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 lin ker. Five single-chain TCR that have different alpha and/or beta varia ble (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 t he mAbs reacted with one or more of the single-chain receptors. Wester n 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 betwee n two related V alpha regions that differed at only six residues. A mo del 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 thi s possibility, site-directed mutagenesis of the unreactive V alpha reg ion demonstrated that the combination of all three residues restored b inding by the anti-V alpha 8 antibodies. In addition, these three comp limentarity determining regions (CDR) residues are likely to be in clo se proximity to the putative CDR3 which also influenced binding of the antibodies. The epitopes recognized by the V alpha-specific antibodie s are thus predicted to reside closer to the putative binding site tha n the epitopes previously determined to be recognized by the anti-V be ta 8 antibodies, KJ16 and F23.1. Finally, the specificities of KT50 an d KT65 as determined with the E. coli expression system suggests an ex planation for previous observations about the differences in the T cel l populations that are recognized by these antibodies. Copyright (C) 1 996 Elsevier Science Ltd.