Assessment of immune competence of HemA, Foxp3-Tg, HemA/Foxp3-Tg, and wt mice. (A-C) Characterization of splenic CD4⁺ T cells isolated from the 4 strains of mice. (A) Foxp3 staining of CD4⁺ T cells. (B) Proliferation assay after in vitro stimulation of CD4⁺ T cells by anti-CD3 antibody. Splenic CD4⁺ T cells were stimulated with plate-bound anti-CD3 antibody. CD4⁺ cells were incubated for 3 days. Proliferation was estimated by [³H]thymidine incorporation. (C) Suppression of proliferative response to anti-CD3 stimulation by the addition of Tregs to the cultures. CD4⁺ responder T cells were isolated from spleens of wt mice, and stimulated with 5 μg/mL anti-CD3 antibody (i). CD4⁺CD25⁺ Tregs from wt mice, and CD4⁺ Tregs from Foxp3-Tg and HemA/Foxp3-Tg mice were added to proliferating responder cells at a ratio of 1:8 (ii-iv). Wild-type CD4⁺CD25⁺ T cells, Foxp3-Tg CD4⁺ T cells, and HemA/Foxp3-Tg CD4⁺ T cells were also stimulated with anti-CD3 antibody to check for Treg proliferation (v-vii). Each column represents the percentage of proliferation relative to that of control CD4⁺ responder cells. (D) Antibody response to immunization with bacteriophage Φx174. HemA and Hem/AFoxp3-Tg mice (n = 4/group) were challenged twice 4 weeks apart with the neoantigen bacteriophage Φx174 (2 × 10⁸ PFU/each challenge). Phage-neutralizing antibody activity was expressed as the rate of phage inactivation (Kv) using a standard formula. Mice not receiving bacteriophage did not produce neutralizing antibody (data not shown).