In vitro assessment of immune responses induced by the TCR-DNA vaccines. (A) Mice were immunized as in Figure 3 with the TCR-β plasmid plus the β-gal plasmid (33 or 166 ng β-gal/shot) or with pcDNA-gag, and tumor cell (MBL)–reactive T cells were detected ex vivo by IFN-γ ELIspot using unfractionated splenocytes. Data (mean ± SEM) are from 3 to 6 mice per group (quadruplicate wells). Target cells presenting β-gal (peptide-pulsed MC-38 or β-gal–transfected Z17 cells) were used to monitor the anti–β-gal response that was induced by the helper plasmid. (B) As in panel A, mice were coimmunized with β-gal, but a mixture of TCR-α and TCR-β plasmids was delivered using β-gal plasmid at a “high dose” (500 ng/shot) or a “low dose” (33 ng/shot). Splenocytes from mice immunized with pcDNA-gag served as a positive control (mean ± SEM of n = 3 mice/group). (C) β-gal–specific T-cell response in mice receiving TCR-β plasmid and titered amounts of β-gal helper plasmid as measured by IFN-γ ELIspot.