tuberculosis induced CD4+ T-cell-dependent responses. The key findings of the present study are that eight of a total of 157 peptides selected for HLA class I binding induce T-cell responses in PBMC from one or several PPD+ donors (Table 2). In contrast, only four of 10 donors, with low PPD reactivity in ELISPOT
assay, reacted with one or more of the eight antigenic peptides indicating the M. tuberculosis specificity of the responses observed. However, instead of being HLA class I restricted, these responses are apparently restricted buy PD98059 by HLA class II molecules because the responses are all blocked by anti-HLA-DR antibody added to the ELISPOT assay culture. In addition, according to results from cell depletion and FACS analyses anti-M. tuberculosis peptide responses are mediated by CD4+ T cells. The
eight epitopes discovered are derived from five different M. tuberculosis proteins. Three of these [Rv1979, Rv3144c (PPE52) and Rv3532 (PPE61)] each express two positive CTL epitopes whereas the remaining two proteins [Rv0284 and Rv3507 (PE_PGRS)] only harbour a single epitope. Interestingly, six of the eight positive epitopes are derived from the PE/PPE gene family of conserved mycobacterial proteins (Table 2). The PE/PPE gene family is interesting from an immunological point of view because they comprise approximately Compound Library 10% of the M. tuberculosis genome and may be a source of antigenic variation, which the bacterium uses to evade the host immune response.34 These proteins are surface-associated cell wall proteins and may also be accessible to antibodies.40 The B-cell and T-cell
immune responses have been reported against both PE and PPE proteins, but their immunological Adenosine triphosphate significance remains largely unknown.41–44 Only a few T-cell epitopes have been identified for the PE/PPE gene family. Two have been found in PE-PGRS proteins (Rv1818c and Rv3812) and one in PPE protein (Rv3018c). In the present study we report five new epitopes for the PE/PPE gene family: a single epitope for the Rv3507 (PE_PGRS) and four new epitopes for the PPE proteins [Rv3144c (PPE52) and Rv3532 (PPE61)]. Regarding the phenotype of M. tuberculosis peptide-responding T cells, our data from T-cell depletion of PBMC before ELISPOT and FACS analyses showed that the responding T cells are indeed CD4+ T cells. In our previous studies 26–28 in which we probed for specific T-cell immunity in PBMC against pox and flu virus-derived HLA-I binding peptides, respectively, HLA-I and HLA-II antibody blocking experiments and CD4+ and CD8+ T-cell depletion experiments showed that peptide reactivity was initiated by either CD4+ or CD8+ T cells but never by both subsets in the same ELISPOT culture. It is generally accepted that HLA class I binding peptides are composed of 8–11 amino acids, whereas HLA class II binding peptides consist of 15–20 amino acids being recognized by CD8+ and CD4+ T cells, respectively.