Reduced proliferative response of CD4⁺ cells to hOKT3γ1(Ala-Ala) stimulation occurs only in the presence of CD8⁺ cells but is not due to lack of IL-2. CFSE-labeled cells were cultured in the presence of hOKT3γ1(Ala-Ala) for 6 days. Cells were stained with PE-conjugated anti-CD4 and anti-CD25 mAbs and analyzed on a FACSCalibur flow cytometer. (A and C) Bulk PBMCs. (B and D) PBMCs depleted of CD8⁺ T cells. Representative results of 6 independent experiments are shown. The numbers over the dots in A and B represent the number of cell divisions. The percentages in C and D represent the percentage of CD4⁺ cells that were CD25⁺. PBMCs were cultured with the anti-CD3 mAb in the absence (E and F) or presence (G and H) of recombinant IL-2 (50 U/ml). The addition of IL-2 to the cultures enhanced proliferation of CD8⁺ T cells (E and G) but did not have an effect on CD4⁺ T cell proliferation in the PBMCs (F and H). Representative results of 4 independent experiments are shown.