Here, we have extended these observations by showing that in silico predicted HLA-I binding 9mer peptides derived from M. tuberculosis proteins induce T-cell-dependent responses that appear to be HLA-II restricted because they are totally blocked by a pan HLA-II antibody as well as by an anti-HLA-DR antibody. As in our previous study

with vaccinia virus-derived peptides,39 there was a trend of correlation between HLA class II restricted antigenicity and a measured high peptide HLA-I binding affinity, so six of eight antigenic M. tuberculosis peptides bind HLA-I with a KD < 50 nm. However, in accordance with our recent observation on flu epitopes,28 Small molecule library chemical structure we found that two of the M. tuberculosis peptides with intermediate binding affinities to HLA class I were also capable of stimulating a

strong HLA-II restricted T-cell responses. As the eight antigenic 9mer epitopes appear to be restricted by HLA-II DR molecules (Fig. 1), we tried to predict the binding of all the 157 9mers used in this study to all DR alleles present among the donors using the publicly available MHC-II predictor NetMHCIIpan48 (http://www.cbs.dtu.dk/services). Forty-eight peptides including the two antigenic peptides LEEIGILLL and IVFATAARY were predicted to be either strong binders (SB, predicted KD < 50nm) or weak binders (WB, predicted KD < 500 nm), respectively, to one or more DR alleles present among the donors, (see Supplementary material Tables S1 and Belnacasan nmr S2). However, the two donors (no. 19 and 32) who reacted with these two peptides did not express the predicted HLA-DR alleles. We have recently developed a technology for assaying the binding of peptides to recombinant HLA-DR molecules.32 However, only three of the eight antigenic M. tuberculosis peptides showed binding to three of the 14 tested HLA-DR molecules, Fossariinae but none of these three HLA-DR molecules were expressed by the two peptide-reactive donors. These negative data might reflect the

fact that the number of assayed HLA-DR molecules only represent one-third of the HLA-DR subtypes expressed by the TB peptide immune donors. In addition, the 10-day peptide exposure period might favour low-affinity interactions that might be missed in our biochemical assay. However, so far we have no definitive proof that the eight antigenic 9mer TB peptides discovered in the present study do bind to HLA-DR. It is well established that CD4+ T cells are instrumental in the control of M. tuberculosis infections.6,7,9–11 For this reason, MHC-II restricted epitopes identified in the present study as capable of stimulating CD4+ T-cell responses may be of importance for the development of effective peptide-based vaccines against TB. In addition, it has been shown that CD4+ T cells are required for priming as well as secondary expansion of CD8+ memory T cells.