T cells from septic mice proliferate poorly to TCR stimulation and display decreased CD3ζ expression. Twenty-four hours after cecal ligation and puncture (CLP) or sham treatment, CD25-depleted CD4⁺ T-effector cells were harvested as described in “Proliferation assay.” T-effector cells (2.5 × 10⁴) were cultured for 72 hours with anti-CD3 (2.5 μg/mL) and anti-CD28 (1 μg/mL; A) or PMA and ionomycin (B). ³H-thymidine was added during the last 14 to 18 hours of culture and incorporation was measured using a liquid scintillation counter. (C) Mice were treated as in panel A, except prior to CLP, mice were given an intraperitoneal injection of anti-GITR or control antibody (300 μg). Mice were given 300 μg anti-GITR or control antibody at the time of CLP surgery. Twenty-four hours after CLP or sham surgery, cells were stained extracellularly for flow cytometry using anti-CD3ϵ Pacific Blue and anti-CD4 allophycocyanin. Cells were then fixed and permeabilized as described in “Flow cytometry” and stained for anti-CD3ζ FITC. (D,E) Histogram overlays showing CD3ζ from CD4⁺ T cells of 1 sham mouse (filled black) versus 1 control antibody–treated CLP mouse (solid black line, no fill) versus 1 anti-GITR–treated CLP mouse (dotted line, no fill) and an isotype control antibody (filled gray) from peripheral lymph nodes (D) or mesenteric lymph nodes (E). The calculated mean fluorescent intensity (MFI) from (F) spleen, (G) peripheral lymph nodes, and (H) mesenteric lymph nodes from n = 3 mice per group. Histogram overlays were made using FCS Express software version 3 (De Novo, Los Angeles, CA). P values are indicative of the difference between groups using Student t test (A) or post hoc analysis using the Fisher LSD method. All error bars indicate SD.