Longitudinal analysis of the EBV-specific CTL response in the peripheral blood of two healthy donors. (A) V(D)J (variable-diversity-joining) junctional TcR sequences from FLRGRAYGL-reactive CTLs from donor 1 during 1987 and 2005, including CTL cell lines generated in 1987 and ex vivo HLA B^*0801-FLRGRAYGL pentamer-sorted CD8⁺ cells in 2005. (B) V(D)J junctional TcR sequences from EENLLDFVRF-reactive CTLs from donor 2 during 1994 and 2005, including CTL cell lines generated in 1994 and ex vivo HLA B^*4405-EENLLDFVRF tetramer-sorted CD8⁺ cells from 1994 and 2005. The TcR extraction procedure from CTL cell lines or pentamer/tetramer-sorted cells was performed as previously described.^6,8  TcR germline sequences are underlined and TcR gene designations follow the nomenclature of the International ImMunoGeneTics Information System.⁹  (C) HLA B^*4405-EENLLDFVRF tetramer-reactive T cells from donor 2 at the time points 1994 and 2005. Peripheral blood mononuclear cells (PBMCs) were stained with tetramer and anti-CD8 monoclonal antibody (mAb). The numbers in the right upper quadrants signify the frequency of CD8^high cells that stained with tetramer. We sorted the tetramer-positive cells by fluorescence-activated cell sorting and performed a switch mechanism at the 5′ end of the RNA transcript (SMART) rapid amplification of complementary DNA ends (RACE) anchor polymerase chain reaction (PCR; Clontech, Mountain View, CA) on the extracted RNA. f values signify the frequency of TcR recovered from either CTL cell lines or multimer-sorted CD8⁺ T cells.