In vitro cytokine secretion, activation, and proliferation by lymphocyte cultures from DO11.10 TCR transgenic BALB/c mice at 30 days after vector administration. Splenocytes or LN cells from naive controls or AAV-CMV-OVA intramuscularly injected mice were cultured in 6-well plates (5 × 10⁶ cells/well) for 3 days and stimulated with 100 μg/mL OVA or mock stimulated. Conditioned media were collected at day 3 of cell culture and analyzed by cytokine-specific ELISA for IL-2 (A,D), IL-10 (B,E), and IFN-γ (C,F). (A-C) Splenocyte cultures. (D-F) LN cell cultures. (G) Summary of percentage CD69⁺ of CD4⁺TCR⁺ cells for lymphoid organs of naive controls and vector-injected mice as determined by flow cytometry. Splenocytes (H) and LN cells (I) were also cultured (5 × 10⁵ cells/well in 96-well plates) overnight with OVA stimulation (100 μL/mL) or without OVA stimulation (mock), and pulsed with ³H-thymidine for an additional 12 hours. Results for ³H-thymidine incorporation are shown in CPM. LNs were inguinal and popliteal LNs. D-LN indicates draining LNs of vector-injected leg muscle; ND-LN, nondraining LN of noninjected contralateral leg. Results from D-LNs are marked by vertical arrow. All experimental conditions for panels A-I (including sizes per group and determination of average ± SD) were as described for Figure 3.