Identification of mycobacterial recombinant antigens recognized by human T cells

The identification of mycobacterial antigens recognized by human T cells is important for the development of new vaccines and diagnostic reagents, and in understanding the mechanisms involved in the pathogenesis of mycobacterial diseases. In this study, Escherichia coli cell lysates, containing mycobacterial recombinant antigens previously identified using antibodies, were tested with peripheral blood mononuclear cells [PBMC], T cell lines and T cell clones to identify the antigens recognized by human T cells. Although the PBMC responded to natural mycobacterial antigens by proliferation, they did not show any specific response to recombinant antigens containing lysates above the background level proliferation induced by control lysates of E coli. With T cell lines raised against whole mycobacteria, the problem of background proliferation was reduced but the specific responses to the lysated containing recombinant antigens were weak. The proliferative response to specific antigens improved by enriching the T cell lines with E. coli lysates containing homologous recombinant antigens. The best response to recombinant antigens was obtained with mycobacterial antigen-specific T cell clones. However, T cell clones did not respond to some of the antigens which induced proliferation of the enriched T cell lines from the same individuals. This could be because of the inherent bias in cloning procedures which may lead to the expansion of selected T cell populations. Our results suggest that enriched T cell lines are best to determine the T cell reactivity of recombinant antigens, but T cell clones are required to demonstrate the existence of species-specific epitopes

Primary structure and MHC restriction of peptide-defined T cell epitopes from recombinant mycobacterial protein antigens

By combining DNA subclone and synthetic peptide approaches, immunogenic epitopes have been mapped from the mycobacterial heat shock proteins [HSP] of 18. 65 and 70 kD, as well as from a novel non-HSP antigen kD recognized by antigen-specific human T cells. In addition the HLA-DR molecules used in antigen presentation of the individual peptide-defined epitopes have been identified. The doner groups used for establishing antigen-specific CD4+ T cell clones and lines were primarily healthy subjects immunized with Mycobacterium bovis BCG and killed Mycobacterium leprae. The results showed that HSP18 contains on epitope [aa 38-50] presented by HLA-DR4; HSP65 contains 4 epitopes presented by DR1 and DR2 and 5 epitopes presented by DR2, DR3, DR5, DR7, and DRw53. The 24-kD non-HSP antigen also displayed one HLA-DRw53-restricted T cell epitopes. In conclusion, the individual HSP T cell epitopes defined here are exclusively presented by only one HLA-DR molecule. However, at the protein level the results suggest that HSP65 and HSP70 are relevant to the subunit vaccine design, since they contain multiple T cell epitopes which can be presented by a spectrum of different HLA-DR molecules