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 c ells of bacillus Calmette-Guerin (BCG)-vaccinated individuals with live Myc obacterium bovis BCG bacilli to establish short-term cell lines and then pu rified the CD8(+) T cells. A highly sensitive enzyme-linked immunospot (ELI SPOT) assay for single cell IFN-gamma release was used to screen CD8(+) T c ells with overlapping peptides spanning the mycobacterial major secreted pr otein, Ag85A, Three peptides consistently induced a high frequency of IFN-g amma responsive CD8(+) T cells, and two HLA-A*0201 binding motifs, P48-56 a nd P242-250 were revealed within the core sequences, CD8(+) T cells respond ing to the 9-mer epitopes were visualized within fresh blood by ELISPOT usi ng free peptide or by binding of HLA-A*0201 tetrameric complexes, The class I-restricted CD8(+) T cells were potent CTL effector cells that efficientl y lysed an HLA-A2-matched monocyte cell line pulsed with peptide as well as autologous macrophages infected with Mycobacterium tuberculosis or recombi nant vaccinia virus er;pressing the whole Ag85A protein. Tetramer assays re vealed a 6-fold higher frequency of peptide-specific T cells than IFN-gamma ELISPOT assays, indicating functional heterogeneity within the CD8(+) T ce ll population. These results demonstrate a previously unrecognized, MHC cla ss I-restricted, CD8(+) CTL response to a major secreted Ag of mycobacteria and supports the use of Ag85A as a candidate vaccine against tuberculosis.