T-cell response to AAV capsid proteins. PBMCs were stimulated for 6 hours with a set of 15-mer peptides covering the AAV capsid protein sequence and cytokine responses assessed by polychromatic flow cytometry. (A) TNF-α response to capsid proteins in CD4⁺ and CD8⁺ T cells. Numbers in the top right corner indicate the percentage of responding CD4⁺ or CD8⁺ T cells. A minimum of 240 000 events were collected for each time point. (B) Immunophenotyping of responding TNF-α⁺ cells (red dots) compared with total CD4⁺ T cells (gray density plots). 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.