Cd8 bw135 80

CD4⁺ T cells and NK cells from fresh or frozen PBMC, pLN and spleen were isolated using,a positive selection with respectively, the anti-human CD4 (CD4 MicroBeads, human Miltenyi Biotec (USA)), or the anti-NKG2a/c monoclonal antibody-PE conjugated (clone Z199 Beckman Coulter (USA)) reveals by anti-PE microbeads (Miltenyi Biotec (USA), the staining and the positive selection of the cells was done according to the supplier’s instructions.
Eight-color panels were used to phenotype, surface stain and sort NK cells from blood and pLNs from chronically infected AGMs. Cells were thawed in 20% FBS-containing media supplemented with benzonase nuclease, and counts and viabilities were performed (Life Technologies). Cells were washed and stained with Aqua Live/Dead stain (Molecular Probes). Cells were washed and blocked using normal mouse IgG (Caltag). For the NKG2_a/c^HIGH/lowCD16^+/−phenotyping panel, PBMCs and pLNs were surface stained for CD3 (SP34.2, 1:10 dilution; BD), CD8 (BW135/80, 1:20 dilution; Miltenyi), CD16 (3G8, 1:20 dilution; Beckman Coulter, Inc.), NKG2a/c (Z199, 1:20 dilution; Beckman Coulter, Inc.), CD20 (2H7, 1:20 dilution 1/20; Biolegend), and CD14 (M5E2, 1:25 dilution; BD). Post-staining, cells were washed, filtered, and sorted on a FACS ARIA II (BD). Cells were directly collected in a lysis buffer that contained TCEP. The purity of the cells was >97%.

CTLs induced by stimulation with the CXorf48-derived peptides were washed with PBS (Sigma), stained with antibodies against CD3 (33-2A3; Immunostep, Salamanca, Spain), CD8 (BW135/80; Miltenyi Biotec), and dextramer specific for HIV-derived peptide or CXorf48-derived peptide (Immudex, Copenhagen, Denmark). The resulting cells were analyzed using flow cytometry (LSR II; BD Biosciences, Franklin Lakes, NJ, USA).