Multi-parameter analysis of skin and blood T cell phenotype was performed on LSR II or BD Fortessa using FACS Diva software (both BD Biosciences, Oxford, UK) and further analyzed using FlowJo software (TreeStar, Inc) as previously described (Agius et al., 2009 (link); Vukmanovic-Stejic et al., 2013 (link)). PBMCs or skin cells were stained with different combinations of antibodies including CD3, CD4, CD8, CD45RA, CD28, CD27 (BD Biosciences) Ki67, CLA, CCR7 and PD-1 (clone EH12.2H7; Biolegend, London, UK). All surface staining was performed for 30 minutes on ice. Isotype control staining and FMO controls were used to set the quadrants. Ki67 staining (clone B56, BD Bioscience) was performed by intracellular staining using the Foxp3 Staining Buffer Set (Miltenyi Biotec, Bisley, UK). For intracellular cytokine staining cells were stimulated with VZV lysate (Virusys corporation, Taneytown, MD) or SEB as positive control and incubated for 15 hours at 37°C, 5% CO2 in the presence of 5 μg/ml Brefeldin A (Sigma-Aldrich, Gillingham, UK). Unstimulated controls were always included. Following stimulations cells were stained for surface markers for 30 minutes at 4°C, washed, fixed and permeabilised (Fix & Perm Cell Permeabilisation Kit, Invitrogen, Paisley, UK) before staining for IL-2, IFN-γ and TNF-α (all from BD Biosciences, Oxford, UK).