Antigen-specific responses in LNs draining SIV gag and RhCMV pp65 immunization sites. (A) Composite result for IFN-γ mRNA levels in LNs draining SIV gag or RhCMV pp65 immunization sites for each animal on indicated time points. Total RNA from LNMCs was collected on the indicated days post immunization and subject to real-time PCR for the quantitation of IFN-γ mRNA. The results are expressed as the number of copies of IFN-γ mRNA per μg total RNA. Standard deviation of the mean for triplicate analyses for Day 3 LNs draining SIV gag and RhCMV immunization sites are shown. (B and D) Intracellular cytokine staining (ICS) for SIV gag-specific CD4+ and CD8+ T cell responses in day 3 LNMCs. Day 3 post immunization LNMCs draining either SIV-gag (top panels, B) or RhCMV-pp65 (bottom panels, B) immunization sites were stimulated with SIV gag 15-mer overlapping peptide pools and stained for IL-2, TNF-α, and IFN-γ. SIV-specific cytokine-producing CD4+ and CD8+ T cells were analyzed by multi-color flow cytometry. (C and E) Intracellular staining of RhCMV pp65-specific CD4+ and CD8+ T cell responses in day 5 LNMCs. Day 5 post immunization LNMCs draining either SIV-gag (top panels, C) or RhCMV-pp65 immunizations (bottom panels, C) were stimulated with RhCMV pp65 15-mer overlapping peptide pools and stained for IL-2, TNF-α, and IFN-γ. The composite results for percent of SIV-specific cytokine producing CD8+ (F) and CD4+ (G) T cells in LNMCs draining SIV gag immunization sites for one macaque at indicated days post immunization are shown. Cytokine producing T cells in LNMCs without stimulation (background) were subtracted from all flow analyses.