Figure 4.

Ex vivo MHC class II tetramer staining of HCV-specific CD4 T cells. (a) PBMC from subject 01-40 with spontaneously resolved HCV viremia were stained with three control tetramers and three HCV tetramers. Cells were positively selected with anti-PE microbeads and analyzed by flow cytometry; the number of CD4⁺/tetramer⁺ cells is indicated in the upper right quadrant. An example of FACS analysis before enrichment for tetramer⁺ cells is shown for control tetramer DR4-CLIP and HCV tetramer DR4–HCV 1248. The frequencies of tetramer-positive cells in the CD4 T cell pool were determined by splitting the sample 9:1 following labeling with anti-PE beads; 90% of the cells were magnetically enriched, while 10% of the cells were analyzed without enrichment to determine the number of CD4 T cells. The frequencies of the tetramer⁺ cells of total CD4 cells are as follows: DR4–HCV 1248: 8.2 per 100,000 (0.008%); DR4–HCV 1579: 1.5 per 100,000 (0.0015%); DR4–HCV 1770: 2.4 per 100,000 (0.0024%). Representative data are shown from two independent experiments with fresh PBMC from subject 01-40. (b) PBMC from a DRB1*0401 HCV-seronegative control were stained with the following MHC class II tetramers: 3 control tetramers (DR4-CLIP, DR4-gp100, and DR4–annexin II), a pool of three HCV tetramers, and an HA (residues 306–318) tetramer. The number of CD4⁺/tetramer⁺ cells following enrichment with anti-PE microbeads is indicated in the upper right quadrant. The frequency of DR4–influenza HA–specific T cells in this subject was 0.9 per 100,000 (0.0009%).