Significant reduction of total spleen and CD4⁺CD25⁻ T cells after sham or CLP surgery. Severe sepsis was induced in C57BL/6 mice by CLP. (A) Total spleen cell number from CLP and sham mice is shown. (B-D) With the use of flow cytometry, the lymphocytes were gated by forward (FSC) and side (SSC) scatter properties, and, then, the expression of CD4 and CD25 molecules were analyzed. CD4⁺ T cells are shown according to their expression level of CD25 at days 1, 3, and 15 after surgery. (E) CD4⁺CD25⁻ T cells that underwent FACS from sham and CLP at day 3 after surgery were stimulated with polyclonal anti-CD3/CD28 for 72 hours, and the levels of IL-10, IFN-γ, IL-13, and IL-4 were measured in the supernatants of these cultures by Bioplex. *P ≤ .05; **P ≤ .01; ***P ≤ .001, compared with sham surgery. (A-D) Data represent mean ± SEM of 3 independent experiments (n = 3-5 mice in both groups), and (E) data show pooled results from 2 independent experiments.