Unique role of TLR9-MyD88 signaling in CD8⁺T-cell responses to the AAV capsid. (A) Dose response of AAV2-SIINFEKL in WT mice (n = 4/group) following IM injection. Anti-capsid CD8⁺ T-cell responses were assessed in peripheral blood on days 7, 10, 14, and 21 by flow cytometry with an H2-K^b–SIINFEKL tetramer. (B) In vivo cell killing assay to demonstrate functionality of anti-capsid CD8⁺ T cells. Representative histograms show preferential killing of SIINFEKL peptide pulsed, 3 μM CTV-labeled splenocytes adoptively transferred into mice positive for anti-capsid CD8⁺ T cells (tetramer⁺). Control unpulsed, 0.3 μM CTV-labeled splenocytes were not killed. No killing was observed in naïve mice that were negative for anti-capsid CD8⁺ T cells. Percentage killing (n = 2/group) of SIINFEKL peptide-pulsed splenocytes by tetramer⁺ or naïve mice is indicated. (C) WT, TLR2^−/−, TLR9^−/−, or MyD88^−/− mice were IM injected with 1 × 10¹¹ vector genomes (vg) of AAV2-SIINFEKL (n = 4/group). Anti-capsid CD8⁺ T-cell responses were assessed in peripheral blood by flow cytometry as a function of time. Significant differences compared with WT mice are indicated. (D) Examples of peak responses for individual mice. (E) WT or AP3^−/− mice were IM injected with AAV2-SIINFEKL, and anti-capsid CD8⁺ T-cell responses were assessed in peripheral blood over time (n = 4/group). (F) WT or STING^−/− mice were IM injected with AAV2-SIINFEKL, and anti-capsid CD8⁺ T-cell responses were assessed in peripheral blood 8 days post-injection (n = 4/group). The dotted line at 0.1% represents the limit of detection of capsid-specific CD8⁺ T cells using the tetramer. Data points are averages ± SEM. Statistically significant decreases compared with responses in WT mice are indicated. P values: (B) **P =.005; (C) day 7, *P =.03 for WT vs MyD88^−/− and *P =.049 for WT vs TLR9^−/−; (C) day 10, **P =.004 for WT vs MyD88^−/− and **P =.006 for WT vs TLR9^−/−; (E) *P =.045. IM, intramuscular(ly).