932318.fig.004a
(a)
932318.fig.004b
(b)
932318.fig.004c
(c)
932318.fig.004d
(d)
Figure 4: Sorting procedure. (a) 107 Chim-AvT dynabeads are coated with HLA-A2-peptide monomers, and coating efficiency is assessed on 105 beads, by labelling with an PE-labelled anti-HLA-A2 mAb (5 μg/mL). (b) Peptide stimulated T cells, containing at least 1% of specific T cells are incubated with Clinimers (ratio 1/1) and recovered on a magnet. After washes in PBS, rosetted T cells are seeded in 96-well plates (103 cells/well), containing irradiated allogeneic feeder cells, for amplification. After 14 days, the specificity of T cells is assessed by double labelling with an anti-CD8 mAb and each specific tetramer. (c) Influence of the number of washes on sorting yields. Sorting yields are calculated by dividing the number of rosetted T lymphocytes counted after the sort by the number of specific T cells in the sorted population estimated by tetramer staining. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones. (d) Influence of the number of washes on purity of selected and amplified T cells. The purity of amplified sorted-T cells is assessed after the 14-day amplification period on feeder cells, by double staining with an anti-CD8 mAb and the specific tetramer. Blue bars represent Melan-A-specific T cells and red bars MELOE-1-specific ones.