Cd8 apc cy7 clone 53 6.7

Lymphocytes were isolated and processed from the peripheral blood as previously described (5 (link)). Cells were stained with CD8, CD44, CD62L, KLRG1, and MHC class I peptide tetramer to LCMV-GP33 (KAVYNFATC) (Beckman Coulter) as described previously (28 (link),29 (link)). Intracellular cytokine staining was performed after 5 hr of ex vivo stimulation with either LCMV epitope D^bNP_396-404 or D^bGP_33–41 peptide, HIV and Ag85B pooled peptides depending on the study as described (5 (link),27 (link)). The following antibodies were used for surface staining: LIVE/DEAD Fixable Violet Dead Cell stain kit (Invitrogen), CD4 (FITC; clone RM4–5; ebioscience), CD8 (APC-Cy7; clone 53–6.7; BD Biosciences); CD44 (A700; clone IM7; Biolegend). For intracellular staining the following antibodies were used: IFN-γ (APC; clone XMG1.2; Biolegend), TNF-α (PE; clone MP6-XT22; ebioscience), CD3 (PerCP/Cy5.5; clone 145-2C11; Biolegend); IL-2 (PeCy7; clone JES6-SH4; ebioscience). All data was collected using a LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR) and SPICE v5.2 (free available from http://exon.niaid.nih.gov/spice/). Boolean gating was performed using FlowJo software to examine the polyfunctionality of the T cells from vaccinated animals.

Mononuclear cell isolation from recipient spleen and liver was carried out as previously described [12 (link)]. Standard flow cytometric surface staining was used. For intracellular cytokine staining, cells were stimulated with 50ng/ml PMA (Sigma-Aldrich), 500 ng/ml ionomycin (Sigma-Aldrich) and 0.7 μl/ml Golgi Plug (BD Biosciences), and incubated at 37 °C for 4 hours before staining as recommended by the manufacturer. The flow antibodies used were as follows: CD4-V450 (clone RM4-5, BD), CD8-APC-Cy7 (clone 53-6.7, BD), B220-PE (clone RA3-6B2, eBioscience), H2K^b-APC (clone AF6-88.5.5.3, eBioscience), H2K^q-Alexa Fluor 647 (clone KH114, Biolegend), CD45.1-FITC (clone A20, BD), IFNγ-Percp-Cy5.5 (clone XMG1.2, eBioscience), IL-4-PE (clone 11B11, BD), IL-5-PE (clone TRFK5, eBioscience), IL-17-PE-Cy7 (clone TC11-18H10.1, Biolegend), CXCR3-Biotin (clone CXCR3-173, eBioscience), PD-1-PE (clone J43, eBioscience), CXCR5-PE-Cy7 (clone SPRCL5, eBioscience), Ly9.1-Biotin (clone 30C7, BD), Streptavidin-PE-Cy7 (BD), and Streptavidin-APC-Cy7 (BD). Stained cells were analyzed using Diva software, LSR II (BD Biosciences, San Jose, CA) and FlowJo (TreeStar, Ashland, OR).