TCR Repertoire Analysis of Epitope-Specific CD8+ T Cells

TCR repertoire analyses were performed on epitope-specific CD8⁺ T cells, which were defined by fluorescence-activated cell sorting (FACS), using the CD4⁻ CD8α⁺ CD44⁺ dextramer-binding (Dextramer⁺) populations (dextramers for NP396, GP33 or NP205). Additionally, the “overall” Vβ/Jβ usage was analyzed by sequencing the dextramer-negative, CD8⁺ T cell population. A detailed description of the procedure was published previously [46 (link),50 (link)]. Brief description of the workflow: RNA was extracted using the RNeasy plus microRNA extraction kit (Qiagen). The cDNA synthesis and amplification PCR were performed using the SMARTer RACE cDNA amplification kit (Clontech) and the Advantage 2PCR kit (Clontech). Amplicon size was determined by running an agarose gel and the respective products were indexed with another PCR (Advantage 2PCR kit (Clontech)) and Nextera primer combinations (Illumina). Finally, all sample concentrations were determined and sequencing was performed with a Miseq (Illumina) using V2 chemistry and 150 bp paired-end sequencing.