Induction of OT-1 anergy by OVA peptide in vivo. C57BL/6 mice that had been received OT-1 T cells were given PBS (naive) or 0.5 mg OVA (anergic) intravenously. Ten days later, the mice were rechallenged with either the control (–) or OVA (+) peptide. Two days following the rechallenge, the mice were killed and pooled spleen and lymph node cells were stained with H-2K^b/OVA tetramer and anti-CD8. (A) The total number of OT-1 cells present in each mouse was calculated and is shown as the mean (± SD) of 3 mice in each group. (B) Tetramer-positive and CD8⁺ cells in the tolerized mice that were positive for both tetramer and CD8 were stained with isotype control (gray) and anti-CD69 or anti-CD25 (black). (C) Ten days following the administration of PBS or OVA, spleens were harvested and restimulated in triplicate in the absence or presence of OVA peptide (1 ng/mL) in 96-well plates. At the time of restimulation, the frequency of OVA-specific T cells was determined by tetramer analysis. OVA-specific IFN-γ production was measured 72 hours later by ELISA. IFN-γ production was determined on a per cell basis as described in “Materials and methods.” Data shown are representative of at least 3 independently performed experiments.