CD137 stimulation reverses OT-1 T-cell anergy in vivo. (A) Anergy was established in OT-1 recipients upon the intravenous administration of OVA peptide as described before. Ten days later, mice were rechallenged with either a control peptide or OVA peptide. The rechallenged mice that were given OVA received either a rat IgG control or anti-CD137 (2A). Mice were killed at the time points indicated, and the total number of OT-1 cells present in each mouse was determined by tetramer analysis, as before. (B) Following the establishment of anergy, mice were rechallenged with various combinations of either the control or OVA peptides and the control rat IgG or anti-CD137 (2A), as indicated. The total number of OT-1 cells present in each mouse 3 days later is shown. Data shown in panels A and B are the mean (± SD) of 3 mice in each group. (C) Two days following OVA challenge and antibody administration (rat IgG or 2A, as indicated), the anergic mice were killed. Cells that were positive for both tetramer and CD8 were stained with either an isotype control (gray) or anti-CD25 (black), as before. (D) OT-1 cells were adoptively transferred into B6 mice. A group of mice was left untreated (nontolerized) while anergy was established as described before in an additional group of mice (tolerized). Ten days later, these mice were challenged with OVA and treated with either control rat IgG or anti-CD137 (2A). Five days following the OVA rechallenge, H-2K^b/OVA-positive OT-1 cells were sorted by FACS and used as effectors in a 4-hour ⁵¹Cr-release assay for cytotoxicity against OVA-pulsed EL4 and unpulsed EL4 at the indicated E/T ratios. Naive OT-1 cells, freshly purified from an OT-1 mouse, were also used as effector cells.