Antibody depletion and adoptive transfer studies to study the role of CD4⁺CD25⁺ T cells in induction of AIHA. (A) A representative flow cytometric analysis of peripheral blood leukocytes obtained from mice before (left) and 4 days after (right) intraperitoneal injection of 500 μg monoclonal anti-CD25 (clone 7D4). Cells were stained with PE-conjugated monoclonal anti-CD4 (GK1.5) and PE-Cy7–conjugated anti-CD25 (clone PC61.5). (B) Levels of IgG-specific autoantibodies on RBCs from mice expressed as percentage of background unstained cells. The difference between rat RBC/anti-CD25 and rat RBC-alone groups was calculated as P < .001, and between rat RBC/anti-CD25 and rat RBC/isotype control groups as P < .001. As expected, the rat RBC/isotype-control group was different from the no-injection control group (P < .001), but no differences were found between the rat RBC/isotype-control and rat RBC-alone groups (P = .9). In addition, there were no differences between no-injection and anti-CD25 alone–injected groups (P = .5). (C) The circulating reticulocyte counts of the mice are expressed as a percentage of total blood. The difference between rat RBC/anti-CD25 and rat RBC-alone groups was P = 4 × 10^-9. No differences were found between the no-injection and anti-CD25 alone groups (P = .2). (D) The relative levels of anti–ds DNA in plasma of mice were measured by ELISA using a commercially available kit and are presented in optical density (OD) units on the y-axis. The difference between rat RBC/anti-CD25 and rat RBC-alone groups was calualted as P = .001. (E) The presence of IgG-specific xenoantibodies to rat RBCs in plasma from mice at 3 weeks after immunization was measured using diluted plasma (1 in 1000) followed by analysis using flow cytometry and is presented as relative fluorescent units on the y-axis. The difference between rat RBC/anti-CD25 and rat RBC-alone groups was calculated as P = .04. Error bars indicate SEM. (F) About 2 × 10⁵ CD4⁺CD25^hi or CD4⁺CD25^- sorted cells were adoptively transferred into naive C57Bl/6 female mice followed by weekly immunization with rat RBCs for 9 weeks. Levels of IgG autoantibodies on mouse RBCs as well as (G) xenoantibodies (IgG) to rat RBCs are shown for individual mice. The difference in autoantibody levels between mice adoptively transferred with CD4⁺CD25^hi and those that received CD4⁺CD25^- was calculated as P = .04. However, there were no differences in the xenoantibody levels between these 2 groups (P = .4). In panels B-D and F-G, the horizontal lines indicate the mean values.