Apoptosis of OVA-reactive CD4⁺ T cells. (A) Relative increase of annexin V⁺ PI⁻ apoptotic CD4⁺ T cells in popliteal LNs of BALB/c (○) or plt/plt (•) mice was kinetically analyzed after immunization. At indicated days after immunization, popliteal LN cells were harvested and stained with anti-CD4 mAb, annexin V, and PI, then analyzed by flow cytometry. Percent increase of apoptotic CD4⁺ T cells was calculated according to the formula: % increase = (% annexin V⁺PI⁻ apoptotic cells at indicated day in CD4⁺ T cells/% annexin V⁺PI⁻ apoptotic cells at day 0 in CD4⁺ T cells) × 100. Mean ± SD from 3 to 5 mice is given. A representative result from 2 independent experiments is shown. (B) Relative increase of annexin V⁺PI⁻ apoptotic CD4⁺ T cells in popliteal LNs of C57BL/6 (○) or CCR7-deficient (•) mice was kinetically analyzed after immunization. Percent increase was calculated as indicated in panel A. Draining popliteal LN cells were pooled from 2 mice and assessed. (C) Annexin V⁺ apoptotic cells in Vβ14⁺CD4⁺ or Vβ6⁺CD4⁺ T cells in BALB/c and plt/plt mouse LN were analyzed. We repeated these experiments at least 2 times and representative results at days 0 and 18 after immunization with OVA in CFA are shown.