Polyfunctional analysis of T-cell responses to the AAV-1 capsid. Concurrent expression of IL-2, IFN-γ, perforin, TNF-α, and CD107 is measured at baseline and after gene transfer. Pie charts represent the proportion of capsid-specific CD4⁺ (left) or CD8⁺ (right) T cells expressing 1, 2, 3, 4, or 5 markers. Cells were gated on forward and side scatter, CD3⁺ cells, CD14⁻CD16⁻CD20⁻ cells. Cells were also stained with an Invitrogen amine reactive live/dead aqua dye. CD4⁺ or CD8⁺ T cells were analyzed for IL-2, IFN-γ, perforin, TNF-α, and CD107 expression in response to AAV-1 peptides against medium control.