Figure 3. Circulating Memory CD8⁺ T Cells Are Recruited to the Site of T_RM Activation and Are the Source of Secondary T_rm CD8⁺ T Cells.

(A–C) Mice were infected on the left ear skin with VACV and received control IgG or CD8 depleting antibody 35 days post-infection. Mice were then challenged with B8R_20–27, and the number of CD8⁺ T cells (A) and B8R_20–27-specific CD8⁺ T cells (B and C) was quantified 40 h after challenge. (B) Representative flow cytometry plots depicting B8R-specific CD8⁺ T cells in the challenged skin. (C) Quantification of (B).

(D) Mice were infected on the skin with VACV, and on day 35 after infection, B8R-specific CD8⁺ T cells in the blood were analyzed for expression of P- and E-selectin ligands. Naive CD8⁺ T cells (CD62L⁺/CD44⁻) were used as a negative control for selectin binding.

(E) Quantification of (D).

(F) Mice were infected on the skin with VACV and received control IgG or P-/E-selectin blocking antibodies before challenge with B8R_20–27 or control peptide on day 40 post-infection. The number of B8R-specific CD8⁺ T cells in the skin 40 h after challenge was determined by flow cytometry (see Figure S3).

(G) Mice were treated as in (A), except they were challenged three times with B8R_20–27 (as in Figure 2E) and analyzed by flow cytometry 10 days after the last peptide challenge.

(H) Quantification of the number of B8R-specific CD8⁺ T cells in (G).

(I) Quantification of CD103 and CD69 expression by B8R-specific CD8⁺ T cells identified in (G).

Statistical significance was determined using an unpaired t test. Error bars represent SEM. Data in (A)–(C) are representative of 2 independent experiments (n = 3–5). Data in (D)–(F) are representative of 3 independent experiments (n = 3–10). Data in (G)-(I) are pooled from 2 independent experiments (n = 4–5).