Addition of IL-2 reverses the effects of IL-10 on the SEB reduction of p27^Kip1 and SEB-induced upregulation of cyclin D2 in CD4⁺ T cells. (A) PBMCs were incubated in 6-well plates at 6 × 10⁵ cells/mL along with IL-10 at 10 U/mL or media only. After 2 days of incubation, media alone, SEB at 100 pg/mL, or SEB and IL-2 (100 U/mL) were added. The cells were further incubated until the indicated harvest day. Cells were harvested and CD4⁺ T cells were separated and collected by negative selection affinity chromatography. Lysates from these cells were subjected to SDS-PAGE and Western analysis using antibodies specific for p27^Kip1 and cyclin D2. The lower band in the cyclin D2 doublet represents the faster-migrating activated form of cyclin D2. (B) Quantitation was performed by densitometric analysis of bands from day 3.5. The differences between SEB treatment and treatment with IL-10 and SEB were found to be statistically significant for both immunoblots (P < .001) by the Student’s t-test. Differences were also statistically significant when comparing IL-10 and SEB treatments with those in which IL-2 was also added: P < .001 for the p27^Kip1 blot and P < .002 for the activated form in the cyclin D2.