Figure 6.

Binding and antigenicity of cysteine-modified NP_218–226. (A) NP_218–226 peptide freshly dissolved in PBS was diluted as indicated in 200 μl of cystine-containing DMEM and incubated for 2 h to produce CysNP_218–226 or incubated in cystine-free DMEM with TCEP as a noncysteinylated control. Alternatively, an old stock consisting primarily of dimeric NP_218–226 was treated in cystine-free DMEM in the same manner in the presence or absence of TCEP. Diluted peptides were then incubated for 1 h at 26°C with T2-K^d cells previously cultured for 14 h at 26°C and shifted to 37°C for 2 h to melt K^d molecules. The cells were then analyzed by flow cytometry after staining with fluorescein-conjugated SF1.1.1 mAb. (B) T_CD8+ specific for unmodified NP_218–226 were tested in a microcytotoxicity assay for their ability to lyse ⁵¹Cr-labeled P815 target cells incubated in the presence of peptides treated as above.