Permeabilisation buffer

All flow cytometry was performed on a Fortessa LSR II flow cytometer (Becton Dickinson) using DIVA software (Becton Dickinson) and analysed using Flow-jo (Treestar, Ashland, OR) software. Prior to surface staining cells were incubated with mouse Fc Block (Biolegend) for 5 minutes in the dark at 4°C, after which 50μL of the relevant antibody cocktail was added, and the cells were left to incubate in the dark at 4°C for 30 minutes. Surface antibodies were FITC – F4/80, APC- CD11b, PE-ABCA1 (Santa Cruz Biotechnology, Heidelberg, Germany) FITC-TF (Biorbyt, Cambridge, United Kingdom) or FITC-TF (American Diagnostica). After surface staining cells were resuspended in 200μl pre-diluted Near IR live/dead stain (Life Technologies) and left to incubate in the dark at 4°C for 15 minutes. For intracellular staining, cells were permeabilised with Foxp3 intracellular staining permeabilisation solution for 30 minutes (e-Bioscience). Intracellular staining was performed using directly conjugated antibodies (BV605-CD206 (Biolegend) and PE-Cy7-iNOS (e-Bioscience)) made up into a staining cocktail using permeabilisation buffer (e-Bioscience), 50μL of staining cocktail was added per well and staining took place at 21 °C in the dark.

HEKn were fixed in 4% PFA, blocked in 10% Goat serum, washed in permeabilisation buffer (eBiosciences, Hatfield, UK) and incubated with the anti-VZV-FITC (Millipore, Watford, UK) for 30 min in the dark. Cells were subsequently washed in permeabilisation buffer, resuspended in PBS, processed by FACS Calibur and analysed using FloJo (v7.6.5).

Flow cytometric analyses were conducted on a Canto II or LSR II (BD Biosciences) and data were analysed using FlowJo software (Tree Star, Ashland, OR). Hoechst 33258 (Sigma) or LIVE/DEAD Fixable Violet or Near-IR Dead Cell Stain kit (Invitrogen) was used to exclude dead cells in all samples analysed. Anti-CD16/32 antibody (2.4G2) was included in each staining at 10 μg/ml to block unspecific antibody binding via Fc receptors. All antibodies were purchased from Biolegend or eBioscience. For intracellular cytokine staining, cells were fixed in 4% PFA and intracellular staining with fluorochrome-labelled antibodies was performed in Permeabilisation Buffer (eBioscience) according to the manufacturer’s protocol. Quantification of T cell numbers was performed using fluorochrome-labeled microbeads (CountBright absolute counting beads, Life Technologies). For intracellular pAkt staining cells were stained with surface markers, fixed in 4% paraformaldehyde, permeabilised in 90% ice-cold methanol and stained with pAkt^S473 (#4060) and pAkt^T308 (#13038) and a secondary anti-rabbit Alexa647 coupled antibody (#4414) (Cell Signalling Technologies). For viability assays, cells were stained with propidium iodide or 7-AAD together with Annexin V. Viable cells were defined as PI/7-AAD negative and AnnV negative. Data were analysed using Flow Jo software (Tree Star Inc.; CA, USA).

For analysis of transcription factors, cells were resuspended in 400 μl fixation/permeabilisation buffer (eBioscience) and incubated at 4 °C for between 1 and 18 h. Following incubation, cells were resuspended and washed twice in 1 ml permeabilisation buffer (eBioscience). In total 50 μl of antibody or isotype control (diluted in permeabilisation buffer) was added to each sample. Cells were resuspended by gentle vortex and incubated at room temperature for 30 min. Finally, cells were washed in 2 ml of FACS buffer and resuspended in 200 μl FACS buffer for acquisition.

KRT5⁺ cells in media (PromoCell, supplemented as previously) were seeded (3.0 ×10⁴/ well) on a collagen I (PureCol, Advanced BioMatrix, #5005) coated 96-well μ-plate (Ibidi). When confluent, the cells were fixed in 4% PFA (Electron Microscopy Sciences, #RT-15710) for 20 min, washed in PBS, then permeabilised with permeabilisation buffer (eBioscience; #00-8333-56) for 20 min (room temperature). The cells were blocked with 2% BSA in PBS for 30 min (room temperature) then incubated with primary conjugated antibodies to KRT5 overnight at 4 °C. Cell nuclei were counterstained with DAPI (Sigma-Aldrich #D9542; 1:2000). Imaging was carried out with a Leica SP5 inverted confocal microscope. Images were acquired using LAS AF (Leica) and analysed using Fiji v1.52p software^{81 (link)}.

We used mAbs to evaluate the expression levels of CD4, CD25, CD69, and Ki-67 in cultured CD4+ T cells. To assess the expression of molecules on cell membranes, the cells were stained for 30 min at 4 °C in the dark. The cells were then fixed with 2% p-formaldehyde in phosphate-buffered saline (PBS, 0 mM sodium phosphate, 0.15 M sodium chloride, pH 7.2). The cells were washed to evaluate intracellular Ki-67 staining. Next, the cell pellet was suspended in a fixation/permeabilisation solution (eBioscience, Thermo Fisher, Waltham, MA, USA) at 4 °C, washed with permeabilisation buffer (eBioscience, Thermo Fisher, Waltham, MA, USA), and stained with Ki-67 for 30 min at 4 °C. Then, it was washed and analysed using flow cytometry. In addition, cells used for the Fluorescence Minus One (FMO) condition were stained and acquired in parallel to identify the background levels of staining.
Data were collected using a FACS Aria II (BD Biosciences, San Jose, CA, USA) and analysed using FlowJo v10.2 (FlowJo LLC, Inc., Ashland, OR, USA). For each case, 50,000 events are recorded. More details on the antibodies used are listed in Table A1.