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 mo nonuclear cells (PBMC) to pools of short synthetic peptides. The lengt h and endings of the peptides used were shown to be critical for succe ss in identifying Th cell epitopes. Many epitopes would be missed if e ither long (31mers) or short (less than 12mers) peptides were used. Po ols 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 s horter peptides spanning this region. Although peptides with either un blocked or blocked ends were stimulatory, peptides with blocked ends w ere generally more efficient. The peptide concentration and number of available APC were also found to affect the efficiency of the prolifer ation assay as a measure of peptide recognition by Th cells. Two scree nings of the entire set of tetanus toxin peptide pools using different samples of PBMC from the same donor identified common major stimulato ry regions. Thus, PBMC and peptide pools can be used for the reproduci ble identification of Th cell epitopes. After immunization with tetanu s toroid (TT), peptide-responsive cells increased in frequency in para llel to the increase in TT responsive cells, indicating that the pepti de-responsive cells were primed by TT.