Fig. 6

Use of mAbs generated from human immunoglobulin transgenic rats immunized with HLA-A2/Pp65 in functional experiments. a T2 cells (HLA-A2⁺) were loaded for 4 h with different peptides at 50 μg/mL or not (unloaded T2 cells), then stained with purified anti-HLA-A or anti-HLA-A2/Pp65 mAbs (clones 1.2, 1.5, 2.7 and 5.6) at 1 μg/mL. A secondary Ab anti-human IgG-PE was added at 1 μg/mL. Cells were analyzed on a BD FACS Canto II cytometer. Specific anti-HLA-A2/Pp65 staining was observed with mAbs 2.7 and 1.5. b One A2/Pp65 specific T cell line, one A2/Pp65 specific T cell clone and one A2/MelA specific T cell line were stimulated with T2 cells loaded O/N respectively with Pp65 or MelA peptides in the presence of 10, 50 or 100 μg/mL of isotype control mAb, purified mAbs to be tested or positive control mAbs (anti-HLA-A2 (clone BB7.2), anti-pan ClassI; BD Biosciences). The percent inhibition of T-cell activation is indicated in the figure (read-out: IFNγ production). Three independent experiments were performed. c MRC-5 cells infected (MOI = 0,1) or not with CMV virus (Toledo strain) were stained 72 h post-infection with purified mAb 1.5 at 2 or 10 μg/mL or not (MRC-5 cells without Ab). A secondary Ab anti-human IgG-PE was added at 1 μg/mL (upper panel). Percentages of PE⁺ cells are indicated on dot plots in black, as well as the geometric mean of PE fluorescence in purple. Staining with an anti-HLA-A2 mAb (BD Biosciences) was also performed on non-infected (grey line) or infected MR-5 cells (black line); dotted lines represents unstained cells respectively (lower panel)