The effect of peptide alteration on the binding of P1A-specific T cells to the H-2L^d:peptide complex. The H-2L^d:Ig, loaded with given peptides, was preincubated with PE-conjugated anti-IgG1 mAb and used to stain transgenic spleen cells in conjunction with FITC-conjugated anti-CD8 mAb. (a) Characterization of transgenic mice expressing a TCR specific for the tumor antigen P1A. Spleen cells isolated from transgenic mice were stained either for coexpression of CD8 and the transgenic TCR (left panel) or for the specificity and sensitivity of dimer binding to the peptide dimer (center and right panels). (b) Dot plots depicting binding of the MHC:peptide complex to transgenic T cells. The peptide:H-2L^dIg complexes were used at 1:1 dilution, which consisted of 1 μg of H-2L^dIg and 0.75 μg of peptides in 50 μl PBS. (c) Quantitative comparison of the avidity of wild-type and mutant H-2L^d:P1A complex. Transgenic spleen cells were stained with given dilutions of H-2L^d loaded with excess wild-type P1A, mutant P1A, or control peptides. Data shown are mean fluorescence intensities of gated CD8 T cells. This experiment was repeated two to four times. Statistical significance was determined by paired t test.