Characterization of the TCR α and β chains of CD1d/GPI dimer⁺ T cells. Flow-sorted CD1d/h-GPI dimer⁺ T-cell fractions were subjected to a 2-step reverse transcription PCR to amplify the whole TCR α- and β-chain mRNA repertoire followed by plasmid cloning and sequencing of individual clones (see also supplemental Figure 4). (A) Nucleotide (top) and amino acid (bottom) sequence of the TCRVα21-Jα31-1 CDR3α chain identified in the CD8⁺ CD1d/GPI dimer⁺ T cells of P5. The last nucleotide triplet in the Vα21 gene, corresponding to arginine, is deleted; there are no Jα segment N insertions or deletions, that is, the TCRVα21Jα31 amino acid sequence is invariant. (B) Nucleotide (top) and amino acid (bottom) sequence of the TCRVβ19-D2-Jβ2.7 chain identified in the CD8⁺ CD1d/GPI dimer⁺ T cells of P5 with a typically diverse CDR3β region as shown. (C) Flow-sorted TCRVβ19⁺ and TCRVβ19⁻ T cells from P5 and 2 healthy controls were placed immediately after sorting into an IFNγ ELISPOT assay against C1R-CD1d cells loaded with Veh or GPI at an E:T ratio of 1:1 (20 × 10³ T cells per well). Data are shown as mean ± SEM of triplicate assays. (D) Bar diagram showing the frequency of the invariant TCRVα21Jα31 mRNA within the whole TCRVα21-Cα repertoire of the flow-sorted T-cell fractions shown (see also Table 1).