Quantification of SIV-specific CD8+ T Cells

SIV-specific CD8⁺ T cells were quantified in mononuclear cells isolated from blood and LN by flow cytometric intracellular cytokine analysis, as previously described in detail^{45 (link)}. Briefly, mixes of sequential (11 amino acid overlapping) 15-mer peptides (AnaSpec) spanning the SIVmac239 Gag, Env, Pol, Nef, Rev/Tat, Vif/Vpr/Vpx proteins were used as antigens in conjunction with anti-CD28 (BD Biosciences) and anti-CD49d co-stimulatory mAb (BD Biosciences). Cells were incubated at 37 °C with peptide mixes/pools and co-stimulatory antibodies for 1 hr, followed by an additional 8 hr. incubation in the presence of Brefeldin A (5 μg/ml, Sigma-Aldrich). Co-stimulation in the absence of peptides served as background control. Cells were stained with fluorochrome-conjugated monoclonal antibodies [CD3 (SP34-2), CD4 (L200), CD8a (SK1), IFN-γ (B27)(BD Biosciences), TNF (MAB11, eBioscience), CD69 (FN50, BD Biosciences)], and data were collected on an LSRII (BD Biosciences) and analyzed using the FlowJo software program (version 8.8.7; Tree Star, Inc.). Response frequencies to each peptide mix were defined by the intracellular expression of TNF and/or IFN-γ by CD3⁺, CD4⁻, CD8⁺ T cells after subtraction of background. Response frequencies to each peptide mix were added to obtain total SIV-specific response frequencies.