Efficient mapping and characterization of a T cell epitope by the simultaneous synthesis of multiple peptides.
Prediction, identification and analysis of T cell epitopes in protein antigens has become a central theme in fundamental and applied immunology. However, while for the characterization of linear B cell epitopes the so-called Pepscan procedure was found to be extremely effective, no such technique has so far been available for T cell studies. Recently, we described the identification and localization of a T cell epitope in a mycobacterial 65-kDa shock protein in the model of adjuvant arthritis. This was done by molecular cloning and conventional solid-phase synthesis techniques. We now show that the delineation of such a T cell epitope and its further characterization can be accomplished in a much more rapid and efficient manner by a modification of the existing Pepscan technique. We show for the first time that several hundreds of peptides, simultaneously synthesized in an automated way on activated polyethylene rods, can be easily recovered from these rods in adequate quantities, enabling a systematic analysis of T cell epitopes. Synthesis of sequentially overlapping peptides along the 65-kDa protein revealed that the adjuvant arthritis T cell clones are fully stimulated by peptides that comprise a minimal sequence of seven residues, corresponding to positions 180-186 in the sequence of the 65-kDa protein of M. bovis Bacillus Calmette Guerin (BCG). Detailed examination of the epitope by peptides containing a single amino acid substitution showed that, apart from one conservative replacement (Glu----Asp), the requirement for the native residue at all positions in peptide 180-186 was absolute for full T cell stimulation. Their indispensability was confirmed with deletion and insertion peptides. It is concluded that the occurrence of indifferent or spacer residues in a minimal stimulatory sequence, as observed by others, is not a general feature of T cell epitopes.
Van der Zee
R
R
Laboratory of Bacteriology, National Institute of Public Health, Bilthoven, The Netherlands.
Van Eden
W
W
Meloen
R H
RH
Noordzij
A
A
Van Embden
J D
JD
D000595:Amino Acid Sequence; D000940:Antigenic Variation; D000942:Antigens, Bacterial / Q000276:immunology / Q000302:isolation & purification; D000945:Antigens, Differentiation, T-Lymphocyte / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000138:chemical synthesis / Q000276:immunology / Q000302:isolation & purification; D000939:Epitopes / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009163:Mycobacterium bovis / Q000032:analysis*; D009842:Oligopeptides / Q000138:chemical synthesis* / Q000276:immunology / Q000302:isolation & purification; D010449:Peptide Mapping
D000942:Antigens, Bacterial; D000945:Antigens, Differentiation, T-Lymphocyte; D001426:Bacterial Proteins; D000939:Epitopes; D009842:Oligopeptides