Figure 3

Higher antigen avidity of CMV-specific CD8⁺ T cells with the CDR3-sequence SVNEAF. CMV_NLV-specific CD8⁺ T cells were stimulated in vitro with CMV_NLV-peptide for two weeks. Donors with a monoclonal profile of BV8⁺ CMV_NLV-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, CMV_NLV-specific-T cells only. (A) CMV_NLV-specific BV8⁺ T cells were stained with different concentrations of CMV_NLV-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) CMV_NLV-specific CD8⁺ T cells were stained with saturated amounts of CMV_NLV-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).