Identification of promiscuous epitopes from the mycobacterial 65-kilodalton heat shock protein recognized by human CD4(+) T cells of the Mycobacterium leprae memory repertoire

By using a synthetic peptide approach, we mapped epitopes from the mycobact erial 65-kDa heat shock protein (HSP65) recognized by human T cells belongi ng to the Mycobacterium leprae memory repertoire. A panel of HSP65 reactive CD4(+) T-cell lines and clones were established from healthy donors 8 year s after immunization with heat-killed M. leprae and then tested for prolife rative reactivity against overlapping peptides comprising both the M. lepra e and Mycobacterium tuberculosis HSP65 sequences. The results showed that t he antigen-specific T-cell lines and clones established responded to 12 myc obacterial HSP65 peptides, of which 9 peptides represented epitopes crossre active between the M tuberculosis and M. leprae HSP65 (amino acids [aa] 61 to 75, 141 to 155, 151 to 165, 331 to 345, 371 to 385, 411 to 425, 431 to 4 45, 441 to 455, and 501 to 515) and 3 peptides (aa 343 to 355, 417 to 429, and 522 to 534) represented M. leprae HSP65-specific epitopes. Major histoc ompatibility complex restriction analysis showed that presentation of 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self HLA-DRB1 genes, whereas 3 peptides with sequences comp letely identical between the M. leprae and M. tuberculosis HSP65 were prese nted to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was pre sented by HLA-DR1, -DR2, and -DR7, peptide (aa 141 to 155) was presented by HLA-DR2, -DR7, and -DR53, whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) wer e able to present peptide (aa 501 to 515) to T cells. In addition, the T-ce ll lines responding to these peptides in proliferation assays showed cytoto xic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion, we demonstrated that promiscuous peptide epi topes from the mycobacterial HSP65 antigen can serve as targets for cytotox ic CD4(+) T cells which belong to the human memory T-cell repertoire agains t M. leprae. The results suggest that such epitopes might be used in the pe ptide-based design of subunit vaccines against mycobacterial diseases.