![Fig. 3. T-HA cells rescued from anti-CD3-induced death have become anergic. / IL-15-conditioned T-HA cells were stimulated for 48 hours with immobilized anti-CD3 mAb or antigen/APC (first stimulus), recovered, and further cultured for 12 days with IL-2 (10 ng/mL) or IL-15 (1 ng/mL) (intermittent culture). On day 14 after the initial stimulation, viable cells were harvested, labeled with the green fluorescent membrane marker PKH2-GL and 1 × 104 stained cells were stimulated with medium (open bars), APC (hatched bars), or antigen/APC (solid bars). (A) Proliferation was measured by addition of [3H]thymidine for the last 12 hours of the 72-hour assay period. Results are expressed as mean cpm from triplicate cultures ± SD. (A, inset) Supernatants were taken from the cultures and IL-2 content was determined. (B) The percentage of apoptotic cells of the stained cell population was determined at the indicated time points by flow cytometry and PI uptake. The background percentage of dead cells present in the population at the time the stimulation was started is represented by the gray bars. Imm indicates immobilized.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/96/3/10.1182_blood.v96.3.1006/5/bloo01527003x.jpeg?Expires=1735872140&Signature=CJ8oToFjd869kSIcaLFnW56sT-jszTjQ6nxe4llSekFL8wTrpmkn8deUYzFWsv6IfEbLJFcfj4IFGsmgdxMInhl7kJivxxUiF1sSOXWs04SSqub~7zAKWDfgdSUtpA21RKrroQ-SXt0Ivcm-vq-ZyzsYnvMz~rJK2fSlXZG9LnHBERTBXScKWNaxjhbBXFQRus-rw91Q3W1oaAoC~F~ghFgu9pPHhDJqPXnxILUOBPchtCl~uCDhCc~THzLaI0z7bP-ahGMPSCXZAXO2CcjUYLorvrHPuu1B3e9Ptuva~GGpWBi6k5JCVRg-AzPKM0bdHA47xPY1M4WwKvSwhXOrpw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
T-HA cells rescued from anti-CD3-induced death have become anergic.
IL-15-conditioned T-HA cells were stimulated for 48 hours with immobilized anti-CD3 mAb or antigen/APC (first stimulus), recovered, and further cultured for 12 days with IL-2 (10 ng/mL) or IL-15 (1 ng/mL) (intermittent culture). On day 14 after the initial stimulation, viable cells were harvested, labeled with the green fluorescent membrane marker PKH2-GL and 1 × 104 stained cells were stimulated with medium (open bars), APC (hatched bars), or antigen/APC (solid bars). (A) Proliferation was measured by addition of [3H]thymidine for the last 12 hours of the 72-hour assay period. Results are expressed as mean cpm from triplicate cultures ± SD. (A, inset) Supernatants were taken from the cultures and IL-2 content was determined. (B) The percentage of apoptotic cells of the stained cell population was determined at the indicated time points by flow cytometry and PI uptake. The background percentage of dead cells present in the population at the time the stimulation was started is represented by the gray bars. Imm indicates immobilized.
