Peptide-loaded HLADR1101-Bir tetramers stain polyclonal T cells specific for p2 and HA. In vitro expanded T cells were stained with HLA-DR*1101-Bir tetramers loaded with either p2 or HA peptides. (a) Staining of PBMCs from a HLA-DR*1101 healthy donor after one round of in vitro stimulation with 5 μM of p2-peptide and 40 u/ml of IL-2. (b) Intracellular production of IFN-γ by T cells contained in the p2-enriched PBMC. T cells were stimulated for 6 hours with p2 in the presence of HLA-DR*1101+ LCL cells at 37°C. Brefeldin A was added after the first hour of stimulation. The cells were then fixed, permeabilised and stained with anti-IFN-γ mAb. Cells unstimulated (nil) and stimulated policlonally with PMA-Ionomycin (PMA/ionomycin) are shown as controls. Numbers in quadrants indicate the percentage of T cells stained with anti-IFN-γ mAb (c) Staining of PBMCs from HLA-DR*1101 healthy donor with HA-loaded HLA-DR*1101-Bir tetramers. T cells were after three rounds of in vitro stimulation with 1 μg/ml of HA peptide and 40 u/ml of IL-2. The staining is performed at the indicated days after the third stimulation. Numbers in quadrants indicate the percentage of T cells stained by HLA-DR tetramers. (d) Intracellular production of IFN-γ by CD4+ T cells contained in the HA-specific T cell line at day 8 from the third in vitro stimulation. Activation and staining of CD4+ T cells was performed as described in (b).