Env-specific CD8 T Cell Responses Analysis

Env-specific CD8 T cell responses were analyzed by ICS assay and flow cytometry using previously described protocols (7 (link), 29 (link)). Briefly, 4 × 10⁶ splenocytes or 10⁶ cells from DLNs were stimulated with 5 μg/ml of clade C HIV-1 Env-1 peptide (a H-2^d-restricted CTL epitope with the sequence PADPNPQEM as previously described (30 (link)) along with 1 μl/ml GolgiPlug (BD Biosciences), anti-CD107a-Alexa 488 (BD Biosciences) and monensin (1X; eBioscience), in RPMI 1640-10% FCS at 37°C for 6 h in a 96-well plate. Next, the cells were washed, stained for the surface markers, fixed, permeabilized (Cytofix/Cytoperm kit; BD Biosciences) and mixed with anti-CD16/CD32 (BD Biosciences) for blocking the Fc receptors. Cells were stained intracellularly with antibodies against intracellular cytokines conjugated with the appropriate fluorochromes. Live cells were selected using the violet LIVE/DEAD stain kit (Invitrogen). Cells were stained with the following conjugated antibodies: CD3-PE-CF594, CD4-APC-Cy7, CD8-V500, IFN-γ-PE-Cy7, IL-2-APC, and TNF-α-PE and then acquired through a GALLIOS flow cytometer (Beckman Coulter). Data analysis was carried out with FlowJo (Version 10.4.2; Tree Star, Ashland, OR) and the response to specific stimuli was obtained after corrections made with values obtained in unstimulated control samples.