Unbiased TCR Profiling of EBV-Specific CD4+ T Cells

Viable EBV-specific tetramer⁺ CD4⁺ T cell populations were sorted at >98% purity directly into RNAlater (Thermo Fisher Scientific) using a custom-modified FACSAria II flow cytometer equipped with DIVA software version 8.0.1 (BD Biosciences). Unbiased amplification of all expressed TRB gene rearrangements was conducted using a template-switch–anchored RT-PCR with a 3′ C region primer (31 ). Amplicons were subcloned, sampled, sequenced, and analyzed as described previously (32 (link)). Gene use was assigned using the International ImMunoGeneTics nomenclature (33 (link)). All functional TCR sequences were deposited online at VDJdb (34 (link)). Expression of defined TCR Vβ segments on the surface of EBV-specific CD4⁺ T cells was assessed using a TCR Vβ Repertoire Kit (Beckman Coulter).