By using a synthetic peptide approach we mapped epitopes from your mycobacterial 65-kDa warmth shock protein (HSP65) recognized by MK-0752 human T cells belonging to the memory repertoire. 9 of the 12 peptides to T cells were restricted by one of the 2 HLA-DR molecules expressed from self genes whereas 3 peptides with sequences completely identical between the and HSP65 were offered to T cells by multiple HLA-DR molecules: peptide (aa 61 to 75) was offered by HLA-DR1 -DR2 and -DR7 peptide (aa 141 to 155) was offered by HLA-DR2 -DR7 and -DR53 whereas both HLA-DR2 and -DR4 (Dw4 and Dw14) were able to present peptide (aa 501 to 515) to T cells. In addition the T-cell lines responding to these peptides in proliferation assays showed cytotoxic activity against autologous monocytes/macrophages pulsed with the same HSP65 peptides. In conclusion we exhibited that promiscuous peptide epitopes from your mycobacterial HSP65 antigen can serve as targets for cytotoxic CD4+ T cells which belong to the human memory T-cell repertoire against immunization in humans (32). Finally immunization with the mycobacterial HSP65 antigen induced protection against and in mouse models of infections (4 54 55 In addition DNA vaccination of mice with the HSP65 antigen provided protection against challenge with (60). These earlier studies suggest that the mycobacterial HSP65 represents a candidate antigen for subunit vaccine design. However immunization with the complete HSP65 molecule may lead to adverse effects such as MK-0752 MK-0752 autoimmune responses and the induction of suppressor T cells (11 37 66 67 PPARG2 An alternative strategy could be to identify and select HSP65 epitopes that are offered to CD4+ Th1 cells in association with multiple HLA class II molecules. Although several epitopes of the mycobacterial HSP65 recognized by human T cells have previously been recognized (2 12 14 43 47 their application to diagnosis or vaccine design is hampered by a stringent HLA-DR restriction requirement. These studies exhibited that all investigated HSP65 epitopes could only be recognized by T cells in association with one of the two HLA-DR molecules expressed from your self-genes (2 12 43 47 By using synthetic peptides covering both the and HSP65 sequences we have in this study identified three novel HSP65 epitopes each offered to CD4+ T cells in association with multiple HLA-DR molecules. The finding that such promiscuous epitopes were targets for acknowledgement by preparations were kindly supplied by R. J. W. Rees from your World Health Business (WHO)/Immunology of Leprosy (IMMLEP) Lender. The recombinant and HSP65 were kindly provided by J. D. A. van Embden from your WHO/IMMLEP Lender. Two units of peptides were used to identify the epitopes recognized by the mycobacterial HSP65-reactive T-cell lines. The first set comprised 50 peptides (P1 to P50) covering the amino acid (aa) sequence of the HSP65 (43). These peptides were 15-mers and overlapped with 5 aa. Another series of 20 peptides (P51 to P70; 13-mers) corresponding to the parts of the HSP65 sequence that differed from your sequence by one or more amino acids were synthesized by the Pepscan method as explained previously (20). Antigen-presenting cells (APC). Heparinized venous blood was MK-0752 obtained from the BCG- and BCG and were in addition typed for Dw4 and Dw14 subtypes of DR4 by using alloreactive T-cell clones (52). All of the donors were HLA class II typed genomically by the hybridization of sequence-specific oligonucleotide probes to PCR-amplified DNA (51). Screening for the presence of the allele (encoding HLA-DR53) was carried out genomically for selected individuals and the vaccinated subjects. HSP65-specific T-cell lines and clones. HSP65-reactive T-cell lines were established from your PBMC of five healthy subjects 8 years after vaccination with killed (28 32 To establish the T-cell lines 2 × 106 PBMC in 1 ml of total medium (RPMI 1640 plus 10% AB serum and 1% penicillin-streptomycin) were cultured with (5 × 107 bacilli/ml) in the wells of 24-well Costar plates (Costar Cambridge Mass.). The plates were incubated at 37°C in an atmosphere of 5% CO2 and 95% air flow. After 6 days of incubation 100 U of recombinant interleukin-2 (Amersham Amersham United Kingdom) was added to the cultures twice a week for 4 weeks (17). To expand the mycobacterial HSP65-reactive T cells the lines were restimulated with HSP65 and autologous APC (15). Phenotypically the T-cell lines were >95% CD4+ and <5% CD8+. HSP65-specific T-cell clones were obtained from the (5 × 107.