A total of 157 peptides were

found to bind to one of the 12 HLA molecules with a measured KD ≤ 500 nm, which is the normally accepted threshold36–38 for being a potential antigenic epitope. The numbers of binding peptides for the individual supertypes are: HLA-A1 (11 peptides), HLA-A2 (15 peptides), HLA-A3 (four peptides), HLA-A24 (14 peptides), HLA-A26 (15 peptides), HLA-B7 (18 peptides), HLA-B8 (seven peptides), HLA-B27 (eight peptides), HLA-B39 (17 peptides), HLA-B44 (20 peptides), HLA-B58 (14 peptides) and HLA-B62 (14 peptides). Consistent with previous classifications, the binding affinity (KD) of the 157 binding peptides can be divided into groups of high-affinity binders (n = 83; KD ≤ 50 nm) and intermediate-affinity binders Ku-0059436 supplier (n = 74; 50 nm < KD ≤ 500 nm). The 157 HLA-I binding peptides were tested for their ability to stimulate T cells from a cohort of healthy PPD+ Danish subjects aged 35–65 years. The peptides were evaluated for their ability to stimulate IFN-γ production

in an ELISPOT assay by PBMC from those HLA-matched donors who reacted most strongly with PPD. Since many donors’ PBMC failed to respond after 2 days of peptide exposure, the Z-VAD-FMK sensitivity of the procedure was increased by exposing PBMC for 10 days to peptides before performing the ELISPOT assays. Positive reactivity towards peptides was confirmed at least twice in the same donor as well as in other HLA supertype matched donors. According to this criterion eight peptides (5%)

belonging to five different supertypes (A1, A26, B7, B44 and B62) were found to be antigenic. An overview of peptide-reactive donors, their HLA class I type, and their reactivity according to ELISPOT data is shown in Table 1. The number of reactive donors and the actual ELISPOT data are shown in Table 2. Each Ureohydrolase of the eight antigenic peptides was also tested in 10 donors with low PPD reactivity. Only four of these donors showed reactivity against one or more of the eight antigenic peptides, an observation, which strongly underscores the M. tuberculosis specificity of the responses observed in the present study. We have previously demonstrated that variola virus-derived 9mer peptides with high HLA-I binding affinity (KD ≤ 5 nm) are able to induce CD4+ T-cell responses from PBMC of vaccinated donors.39 Likewise, we showed that influenza A virus-derived 9mer peptides with binding affinities for HLA-I allele are capable of stimulating strong CD4+ T-cell responses.28 To ascertain whether, or not, CD4+ T cells are involved in the anti-M. tuberculosis responses documented above, a pan-specific anti-HLA-II blocking antibody IVA12 as well as anti-DP, -DQ and -DR blocking antibodies were added into ELISPOT microcultures (see Materials and methods section). Similarly, cultures were exposed to the pan-specific anti-HLA class I antibody W6/32. As shown in Fig.