Comparative analysis of various Treg blockade strategies on the induction of specific anti-self CD8⁺ T cells. Neu^OT-I/OT-II TG mice were immunized intraperitoneally twice (day 0 and day 14) with STxB-OVA/αGalCer alone or combined with anti-CD25 (500 μg at day −3) or cyclophosphamide (4 mg at day −3) or the antagonist of CCR4 (1.5 μg) mixed with the vaccine at day 0. One week later, splenocytes were harvested, purified with anti-CD8–coated magnetic beads, and stained with K^b-OVA_257-264 tetramer or irrelevant K^b-VSV tetramer (A) or cocultured for 18 hours with CD8⁻ cells as APCs sensitized or not with the OVA_257-264 and IFNγ shown by ELISPOT (B). Percentages shown in panel A correspond to specific values after subtracting the percentage observed with irrelevant tetramer (always < 0.1%). Each symbol corresponds to one mouse. The number of spots (B) was calculated after subtracting background (APCs incubated with medium alone). Values of P were calculated by the Mann-Whitney U test.