Figure 2.

gp100 APLs with single amino acid substitutions function as full agonists except for APL A3, which functions as a null ligand. Human T cells transduced with gp100/HLA-A2 TCR genes were tested in (A) ⁵¹Cr-release, (B) TNFα production, and (C) NFAT reporter gene assays. Target cells used were T2 cells that were pre-incubated with 1 μM of gp100 wt peptide (wt), gp100 APLs A1–A8, and G9, or BMLF-1 wt peptide (irr. pept.). gp100 APLs are encoded as indicated in the Section “Materials and Methods.” In the ⁵¹Cr-release, TNFα production, and NFAT reporter gene assays, the effector to target cell ratios were 15:1, 3:1, and 2:1, respectively. T cell responses in all three assays are expressed relative to T2 target cells pulsed with gp100 wt peptide (specific lysis: 80%; production of TNFα: 1015 pg/ml; and activation of NFAT: 34.73 RLU, all set to 100%). Mock-transduced human T cells did not show activity in response to gp100 APLs (data not shown). Results of one (out of three) representative experiment are shown based on the mean value of triplicate data points. Note the null responses of the gp100 APL A3.