In vitro cytokine secretion, activation, and proliferation by lymphocyte cultures from DO11.10 TCR transgenic BALB/c mice at 10 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. (G) Summary of percent CD69⁺ of CD4⁺TCR⁺ cells for lymphoid organs of naive controls and vector-injected mice as determined by flow cytometry. Spleens from each animal were processed and analyzed individually. Results are average (5/group) ± SD. Results from LN cells represent average ± SD for 3 counts by flow cytometry with each count representing LN cells pooled from 1 to 2 animals. 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 counts per minute (CPM). All cell cultures were set up in quadruplicate. LNs were inguinal and popliteal LNs. D-LN indicates draining LNs of vector-injected leg muscle; ND-LN, nondraining LNs of noninjected contralateral leg. Results from D-LNs are marked by vertical arrow. Spleens from each animal were processed and analyzed individually. Results are average (5/group) ± SD. Results from LN cells represent average ± SD for 3 wells with each well representing LN cells pooled from 1 to 2 animals.