PMC

For TCR analysis, RNA was used as the starting material and gene-specific cDNA synthesis was performed using a biotinylated primer located in the constant (C) gene of the TCR chains. The resulting full-length cDNA fragments were magnetically captured on streptavidin beads and ligated to a known single-stranded linker cassette (ssLC). After exponential PCR amplification, sequencing adaptors were added and the PCR products were deep sequenced. C, constant gene; BC, sample-specific barcode.

The figure shows the contributions of the CDR3 aa sequences to the αβ TCR repertoire in HDs and in 10 Sézary patients. Each bar represents an individual CDR3 aa clonotype, with red and violet indicating the first and tenth most predominant sequences, respectively. Grey bars indicate the contribution of the remaining sequences identified in the analysed sample. TCR-LA-MC PCR results show that the stage of the disease correlated well with the observed TCR clonality. These results were confirmed by calculating SI and SA diversity indices (DI). The higher is the SA or the SI, the more diverse is the population. Two replicates were performed for each TCR chain of each patient and sequencing results were combined. aa, amino acid; CDR3, complementarity-determining region 3; HD, healthy donor; P, patient; SA, Shannon index; SI, Simpson index.

TCR-LA-MC PCR was performed on RNA samples for two CMV-seropositive donors ((a) donor 1; (b) donor 2; upper panel, α-chain; lower panel, β-chain) at different time points after CMV stimulation. At day 15, an additional enrichment of the IFNγ-secreting fraction was performed. The 10 most predominant CMV-reactive TCR sequences identified at day 15 and their relative contribution to the total repertoire (sequence count) are shown. Retrieval frequency is indicated with a colour code ranging from light pink (0.01–0.1%) to black (>40%). We observed enrichment of defined TCR specificities over time. The number of T-cell clones (shown as %) contributing to the generation of those specificities also increased, indicating that a portion of the T-cell population is committed to the generation of specific TCR molecules. The CMV specificity of the analysed sequences was further confirmed by analysis of the enriched fraction. Two replicates were performed for day 0, 9 and 15 of each donor and sequencing results were combined. ✓, sequence was identified in the CMV enriched fraction; aa, amino acid; CDR3, complementarity-determining region 3; d, day; IFN, interferon; Seq, sequence.