A safeguard eliminates T cell receptor gene-modified autoreactive T cells after adoptive transfer

Kieback et al. 10.1073/pnas.0710198105.

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Fig. 7. P14/TCRmyc is expressed and functions comparably to P14/TCRwt. (A) The TCR-deficient mouse T cell line 58 was transduced with the P14/TCRwt or P14/TCRmyc, and cells were enriched with vb-chain-specific antibodies. Vb8- and va2-specific antibodies were used to analyze the TCR expression level by flow cytometry. Untransduced cells (neg) served as a negative control. (B) P14/TCRmyc-transduced 58 cells were stained with a myc-specific antibody and analyzed by flow cytometry. 58 cells transduced with the wild-type receptor served as a negative control. (C) B6 splenocytes transduced with either P14/TCRwt or P14/TCRmyc were stained with a CD8-specific antibody, a gp33-specific tetramer, and an irrelevant tetramer (irr) 72 h after transduction. Flow-cytometry staining shows cells gated on CD8 expression. Numbers indicate MFI of the tetramer staining. (D) One hundred thousand P14/TCRwt- and P14/TCRmyc-transduced 58 (CD8a+) cells or untransduced cells (neg) were cocultured with 1 ´ 10⁵ B6 splenocytes loaded with gp33-peptide for 24 h. Cytokine production was analyzed by determining the IL-2 concentration of the culture supernatant. Unloaded splenocytes (w/o) or splenocytes loaded with irrelevant peptide (irr) served as negative controls. The data represent mean values of duplicates, and error bars indicate SD. The experiment was performed twice with reproducible results. (E) 58 cells transduced and enriched for P14/TCRmyc or P14/TCRwt expression were incubated with myc-specific antibody rabbit complement factors. 7-AAD was used to discriminate between living and dead cells. Numbers indicate specific lysis. Results show data from one of at least two independent experiments with comparable results.

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