Human CD8+ CTL Specific for the Mycobacterial Major Secreted Antigen 85A

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Abstract
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-γ 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-γ responsive CD8+ T cells, and two HLA-A*0201 binding motifs, P48–56 and P242–250, 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-γ 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.