Involvement of recycling pathway in NS3 cross-presentation by IFN-DCs. (A) IFN-γ ELISPOT analysis of DCs pulsed with NS3 protein for 1 hour and then washed, fixed with 0.05% glutaraldehyde, and used in a cross-presentation assay with NS3_1406-1415–specific CD8⁺ clone. The results are expressed as mean ± SD of 4 independent experiments. (B) CLSM detection of MHC class-I molecules (green) and Rab11 (red) in unstimulated DCs (CTR) and DCs allowed to internalize NS3 protein (15-minute pulse, 30-minute chase). Costaining of MHC-I with Rab11 was detected in yellow. Insets represent separate channel images. A representative experiment of 3 is shown. (C-D) Evaluation of IFN-γ–forming spots in cocultures of DCs treated with primaquine either before and during 16 hours of NS3 pulsing and then washed and used in a cross-presentation assay with NS3_1406-1415–specific CD8⁺ clone (mean ± SD of 5 independent experiments). Statistical analysis in panels A, C, and D was performed by Mann-Whitney test (*P < .05).