![Fig. 2. Higher [3H]thymidine uptake, proliferation, activation, and AICD sensitivity of GITR−/− cells upon anti-CD3 triggering. / (A) [3H]thymidine uptake of splenocytes and total lymphocytes from lymph nodes (5 × 105 cells/mL) activated by plate-bound anti-CD3 mAb (clone 145-2C-11, Pharmingen) is shown. GITR−/− cells (filled columns) have a [3H]thymidine uptake significantly higher thanGITR+/+ cells (empty columns) (**P < .01 and ***P < .001). Each value is the mean ± SD of at least 3 independent experiments. A total of 2.5 μCi [3H]thymidine (Amersham International, Amersham, United Kingdom) per well was added to the cultures. (B) One of 5 independent experiments of [3H]thymidine uptake of purified T lymphocytes activated by soluble anti-CD3 mAb (0.2 μg/mL) on a feeder of irradiated (20 Gy, 5 minutes) splenocytes is shown. GITR−/− cells (filled circles) have a [3H]thymidine uptake significantly higher than GITR+/+ cells (empty triangles) (***P < .001 and **P < .01), in the presence (dashed lines) and in the absence (solid lines) of murine IL-2 (50 U/mL). UntreatedGITR−/− and GITR+/+cells incorporate less than 1000 cpm and are not shown. Each value is the mean ± SD of 4 counts. (C) Dose dependence of anti-CD3 mAb activation of purified T lymphocytes cultured on a feeder of irradiated splenocytes for 36 hours. One of 3 independent experiments is shown. Each value is the mean ± SD of 4 counts. The difference of incorporation between GITR−/− (filled circles) and GITR+/+ (empty triangles) cells was significant (***P < .001 and **P < .01). (D) T lymphocytes cultured on a feeder of irradiated splenocytes activated with soluble anti-CD3 (0.01 μg/mL) or with soluble anti-CD3 plus anti-CD28 (Pharmingen) (0.01 μg/mL) for 24 hours. One of 3 independent experiments is shown. Each value is the mean ± SD of 4 counts. GITR−/−cells (filled columns) have a [3H]thymidine uptake significantly higher than GITR+/+ cells (empty columns) (*P < .05). (E) GITR−/−cells enter into the cell cycle as a significantly higher number (**P < .01 and *P < .05) upon activation. Purified T lymphocytes from lymph nodes were activated by plate-bound anti-CD3 mAb. The percentage of T cells in S/G2/M phases is shown. Activated splenocytes showed similar results (not shown). Empty and filled columns represent GITR+/+ andGITR−/− lymphocytes, respectively. The values represent the mean ± SD of 3 independent experiments. (F) IL-2R expression, revealed by anti-CD25 phycoerythrin-conjugated mAb (Pharmingen), is significantly higher inGITR−/−–activated T lymphocytes (40% inGITR−/− vs 30% inGITR+/+ cells). T lymphocytes were activated by plate-bound anti-CD3 mAb for 24 hours. The thin line curve represents control sample. Basal values in unstimulated T lymphocytes (5.0% ± 1.3% in wild-type vs 3.1% ± 1.1% in GITR−/−) were similar (P > .05) (not shown). One of 3 independent experiments with similar results is shown. (G) IL-2 produced by GITR−/− T cells (filled columns) activated by platebound anti-CD3 mAb (10 μg/mL) 48 hours after activation is significantly higher (**P < .01) compared with GITR+/+–activated T cells (empty columns). Values represent the mean ± SD of 3 ELISA assays performed for each experimental condition. (H) Higher AICD in GITR−/− T lymphocytes (*P < .05). Cells were stimulated for 2 days with ConA and then plated at a density of 1 × 106/mL on wells that had been coated with anti-CD3 mAb (10 μg/mL), either in the absence or presence of murine IL-2 (50 U/mL). After 24 hours cells were harvested and stained with propidium iodide for evaluation of apoptosis. Empty and filled columns representGITR+/+ and GITR−/−splenocytes, respectively. Values represent the mean ± SD of 3 independent experiments.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/100/1/10.1182_blood-2001-12-0276/7/h81322757002.jpeg?Expires=1736055512&Signature=z98mmBQAQaYkA9-zHKzAOk3gvRKMOL4~YVnoI15RDYclTlHz-6Uo-TdbSGMioNB~R-dljcWOk~Si12Z92n2qo1RM0NbdBgckz83WPIkAlHGrf~6IxpWwlcVYQqStX-vf8epYpPpLa9dzBdlH6QZdwQI5RVphrb-l3yASHZkzJPR-f87qD11fTX55jgXiUFJYU-tBp60ANMdn-D8yRi5IzNrGmxfMjvDV3IOVOjm3rn6oLuWFvQeOKeikJJ-l6rcyYv4dFTHKgmhpeH3jYc2tRoFaeFHdyVuMYTQimlNhv4C1HDGQQhT8IG3TQX-LJGWIjyznxHO7EixDf8~HoIhAKg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Higher [3H]thymidine uptake, proliferation, activation, and AICD sensitivity of GITR−/− cells upon anti-CD3 triggering.
