C57BL/6 mice were transduced with 5 × 10¹¹ vgc AAV-K^d or K^d-D227K. At intervals between 48 hours and 10 days after inoculation, leucocytes were isolated from liver, draining LN, and spleen, and CD3⁺CD8⁺ T cells were analyzed for expression of the activation markers CD44 and PD-1. (A) The frequency of PD-1^hi cells among CD8⁺ T cells from liver increased substantially during the first 10 days after inoculation in mice treated with AAV-K^d and, to a lesser extent, in mice transduced with K^d-D227K. n = 3–6 mice/group; representative plots shown. (B) Data were analyzed using a 2-way ANOVA with Sidak’s and Tukey’s post tests; the proportion of activated (CD44⁺PD-1^hi) CD8⁺ T cells in liver peaked at 36.8% ± 2.0% on d10 after inoculation with AAV-K^d compared with 1.2% ± 0.3% on d2 (P < 0.0001). In mice that had received AAV-K^d-D227K, the proportion of CD44⁺PD-1^Hi CD8⁺ T cells was 9.5% ± 3.8% on d10, compared with 0.95% ± 0.52% on d2 (P = 0.014, n = 3–6/group). From d4–10, the proportion of activated CD8⁺ T cells was significantly greater in the livers of mice transduced with AAV-K^d than those treated with AAV-K^d-D227K (P < 0.0001). (C) Parallel changes were noted in splenocytes and LN cells (not shown) from inoculated mice (n = 3–6/group), although the proportions of activated CD8⁺ T cells in spleen or LN were much smaller than those in liver, and the peak proportions of activated CD8⁺ T cells were attained on d7 (0.85% ± 0.08% for AAV-K^d–transduced mice, 0.34% ± 0.7% for mice receiving AAV-K^d-D227K, and 0.11% ± 0.02% in untransduced mice). Statistical analysis employed 2-way ANOVA with Sidak’s and Tukey’s post tests. Box shows minimum to maximum with a line at the mean. Data are described as mean ± SEM.