Staining and sorting of CD4 T cells by DR*0401/BHRF1 multimers. (A) Clone 4.19 (right) was stained by the DR*0401/BHRF1 tetramer (bottom) but not by the irrelevant DR*0401/HA tetramer (top). Another clone from donor RA1, clone 4.4 (left), that did not recognize BHRF1 in the EBV vaccinia assay, was not stained by the DR*0401/BHRF1 tetramer. Cells were double-stained with 20 μg/mL of DR*0401 tetramers and with 10 μg/mL monoclonal antibody against CD4. (B) Yield of sorting of BHRF1-specific cells by using DR*0401/BHRF1 monomers coated on magnetic beads. Synovial CD4 T cells from patient RA1 and CD4 PBLs from healthy donor D3 were stained by DR*0401/BHRF1 tetramers before (left) and after (right) sorting with DR*0401/BHRF1 monomers coated on magnetic beads. Sorted cells were expanded for 3 weeks under polyclonal stimulation before being stained with DR*0401/BHRF1 tetramers. Percentage of cells staining with tetramers is indicated. Bar indicates the range of fluorescence where the cells were scored positive.