CD137 agonistic mAb reverses established anergy of alloreactive 2C T cells in bone marrow chimera. BDF1 mice were exposed to a lethal dose of irradiation (11 Gy) followed by an intravenous transfer of 5 × 10⁶ bone marrow cells and 1 × 10⁷ spleen cells from 2C TCR transgenic mice. (A) Ten days after BMT, spleen cells were prepared from the recipient mice and subjected to analysis by a flow cytometry. The percentage of 1B2-positive CD8⁺ 2C T cells and 1B2-negative CD8⁺ T cells is shown in the corresponding quadrants. (B) Ten days after BMT, 2C T cells were purified from the recipient spleen cells by an isolation of CD8⁺ T cells. The cells (1 × 10⁵ cells per well) were subsequently cultured with irradiated (30 Gy) DBA/2 spleen cells (5 × 10⁵ cells per well) or alone (left panel), and in the presence or absence of immobilized 1 μg/mL anti-CD3 mAb (right panel). CD8⁺ T cells isolated from naive 2C mice were cultured similarly as control. Proliferative activity was determined by ³H-thymidine incorporation on day 3. The data are shown as an average ± SD of triplicate wells. (C) Ten days after BMT, the recipient mice were further transferred intravenously with CFSE-labeled BDF1 spleen cells (2.5 × 10⁷ cells) or were untreated. In the groups with cell transfer, 100 μg of either control rat IgG or CD137 mAb (2A) was injected intraperitoneally on the same day. After 5 days, spleen cells from the recipient mice were analyzed by a flow cytometry. The percentage of 1B2-positive 2C T cells and the transferred BDF1 cells (CFSE^high cells) are indicated.