Deconvolution strategy to define Be-dependent peptides that stimulate T cell hybridomas AV22 and AV9. (A) AV22 cells were stimulated with selected decapeptide mixtures with three positions fixed in the presence of 75 µM BeSO4. Two concentrations of mixtures were used, and IL-2 response (mean ± SEM pg/ml) was measured by ELISA. Mixtures with two fixed positions (D4L5 and D5E8) were included as positive controls. Data are representative of three separate experiments performed in triplicate. (B) IL-2 response of three separate experiments (mean ± SEM) of AV22 and AV9 to a biased decapeptide positional scanning library (W2D4L5) is shown. Peptide mixtures were fixed at W2, D4, and L5, and one additional peptide position (p1, p3, p6, p7, p8, p9, and p10) was scanned with each of 20 amino acids (140 mixtures). Each panel shows the response of hybridomas to 20 µg/ml mixtures and 75 µM BeSO4 compared with the W2D4L5 mixture with no additional fixed positions (Ct). For A and B, the x axis denotes the amino acid (single letter code) fixed at each defined position, and mixtures did not stimulate hybridomas in the absence of BeSO4. (C) Selection of amino acids for each peptide position based on the most active mixtures in the presence of BeSO4.
