title: Use of HLA-DR*08032/E7 and HLA-DR*0818/E7 tetramers in tracking of epitope-specific CD4+ T cells in active and convalescent tuberculosis patients compared with control donors.
abstract: Comparative tracking of tetramer-positive and epitope-specific CD4(+) T cells in blood and other tissues from tuberculosis (TB) patients during TB development and treatment using control donor samples is not well characterized. In this study, a novel HLA-DR-restricted peptide E7 from the ESAT-6 protein of Mycobacterium tuberculosis (MTB) was used to prepare modified HLA-DR*08032/E7 tetramer (tetramer 1) and HLA-DR*0818/E7 tetramer (tetramer 2) to monitor a series of samples from TB patients and control donors. Tetramer staining showed that (1) by direct staining of single sample and flow cytometric analyses, detection of tetramer-positive CD4(+) T cells ranged from 0.1% to 8.8% (median 0.67% in tetramer 1 and 0.5% in tetramer 2), 0.1 to 10.7% (0.74% and 0.71%), 0.02 to 2.2% (0.25% and 0.25%), 0.02 to 0.48% (0.2% and 0.2%) and most at under 0-0.2% (0.2% and 0.16%) in the initial pulmonary TB (PTB) patients' blood, pleural fluid (PLF) of initial tuberculous pleuritis patients, non-TB patients' blood, healthy donors' blood and umbilical cord blood, respectively; significantly higher levels of CD4(+) T cells were detected in samples of TB patients than in three control donor groups; (2) by direct staining of time point TB samples and flow cytometric analyses, along with TB symptom amendment at day 60, tetramer-positive CD4(+) T cells began to decrease, until after 90-120 days, reached and kept at a relatively low even normal level about at 0.03-0.3%; (3) by enrichment approach, at least 10-fold increased memory tetramer-positive CD4(+) T cells were seen; (4) by in situ staining, tetramer-positive, IFN-γ-producing and/or TNF-α-producing CD4(+) T cells in the lymph node and lung granuloma and cavernous tissues of TB patients could be determined. Therefore, by further increasing the sample size tested to confirm the specificity and sensitivity of tetrameric molecules, it should be possible to develop them for use as research and diagnostic reagents.
authors.affiliation: Department of Microbiology, Ministry of Education Key Laboratory of Tropical Diseases Control, Gangdong Provincial Research Center for Severe Infectious Disease Prevention and Control Technology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan RD. II, Guangzhou 510080, Guangdong, PR China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhu
authors.forename: Lin
authors.initials: L
authors.lastname: Zhou
authors.forename: Yimin
authors.initials: Y
authors.lastname: Fang
authors.forename: Lirong
authors.initials: L
authors.lastname: Huang
authors.forename: Liangliang
authors.initials: L
authors.lastname: Ren
authors.forename: Yi
authors.initials: Y
authors.lastname: Peng
authors.forename: Yifen
authors.initials: Y
authors.lastname: Li
authors.forename: Fangfang
authors.initials: F
authors.lastname: Yang
authors.forename: Dan
authors.initials: D
authors.lastname: Xie
authors.forename: Wenzheng
authors.initials: W
authors.lastname: Tang
authors.forename: Na
authors.initials: N
authors.lastname: Zhang
authors.forename: Qiu
authors.initials: Q
authors.lastname: Zhong
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Lai
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D003001:Cloning, Molecular; D003289:Convalescence; D004331:Drosophila melanogaster; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D004926:Escherichia coli; D005260:Female; D005434:Flow Cytometry; D006684:HLA-DR Antigens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007118:Immunoassay; D018655:Lymphocyte Count; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D058889:Molecular Typing; D009169:Mycobacterium tuberculosis / Q000737:chemistry* / Q000276:immunology; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014397:Tuberculosis, Pulmonary / Q000097:blood / Q000175:diagnosis* / Q000276:immunology / Q000473:pathology
chemical_list: D000942:Antigens, Bacterial; D018984:Epitopes, T-Lymphocyte; D006684:HLA-DR Antigens; D011994:Recombinant Proteins