Functional analysis and specificity of the recombinant DR1-A12. Tetrameric DR1-A12 was tested for its ability to bind specifically to DR1-restricted, CII-specific T cells. A panel of T-cell hybridomas was cultured with the tetramers in saturating concentrations at 37°C for 3 hours. An anti-CD4 antibody was added in the last 30 minutes before the cells were washed and analyzed on the flow cytometer. The left panel demonstrates an unstained control. The middle panel shows hybridoma DR1.E174, which is representative of tests performed on nine individual CII-specific DR1-restricted hybridomas. The right panel (DR1.HA.8) is representative of four control T-cell hybridomas specific for an HA peptide in the context of DR1. Previous testing has shown that none of these hybridomas is bound by the irrelevant tetramer DR1-HA, which contains the immunodominant determinant of hemagglutinin ). These data confirm that that our A12/DR1-tetramer recognizes only the collagen-specific, DR1-restricted T cells in our panel.