Cell enumeration was performed in whole blood samples using Flow-Count™ beads (Beckman Coulter, UK) according to manufacturer’s instructions. After red blood cell lysis, mononuclear cells were stained with anti-CD3/eFluor 450, anti-CD4/FITC and anti-CD8a/PerCP-Cy5.5 monoclonal antibodies (mAb) (all eBioscience, USA). Expression of PD-1, KLRG1 and LAG-3 was assessed in whole blood samples after staining with anti-CD3/eFluor 450, anti-CD8a/PerCP-Cy5.5, anti-PD-1/FITC, KLRG-1/APC and anti-LAG-3/PE mAbs (all eBioscience). Pentamer analysis was performed as previously described [14 (link)], using H-2Kb/SIINFEKL or H-2Kb/SVYDFFVWL Pro5 pentamer/PE (ProImmune, UK).
To assess peptide-induced intracellular accumulation of IFN-γ by CD8+ T cells, splenocytes were stimulated with 1 μg/mL SVL peptide, 0.5 μg/mL co-stimulatory anti-CD28 antibody (eBioscience) in the presence of GolgiPlug (BD Biosciences, Belgium) for 5 h prior to fixation, permeabilization, and staining with anti-CD3/eFluor 450, anti-CD8a/PerCP-Cy5.5 and anti-IFN-γ/PE mAbs (eBioscience).
Samples were analysed using a FACSCantoII (BD Biosciences) and FACSDiva (BD Biosciences) or FlowJo (Treestar, OR) software.
To assess peptide-induced intracellular accumulation of IFN-γ by CD8+ T cells, splenocytes were stimulated with 1 μg/mL SVL peptide, 0.5 μg/mL co-stimulatory anti-CD28 antibody (eBioscience) in the presence of GolgiPlug (BD Biosciences, Belgium) for 5 h prior to fixation, permeabilization, and staining with anti-CD3/eFluor 450, anti-CD8a/PerCP-Cy5.5 and anti-IFN-γ/PE mAbs (eBioscience).
Samples were analysed using a FACSCantoII (BD Biosciences) and FACSDiva (BD Biosciences) or FlowJo (Treestar, OR) software.