Scanning for T helper epitopes with human PBMC using pools of short synthetic peptides

Major T helper epitopes of medically important antigens can be located by measuring the proliferative responses of human peripheral blood mononuclear cells (PBMC) to pools of short synthetic peptides. The length and endings of the peptides used were shown to be critical for success in identifying Th cell epitopes. Many epitopes would be missed if either long (31mers) or short (less than 12mers) peptides were used. Pools of 14 and 16mers were more efficient than 12mers spanning the same region, however, for a promiscuous Th cell epitope of tetanus toxin (tt 947-967), two of three donors tested did not respond to 18mers or shorter peptides spanning this region. Although peptides with either unblocked or blocked ends were stimulatory, peptides with blocked ends were generally more efficient. The peptide concentration and number of available APC were also found affect the efficiency of the proliferation assay as a measure of peptide recognition by Th cells. Two screenings of the entire set of tetanus toxin peptide pools using different samples of PBMC from the same donor identified common major stimulatory regions. Thus, PBMC and peptide pools can be used for the reproducible identification of Th cell epitopes. After immunization with tetanus toxoid (TT), peptide-responsive cells increased in frequency in parallel to the increase in TT responsive cells, indicating that the peptide-responsive cells were primed by TT.