Human CD8+ CTL specific for the mycobacterial major secreted antigen 85A.
The role of CD8(+) CTL in protection against tuberculosis in human disease is unclear. In this study, we stimulated the peripheral blood mononuclear cells of bacillus Calmette-Guérin (BCG)-vaccinated individuals with live Mycobacterium bovis BCG bacilli to establish short-term cell lines and then purified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELISPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T cells with overlapping peptides spanning the mycobacterial major secreted protein, Ag85A. Three peptides consistently induced a high frequency of IFN-gamma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P(48-56) and P(242-250), were revealed within the core sequences. CD8(+) T cells responding to the 9-mer epitopes were visualized within fresh blood by ELISPOT using free peptide or by binding of HLA-A*0201 tetrameric complexes. The class I-restricted CD8(+) T cells were potent CTL effector cells that efficiently lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombinant vaccinia virus expressing the whole Ag85A protein. Tetramer assays revealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T cell population. These results demonstrate a previously unrecognized, MHC class I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.
Smith
S M
SM
Immunology Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, United Kingdom. Steven.Smith@bristol.ac.uk
Brookes
R
R
Klein
M R
MR
Malin
A S
AS
Lukey
P T
PT
King
A S
AS
Ogg
G S
GS
Hill
A V
AV
Dockrell
H M
HM
D000217:Acyltransferases; D017951:Antigen Presentation; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000276:immunology* / Q000378:metabolism; D001500:BCG Vaccine / Q000276:immunology; D002460:Cell Line; D003601:Cytotoxicity Tests, Immunologic; D018604:Epitope Mapping; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D005714:Gambia; D006801:Humans; D016130:Immunophenotyping; D007371:Interferon-gamma / Q000096:biosynthesis; D015847:Interleukin-4 / Q000096:biosynthesis; D018655:Lymphocyte Count; D009169:Mycobacterium tuberculosis / Q000276:immunology*; D009842:Oligopeptides / Q000276:immunology / Q000378:metabolism; D016176:T-Lymphocyte Subsets / Q000276:immunology / Q000378:metabolism / Q000382:microbiology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology* / Q000378:metabolism / Q000382:microbiology*; D014407:Tumor Cells, Cultured; D006113:United Kingdom
D000942:Antigens, Bacterial; D001500:BCG Vaccine; D018984:Epitopes, T-Lymphocyte; D009842:Oligopeptides; D015847:Interleukin-4; D007371:Interferon-gamma; D000217:Acyltransferases; C112580:antigen 85A, Mycobacterium tuberculosis