Anti cd4 bv711 clone rpa t4

To assess cell surface expression of proteins, flow cytometry was performed. Cells were washed with PBS and subsequently stained with the viability dye LIVE/DEAD Fixable Near-IR (Life Technologies) and with the antibodies anti-CD3-BUV395 (clone UCHT1, BD), anti-CD4-BV711 (clone RPA-T4, Biolegend), anti-CD155-PE (clone SKIL4, Biolegend), anti-HLA-ABC-Pe-Cy7 (clone W6/32, Biolegend), anti-HLA-E-BV421 (clone 3D12, Biolegend), anti-tetherin-APC (clone RS38E, Biolegend) and anti-IgG1-PE isotype control (clone MOPC21, Biolegend) for 15 min at RT. After washing the cells with PBS, an intracellular staining was performed. In brief, cells were incubated in BD Cytofix/Cytoperm for 20 min at 4°C, washed with BD Perm/Wash buffer and stained with anti-p24-FITC (clone KC57, Beckman Coulter) for 20 min. After another washing step, cells were fixed in BD Cellfix and analyzed by flow cytometry (BD LSR Fortessa). HIV-1-infected cells were defined as p24 Gag⁺ CD4^dim and uninfected as p24 Gag^- CD4⁺ cells. Cells infected with HIV-1 ΔNef mutant viruses were defined as p24 Gag⁺ and tetherin^- cell, as Nef-deficient viruses are not able to downregulate CD4.

PBMCs were suspended at 1 x 10⁶/mL in PBS and incubated at 37^°C for 20 min with 0.5 uM carboxyfluorescein succinimidyl ester (CFSE; Life Technologies). After the addition of serum and washes with PBS, cells were resuspended at 1 x 10⁶/mL and plated into 96-well U-bottom plates (Corning) at 200 uL volumes. Peptide pools were added at a final concentration of 0.25 ug/mL. On day 6, cells were harvested, washed with PBS + 2% Fetal Bovine Serum, and stained with anti-CD3-PE-Cy7 (clone SK7; BioLegend), anti-CD8 APC (clone SK1; BioLegend), anti-CD4 BV711 (clone RPA-T4; BioLegend) and LIVE/DEAD violet viability dye (Life Technologies). Cells were washed and fixed in 2% paraformaldehyde, prior to flow cytometric analysis on a BD LSR II (BD Biosciences). A positive response was defined as one with a percentage of CD3⁺ CD8⁺ or CD3⁺ CD4⁺ CFSE low cells at least 1.5x greater than the highest of two negative-control wells and greater than 0.2% CD8⁺ or CD4⁺ CFSE low cells in magnitude following background subtraction. For graphical analyses, responses are plotted at a value of 0.1% CD8⁺ or CD4⁺ CFSE low cells.