Fig. 1.

Overview of single-cell multiplex clonotypic analysis of epitope-specific T cells. Single epitope-specific CD8+ T cells are sorted on a flow cytometric cell sorter into 96-well PCR plates. RT-PCR is performed on the individual cells. The resultant cDNA is subjected to two rounds of nested PCR. In the first round, CDR3α and CDR3β transcript amplification is achieved with the use of a multiplexed, comprehensive panel of external sense Vα and Vβ and antisense Cα and Cβ segment-specific primers. First-round PCR products are subjected to two separate second-round PCRs, incorporating, respectively, a multiplexed panel of external sense Vα and antisense Cα or external sense Vβ and antisense Cβ segment-specific primers. PCR products thus derived are sequenced and translated to yield paired CDR3αβ repertoire data. Inset: Nucleotide products from nested PCR performed on cDNA derived from single CMV-NLV-specific CD8+ T cells from a young adult donor, incorporating TCRα- (top 2 rows) and TCRβ-specific primers (bottom 2 rows).