Efficient and preferential pairing of cysteine-modified TCRs

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Efficient and preferential pairing of cysteine-modified TCRs Efficient and preferential pairing of cysteine-modified TCRs. A, TCR titration. Efficient and preferential pairing of cysteine-modified TCRs. A, TCR titration. OKT3-stimulated PBLs, electroporated with different amount of mRNA (0.2, 0.6, 2, and 6 μg total) encoding either the wild-type receptor F5 or its cysteine-modified version F5-Cys, were cocultured with the melanoma tumor 526. Twenty-four hours after the beginning of the coculture, the concentration of IFN-γ secreted in the medium was measured using an ELISA procedure. B, TCR competition assay. TCR-deficient Jurkat/RT3-T3.5 cells were electroporated with 1 μg of each chain of F4 (white) or F4-Cys (black) along with 1 μg of each chain of the competitor TCR: 1G4, TE-8 (an NY-ESO-1 class I–restricted TCR), and p53-H [for p53-H, we also used an additional amount (i.e., 0.5 μg) indicated as p53-H (1:0.5)]. Twenty-four hours after electroporation, the cells were stained with MART-1 tetramer, and the percentage of MART-1 TCR relative expression was calculated by dividing percentage of tetramer-positive cells of a given sample by that of the control sample (without competitor TCR). All the differences were found to be statistically significant based on Student's t test (P < 0.001). Cyrille J. Cohen et al. Cancer Res 2007;67:3898-3903 ©2007 by American Association for Cancer Research