Figure 4
Analysis of CMV NLV specific T cells ex vivo and after 14 days in culture. PBMC as well as femur derived BMMC from HLA-A2 positive donors were cultivated in the presence of CMVNLV peptide (0.1 μg/m) and IL-2 (20 ng/ml) for two weeks. A, T cells were stained with PerCP-conjugated anti-CD8 antibody and APC-conjugated pentamers containing the CMVNLV peptide. Gating on CD8+ T cells was performed. A representative example is shown for CMVNLV peptide specific CD8+ T cells from the BM (left panel) and PB (right panel) before culture as well as after two weeks of culture with the peptide CMVNLV and IL-2 followed by the purification of the CMVNLV specific population with magnetic beads as described in Materials and Methods. The staining depicts one of three similar experiments. B, Spectratyping of the CDR3 region was performed on purified CMVNLV specific T cells derived from BM and PB. Representative results comparing the clonal composition of CMVNLV specific T cells derived from BM and PB for selected Vβ families are shown. The relative size of the CDR3-region fragments is indicated below the peaks. C, Clonality and intensity scores were determined for spectratyping results of 24 individual Vβ families for CMVNLV specific T cells derived from BM (black) and PB (white). Results are depicted for three individual donors. The clonality score (1 = Gaussian distribution; 2 = several peaks; 3 = one peak) and intensity score (0 = <500 RFU [relative fluorescence units]; 1 = 500-3000 RFU; 2 = 3000-8000 RFU; 3= >8000 RFU) were added.