(A) [3H]thymidine uptake of splenocytes and total lymphocytes from lymph nodes (5 × 105 cells/mL) activated by plate-bound anti-CD3 mAb (clone 145-2C-11, Pharmingen) is shown. GITR−/− cells (filled columns) have a [3H]thymidine uptake significantly higher thanGITR+/+ cells (empty columns) (**P < .01 and ***P < .001). Each value is the mean ± SD of at least 3 independent experiments. A total of 2.5 μCi [3H]thymidine (Amersham International, Amersham, United Kingdom) per well was added to the cultures. (B) One of 5 independent experiments of [3H]thymidine uptake of purified T lymphocytes activated by soluble anti-CD3 mAb (0.2 μg/mL) on a feeder of irradiated (20 Gy, 5 minutes) splenocytes is shown. GITR−/− cells (filled circles) have a [3H]thymidine uptake significantly higher than GITR+/+ cells (empty triangles) (***P < .001 and **P < .01), in the presence (dashed lines) and in the absence (solid lines) of murine IL-2 (50 U/mL). UntreatedGITR−/− and GITR+/+cells incorporate less than 1000 cpm and are not shown. Each value is the mean ± SD of 4 counts. (C) Dose dependence of anti-CD3 mAb activation of purified T lymphocytes cultured on a feeder of irradiated splenocytes for 36 hours. One of 3 independent experiments is shown. Each value is the mean ± SD of 4 counts. The difference of incorporation between GITR−/− (filled circles) and GITR+/+ (empty triangles) cells was significant (***P < .001 and **P < .01). (D) T lymphocytes cultured on a feeder of irradiated splenocytes activated with soluble anti-CD3 (0.01 μg/mL) or with soluble anti-CD3 plus anti-CD28 (Pharmingen) (0.01 μg/mL) for 24 hours. One of 3 independent experiments is shown. Each value is the mean ± SD of 4 counts. GITR−/−cells (filled columns) have a [3H]thymidine uptake significantly higher than GITR+/+ cells (empty columns) (*P < .05). (E) GITR−/−cells enter into the cell cycle as a significantly higher number (**P < .01 and *P < .05) upon activation. Purified T lymphocytes from lymph nodes were activated by plate-bound anti-CD3 mAb. The percentage of T cells in S/G2/M phases is shown. Activated splenocytes showed similar results (not shown). Empty and filled columns represent GITR+/+ andGITR−/− lymphocytes, respectively. The values represent the mean ± SD of 3 independent experiments. (F) IL-2R expression, revealed by anti-CD25 phycoerythrin-conjugated mAb (Pharmingen), is significantly higher inGITR−/−–activated T lymphocytes (40% inGITR−/− vs 30% inGITR+/+ cells). T lymphocytes were activated by plate-bound anti-CD3 mAb for 24 hours. The thin line curve represents control sample. Basal values in unstimulated T lymphocytes (5.0% ± 1.3% in wild-type vs 3.1% ± 1.1% in GITR−/−) were similar (P > .05) (not shown). One of 3 independent experiments with similar results is shown. (G) IL-2 produced by GITR−/− T cells (filled columns) activated by platebound anti-CD3 mAb (10 μg/mL) 48 hours after activation is significantly higher (**P < .01) compared with GITR+/+–activated T cells (empty columns). Values represent the mean ± SD of 3 ELISA assays performed for each experimental condition. (H) Higher AICD in GITR−/− T lymphocytes (*P < .05). Cells were stimulated for 2 days with ConA and then plated at a density of 1 × 106/mL on wells that had been coated with anti-CD3 mAb (10 μg/mL), either in the absence or presence of murine IL-2 (50 U/mL). After 24 hours cells were harvested and stained with propidium iodide for evaluation of apoptosis. Empty and filled columns representGITR+/+ and GITR−/−splenocytes, respectively. Values represent the mean ± SD of 3 independent experiments.
