EPITOPE REPERTOIRE OF HUMAN CD4(+) LINES PROPAGATED WITH TETANUS TOROID OR WITH SYNTHETIC TETANUS TOXIN SEQUENCES

The use of synthetic antigen sequences allows propagation in vitro of T cell lines and clones specific for rare antigens, or for individual epitopes. In the present study we investigated the extent of similarit y of the epitope repertoire of CD4(+) T cell line specific for the ant igen tetanus toxin (TTX), propagated with the complete molecule of tet anus toroid (TTD)I and with the synthetic TTX peptides. We propagated from two healthy subjects CD4(+) T cell lines specific for TTD, by cyc les of stimulation in vitro with TTD or with pools of overlapping synt hetic peptides, 20 residues long and overlapping by five residues, cor responding to all or part of the tetanus toxin (TTX) sequence. One poo l corresponded to the complete TTX sequence (peptide pool). Two other pools corresponded to residues 1-305 of the TTX light chain and 476-78 0 of the TTX heavy chain (peptide minipools). The peptide pool-propaga ted lines recognized TTD vigorously, at levels comparable with those o f the TTD-propagated lines. They recognized several peptides, most of which were also recognized by the TTD-propagated line from the same su bject. They also recognized to a low extent a few peptides not recogni zed by the corresponding TTD-propagated line, which might contain cryp tic epitopes. The TTD-propagated lines recognized also several peptide s that did not elicit a detectable response by the lines propagated wi th the complete peptide pool. The peptide minipool propagated lines re cognized most of the peptides recognized by the TTD-propagated lines. They also recognized several peptides that did not elicit a measurable response of the TTD-propagated line from the same subject, which migh t contain cryptic epitopes. Very few peptides recognized by the TTD-pr opagated line did not evoke a response from the peptide minipool propa gated lines. To verify that the response to the TTD molecule of the li nes propagated with the peptide pools reflected the response of clones recognizing different epitopes produced upon in vitro processing of t he TTD molecule, we propagated from each of the two subjects CD4(+) T cell Lines by stimulation with individual peptide recognized by the TT D-specific lines of that subject (13 peptide-specific lines from subje ct #1, and 15 from subject #2). All lines responded to the presence of TTD to an extent comparable to the response induced by the same conce ntration of the relevant peptide, demonstrating that propagation by sy nthetic epitope sequences allows expansion of T cell clones specific f or epitopes which result from processing of the complete TTD molecule. Therefore, whereas the use of very large pools of synthetic antigen p eptides for propagation of antigen specific human CD4(+) cell lines mi ght lead to loss of clones recognizing less immunogenic sequence regio ns, peptide pools comprising a relatively limited number of synthetic sequences allow propagation of the majority of the antigen specific T cell clones. The use of peptide pools, and especially of limited pepti de pools, results in propagation of polyclonal T cell lines having a m ore diverse repertoire than the lines propagated by stimulation with t he complete antigen molecule. The T clones propagated by the use of sh ort peptide sequences, which are not expanded when the complete antige n molecule is used, may recognize poorly processed, cryptic epitopes. This approach may be adopted to propagate and detect minor clonal popu lations, recognizing less, immunogenic parts of the antigen. (C) 1996 Academic Press Limited