Antigen in nonlymphoid tissue regulates expression of CD69 during VacV infection. (A) Naive Thy1.1 P14 CD8⁺ T cells were transferred into B6 mice and co-infected with VacV-GP33 on the left ear and VacV-OVA on the right ear. On days 7, 10, and 15 after infection, P14 CD8⁺ T cells in the left ear, right ear, and spleen were analyzed for frequency of CD127^Hi/KLRG1^Lo cells. (B) Quantification of the percentage of CD127^Hi, KLRG1^Lo cells from A. (C) Same as A, except frequency of P14 CD8⁺ T cells expressing CD69 was determined on days 10, 15, and 40 after infection. (D) Quantification of C. (E) Same as C, except frequency of CD103-expressing cells was determined. (F) Quantification of E. Data are representative of three independent experiments of three to five mice per group. *, P < 0.001; **, P < 0.0001, paired Student’s t test.