Challenge of tolerized mice with unrelated T-dependent and T-independent antigens. Tolerized mice (n = 2) or naive mice (n = 2) were challenged with the T-dependent antigen, bacteriophage Φx174 twice 4 weeks apart starting at week 24 after plasmid injection; antibody titers, expressed as Kv, were monitored as described in “Immunization of mice with bacteriophage Φx174.” (A) Kv value over time after primary and secondary bacteriophage Φx174 immunization. The proportion of phage-neutralizing antibody of the IgG isotype was measured 2 weeks after secondary immunization by treating with 2-mercaptoethanol and is provided in panel A. We used TNP-Ficoll (20 μg) as T-independent antigen to immunize tolerized (n = 4) or FVIII plasmid only–treated hemophilia A mice (n = 3) at week 24 after plasmid injection. Serum was collected at day 10 after challenge and assessed for (B) anti-TNP IgM, and (C) anti-TNP IgG3. No significant differences were found between the groups for Kv, anti-TNP IgM, and anti-TNP IgG3 levels. Data are mean ± SD.