces transient down-regulation of CD28 synthesis and prolonged unresponsiveness to CD28 signaling","volume":"150","author":"Linsley","year":"1993","journal-title":"J. Immunol."},{"key":"2021113011051270300_B49","doi-asserted-by":"crossref","first-page":"6107","DOI":"10.4049\/jimmunol.172.10.6107","article-title":"The size and phenotype of virus-specific T cell populations is determined by repetitive antigenic stimulation and environmental cytokines","volume":"172","author":"Gamadia","year":"2004","journal-title":"J. Immunol."},{"key":"2021113011051270300_B50","doi-asserted-by":"crossref","first-page":"1191","DOI":"10.1038\/ni1009","article-title":"Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells","volume":"4","author":"Kaech","year":"2003","journal-title":"Nat. Immunol."},{"key":"2021113011051270300_B51","doi-asserted-by":"crossref","first-page":"2091","DOI":"10.1182\/blood-2005-02-0449","article-title":"IL-7 receptor chain expression distinguishes functional subsets of virus-specific human CD8+ T cells","volume":"106","author":"van Leeuwen","year":"2005","journal-title":"Blood"},{"key":"2021113011051270300_B52","doi-asserted-by":"crossref","first-page":"2222","DOI":"10.4049\/jimmunol.1101122","article-title":"Functional capacity of Mycobacterium tuberculosis-specific T cell responses in humans is associated with Mycobacterial load","volume":"187","author":"Day","year":"2011","journal-title":"J. Immunol."},{"key":"2021113011051270300_B53","doi-asserted-by":"crossref","first-page":"e1005490","DOI":"10.1371\/journal.ppat.1005490","article-title":"TIM3 mediates T cell exhaustion during mycobacterium tuberculosis infection","volume":"12","author":"Jayaraman","year":"2016","journal-title":"PLoS Pathog."},{"key":"2021113011051270300_B54","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1080\/21645515.2016.1261229","article-title":"Heterologous boosting with recombinant VSV-846 in BCG-primed mice confers improved protection against Mycobacterium infection","volume":"13","author":"Zhang","year":"2017","journal-title":"Hum. Vaccines Immunother."},{"key":"2021113011051270300_B55","doi-asserted-by":"crossref","first-page":"378","DOI":"10.4161\/hv.27121","article-title":"A novel vaccine p846 encoding Rv3615c, Mtb10.4, and Rv2660c elicits robust immune response and alleviates lung injury induced by Mycobacterium infection","volume":"10","author":"Kong","year":"2014","journal-title":"Hum. Vaccines Immunother."}],"container-title":["Clinical Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/clinsci\/article-pdf\/131\/15\/1859\/828727\/cs-2017-0205.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/clinsci\/article-pdf\/131\/15\/1859\/828727\/cs-2017-0205.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2023,8,23]],"date-time":"2023-08-23T22:57:01Z","timestamp":1692831421000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/clinsci\/article\/131\/15\/1859\/72313\/Mycobacterium-tuberculosis-Rv3615c-is-a-highly"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,7,5]]},"references-count":55,"journal-issue":{"issue":"15","published-print":{"date-parts":[[2017,8,1]]}},"URL":"https:\/\/doi.org\/10.1042\/cs20170205","relation":{},"ISSN":["0143-5221","1470-8736"],"issn-type":[{"value":"0143-5221","type":"print"},{"value":"1470-8736","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,7,5]]}}}