Characterizing SIV Env-specific CD4 T Cells

SIV Env-specific CD4 T cells were characterized as previously described (33 (link)). Briefly, 1 × 10⁶ to 2 × 10⁶ splenic and iliac LN mononuclear cells were stimulated for 5 h with 1 μg/ml of an overlapping SIV_mac239 Env peptide pool (NIH AIDS Reagent Program) in the presence of GolgiStop and GolgiPlug (BD Biosciences). Cells were washed three times in autoMACS rinsing solution (Miltenyi Biotec) and stained with vital exclusion dye (Life Technologies) for 10 min at 4°C. Cells were again washed and treated with Cytofix/Cytoperm (BD Biosciences) for 20 min at 4°C. All subsequent washes and stainings were performed by using BD Perm/Wash (BD Biosciences). Cells were incubated with an interleukin-21 (IL-21) receptor (IL-21R)/Fc fusion protein (R&D Systems) for 30 min at 4°C, washed three times, and stained with goat anti-human Fcγ antibody (Jackson ImmunoResearch Laboratories) for 30 min at 4°C. Cells were subsequently washed three times and stained with anti-CD4 (RM4-5), anti-CD8a (53-6.7), anti-CD44 (IM7), anti-gamma interferon (IFN-γ) (XMG1.2), and anti-IL-2 (JES6-5H4) for 30 min at 4°C. Samples were washed three additional times, fixed in 2% formaldehyde, and stored at 4°C. Samples were acquired on an LSR II flow cytometer (BD Biosciences) and analyzed by using FlowJo v9.8.3 (TreeStar).