OT-1 activation following intravenous administration of OVA peptide. C57BL/6 mice were given OT-1 cells intravenously. Twenty-four hours later, the mice were given 0.5 mg of either a control peptide or OVA peptide intravenously. (A) Two days following the administration of peptide, 2 mice in each group were killed and the pooled spleen and lymph node cells were stained with both H-2K^b/OVA tetramer and anti-CD8. The percent of CD8⁺ cells that were tetramer-positive (gates as indicated) is shown. Cells were subsequently stained with anti-CD69 or anti-CD25 (black) and an isotype control (gray). Forward scatter (FSC), CD69, and CD25 expression was analyzed in the gated cells. (B) Ten days following the control or OVA peptide injection, spleen cells harvested from the recipient mice were stained with anti–VLA-4 (black) or an isotype control (gray) in conjunction with both H-2K^b/OVA tetramer and anti-CD8. The expression of VLA-4 was analyzed in the gate of tetramer-positive CD8⁺ OT-1 T cells.