Enumeration of cells secreting Ag-specific IFN-γ of T cells from MBPGP68–85- and MBPRAT68–85-primed LEW rats that had received different DNA constructs 4 weeks before immunization i.m. as indicated in Materials and Methods. Responses are shown after DNA vaccination with pZZ (A and B), after vaccination with pZZ/MBPGP68–85 (C and D), after vaccination with pZZ/MBPRAT68–85 (E and F), and after vaccination with pZZ/MBPRAT21.5 (G and H). Each value represents data from four LEW rats. Spleens were obtained on day 12 postimmunization, and MNC were cultured for 48 h in triplicate in the presence of either MBPGP68–85, MBPGP63–88, MBPRAT68–85, MBPRAT63–88, MBP89–101, or MOG37–54 at a concentration 10 μg/ml peptide, medium alone, or Con A at a concentration of 3 μg/ml. Asterisks indicate significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001).