Hepatic gene transfer (3 × 10¹² vg's AAV-EF1α-ova/animal, n = 4) in BALB/c mice 24 hours after adoptive transfer of 5 × 10⁶ CD4⁺ T cells from DO11.10 transgenic mice. Mice were killed at day 10, and pooled splenocytes were cultured. (A) IL-2 cytokine release upon in vitro stimulation with ova peptide ISQAVHAAHAEINEAGR as a function of time. Control cells were from mice (n = 4) that received vehicle control (5% sorbitol in HBS) instead of vector 24 hours after adoptive T-cell transfer. (B) TUNEL assay to measure apoptosis of DO11.10 TCR⁺ cells after 36 hours of in vitro culture. Shown are cells from AAV-EF1α-ova–transduced mice. Gated lymphocytes were analyzed for TCR⁺ and Fas⁺ (CD95⁺) cells. TCR⁺ were subsequently analyzed for incorporation of Cy5-labeled nucleotides (terminal transferase reaction to detect DNA fragmentation) to determine percent apoptotic cells. (C) Summary of flow cytometry results. Percent apoptotic cells of TCR⁺ cells after in vitro culture in mock, ova, ova peptide (Peptide), or dexamethasone (Dexa, positive control for induction of apoptosis) containing media are shown.