Recently activated T cells have a defect in store-operated calcium entry. (A and B) GFP⁺ OT-I CD8⁺ T cells were adoptively transferred, and recipients were stimulated by an i.v. injection of OVAp or left unstimulated. After 48 or 72 h, lymph node cells were harvested and stained with the calcium-sensitive dye Indo-1. Cells were stimulated with OVAp, Con A, or thapsigargin, and calcium responses were analyzed by flow cytometry. (A) Kinetics of calcium responses are shown for activated T cells (48 or 72 h) or naive T cells isolated ex vivo. Results are representative of three independent experiments. (B) Maximal value from Indo-1 ratio is shown for naive and activated T cells stimulated with the indicated concentration of OVAp, Con A, or thapsigargin. Results are pooled from three independent experiments. Data are shown as mean ± SEM. Significance testing were performed by using an unpaired t test. *, P < 0.05; **, P < 0.01. (C) GFP⁺ OT-I CD8⁺ T cells were labeled with SNARF and transferred into recipient mice. DCs pulsed with OVAp were injected in the footpad at the same time. At 48 h, cells from the draining lymph node were stained with Indo-1 and stimulated by Con A or thapsigargin. Kinetics of calcium responses analyzed by flow cytometry are shown for undivided (SNARF^high) and divided (SNARF^low) T cells recovered at 48 h. Results are representative of two independent experiments.