Figure 6.

NY-ESO-1-reactive CD4 and CD8 T cell frequencies. Frequency analysis of peptide-specific CD4 T cells (A) and tetramer staining of CD8 T cells (B) are shown. (A) A limited number (2 x 10⁴) of CD4 T cells were seeded in duplicate in 96-well plates and cultured with irradiated (40 Gy) autologous CD4- and CD8-depleted PBMCs (2 x 10⁴) as APCs in the presence of peptides 16 (aa 91-108) (1 µg/ml) (top) and 21 (aa 121-138) (1 µg/ml) (bottom) for 14 days. On the twenty-sixth day after stimulating twice, IFNγ production by the cells in each well was determined against each peptide (1 µg/ml) using autologous EBV-B cells (1 x 10⁴) as APCs by ELISA after incubation for 18 h. An O.D. value exceeding 0.3 after subtraction of the background (without peptide) was taken as positive. The number of positive wells was 6 in both cultures. The peptide-specific CD4 T cell frequency was calculated to be 3.2 x 10^-6 for both peptides. (B) Four tetramers were prepared. The patient HLA class I genotype was HLA-A*0206, A*2402, B27, B54, and Cw1. The sequence SLLMWTQC (aa 157-165) used to prepare the A*0206-tetramers lies in peptide 27 (aa 153-170) recognized by the patient's CD8 T cells, as shown in Figure 4.