Effects of AAV capsid-specific CD8⁺ T cells on systemic liver-derived hF.IX expression in vivo. (A) Diagram of in vivo model and experimental outline: In vitro expanded capsid-specific, L^d-restricted CD8⁺ T cells (cap-CD8) isolated from BALB/c mice were adoptively transferred by tail vein injection into Rag-1^−/− BALB/c mice 24 hours after administration of AAV-ApoE/hAAT-hF.IX vector (1 × 10¹¹ vg per mouse; n = 4 per experimental group). As negative control cells, CD8⁺ T cells specific for the influenza dominant epitope (con-CD8) were generated in BALB/c mice and in vitro expanded in parallel. Mice received cap-CD8, con-CD8, or no cells as indicated in each panel. Following T- cell transfer, mice were treated with LPS on days 0, 1, and 2 to provide an additional activation signal. Systemic hF.IX levels were measured as a function of time after administration of the following vectors: AAV2 (B), AAV2 (without LPS administration) (C), AAV2 in absence or presence of proteasome inhibitor bortezomib (PI) (D), or AAV2(Y-F) in absence or presence of PI (E). Fold differences of hF.IX levels for mice treated with con-CD8 and cap-CD8 are indicated (except for panel B, which is no CD8 compared with cap-CD8). Data are average ± standard deviation (SD) for each time point with n = 5 per experimental group. *P < .05; **P < .01 using Student t test for each time point. (F) Systemic hF.IX levels as a function of time after AAV2(Y-F) vector administration to Rag-1^−/− BALB/c mice that received cap-CD8 cells at the indicated time points or no cells (control; n = 4 per group).