Clonotypic analysis of the TCR repertoire for TRAT-specific CD8⁺ T cells. (A) HLA Cw*0602-restricted TRAT-specific CD8⁺ T-cell clones were expanded by use of the agarose cloning method and then assessed for TRBV expression by the use of specific monoclonal antibodies. Representative data from 4 of the 25 clones are presented. (B) These T-cell clones were then assessed for reactivity against peptide-coated and -uncoated HLA Cw*0602⁺ and DRB*0401⁺ LCLs by the use of ICS assays. Clones 2G8 and ID3 showed reactivity against peptide-coated and -uncoated LCLs, whereas clones 2B11 and IE3 showed reactivity against peptide-coated LCLs only. (C) Amino acid sequences of CDR3 regions of TRAV and TRBV chains expressed by TRAT-specific clones. (D) T-cell recognition of alanine analogs of the TRAT peptide epitope. HLA-Cw*0602⁺ LCLs were presensitized with the individual peptides (0.1 μg/mL) and then exposed to TRAT-specific CTL clones. An effector:target ratio of 5:1 was used in the assay. The T-cell reactivity was assessed by the use of standard ICS assays. Data presented in each of the subpanels show relative IFN-γ expression in the presence of alanine analogs compared with the wild-type peptide.