HLA-DR*1101-Bir, but not HLA-DR*1101-Ig, stain specific CD4+ T cells, and display a temperature-dependence. CD4+ TCC specific for p2, endowed with different affinity of recognition, and an irrelevant CD4+ TCC were stained with p2-loaded HLA-DR*1101-Bir tetramers at different temperature. (a) The relative affinity for the p2-HLA-DR*1101 displayed by the two TCC clones 162 and 51 is determined in an IFN-γ-releasing assay following production in response to different doses of p2 peptide. 104 T cells are incubated with 5 × 104 HLA-DR*1101 LCL cells and the indicated doses of p2 peptide. After 48 hours, the concentration of IFN-γ in the culture supernatant is measured by ELISA. Indicated are the concentration of p2 peptide required to elicit half-maximum release of IFN-γ in the two TCC. (b) Staining of TCC162, TCC51 and the irrelevant TCC with either p2-loaded HLA-DR*1101-Ig or p2-loaded HLA-DR*1101-Bir tetramers. Staining is performed for 2 hours at the indicated temperatures with 10 μg of tetramer per sample. The tetramer staining on CD3+CD4+ gated cells is shown. The amount of surface TCR, obtained by staining with anti-CD3 mAb, expressed by the TCC in the different conditions is shown by the mean fluorescent intensity (CD3 mfi).