Anti cd4 qdot655

Epitope-specific CD8 T cells were expanded as described above and rested in R-10 media without IL-2 for 24 hours at 37°C/5%CO₂. Target CD4 T cells were isolated from HIV-1 seronegative individual matched for the HLA-I allele of interest and were activated with PHA (5μg/ml) and IL2 (50U/ml) for two days. Activated target cells were cultured with or without cognate NAE or AE peptide for one hour. Next, NAE or AE specific CD8 T cell lines were co-cultured with CD4 T cell targets in duplicate at 0:1, 1:1, and 3:1 effector to target (E/T) ratios for 24 hours. After incubation, the co-cultured cells were surface stained with anti-CD3-Pacific Blue and anti-CD4-Qdot655 (all Invitrogen) at 4°C for 30 min. The cells were then labeled with 7-aminoactinomycin D (7AAD, BD Biosciences) at 5pg/μl for 30 min at 4°C. Events were acquired on an LSR II flow cytometer to detect the apoptosis of CD4 T cells (7AAD⁺ CD4 T cells) as an indication of CD8 T cell mediated killing. Data for each E:T ratio was normalized to corresponding negative control at the same E:T ratio and then normalized to E:T at 0:1 for each line. The area under curve (AUC) value was calculated for the 7-AAD expression using GraphPad Prism (version 7.0) and was used for statistical analysis.

PBMCs with dual NAE and AE specific CD8 T cell responses were labeled with tetramers at room temperature for 30 min and then were stained at 4°C for 30 min with dead cell dye (Invitrogen), anti-CD3-Alexa 780 (eBioscience), anti-CD4-Qdot655 (Invitrogen), and anti-CD8-V500 (BD Pharmingen). At least 10⁶ total events were acquired on an LSR II flow cytometer (BD Immunocytometry Systems), and data were analyzed using FlowJo (version 9.6.4; TreeStar Inc.).