In vitro killing of AAV-pulsed murine hepatocytes by capsid-specific CD8⁺ T cells. (A) Percent death of target cells (adult BALB/c hepatocyte cell line H2.35), pulsed with either AAV2 or AAV2(Y-F), as a function of MOI (at effector to target ratio of 80:1). (B) Percent surviving hF.IX⁺ H2.35 hepatocytes (as determined by flow cytometry after completion of in vitro killing assay) pulsed with either AAV2 or AAV2(Y-F), as a function of MOI (at effector to target ratio of 80:1). (C) Percent hF.IX⁺ H2.35 hepatocytes transduced with either AAV2 or AAV2(Y-F) (in the absence of effector cells) as a function of MOI. Data are average ± SD for quadruplicate measurements. Panels are representative examples of at least 2 experiments. Statistically significant differences between AAV2 and AAV2(Y-F) for a specific target:effector ratio: *P < .05; **P < .01, unpaired, 2-tailed t test. (D-E) Effects of proteasomal inhibition on liver target cell death. Shown are percent dead H2.35 target cells that had been pulsed with either AAV2 or AAV2(Y-F) at MOI of 10⁴ vg per cell and treated with proteasomal inhibitor (PI). Liver cells were treated with 100 nM bortezomib (D) or 300 nM MG-132 (E). Controls include mock-transduced target cells and cells that were not treated with PI. Assays were again performed in quadruplicate.