Reactivity and epitope mapping of single-chain T cell receptors with monoclonal antibodies.
To examine further the structure of the T cell receptor (TCR) and the specificity of mAbs generated against the native protein, the TCR was expressed in Escherichia coli as a single chain in which the variable regions of the alpha and beta chains are joined by a 25 amino acid linker. Five single-chain TCR that have different alpha and/or beta variable (V) regions were examined with the anti-V beta 8 region mAbs KJ16 and F23.1 and the anti-V alpha 8 mAbs KT50, KT65 and B21.14. Each of the mAbs reacted with one or more of the single-chain receptors. Western blot analysis demonstrated that the intrachain disulfide bonds were required for proper epitope conformation and recognition of the TCR by the antibodies. KT50, KT65 and B21.14 antibodies distinguished between two related V alpha regions that differed at only six residues. A model of the V regions of the TCR based on immunoglobulin (Ig) structure suggests that three of these six variant residues are in the putative CDR1 of the receptor and possibly accessible to antibody. To test this possibility, site-directed mutagenesis of the unreactive V alpha region demonstrated that the combination of all three residues restored binding by the anti-V alpha 8 antibodies. In addition, these three complimentarity determining regions (CDR) residues are likely to be in close proximity to the putative CDR3 which also influenced binding of the antibodies. The epitopes recognized by the V alpha-specific antibodies are thus predicted to reside closer to the putative binding site than the epitopes previously determined to be recognized by the anti-V beta 8 antibodies, KJ16 and F23.1. Finally, the specificities of KT50 and KT65 as determined with the E. coli expression system suggests an explanation for previous observations about the differences in the T cell populations that are recognized by these antibodies.
Brodnicki
T C
TC
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
Holman
P O
PO
Kranz
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics; D000937:Antigen-Antibody Reactions / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody / Q000235:genetics; D018604:Epitope Mapping; D005822:Genetic Vectors / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000276:immunology; D010084:Oxidation-Reduction; D051381:Rats; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
D000911:Antibodies, Monoclonal; D016693:Receptors, Antigen, T-Cell, alpha-beta