Anti cd8 af700

The PSC7A nanovaccine was made by physically mixing E7 peptide and PSC7A NPs. Non-degradable PC7A based nanovaccine was used for comparison. Six- to eight-week-old C57BL/6 mice were injected subcutaneously at the tail base with PBS, E7p only (0.5 μg), PSC7A NP only (30 μg), PC7A nanovaccine (0.5 μg E7p in 30 μg PC7A NP), or PSC7A nanovaccine (0.5 μg E7p in 30 μg PSC7A NP) three times in 6 day intervals. At 24 h after the last administration (on day 13), the mice were sacrificed. Their inguinal lymph nodes were harvested, dispersed into single-cell suspensions, and stained with the indicated antibodies for flow cytometry analysis. The following primary antibodies were used for staining: anti-CD3-APC (Biolegend, cat. No. 100235, clone 17A2), anti-CD45-PerCP (Biolegend, cat. no. 103129, clone 30-F11), anti-CD4-FITC (Biolegend, cat. no. 100405, clone GK1.5), anti-CD8-AF700 (Biolegend, cat. no. 100729, clone 53-6.7), anti-H-2D^b/HPV16 E7 (RAHYNIVTF) MHC Tetramer-PE (Immudex, cat. no. JA2195), anti-CD11c-FITC (Biolegend, cat. no. 117305, clone N418), and anti-CD80-PE/Cy7 (Biolegend, cat. no. 104733, clone 16-10A1). Flow data were acquired on a BD LSRFortessa™ Flow Cytometer and analyzed using FACSDiva 8.0.1 software.

After fixation, cells were collected by centrifugation at 1200 rpm for 5 min, formaldehyde blocked by washing the cells with 200 μL of PBS containing 5 mg/ml BSA (PBSA) and collected again by centrifugation at 1200 rpm for 5 min. Then, cells were resuspended and permeabilized in ice-cold methanol for 20 minutes on ice. Cells were then fluorescently barcoded (Krutzik and Nolan, 2006 (link)) using a combination of different concentrations of amino-acid reactive dyes (PacificBlue #10163, DyLight800 #46421, Thermo Scientific). Finally, cells were pooled and stained with anti-CD3^BV510 (Biolegend, #300448), anti-CD4^PE (Biolegend, #357404), anti-CD8^AF700 (Biolegend, #300920), anti-pSTAT1-Tyr701^AF647 (Cell Signaling, #8009S), anti-pSTAT1-Ser727^AF488 (Biolegend, #686410), anti-pSTAT3-Tyr705^AF488 (Biolegend, #651006) and anti-pSTAT3-Ser727^AF647 (Biolegend, #698914). Cells were analyzed in a CytoFlex S flow cytometer (Beckman Coulter) with the individual cell populations being identified by their barcoding pattern and mean fluorescence intensity (MFI) for the different forms of STAT1 or STAT3 measured.