Higher antigen avidity of CMV-specific CD8+ T cells with the CDR3-sequence SVNEAF. CMVNLV-specific CD8+ T cells were stimulated in vitro with CMVNLV-peptide for two weeks. Donors with a monoclonal profile of BV8+ CMVNLV-specific T cells with the CDR3-sequence SANYGYT or SVNEAF respectively were selected. BV8+ T cells were identified by FACS-staining and results are shown for BV8+ gated, CMVNLV-specific-T cells only. (A) CMVNLV-specific BV8+ T cells were stained with different concentrations of CMVNLV-pentamer. The mean fluorescence intensity (MFI; note the logarithmic scale) of bound pentamer is indicated for donors with the CDR3-sequence SANYGYT (light grey) or SVNEAF (dark grey). n = 3; mean ± SEM. * p < 0.05 (Student's t-test for unpaired data). (B) CMVNLV-specific CD8+ T cells were stained with saturated amounts of CMVNLV-pentamer (1.25 μg/ml) and the dissociation rates of pentamers from CMV-specific CD8+ T cells were determined for donors with the CDR3-sequence SANYGYT (light grey) or SVNEAF (dark grey). Dissociation of pentamers was assessed by FACS analysis at different time points (range 0–180 min). Mean fluorescence intensity (MFI) of pentamer-positive T cells at time point 0 (maximum pentamer binding) was considered as 100%. n = 3; mean ± SEM. * p < 0.05 (Student's t-test for unpaired data).